Instructions for use

Title Adiponectin Inhibits LPS-Induced HMGB1 Release through an AMP Kinase and Heme Oxygenase-1-DependentPathway in RAW 264 Macrophage Cells

Author(s) Elfeky Mohamed Kaede Ryuji Okamatsu-Ogura Yuko Kimura Kazuhiro

Citation Mediators of inflammation 2016 5701959httpsdoiorg10115520165701959

Issue Date 2016-05-10

Doc URL httphdlhandlenet211562602

Rights(URL) httpscreativecommonsorglicensesby40

Type article

File Information 5701959pdf

Hokkaido University Collection of Scholarly and Academic Papers HUSCAP

Research ArticleAdiponectin Inhibits LPS-Induced HMGB1 Release throughan AMP Kinase and Heme Oxygenase-1-Dependent Pathway inRAW 264 Macrophage Cells

Mohamed Elfeky12 Ryuji Kaede1 Yuko Okamatsu-Ogura1 and Kazuhiro Kimura1

1Department of Biomedical Sciences Graduate School of Veterinary Medicine Hokkaido University Kita 18 Nishi 9 Kita-kuSapporo 060-0818 Japan2Department of Biochemistry Faculty of Veterinary Medicine Alexandria University Edfina Behera 22785 Egypt

Correspondence should be addressed to Kazuhiro Kimura k-kimuravetmedhokudaiacjp

Received 9 February 2016 Accepted 10 May 2016

Academic Editor Denis Girard

Copyright copy 2016 Mohamed Elfeky et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

High mobility group protein B1 (HMGB1) is a late inflammatory mediator that exaggerates septic symptoms Adiponectinan adipokine has potent anti-inflammatory properties However possible effects of adiponectin on lipopolysaccharide- (LPS-)inducedHMGB1 release are unknownThe aimof this studywas to investigate effects of full length adiponectin onHMGB1 release inLPS-stimulated RAW 264 macrophage cells Treatment of the cells with LPS alone significantly induced HMGB1 release associatedwith HMGB1 translocation from the nucleus to the cytosol However prior treatment with adiponectin suppressed LPS-inducedHMGB1 release and translocationThe anti-inflammatory cytokine interleukin- (IL-) 10 similarly suppressed LPS-inducedHMGB1release Adiponectin treatment decreased toll-like receptor 4 (TLR4) mRNA expression and increased heme oxygenase- (HO-) 1mRNA expression without inducing IL-10 mRNA while IL-10 treatment decreased TLR2 and HMGB1 mRNA expression andincreased the expression of IL-10 andHO-1mRNA Treatment with the HO-1 inhibitor ZnPP completely prevented the suppressionof HMGB1 release by adiponectin but only partially inhibited that induced by IL-10 Treatment with compound C an AMP kinase(AMPK) inhibitor abolished the increase in HO-1 expression and the suppression of HMGB1 release mediated by adiponectinIn conclusion our results indicate that adiponectin suppresses HMGB1 release by LPS through an AMPK-mediated and HO-1-dependent IL-10-independent pathway

1 Introduction

Sepsis an almost universally fatal clinical syndrome that iscaused bymicrobial infection results from excess stimulationof the host immune system by pathogen components to pro-duce various proinflammatory cytokines [1] Overproductionof these cytokines causes systemic inflammation that can leadto tissue damage multiple organ failure and death [2 3] Forexample bacterial lipopolysaccharides (LPS) a cell wall com-ponent of gram-negative bacteria induces an acute inflam-matory response initiated by its interaction with toll-likereceptor 4 (TLR4) resulting in sequential release of ldquoearlyrdquo(eg tumor necrosis factor- (TNF-) 120572 interleukin- (IL-) 1and IL-6) and ldquolaterdquo (eg high mobility group protein B1

(HMGB1)) proinflammatory cytokines [4ndash6] However ther-apies designed to block early released cytokines such as TNF-120572 or IL-1120573 have shown limited efficacy due to the early andtransient kinetics of the production of these inflammatorycytokines [7 8]

HMGB1 is a highly conserved ubiquitous nonhistonenuclear protein that exhibits diverse functions accordingto its cellular location Nuclear HMGB1 participates inDNA replication recombination transcription and repair Inresponse to infection or injury HMGB1 is actively secreted byinnate immune cells andor passively released by injured ordamaged cells Once releasedHMGB1 bindswith cell-surfacereceptors such as the receptor for advanced glycation endproducts (RAGE) andor TLRs including TLR2 and TLR4

Hindawi Publishing CorporationMediators of InflammationVolume 2016 Article ID 5701959 9 pageshttpdxdoiorg10115520165701959

2 Mediators of Inflammation

and mediates various cellular responses infiltration of innateimmune cells and subsequent release of various proinflam-matory cytokines [9ndash12] Administration of recombinantHMGB1 to mice is lethal while administration of anti-HMGB1 antibodies or inhibitors provides protection againstLPS-induced acute tissue damage and lethal endotoxaemia[4 11 13 14] Therefore targeting HMGB1 release providesa wide window for clinical intervention against systemicinflammatory diseases

Adiponectin which is also known as adipocyte comple-ment-related protein (Acrp30) is one of the most abundantones of the bioactive molecules called adipokines that aresecreted from adipose tissue [15] Adiponectin plays animportant role in various physiological processes includinglipid metabolism insulin sensitization and anti-inflamma-tory responses [16ndash18] Evidence indicates that adiponectinsuppresses the ldquoearlyrdquo phase of macrophage inflammatoryresponses For example adiponectin reduces macrophagedifferentiation and migration [19] and promotes macrophagepolarization toward an anti-inflammatoryM2phenotype bothin vivo and in cultured macrophages [20 21] Adiponectinalso inhibits the upregulation of the expression of adhesionmolecules and the enhancement of phagocytic activity andcytokine production in LPS-stimulated macrophages [1922] whereas it increases the release of anti-inflammatorymediators such as IL-10 and IL-1 receptor antagonist frommacrophages [23]

A number of animal studies show that adiponectin hasa protective effect against the development of inflammationrelated disorders For example treatment with adiponectinimproves atherosclerosis through inhibition of macrophageaggregation [19] and improves nonalcoholic steatohepatitisvia inhibition of lipogenic factors and TNF-120572 [24] Moreoveradiponectin protects from endotoxin-induced disorders oforgans including the liver [25] the lung [26] and the heart[27] although its deficiency is associated with severe polymi-crobial sepsis with high mortality [28] However there hasbeen no published report regarding the effects of adiponectinon the regulation of endotoxin-mediated release of ldquolaterdquoproinflammatory mediators such as HMGB1 Therefore inthis study we investigated the effect of adiponectin on LPS-induced HMGB1 release in murine RAW 264 macrophagecells

2 Materials and Methods

21 Materials Rabbit anti-HMGB1 antibody was purchasedfrom Cell Signaling Technology (CST) (Beverly MA USA)Recombinant mouse full length adiponectin expressed inHEK293 cells was purchased from Biovendor (AshevilleNC USA) Recombinant murine IL-10 was purchased fromPeproTech (Rocky Hill NJ USA) Zinc protoporphyrin IX(ZnPP) was purchased from Frontier Scientific (Logan UTUSA) SB203580 compound C (dorsomorphin) wortman-nin and bovine serum albumin (BSA) were purchased fromSigma-Aldrich Fine Chemicals (St Louis MO USA) OPTI-MEM Iwas purchased from Invitrogen (Carlsbad CA USA)

22 Cell Culture Cells of the murine macrophage-like cellline RAW 264 (RCB0535 RIKEN Cell Bank Japan) weremaintained in Dulbeccorsquos modified Eaglersquos medium (DMEMWako Pure Chemicals Osaka Japan) and supplemented with10 fetal bovine serum (FBS Trace Scientific LtdMelbourneAustralia) 100UmL penicillin and 100 120583gmL streptomycinin an atmosphere of humidified 5 CO

2at 37∘C When the

cells reached 80ndash90 confluence they were washed twicewith and subsequently cultured in serum-free OPTI-MEMI for 12 h before all treatments The cells were treated withor without increasing concentrations of adiponectin or IL-10 for 18 h and were then stimulated with LPS (Escherichiacoli O55B5 Sigma-Aldrich) dissolved in phosphate buffersaline (PBS) at a concentration of 200 ngmL for another24 h When included the cells were treated with or withoutZnPP SB203580 compound C or wortmannin 1 h beforeadiponectin (10120583gmL) or IL-10 (100 ngmL) addition

23Western BlottingAnalysis The level ofHMGB1 in the cul-ture medium was determined by western blotting analysis aspreviously reported [29ndash32] Briefly culturemedium sampleswere centrifuged to remove cellular debris then concentrated60-fold with the Amicon Ultra-4-10000 NMWL (MilliporeBillerica MA USA) The concentrated samples were mixedwith SDS loading buffer (500mM Tris-HCl 10 SDS 05bromophenol blue and 5 2-mercaptoethanol) boiled at100∘C for 5min separated on 15 SDS-polyacrylamide gelsand transferred onto a polyvinylidene fluoride membrane(Immobilon Millipore) The membrane was incubated in ablocking buffer (20mM Tris-HCl (pH 75) 150mM NaCl01 Tween 20 (TBS-T) and 5 skimmed milk) and thenwith rabbit anti-HMGB1 polyclonal antibody (1 2000 dilu-tion in the blocking buffer) overnight at 4∘C Subsequentlythe membrane was washed with TBS-T for 15min andincubated with horseradish peroxidase-linked goat anti-rabbit immunoglobulin (CST) (1 5000 dilution in the block-ing buffer) for 1 h at room temperature The signals werevisualized using chemiluminescent HRP Substrate (Milli-pore) according to the manufacturerrsquos instructions and weredetected using the ImageQuant LAS 500 system (GE Health-care Buckinghamshire UK)The intensity of chemilumines-cence of the corresponding bands was quantified using ImageJ software (v 148 httpimagejnihgovij)

24 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted from RAW 264 cells using the RNAiso reagent(Takara Bio Shiga Japan) according to the manufacturerrsquosprotocol Total RNA (2120583g) was reverse transcribed using a 15-mer oligo (dT) adaptor primer andM-MLVreverse transcrip-tase (Invitrogen) Quantitative real-time PCR was performedon a fluorescence thermal cycler (Light Cycler system RocheDiagnostics Mannheim Germany) using FastStart EssentialDNA Green Master PCR kits (Roche Diagnostics) Expres-sion levels were determined using the standard curvemethodwith respective cDNA fragments as standards The levels arereported relative to Gapdh expression as an internal controlThe primer sequences used in this study and the length ofeach PCR product are listed in Table 1

Mediators of Inflammation 3

Table 1 Primer sequences for quantitative real-time PCR and the length of each PCR product

Mouse gene Gene product Foreword primer Reverse primer Product size (bp)Gapdh GAPDH GAAGGTCGGTGTGAACGGATT GAAGACACCAGTAGACTCCAC 294Hmgb1 HMGB1 GGGAGACCAAAAAGAAGTTC GGCAGCTTTCTTCTCATAGG 200Hmox1 HO-1 TTCAGAAGGGTCAGGTGTCC CAGTGAGGCCCATACCAGAA 193Il-10 IL-10 GCCAAGCCTTATCGGAAATG TTTTCACAGGGGAGAAATCG 163Ly96 MD2 ACGCTGCTTTCTCCCATATT CATTGGTTCCCCTCAGTCTT 150Nfe2l2 Nrf2 ACATGGAGCAAGTTTGGCAG TGGAGAGGATGCTGCTGAAA 235Sirtuin1 SIRT1 AGGGAACCTTTGCCTCATCT GAGGTGTTGGTGGCAACTCT 159Sirtuin6 SIRT6 ACCTGCAACCCACAAAACAT GGCTCAGCCTTGAGTGCTAC 178Tlr2 TLR2 CGGAGGTAGAGTTCGACGAC AACTGGGGGATATGCAACCT 127Tlr4 TLR4 CAGCAAAGTCCCTGATGACA AGAGGTGGTGTAAGCCATGC 179

25 Immunofluorescence Thecellular localization ofHMGB1was investigated using an immunofluorescence stainingassay RAW264 cells (5times 104 cellswell) were cultured on glasscoverslips in 6-well plates The cells were washed twice withPBS and then fixed with 4 paraformaldehyde for 30minat room temperature Subsequently the cells were permeabi-lized with 10 Triton X-100 in PBS supplemented with 05BSA and 015 glycine for 10min following which they wereblocked in PBS containing 5 BSA and 03 Triton X-100 for60min The glass coverslips were then incubated with rabbitanti-HMGB1 antibody (1 100 dilution in PBS containing 1BSA and 03 Triton X-100) overnight at 4∘C followed bygoat anti-rabbit Alexa flour 488 (1 400 dilution) (Invitrogen)in the dark for 1 h at room temperature Cells were washedwith PBS containing 01 Triton X-100 between all incuba-tions steps followed by a final wash in PBS Nuclei werelabeled by incubation with 410158406-diamidino-2-phenylindole(DAPI Invitrogen) for 10min The cells were washed threetimes for 5min with PBS The coverslips were mounted onslides using ProlongAntifade Reagents (Invitrogen) Imageswere captured using a fluorescence microscope (BiorevoBZ-9000 Keyence Japan Osaka Japan) with a times100 oil-immersion lens No fluorescence was detected in control cellsprocessed without the primary antibody The fluorescenceintensities of cytosolic and nuclear HMGB1 were quantifiedusing Image J software

26 Statistical Analysis IBMSPSS Statistics version 220 soft-ware (SPSS Chicago IL USA) was used for statistical analy-sis Data are presented as means plusmn standard error (SE) Statis-tical comparisons between multiple groups were performedwith one-way analysis of variance (ANOVA) followed byeither Dunnettrsquos or a Tukey HSD post hoc test A 119901 value of lt005 was considered statistically significant

3 Results

RAW 264 cells released a small amount of HMGB1 into themedium under the culture conditions without any stimula-tion The amount of HMGB1 that was released increased 6-fold upon stimulation of the cells with 200 ngmL of LPS

(Figure 1(a)) Cell viability was almost 100 even after treat-ment with 1 120583gmL of LPS (data not shown) The increasedrelease of HMGB1 with LPS treatment was accompaniedby HMGB1 translocation from the nucleus to the cytosol(Figures 1(b) and 1(c)) These results suggested that HMGB1release was under the control of LPS signaling rather thanbeing passive release due to LPS cytotoxicity Prior treatmentof the cells with full length adiponectin failed to affectbasal HMGB1 release but dose dependently suppressed LPS-induced HMGB1 release and was accompanied by nuclearlocalization of most of the HMGB1 (Figures 1(a)ndash1(c))

As it has been reported that globular adiponectin exertsits anti-inflammatory actions through induction of IL-10 [21]we next examined the effect of IL-10 on LPS induction ofHMGB1 release Prior treatment of the cells with IL-10 alsofailed to enhance basal HMGB1 release However IL-10 atdoses of 50 and 100 ngmL greatly decreased the HMGB1release into the medium that was induced by LPS (Figure 2)

To further examine the mechanism behind the suppres-sive effect of adiponectin on LPS-induced HMGB1 releasewe compared mRNA expression in cells treated with eitherfull length adiponectin or IL-10 Among the genes quantifiedRAW 264 cells constitutively expressed TLR4 mRNA TLR2mRNA and myeloid differentiation factor 2 (MD2) mRNA(Figures 3(a)ndash3(c)) all of which are plasma membranecomponents responsible for LPS binding and signalingTreatment of the cells with adiponectin selectively decreasedexpression of TLR4 mRNA while IL-10 treatment reducedonly TLR2 mRNA expression Distinct differences betweenfull length adiponectin and IL-10 treatments were alsoobserved in the expression of HMGB1 and IL-10 genes Thecells constitutively expressedHMGB1mRNAwhichwas sup-pressed only by IL-10 treatment and not by adiponectin treat-ment (Figure 3(d)) On the other hand the cells expressedvery low levels of IL-10 mRNA which was enhanced only byIL-10 treatment but not by full length adiponectin treatment(Figure 3(e))Thus it was unlikely that full length adiponectinexerted its suppressive effect on LPS-inducedHMGB1 releasethrough induction of IL-10

Interestingly both IL-10 and full length adiponectintreatments enhanced the mRNA expression of HO-1 adownstream anti-inflammatory effector of IL-10 signaling

4 Mediators of Inflammation

0

1

2

3

4

5

6

7lowastlowast

HM

GB1

rele

ase (

AU)

minus

minus

minus

5

+

minus

+

5

+

1

+

10

Adiponectin(120583gmL)

LPS(200ngmL)

(a)LP

S +

adip

onec

tinLP

SC

ontro

l

Nucleus HMGB1 Merge

(b)

LPS + adiponectinLPSControl

lowastlowast

0

10

20

30

40

Ratio

of c

ytos

olic

to n

ucle

ar H

MG

B1

(c)

Figure 1 Effect of recombinant adiponectin on LPS-induced HMGB1 release and HMGB1 cellular translocation Raw 264 cells were culturedin DMEM supplemented with 10 FBS and were cultured in serum-free OPTI-MEM Imedium for additional 12 hThe cells were treated withincreasing concentrations of adiponectin for 18 h then stimulated with LPS (200 ngmL) for another 24 h (a) Culture medium was collectedand analyzed by HMGB1 western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 005 significance comparedwith LPS treated cells) (b and c) Cellular HMGB1 was immunostained with an anti-HMGB1 rabbit primary and Alexa Fluor 488 anti-rabbitsecondary antibodies The nucleus was stained with DAPI Merge indicates the combination of both HMGB1 (Green) and nuclear (Blue)fluorescence The fluorescence intensities of cytosolic and nuclear HMGB1 in (b) were separately analyzed and the ratio of cytosolic HMGB1to nuclear HMGB1 is shown in (c) (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 significance compared with LPS treated cells)

(Figure 3(f)) while neither treatment affected the mRNAexpression of nuclear factor erythroid-derived 2 related factor2 (Nrf2) a transcription factor relatedwithHO-1 gene expres-sion (Figure 3(g)) In addition neither IL-10 nor full lengthadiponectin treatment increased the mRNA expression ofSirt1 or Sirt6 which are histone deacetylases that function asa chromatin silencer to regulate recombination and genomicstability (Figures 3(h) and 3(i)) We therefore next examined

the involvement of HO-1 in the suppressive effect of fulllength adiponectin on LPS-induced HMGB1 release Treat-ment of the cells with zinc protoporphyrin (ZnPP) a HO-1 inhibitor did not have any effect on HMGB1 release fromeither LPS-stimulated or control cells (Figure 4) Howevertreatment with ZnPP for 1 h before full length adiponectintreatment almost completely abolished adiponectin sup-pression of LPS-induced HMGB1 release although it only

Mediators of Inflammation 5

0

1

2

3

4

5

6

7

HM

GB1

rele

ase (

AU)

minus

minus

minus

50

+

minus

+

50

+

10

+

100IL-10(ngmL)

LPS(200ngmL)

lowastlowast

Figure 2 Effect of recombinant IL-10 on LPS-induced HMGB1 release Raw 264 cells were cultured as described in Figure 1 legend and weretreated with IL-10 for 18 h then stimulated with LPS (200 ngmL) for another 24 h Culture medium was collected and analyzed by HMGB1western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results are expressed as means plusmn SEof three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 and

119901 lt 005 significance compared with LPStreated cells)

slightly inhibited the suppression by IL-10 These resultsindicate that increased expression of HO-1 in response tofull length adiponectin is necessary for adiponectin-mediatedprevention of LPS-induced HMGB1 release

We then examined whether increased expression of HO-1 mRNA by full length adiponectin was mediated throughAMP-activated kinase (AMPK) a main signaling pathwayof adiponectin action [33ndash35] Treatment of the cells withcompound C an AMPK inhibitor abolished the increase inexpression ofHO-1mRNAby full length adiponectin whereastreatment of the cells with wortmannin a phosphatidylinosi-tol-3-kinase (PI3K) inhibitor or with SB203580 a p38mitogen-activated protein kinase (p38MAPK) inhibitor didnot affect this increase (Figure 5) Consistent with theseresults treatment of the cells with compound C but notwith SB203580 abolished adiponectin-mediated suppressionof LPS-induced HMGB1 release (Figure 6)

4 Discussion

In the present study we demonstrated for the first timethat full length adiponectin prevents LPS-induced HMGB1translocation from the nucleus to the cytosol and its subse-quent release from Raw 264 mouse macrophage cells Thisprocess is most probably mediated by AMPK-dependentHO-1 induction as evidenced by the following results BothAMPK and HO-1 inhibitors prevented the suppression ofLPS-induced HMGB1 release by full length adiponectinand the AMPK inhibitor also prevented induction of HO-1mRNA by full length adiponectin Furthermore the mech-anism of the full length adiponectin effect is supported byprevious reports that showed that full length adiponectinactivates AMPK activity [33ndash35] that activation of AMPKby metformin or dehydrodiconiferyl alcohol enhances HO-1 expression and its activity [36 37] and that HO-1 isindispensable for the prevention of HMGB1 release [3839] Of course other events such as selective reduction in

TLR4 mRNA expression by full length adiponectin mightcontribute at least in part to the suppression of LPS-induced HMGB1 release since TLR4 is the predominantreceptor for LPS [40 41] and a similar decrease in cell-surfaceTLR4 expression is seen in macrophage cells treated withglobular adiponectin [42] However other intracellular sig-naling pathways related to p38MAPK PI3K and the nuclearhistone deacetylase sirtuin are unlikely to be involved in themechanism although they have been reported to be involvedin some adiponectin functions [43ndash46] or in the processes ofHO-1 induction and LPS-induced HMGB1 release [47ndash49]

We have also demonstrated that IL-10 is a potent inhibitorof LPS-induced HMGB1 release However the fact that fulllength adiponectin failed to induce IL-10 mRNA suggestedthat the suppression by full length adiponectin might not beattributed to IL-10 production This hypothesis is supportedby previous findings that the effects of full length adiponectinon macrophage function are independent of IL-10 [42 44]although anti-inflammatory effects of globular adiponectinare mediated by IL-10 [21 23 50ndash52] The discrepancybetween the role of IL-10 in the effects of full length andglobular adiponectin has not been explored but is possiblydue to different signals mediated through adipoR2 andadipoR1 respectively [42]

Accumulating evidence indicates that HO-1 plays a piv-otal role in the anti-inflammatory cytoprotective effects of awide variety of compounds including statins phytochemicalssuch as resveratrol and aspirin [53] HO-1 is a microsomalenzyme that catalyzes the degradation of proinflammatoryfree heme and produces equimolar amounts of carbonmonoxide bilirubin and iron [54] The mechanisms thatmediate the anti-inflammatory effects of HO-1 are not fullyunderstood but the potent antioxidant activity of bilirubinand the signaling gas activity of carbon monoxide arereported to suppress apoptosis necrosis inflammation andoxidative stress Interestingly HO-1 is induced by pathophys-iological stimuli including LPS and hemodynamic changes

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

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Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Mediators of Inflammation

and mediates various cellular responses infiltration of innateimmune cells and subsequent release of various proinflam-matory cytokines [9ndash12] Administration of recombinantHMGB1 to mice is lethal while administration of anti-HMGB1 antibodies or inhibitors provides protection againstLPS-induced acute tissue damage and lethal endotoxaemia[4 11 13 14] Therefore targeting HMGB1 release providesa wide window for clinical intervention against systemicinflammatory diseases

Adiponectin which is also known as adipocyte comple-ment-related protein (Acrp30) is one of the most abundantones of the bioactive molecules called adipokines that aresecreted from adipose tissue [15] Adiponectin plays animportant role in various physiological processes includinglipid metabolism insulin sensitization and anti-inflamma-tory responses [16ndash18] Evidence indicates that adiponectinsuppresses the ldquoearlyrdquo phase of macrophage inflammatoryresponses For example adiponectin reduces macrophagedifferentiation and migration [19] and promotes macrophagepolarization toward an anti-inflammatoryM2phenotype bothin vivo and in cultured macrophages [20 21] Adiponectinalso inhibits the upregulation of the expression of adhesionmolecules and the enhancement of phagocytic activity andcytokine production in LPS-stimulated macrophages [1922] whereas it increases the release of anti-inflammatorymediators such as IL-10 and IL-1 receptor antagonist frommacrophages [23]

A number of animal studies show that adiponectin hasa protective effect against the development of inflammationrelated disorders For example treatment with adiponectinimproves atherosclerosis through inhibition of macrophageaggregation [19] and improves nonalcoholic steatohepatitisvia inhibition of lipogenic factors and TNF-120572 [24] Moreoveradiponectin protects from endotoxin-induced disorders oforgans including the liver [25] the lung [26] and the heart[27] although its deficiency is associated with severe polymi-crobial sepsis with high mortality [28] However there hasbeen no published report regarding the effects of adiponectinon the regulation of endotoxin-mediated release of ldquolaterdquoproinflammatory mediators such as HMGB1 Therefore inthis study we investigated the effect of adiponectin on LPS-induced HMGB1 release in murine RAW 264 macrophagecells

2 Materials and Methods

21 Materials Rabbit anti-HMGB1 antibody was purchasedfrom Cell Signaling Technology (CST) (Beverly MA USA)Recombinant mouse full length adiponectin expressed inHEK293 cells was purchased from Biovendor (AshevilleNC USA) Recombinant murine IL-10 was purchased fromPeproTech (Rocky Hill NJ USA) Zinc protoporphyrin IX(ZnPP) was purchased from Frontier Scientific (Logan UTUSA) SB203580 compound C (dorsomorphin) wortman-nin and bovine serum albumin (BSA) were purchased fromSigma-Aldrich Fine Chemicals (St Louis MO USA) OPTI-MEM Iwas purchased from Invitrogen (Carlsbad CA USA)

22 Cell Culture Cells of the murine macrophage-like cellline RAW 264 (RCB0535 RIKEN Cell Bank Japan) weremaintained in Dulbeccorsquos modified Eaglersquos medium (DMEMWako Pure Chemicals Osaka Japan) and supplemented with10 fetal bovine serum (FBS Trace Scientific LtdMelbourneAustralia) 100UmL penicillin and 100 120583gmL streptomycinin an atmosphere of humidified 5 CO

2at 37∘C When the

cells reached 80ndash90 confluence they were washed twicewith and subsequently cultured in serum-free OPTI-MEMI for 12 h before all treatments The cells were treated withor without increasing concentrations of adiponectin or IL-10 for 18 h and were then stimulated with LPS (Escherichiacoli O55B5 Sigma-Aldrich) dissolved in phosphate buffersaline (PBS) at a concentration of 200 ngmL for another24 h When included the cells were treated with or withoutZnPP SB203580 compound C or wortmannin 1 h beforeadiponectin (10120583gmL) or IL-10 (100 ngmL) addition

23Western BlottingAnalysis The level ofHMGB1 in the cul-ture medium was determined by western blotting analysis aspreviously reported [29ndash32] Briefly culturemedium sampleswere centrifuged to remove cellular debris then concentrated60-fold with the Amicon Ultra-4-10000 NMWL (MilliporeBillerica MA USA) The concentrated samples were mixedwith SDS loading buffer (500mM Tris-HCl 10 SDS 05bromophenol blue and 5 2-mercaptoethanol) boiled at100∘C for 5min separated on 15 SDS-polyacrylamide gelsand transferred onto a polyvinylidene fluoride membrane(Immobilon Millipore) The membrane was incubated in ablocking buffer (20mM Tris-HCl (pH 75) 150mM NaCl01 Tween 20 (TBS-T) and 5 skimmed milk) and thenwith rabbit anti-HMGB1 polyclonal antibody (1 2000 dilu-tion in the blocking buffer) overnight at 4∘C Subsequentlythe membrane was washed with TBS-T for 15min andincubated with horseradish peroxidase-linked goat anti-rabbit immunoglobulin (CST) (1 5000 dilution in the block-ing buffer) for 1 h at room temperature The signals werevisualized using chemiluminescent HRP Substrate (Milli-pore) according to the manufacturerrsquos instructions and weredetected using the ImageQuant LAS 500 system (GE Health-care Buckinghamshire UK)The intensity of chemilumines-cence of the corresponding bands was quantified using ImageJ software (v 148 httpimagejnihgovij)

24 Quantitative Real-Time PCR (qRT-PCR) Total RNA wasextracted from RAW 264 cells using the RNAiso reagent(Takara Bio Shiga Japan) according to the manufacturerrsquosprotocol Total RNA (2120583g) was reverse transcribed using a 15-mer oligo (dT) adaptor primer andM-MLVreverse transcrip-tase (Invitrogen) Quantitative real-time PCR was performedon a fluorescence thermal cycler (Light Cycler system RocheDiagnostics Mannheim Germany) using FastStart EssentialDNA Green Master PCR kits (Roche Diagnostics) Expres-sion levels were determined using the standard curvemethodwith respective cDNA fragments as standards The levels arereported relative to Gapdh expression as an internal controlThe primer sequences used in this study and the length ofeach PCR product are listed in Table 1

Mediators of Inflammation 3

Table 1 Primer sequences for quantitative real-time PCR and the length of each PCR product

Mouse gene Gene product Foreword primer Reverse primer Product size (bp)Gapdh GAPDH GAAGGTCGGTGTGAACGGATT GAAGACACCAGTAGACTCCAC 294Hmgb1 HMGB1 GGGAGACCAAAAAGAAGTTC GGCAGCTTTCTTCTCATAGG 200Hmox1 HO-1 TTCAGAAGGGTCAGGTGTCC CAGTGAGGCCCATACCAGAA 193Il-10 IL-10 GCCAAGCCTTATCGGAAATG TTTTCACAGGGGAGAAATCG 163Ly96 MD2 ACGCTGCTTTCTCCCATATT CATTGGTTCCCCTCAGTCTT 150Nfe2l2 Nrf2 ACATGGAGCAAGTTTGGCAG TGGAGAGGATGCTGCTGAAA 235Sirtuin1 SIRT1 AGGGAACCTTTGCCTCATCT GAGGTGTTGGTGGCAACTCT 159Sirtuin6 SIRT6 ACCTGCAACCCACAAAACAT GGCTCAGCCTTGAGTGCTAC 178Tlr2 TLR2 CGGAGGTAGAGTTCGACGAC AACTGGGGGATATGCAACCT 127Tlr4 TLR4 CAGCAAAGTCCCTGATGACA AGAGGTGGTGTAAGCCATGC 179

25 Immunofluorescence Thecellular localization ofHMGB1was investigated using an immunofluorescence stainingassay RAW264 cells (5times 104 cellswell) were cultured on glasscoverslips in 6-well plates The cells were washed twice withPBS and then fixed with 4 paraformaldehyde for 30minat room temperature Subsequently the cells were permeabi-lized with 10 Triton X-100 in PBS supplemented with 05BSA and 015 glycine for 10min following which they wereblocked in PBS containing 5 BSA and 03 Triton X-100 for60min The glass coverslips were then incubated with rabbitanti-HMGB1 antibody (1 100 dilution in PBS containing 1BSA and 03 Triton X-100) overnight at 4∘C followed bygoat anti-rabbit Alexa flour 488 (1 400 dilution) (Invitrogen)in the dark for 1 h at room temperature Cells were washedwith PBS containing 01 Triton X-100 between all incuba-tions steps followed by a final wash in PBS Nuclei werelabeled by incubation with 410158406-diamidino-2-phenylindole(DAPI Invitrogen) for 10min The cells were washed threetimes for 5min with PBS The coverslips were mounted onslides using ProlongAntifade Reagents (Invitrogen) Imageswere captured using a fluorescence microscope (BiorevoBZ-9000 Keyence Japan Osaka Japan) with a times100 oil-immersion lens No fluorescence was detected in control cellsprocessed without the primary antibody The fluorescenceintensities of cytosolic and nuclear HMGB1 were quantifiedusing Image J software

26 Statistical Analysis IBMSPSS Statistics version 220 soft-ware (SPSS Chicago IL USA) was used for statistical analy-sis Data are presented as means plusmn standard error (SE) Statis-tical comparisons between multiple groups were performedwith one-way analysis of variance (ANOVA) followed byeither Dunnettrsquos or a Tukey HSD post hoc test A 119901 value of lt005 was considered statistically significant

3 Results

RAW 264 cells released a small amount of HMGB1 into themedium under the culture conditions without any stimula-tion The amount of HMGB1 that was released increased 6-fold upon stimulation of the cells with 200 ngmL of LPS

(Figure 1(a)) Cell viability was almost 100 even after treat-ment with 1 120583gmL of LPS (data not shown) The increasedrelease of HMGB1 with LPS treatment was accompaniedby HMGB1 translocation from the nucleus to the cytosol(Figures 1(b) and 1(c)) These results suggested that HMGB1release was under the control of LPS signaling rather thanbeing passive release due to LPS cytotoxicity Prior treatmentof the cells with full length adiponectin failed to affectbasal HMGB1 release but dose dependently suppressed LPS-induced HMGB1 release and was accompanied by nuclearlocalization of most of the HMGB1 (Figures 1(a)ndash1(c))

As it has been reported that globular adiponectin exertsits anti-inflammatory actions through induction of IL-10 [21]we next examined the effect of IL-10 on LPS induction ofHMGB1 release Prior treatment of the cells with IL-10 alsofailed to enhance basal HMGB1 release However IL-10 atdoses of 50 and 100 ngmL greatly decreased the HMGB1release into the medium that was induced by LPS (Figure 2)

To further examine the mechanism behind the suppres-sive effect of adiponectin on LPS-induced HMGB1 releasewe compared mRNA expression in cells treated with eitherfull length adiponectin or IL-10 Among the genes quantifiedRAW 264 cells constitutively expressed TLR4 mRNA TLR2mRNA and myeloid differentiation factor 2 (MD2) mRNA(Figures 3(a)ndash3(c)) all of which are plasma membranecomponents responsible for LPS binding and signalingTreatment of the cells with adiponectin selectively decreasedexpression of TLR4 mRNA while IL-10 treatment reducedonly TLR2 mRNA expression Distinct differences betweenfull length adiponectin and IL-10 treatments were alsoobserved in the expression of HMGB1 and IL-10 genes Thecells constitutively expressedHMGB1mRNAwhichwas sup-pressed only by IL-10 treatment and not by adiponectin treat-ment (Figure 3(d)) On the other hand the cells expressedvery low levels of IL-10 mRNA which was enhanced only byIL-10 treatment but not by full length adiponectin treatment(Figure 3(e))Thus it was unlikely that full length adiponectinexerted its suppressive effect on LPS-inducedHMGB1 releasethrough induction of IL-10

Interestingly both IL-10 and full length adiponectintreatments enhanced the mRNA expression of HO-1 adownstream anti-inflammatory effector of IL-10 signaling

4 Mediators of Inflammation

0

1

2

3

4

5

6

7lowastlowast

HM

GB1

rele

ase (

AU)

minus

minus

minus

5

+

minus

+

5

+

1

+

10

Adiponectin(120583gmL)

LPS(200ngmL)

(a)LP

S +

adip

onec

tinLP

SC

ontro

l

Nucleus HMGB1 Merge

(b)

LPS + adiponectinLPSControl

lowastlowast

0

10

20

30

40

Ratio

of c

ytos

olic

to n

ucle

ar H

MG

B1

(c)

Figure 1 Effect of recombinant adiponectin on LPS-induced HMGB1 release and HMGB1 cellular translocation Raw 264 cells were culturedin DMEM supplemented with 10 FBS and were cultured in serum-free OPTI-MEM Imedium for additional 12 hThe cells were treated withincreasing concentrations of adiponectin for 18 h then stimulated with LPS (200 ngmL) for another 24 h (a) Culture medium was collectedand analyzed by HMGB1 western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 005 significance comparedwith LPS treated cells) (b and c) Cellular HMGB1 was immunostained with an anti-HMGB1 rabbit primary and Alexa Fluor 488 anti-rabbitsecondary antibodies The nucleus was stained with DAPI Merge indicates the combination of both HMGB1 (Green) and nuclear (Blue)fluorescence The fluorescence intensities of cytosolic and nuclear HMGB1 in (b) were separately analyzed and the ratio of cytosolic HMGB1to nuclear HMGB1 is shown in (c) (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 significance compared with LPS treated cells)

(Figure 3(f)) while neither treatment affected the mRNAexpression of nuclear factor erythroid-derived 2 related factor2 (Nrf2) a transcription factor relatedwithHO-1 gene expres-sion (Figure 3(g)) In addition neither IL-10 nor full lengthadiponectin treatment increased the mRNA expression ofSirt1 or Sirt6 which are histone deacetylases that function asa chromatin silencer to regulate recombination and genomicstability (Figures 3(h) and 3(i)) We therefore next examined

the involvement of HO-1 in the suppressive effect of fulllength adiponectin on LPS-induced HMGB1 release Treat-ment of the cells with zinc protoporphyrin (ZnPP) a HO-1 inhibitor did not have any effect on HMGB1 release fromeither LPS-stimulated or control cells (Figure 4) Howevertreatment with ZnPP for 1 h before full length adiponectintreatment almost completely abolished adiponectin sup-pression of LPS-induced HMGB1 release although it only

Mediators of Inflammation 5

0

1

2

3

4

5

6

7

HM

GB1

rele

ase (

AU)

minus

minus

minus

50

+

minus

+

50

+

10

+

100IL-10(ngmL)

LPS(200ngmL)

lowastlowast

Figure 2 Effect of recombinant IL-10 on LPS-induced HMGB1 release Raw 264 cells were cultured as described in Figure 1 legend and weretreated with IL-10 for 18 h then stimulated with LPS (200 ngmL) for another 24 h Culture medium was collected and analyzed by HMGB1western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results are expressed as means plusmn SEof three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 and

119901 lt 005 significance compared with LPStreated cells)

slightly inhibited the suppression by IL-10 These resultsindicate that increased expression of HO-1 in response tofull length adiponectin is necessary for adiponectin-mediatedprevention of LPS-induced HMGB1 release

We then examined whether increased expression of HO-1 mRNA by full length adiponectin was mediated throughAMP-activated kinase (AMPK) a main signaling pathwayof adiponectin action [33ndash35] Treatment of the cells withcompound C an AMPK inhibitor abolished the increase inexpression ofHO-1mRNAby full length adiponectin whereastreatment of the cells with wortmannin a phosphatidylinosi-tol-3-kinase (PI3K) inhibitor or with SB203580 a p38mitogen-activated protein kinase (p38MAPK) inhibitor didnot affect this increase (Figure 5) Consistent with theseresults treatment of the cells with compound C but notwith SB203580 abolished adiponectin-mediated suppressionof LPS-induced HMGB1 release (Figure 6)

4 Discussion

In the present study we demonstrated for the first timethat full length adiponectin prevents LPS-induced HMGB1translocation from the nucleus to the cytosol and its subse-quent release from Raw 264 mouse macrophage cells Thisprocess is most probably mediated by AMPK-dependentHO-1 induction as evidenced by the following results BothAMPK and HO-1 inhibitors prevented the suppression ofLPS-induced HMGB1 release by full length adiponectinand the AMPK inhibitor also prevented induction of HO-1mRNA by full length adiponectin Furthermore the mech-anism of the full length adiponectin effect is supported byprevious reports that showed that full length adiponectinactivates AMPK activity [33ndash35] that activation of AMPKby metformin or dehydrodiconiferyl alcohol enhances HO-1 expression and its activity [36 37] and that HO-1 isindispensable for the prevention of HMGB1 release [3839] Of course other events such as selective reduction in

TLR4 mRNA expression by full length adiponectin mightcontribute at least in part to the suppression of LPS-induced HMGB1 release since TLR4 is the predominantreceptor for LPS [40 41] and a similar decrease in cell-surfaceTLR4 expression is seen in macrophage cells treated withglobular adiponectin [42] However other intracellular sig-naling pathways related to p38MAPK PI3K and the nuclearhistone deacetylase sirtuin are unlikely to be involved in themechanism although they have been reported to be involvedin some adiponectin functions [43ndash46] or in the processes ofHO-1 induction and LPS-induced HMGB1 release [47ndash49]

We have also demonstrated that IL-10 is a potent inhibitorof LPS-induced HMGB1 release However the fact that fulllength adiponectin failed to induce IL-10 mRNA suggestedthat the suppression by full length adiponectin might not beattributed to IL-10 production This hypothesis is supportedby previous findings that the effects of full length adiponectinon macrophage function are independent of IL-10 [42 44]although anti-inflammatory effects of globular adiponectinare mediated by IL-10 [21 23 50ndash52] The discrepancybetween the role of IL-10 in the effects of full length andglobular adiponectin has not been explored but is possiblydue to different signals mediated through adipoR2 andadipoR1 respectively [42]

Accumulating evidence indicates that HO-1 plays a piv-otal role in the anti-inflammatory cytoprotective effects of awide variety of compounds including statins phytochemicalssuch as resveratrol and aspirin [53] HO-1 is a microsomalenzyme that catalyzes the degradation of proinflammatoryfree heme and produces equimolar amounts of carbonmonoxide bilirubin and iron [54] The mechanisms thatmediate the anti-inflammatory effects of HO-1 are not fullyunderstood but the potent antioxidant activity of bilirubinand the signaling gas activity of carbon monoxide arereported to suppress apoptosis necrosis inflammation andoxidative stress Interestingly HO-1 is induced by pathophys-iological stimuli including LPS and hemodynamic changes

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 3

Table 1 Primer sequences for quantitative real-time PCR and the length of each PCR product

Mouse gene Gene product Foreword primer Reverse primer Product size (bp)Gapdh GAPDH GAAGGTCGGTGTGAACGGATT GAAGACACCAGTAGACTCCAC 294Hmgb1 HMGB1 GGGAGACCAAAAAGAAGTTC GGCAGCTTTCTTCTCATAGG 200Hmox1 HO-1 TTCAGAAGGGTCAGGTGTCC CAGTGAGGCCCATACCAGAA 193Il-10 IL-10 GCCAAGCCTTATCGGAAATG TTTTCACAGGGGAGAAATCG 163Ly96 MD2 ACGCTGCTTTCTCCCATATT CATTGGTTCCCCTCAGTCTT 150Nfe2l2 Nrf2 ACATGGAGCAAGTTTGGCAG TGGAGAGGATGCTGCTGAAA 235Sirtuin1 SIRT1 AGGGAACCTTTGCCTCATCT GAGGTGTTGGTGGCAACTCT 159Sirtuin6 SIRT6 ACCTGCAACCCACAAAACAT GGCTCAGCCTTGAGTGCTAC 178Tlr2 TLR2 CGGAGGTAGAGTTCGACGAC AACTGGGGGATATGCAACCT 127Tlr4 TLR4 CAGCAAAGTCCCTGATGACA AGAGGTGGTGTAAGCCATGC 179

25 Immunofluorescence Thecellular localization ofHMGB1was investigated using an immunofluorescence stainingassay RAW264 cells (5times 104 cellswell) were cultured on glasscoverslips in 6-well plates The cells were washed twice withPBS and then fixed with 4 paraformaldehyde for 30minat room temperature Subsequently the cells were permeabi-lized with 10 Triton X-100 in PBS supplemented with 05BSA and 015 glycine for 10min following which they wereblocked in PBS containing 5 BSA and 03 Triton X-100 for60min The glass coverslips were then incubated with rabbitanti-HMGB1 antibody (1 100 dilution in PBS containing 1BSA and 03 Triton X-100) overnight at 4∘C followed bygoat anti-rabbit Alexa flour 488 (1 400 dilution) (Invitrogen)in the dark for 1 h at room temperature Cells were washedwith PBS containing 01 Triton X-100 between all incuba-tions steps followed by a final wash in PBS Nuclei werelabeled by incubation with 410158406-diamidino-2-phenylindole(DAPI Invitrogen) for 10min The cells were washed threetimes for 5min with PBS The coverslips were mounted onslides using ProlongAntifade Reagents (Invitrogen) Imageswere captured using a fluorescence microscope (BiorevoBZ-9000 Keyence Japan Osaka Japan) with a times100 oil-immersion lens No fluorescence was detected in control cellsprocessed without the primary antibody The fluorescenceintensities of cytosolic and nuclear HMGB1 were quantifiedusing Image J software

26 Statistical Analysis IBMSPSS Statistics version 220 soft-ware (SPSS Chicago IL USA) was used for statistical analy-sis Data are presented as means plusmn standard error (SE) Statis-tical comparisons between multiple groups were performedwith one-way analysis of variance (ANOVA) followed byeither Dunnettrsquos or a Tukey HSD post hoc test A 119901 value of lt005 was considered statistically significant

3 Results

RAW 264 cells released a small amount of HMGB1 into themedium under the culture conditions without any stimula-tion The amount of HMGB1 that was released increased 6-fold upon stimulation of the cells with 200 ngmL of LPS

(Figure 1(a)) Cell viability was almost 100 even after treat-ment with 1 120583gmL of LPS (data not shown) The increasedrelease of HMGB1 with LPS treatment was accompaniedby HMGB1 translocation from the nucleus to the cytosol(Figures 1(b) and 1(c)) These results suggested that HMGB1release was under the control of LPS signaling rather thanbeing passive release due to LPS cytotoxicity Prior treatmentof the cells with full length adiponectin failed to affectbasal HMGB1 release but dose dependently suppressed LPS-induced HMGB1 release and was accompanied by nuclearlocalization of most of the HMGB1 (Figures 1(a)ndash1(c))

As it has been reported that globular adiponectin exertsits anti-inflammatory actions through induction of IL-10 [21]we next examined the effect of IL-10 on LPS induction ofHMGB1 release Prior treatment of the cells with IL-10 alsofailed to enhance basal HMGB1 release However IL-10 atdoses of 50 and 100 ngmL greatly decreased the HMGB1release into the medium that was induced by LPS (Figure 2)

To further examine the mechanism behind the suppres-sive effect of adiponectin on LPS-induced HMGB1 releasewe compared mRNA expression in cells treated with eitherfull length adiponectin or IL-10 Among the genes quantifiedRAW 264 cells constitutively expressed TLR4 mRNA TLR2mRNA and myeloid differentiation factor 2 (MD2) mRNA(Figures 3(a)ndash3(c)) all of which are plasma membranecomponents responsible for LPS binding and signalingTreatment of the cells with adiponectin selectively decreasedexpression of TLR4 mRNA while IL-10 treatment reducedonly TLR2 mRNA expression Distinct differences betweenfull length adiponectin and IL-10 treatments were alsoobserved in the expression of HMGB1 and IL-10 genes Thecells constitutively expressedHMGB1mRNAwhichwas sup-pressed only by IL-10 treatment and not by adiponectin treat-ment (Figure 3(d)) On the other hand the cells expressedvery low levels of IL-10 mRNA which was enhanced only byIL-10 treatment but not by full length adiponectin treatment(Figure 3(e))Thus it was unlikely that full length adiponectinexerted its suppressive effect on LPS-inducedHMGB1 releasethrough induction of IL-10

Interestingly both IL-10 and full length adiponectintreatments enhanced the mRNA expression of HO-1 adownstream anti-inflammatory effector of IL-10 signaling

4 Mediators of Inflammation

0

1

2

3

4

5

6

7lowastlowast

HM

GB1

rele

ase (

AU)

minus

minus

minus

5

+

minus

+

5

+

1

+

10

Adiponectin(120583gmL)

LPS(200ngmL)

(a)LP

S +

adip

onec

tinLP

SC

ontro

l

Nucleus HMGB1 Merge

(b)

LPS + adiponectinLPSControl

lowastlowast

0

10

20

30

40

Ratio

of c

ytos

olic

to n

ucle

ar H

MG

B1

(c)

Figure 1 Effect of recombinant adiponectin on LPS-induced HMGB1 release and HMGB1 cellular translocation Raw 264 cells were culturedin DMEM supplemented with 10 FBS and were cultured in serum-free OPTI-MEM Imedium for additional 12 hThe cells were treated withincreasing concentrations of adiponectin for 18 h then stimulated with LPS (200 ngmL) for another 24 h (a) Culture medium was collectedand analyzed by HMGB1 western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 005 significance comparedwith LPS treated cells) (b and c) Cellular HMGB1 was immunostained with an anti-HMGB1 rabbit primary and Alexa Fluor 488 anti-rabbitsecondary antibodies The nucleus was stained with DAPI Merge indicates the combination of both HMGB1 (Green) and nuclear (Blue)fluorescence The fluorescence intensities of cytosolic and nuclear HMGB1 in (b) were separately analyzed and the ratio of cytosolic HMGB1to nuclear HMGB1 is shown in (c) (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 significance compared with LPS treated cells)

(Figure 3(f)) while neither treatment affected the mRNAexpression of nuclear factor erythroid-derived 2 related factor2 (Nrf2) a transcription factor relatedwithHO-1 gene expres-sion (Figure 3(g)) In addition neither IL-10 nor full lengthadiponectin treatment increased the mRNA expression ofSirt1 or Sirt6 which are histone deacetylases that function asa chromatin silencer to regulate recombination and genomicstability (Figures 3(h) and 3(i)) We therefore next examined

the involvement of HO-1 in the suppressive effect of fulllength adiponectin on LPS-induced HMGB1 release Treat-ment of the cells with zinc protoporphyrin (ZnPP) a HO-1 inhibitor did not have any effect on HMGB1 release fromeither LPS-stimulated or control cells (Figure 4) Howevertreatment with ZnPP for 1 h before full length adiponectintreatment almost completely abolished adiponectin sup-pression of LPS-induced HMGB1 release although it only

Mediators of Inflammation 5

0

1

2

3

4

5

6

7

HM

GB1

rele

ase (

AU)

minus

minus

minus

50

+

minus

+

50

+

10

+

100IL-10(ngmL)

LPS(200ngmL)

lowastlowast

Figure 2 Effect of recombinant IL-10 on LPS-induced HMGB1 release Raw 264 cells were cultured as described in Figure 1 legend and weretreated with IL-10 for 18 h then stimulated with LPS (200 ngmL) for another 24 h Culture medium was collected and analyzed by HMGB1western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results are expressed as means plusmn SEof three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 and

119901 lt 005 significance compared with LPStreated cells)

slightly inhibited the suppression by IL-10 These resultsindicate that increased expression of HO-1 in response tofull length adiponectin is necessary for adiponectin-mediatedprevention of LPS-induced HMGB1 release

We then examined whether increased expression of HO-1 mRNA by full length adiponectin was mediated throughAMP-activated kinase (AMPK) a main signaling pathwayof adiponectin action [33ndash35] Treatment of the cells withcompound C an AMPK inhibitor abolished the increase inexpression ofHO-1mRNAby full length adiponectin whereastreatment of the cells with wortmannin a phosphatidylinosi-tol-3-kinase (PI3K) inhibitor or with SB203580 a p38mitogen-activated protein kinase (p38MAPK) inhibitor didnot affect this increase (Figure 5) Consistent with theseresults treatment of the cells with compound C but notwith SB203580 abolished adiponectin-mediated suppressionof LPS-induced HMGB1 release (Figure 6)

4 Discussion

In the present study we demonstrated for the first timethat full length adiponectin prevents LPS-induced HMGB1translocation from the nucleus to the cytosol and its subse-quent release from Raw 264 mouse macrophage cells Thisprocess is most probably mediated by AMPK-dependentHO-1 induction as evidenced by the following results BothAMPK and HO-1 inhibitors prevented the suppression ofLPS-induced HMGB1 release by full length adiponectinand the AMPK inhibitor also prevented induction of HO-1mRNA by full length adiponectin Furthermore the mech-anism of the full length adiponectin effect is supported byprevious reports that showed that full length adiponectinactivates AMPK activity [33ndash35] that activation of AMPKby metformin or dehydrodiconiferyl alcohol enhances HO-1 expression and its activity [36 37] and that HO-1 isindispensable for the prevention of HMGB1 release [3839] Of course other events such as selective reduction in

TLR4 mRNA expression by full length adiponectin mightcontribute at least in part to the suppression of LPS-induced HMGB1 release since TLR4 is the predominantreceptor for LPS [40 41] and a similar decrease in cell-surfaceTLR4 expression is seen in macrophage cells treated withglobular adiponectin [42] However other intracellular sig-naling pathways related to p38MAPK PI3K and the nuclearhistone deacetylase sirtuin are unlikely to be involved in themechanism although they have been reported to be involvedin some adiponectin functions [43ndash46] or in the processes ofHO-1 induction and LPS-induced HMGB1 release [47ndash49]

We have also demonstrated that IL-10 is a potent inhibitorof LPS-induced HMGB1 release However the fact that fulllength adiponectin failed to induce IL-10 mRNA suggestedthat the suppression by full length adiponectin might not beattributed to IL-10 production This hypothesis is supportedby previous findings that the effects of full length adiponectinon macrophage function are independent of IL-10 [42 44]although anti-inflammatory effects of globular adiponectinare mediated by IL-10 [21 23 50ndash52] The discrepancybetween the role of IL-10 in the effects of full length andglobular adiponectin has not been explored but is possiblydue to different signals mediated through adipoR2 andadipoR1 respectively [42]

Accumulating evidence indicates that HO-1 plays a piv-otal role in the anti-inflammatory cytoprotective effects of awide variety of compounds including statins phytochemicalssuch as resveratrol and aspirin [53] HO-1 is a microsomalenzyme that catalyzes the degradation of proinflammatoryfree heme and produces equimolar amounts of carbonmonoxide bilirubin and iron [54] The mechanisms thatmediate the anti-inflammatory effects of HO-1 are not fullyunderstood but the potent antioxidant activity of bilirubinand the signaling gas activity of carbon monoxide arereported to suppress apoptosis necrosis inflammation andoxidative stress Interestingly HO-1 is induced by pathophys-iological stimuli including LPS and hemodynamic changes

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

4 Mediators of Inflammation

0

1

2

3

4

5

6

7lowastlowast

HM

GB1

rele

ase (

AU)

minus

minus

minus

5

+

minus

+

5

+

1

+

10

Adiponectin(120583gmL)

LPS(200ngmL)

(a)LP

S +

adip

onec

tinLP

SC

ontro

l

Nucleus HMGB1 Merge

(b)

LPS + adiponectinLPSControl

lowastlowast

0

10

20

30

40

Ratio

of c

ytos

olic

to n

ucle

ar H

MG

B1

(c)

Figure 1 Effect of recombinant adiponectin on LPS-induced HMGB1 release and HMGB1 cellular translocation Raw 264 cells were culturedin DMEM supplemented with 10 FBS and were cultured in serum-free OPTI-MEM Imedium for additional 12 hThe cells were treated withincreasing concentrations of adiponectin for 18 h then stimulated with LPS (200 ngmL) for another 24 h (a) Culture medium was collectedand analyzed by HMGB1 western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 005 significance comparedwith LPS treated cells) (b and c) Cellular HMGB1 was immunostained with an anti-HMGB1 rabbit primary and Alexa Fluor 488 anti-rabbitsecondary antibodies The nucleus was stained with DAPI Merge indicates the combination of both HMGB1 (Green) and nuclear (Blue)fluorescence The fluorescence intensities of cytosolic and nuclear HMGB1 in (b) were separately analyzed and the ratio of cytosolic HMGB1to nuclear HMGB1 is shown in (c) (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 significance compared with LPS treated cells)

(Figure 3(f)) while neither treatment affected the mRNAexpression of nuclear factor erythroid-derived 2 related factor2 (Nrf2) a transcription factor relatedwithHO-1 gene expres-sion (Figure 3(g)) In addition neither IL-10 nor full lengthadiponectin treatment increased the mRNA expression ofSirt1 or Sirt6 which are histone deacetylases that function asa chromatin silencer to regulate recombination and genomicstability (Figures 3(h) and 3(i)) We therefore next examined

the involvement of HO-1 in the suppressive effect of fulllength adiponectin on LPS-induced HMGB1 release Treat-ment of the cells with zinc protoporphyrin (ZnPP) a HO-1 inhibitor did not have any effect on HMGB1 release fromeither LPS-stimulated or control cells (Figure 4) Howevertreatment with ZnPP for 1 h before full length adiponectintreatment almost completely abolished adiponectin sup-pression of LPS-induced HMGB1 release although it only

Mediators of Inflammation 5

0

1

2

3

4

5

6

7

HM

GB1

rele

ase (

AU)

minus

minus

minus

50

+

minus

+

50

+

10

+

100IL-10(ngmL)

LPS(200ngmL)

lowastlowast

Figure 2 Effect of recombinant IL-10 on LPS-induced HMGB1 release Raw 264 cells were cultured as described in Figure 1 legend and weretreated with IL-10 for 18 h then stimulated with LPS (200 ngmL) for another 24 h Culture medium was collected and analyzed by HMGB1western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results are expressed as means plusmn SEof three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 and

119901 lt 005 significance compared with LPStreated cells)

slightly inhibited the suppression by IL-10 These resultsindicate that increased expression of HO-1 in response tofull length adiponectin is necessary for adiponectin-mediatedprevention of LPS-induced HMGB1 release

We then examined whether increased expression of HO-1 mRNA by full length adiponectin was mediated throughAMP-activated kinase (AMPK) a main signaling pathwayof adiponectin action [33ndash35] Treatment of the cells withcompound C an AMPK inhibitor abolished the increase inexpression ofHO-1mRNAby full length adiponectin whereastreatment of the cells with wortmannin a phosphatidylinosi-tol-3-kinase (PI3K) inhibitor or with SB203580 a p38mitogen-activated protein kinase (p38MAPK) inhibitor didnot affect this increase (Figure 5) Consistent with theseresults treatment of the cells with compound C but notwith SB203580 abolished adiponectin-mediated suppressionof LPS-induced HMGB1 release (Figure 6)

4 Discussion

In the present study we demonstrated for the first timethat full length adiponectin prevents LPS-induced HMGB1translocation from the nucleus to the cytosol and its subse-quent release from Raw 264 mouse macrophage cells Thisprocess is most probably mediated by AMPK-dependentHO-1 induction as evidenced by the following results BothAMPK and HO-1 inhibitors prevented the suppression ofLPS-induced HMGB1 release by full length adiponectinand the AMPK inhibitor also prevented induction of HO-1mRNA by full length adiponectin Furthermore the mech-anism of the full length adiponectin effect is supported byprevious reports that showed that full length adiponectinactivates AMPK activity [33ndash35] that activation of AMPKby metformin or dehydrodiconiferyl alcohol enhances HO-1 expression and its activity [36 37] and that HO-1 isindispensable for the prevention of HMGB1 release [3839] Of course other events such as selective reduction in

TLR4 mRNA expression by full length adiponectin mightcontribute at least in part to the suppression of LPS-induced HMGB1 release since TLR4 is the predominantreceptor for LPS [40 41] and a similar decrease in cell-surfaceTLR4 expression is seen in macrophage cells treated withglobular adiponectin [42] However other intracellular sig-naling pathways related to p38MAPK PI3K and the nuclearhistone deacetylase sirtuin are unlikely to be involved in themechanism although they have been reported to be involvedin some adiponectin functions [43ndash46] or in the processes ofHO-1 induction and LPS-induced HMGB1 release [47ndash49]

We have also demonstrated that IL-10 is a potent inhibitorof LPS-induced HMGB1 release However the fact that fulllength adiponectin failed to induce IL-10 mRNA suggestedthat the suppression by full length adiponectin might not beattributed to IL-10 production This hypothesis is supportedby previous findings that the effects of full length adiponectinon macrophage function are independent of IL-10 [42 44]although anti-inflammatory effects of globular adiponectinare mediated by IL-10 [21 23 50ndash52] The discrepancybetween the role of IL-10 in the effects of full length andglobular adiponectin has not been explored but is possiblydue to different signals mediated through adipoR2 andadipoR1 respectively [42]

Accumulating evidence indicates that HO-1 plays a piv-otal role in the anti-inflammatory cytoprotective effects of awide variety of compounds including statins phytochemicalssuch as resveratrol and aspirin [53] HO-1 is a microsomalenzyme that catalyzes the degradation of proinflammatoryfree heme and produces equimolar amounts of carbonmonoxide bilirubin and iron [54] The mechanisms thatmediate the anti-inflammatory effects of HO-1 are not fullyunderstood but the potent antioxidant activity of bilirubinand the signaling gas activity of carbon monoxide arereported to suppress apoptosis necrosis inflammation andoxidative stress Interestingly HO-1 is induced by pathophys-iological stimuli including LPS and hemodynamic changes

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

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Diabetes ResearchJournal of

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 5

0

1

2

3

4

5

6

7

HM

GB1

rele

ase (

AU)

minus

minus

minus

50

+

minus

+

50

+

10

+

100IL-10(ngmL)

LPS(200ngmL)

lowastlowast

Figure 2 Effect of recombinant IL-10 on LPS-induced HMGB1 release Raw 264 cells were cultured as described in Figure 1 legend and weretreated with IL-10 for 18 h then stimulated with LPS (200 ngmL) for another 24 h Culture medium was collected and analyzed by HMGB1western blotting followed by quantification of the intensity of the chemiluminescent HMGB1 band The results are expressed as means plusmn SEof three independent experiments (lowastlowast119901 lt 001 significance compared with control 119901 lt 001 and

119901 lt 005 significance compared with LPStreated cells)

slightly inhibited the suppression by IL-10 These resultsindicate that increased expression of HO-1 in response tofull length adiponectin is necessary for adiponectin-mediatedprevention of LPS-induced HMGB1 release

We then examined whether increased expression of HO-1 mRNA by full length adiponectin was mediated throughAMP-activated kinase (AMPK) a main signaling pathwayof adiponectin action [33ndash35] Treatment of the cells withcompound C an AMPK inhibitor abolished the increase inexpression ofHO-1mRNAby full length adiponectin whereastreatment of the cells with wortmannin a phosphatidylinosi-tol-3-kinase (PI3K) inhibitor or with SB203580 a p38mitogen-activated protein kinase (p38MAPK) inhibitor didnot affect this increase (Figure 5) Consistent with theseresults treatment of the cells with compound C but notwith SB203580 abolished adiponectin-mediated suppressionof LPS-induced HMGB1 release (Figure 6)

4 Discussion

In the present study we demonstrated for the first timethat full length adiponectin prevents LPS-induced HMGB1translocation from the nucleus to the cytosol and its subse-quent release from Raw 264 mouse macrophage cells Thisprocess is most probably mediated by AMPK-dependentHO-1 induction as evidenced by the following results BothAMPK and HO-1 inhibitors prevented the suppression ofLPS-induced HMGB1 release by full length adiponectinand the AMPK inhibitor also prevented induction of HO-1mRNA by full length adiponectin Furthermore the mech-anism of the full length adiponectin effect is supported byprevious reports that showed that full length adiponectinactivates AMPK activity [33ndash35] that activation of AMPKby metformin or dehydrodiconiferyl alcohol enhances HO-1 expression and its activity [36 37] and that HO-1 isindispensable for the prevention of HMGB1 release [3839] Of course other events such as selective reduction in

TLR4 mRNA expression by full length adiponectin mightcontribute at least in part to the suppression of LPS-induced HMGB1 release since TLR4 is the predominantreceptor for LPS [40 41] and a similar decrease in cell-surfaceTLR4 expression is seen in macrophage cells treated withglobular adiponectin [42] However other intracellular sig-naling pathways related to p38MAPK PI3K and the nuclearhistone deacetylase sirtuin are unlikely to be involved in themechanism although they have been reported to be involvedin some adiponectin functions [43ndash46] or in the processes ofHO-1 induction and LPS-induced HMGB1 release [47ndash49]

We have also demonstrated that IL-10 is a potent inhibitorof LPS-induced HMGB1 release However the fact that fulllength adiponectin failed to induce IL-10 mRNA suggestedthat the suppression by full length adiponectin might not beattributed to IL-10 production This hypothesis is supportedby previous findings that the effects of full length adiponectinon macrophage function are independent of IL-10 [42 44]although anti-inflammatory effects of globular adiponectinare mediated by IL-10 [21 23 50ndash52] The discrepancybetween the role of IL-10 in the effects of full length andglobular adiponectin has not been explored but is possiblydue to different signals mediated through adipoR2 andadipoR1 respectively [42]

Accumulating evidence indicates that HO-1 plays a piv-otal role in the anti-inflammatory cytoprotective effects of awide variety of compounds including statins phytochemicalssuch as resveratrol and aspirin [53] HO-1 is a microsomalenzyme that catalyzes the degradation of proinflammatoryfree heme and produces equimolar amounts of carbonmonoxide bilirubin and iron [54] The mechanisms thatmediate the anti-inflammatory effects of HO-1 are not fullyunderstood but the potent antioxidant activity of bilirubinand the signaling gas activity of carbon monoxide arereported to suppress apoptosis necrosis inflammation andoxidative stress Interestingly HO-1 is induced by pathophys-iological stimuli including LPS and hemodynamic changes

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

6 Mediators of Inflammation

0

002

004

006

008

01

012

014

Cont APN IL-10

Tlr4

Gap

dh

TLR4

lowast

(a)

0

002

004

006

008

01

Cont APN IL-10

Tlr2

Gap

dh

lowast

TLR2

(b)

0

0005

001

0015

002

Cont APN IL-10

Ly96

Gap

dh

MD2

(c)

0

001

002

003

004

005

006

007

Cont APN IL-10

Hm

gb1

Gap

dh

HMGB1

lowast

(d)

0

005

01

015

02

025

03

Cont APN IL-10

IL-10

IL-10

Gap

dh (times

10minus4)

lowast

(e)

0

002

004

006

008

01

Cont APN IL-10

Hm

ox1

Gap

dh

HO-1

lowast

lowastlowastlowast

(f)

0

005

01

015

02

Nfe

212

Gap

dh

Nrf2

Cont APN IL-10(g)

0

02

04

06

08

1

12

14

16 Sirt1

Cont APN IL-10

Sirt

uin1

Gap

dh (times

10minus3)

(h)

0

01

02

03

04

05 Sirt6

Cont APN IL-10

Sirt

uin6

Gap

dh (times

10minus2)

(i)

Figure 3 Effects of adiponectin and IL-10 on mRNA expression in RAW 264 cells Raw 264 cells were cultured as described in Figure 1legend and were treated with adiponectin (APN 10120583gmL) and IL-10 (100 ngmL) for 18 h RNA was extracted and expressions of (a) TLR4(b) TLR2 (c)MD2 (d) HMGB1 (e) IL-10 (f) HO-1 (g) Nrf2 (h) Sirt1 (i) Sirt6 and Gapdh (control) mRNAs weremeasured using qRT-PCRThe results are expressed as means plusmn SE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowast119901 lt 005 significance compared with control(Cont))

but in most cases pathophysiological activation of HO-1results in only a transient or marginal increase in HO-1that falls below the threshold necessary to activate down-stream components such as carbon monoxide [53] In thepresent study a fourfold increase in HO-1 mRNA expression

compared to its basal expression was induced by full lengthadiponectin whereas only a twofold increase was inducedby IL-10 Combined with the result that the HO-1 inhibitoronly partially abrogated the suppression by IL-10 of LPS-induced HMGB1 release these findings suggested that IL-10

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 7

HM

GB1

rele

ase (

AU)

02468

101214

1 2 3 4 5 6 7 8

+

minus

minus

minus

minus

minus

+

minus

minus

+

minus

minus

minus

+

+

minus

minus

+

minus

+

minus

+

+

+

minus

+

minus

minus

+

+

+

minus

+IL-10(100ngmL)

Adiponectin(10120583gmL)

ZnPP(1120583M)

LPS(200ngmL)

lowastlowast

lowastlowast

1111111111111 2222222222222 333333333333333333333333333 444444444444444444444444444 5555555555555555555555555 66666666666666666666666666 7777777777777777777777 888888888888888888888888888888888

Figure 4 Effect of an HO-1 inhibitor on the anti-inflammatoryaction of adiponectin or IL-10 on LPS-induced HMGB1 releaseRaw 264 cells were cultured as described in Figure 1 legend andwere treated with dimethyl sulfoxide (DMSO control) or ZnPP(1 120583M) for 1 h before treatment with adiponectin (10120583gmL) orIL-10 (100 ngmL) for 18 h Cells were then stimulated with LPS(200 ngmL) for another 24 h Culture medium was collected andanalyzed by HMGB1 western blotting followed by quantification ofthe intensity of the chemiluminescent HMGB1 bandThe results areexpressed as means plusmn SE of three independent experiments (lowastlowast119901 lt001 significance compared with control 119901 lt 001 and

119901 lt 005

significance compared with LPS treated cells +119901 lt 005 significancecompared with LPS plus ZnPP treated cells)

SB203580 WortmanninCompound CminusAdiponectin + minus + minus + minus +

lowastlowastlowastlowast

lowastlowastlowast

0

01

02

03

04

05

06

Hm

ox1

Gap

dh

Figure 5 Effect of kinase inhibitors on adiponectin-induced HO-1mRNA expression in RAW 264 cells Raw 264 cells were culturedas described in Figure 1 legend and were treated with adiponectin(10120583gmL) for 18 h in the presence of DMSO (control) compoundC (10 120583M) wortmannin (1 120583M) or SB203580 (10120583M) RNA wasextracted and expressions of Hmox1 (HO-1) and Gapdh mRNAsweremeasured using qRT-PCRThe results are expressed asmeansplusmnSE of three independent experiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001significance compared with control)

induces only a marginal increase in HO-1 mRNA and mainlyutilizes an HO-1-independent pathway for the suppression ofHMGB1 release

In summary we provide the novel finding that full lengthadiponectin suppresses HMGB1 release by LPS throughan AMPK-HO-1-dependent pathway Therefore adiponectinplays an important role as a regulator of inflammation

minus

minus

minus

+

minus

minus

+

minus

+

+

+

minus

+

+

+Adiponectin(10120583gmL)

Compound C(10120583M)

LPS(200ngmL)

lowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(a)

minus

minus

minus

+

minus

minus

+

+

minus

+

minus

+

+

+

+

minus

+

minus

+

SB203580(10120583M)

Adiponectin(10120583gmL)

LPS(200ngmL)

lowastlowastlowast lowastlowastlowast

0

2

4

6

8

10

HM

GB1

rele

ase (

AU)

(b)

Figure 6 Effect of kinase inhibitors on the suppression byadiponectin of LPS-induced HMGB1 release Raw 264 cells werecultured as described in Figure 1 legend and were treated withcompound C (a) or SB203580 (b) for 1 h before treatment withadiponectin (10120583gmL) for 18 h following which they were stimu-lated with LPS (200 ngmL) for another 24 h Culture media werecollected and analyzed by HMGB1 western blotting followed byquantification of the intensity of the chemiluminescent HMGB1band The results are expressed as means plusmn SE of three independentexperiments (lowastlowastlowast119901 lt 0001 and lowastlowast119901 lt 001 significance comparedwith control 119901 lt 005 significance comparedwith LPS treated cells+119901 lt 005 significance compared with LPS plus SB203580 treatedcells)

through inhibition of both early and late proinflammatorymediators under pathological conditions such as sepsisThus it is possible that adiponectin might be a target fordevelopment of therapeutic agents against sepsis and othersystemic inflammatory disorders

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

8 Mediators of Inflammation

Competing Interests

The authors declare that there are no competing interestsregarding the publication of this paper

References

[1] G S Martin D M Mannino S Eaton and M Moss ldquoTheepidemiology of sepsis in the United States from 1979 through2000rdquoTheNew England Journal of Medicine vol 348 no 16 pp1546ndash1554 2003

[2] N C Riedemann R-F Guo and P A Ward ldquoThe enigma ofsepsisrdquo The Journal of Clinical Investigation vol 112 no 4 pp460ndash467 2003

[3] A Oberholzer C Oberholzer and L L Moldawer ldquoSepsissyndromes understanding the role of innate and acquiredimmunityrdquo Shock vol 16 no 2 pp 83ndash96 2001

[4] HWang O BloomM Zhang et al ldquoHMG-1 as a late mediatorof endotoxin lethality in micerdquo Science vol 285 no 5425 pp248ndash251 1999

[5] M Guha and N Mackman ldquoLPS induction of gene expressionin human monocytesrdquo Cellular Signalling vol 13 no 2 pp 85ndash94 2001

[6] B Beutler and E T Rietschel ldquoInnate immune sensing and itsroots the story of endotoxinrdquoNature Reviews Immunology vol3 no 2 pp 169ndash176 2003

[7] E Abraham A Anzueto G Gutierrez et al ldquoDouble-blind randomised controlled trial of monoclonal antibody tohuman tumour necrosis factor in treatment of septic shockNORASEPT II Study GrouprdquoThe Lancet vol 351 no 9107 pp929ndash933 1998

[8] A Leelahavanichkul H Yasuda K Doi et al ldquoMethyl-2-acetamidoacrylate an ethyl pyruvate analog decreases sepsis-induced acute kidney injury in micerdquo American Journal ofPhysiologymdashRenal Physiology vol 295 no 6 pp F1825ndashF18352008

[9] U Andersson H Wang K Palmblad et al ldquoHigh mobilitygroup 1 protein (HMG-1) stimulates proinflammatory cytokinesynthesis in human monocytesrdquo Journal of ExperimentalMedicine vol 192 no 4 pp 565ndash570 2000

[10] J S Park D Svetkauskaite Q He et al ldquoInvolvement of toll-likereceptors 2 and 4 in cellular activation by high mobility groupbox 1 proteinrdquoThe Journal of Biological Chemistry vol 279 no9 pp 7370ndash7377 2004

[11] M T Lotze and K J Tracey ldquoHigh-mobility group box 1 protein(HMGB1) nuclear weapon in the immune arsenalrdquo NatureReviews Immunology vol 5 no 4 pp 331ndash342 2005

[12] A Rouhiainen S Tumova L ValmuNKalkkinen andH Rau-vala ldquoPivotal Advance analysis of proinflammatory activity ofhighly purified eukaryotic recombinantHMGB1 (amphoterin)rdquoJournal of Leukocyte Biology vol 81 no 1 pp 49ndash58 2007

[13] H Wang H Yang and K J Tracey ldquoExtracellular roleof HMGB1 in inflammation and sepsisrdquo Journal of InternalMedicine vol 255 no 3 pp 320ndash331 2004

[14] H Yang M Ochani J Li et al ldquoReversing established sepsiswith antagonists of endogenous high-mobility group box 1rdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 101 no 1 pp 296ndash301 2004

[15] Y Arita S Kihara N Ouchi et al ldquoParadoxical decrease of anadipose-specific protein adiponectin in obesityrdquo Biochemicaland Biophysical Research Communications vol 257 no 1 pp79ndash83 1999

[16] H Tilg and A M Wolf ldquoAdiponectin a key fat-derivedmolecule regulating inflammationrdquo Expert Opinion on Thera-peutic Targets vol 9 no 2 pp 245ndash251 2005

[17] N Ouchi and K Walsh ldquoA novel role for adiponectin in theregulation of inflammationrdquo Arteriosclerosis Thrombosis andVascular Biology vol 28 no 7 pp 1219ndash1221 2008

[18] N Ouchi J L Parker J J Lugus and K Walsh ldquoAdipokines ininflammation andmetabolic diseaserdquoNature Reviews Immunol-ogy vol 11 no 2 pp 85ndash97 2011

[19] T Yokota K Oritani I Takahashi et al ldquoAdiponectin a newmember of the family of soluble defense collagens negativelyregulates the growth of myelomonocytic progenitors and thefunctions of macrophagesrdquo Blood vol 96 no 5 pp 1723ndash17322000

[20] K Ohashi J L Parker N Ouchi et al ldquoAdiponectin promotesmacrophage polarization toward an anti-inflammatory pheno-typerdquo Journal of Biological Chemistry vol 285 no 9 pp 6153ndash6160 2010

[21] P Mandal B T Pratt M Barnes M R McMullen and LE Nagy ldquoMolecular mechanism for adiponectin-dependentm2 macrophage polarization link between the metabolic andinnate immune activity of full-length adiponectinrdquoThe Journalof Biological Chemistry vol 286 no 15 pp 13460ndash13469 2011

[22] M C Wulster-Radcliffe K M Ajuwon J Wang J A Chris-tian and M E Spurlock ldquoAdiponectin differentially regulatescytokines in porcinemacrophagesrdquoBiochemical andBiophysicalResearch Communications vol 316 no 3 pp 924ndash929 2004

[23] A M Wolf D Wolf H Rumpold B Enrich and H TilgldquoAdiponectin induces the anti-inflammatory cytokines IL-10and IL-1RA in human leukocytesrdquo Biochemical and BiophysicalResearch Communications vol 323 no 2 pp 630ndash635 2004

[24] A Xu Y Wang H Keshaw L Y Xu K S L Lam and GJ S Cooper ldquoThe fat-derived hormone adiponectin alleviatesalcoholic and nonalcoholic fatty liver diseases in micerdquo Journalof Clinical Investigation vol 112 no 1 pp 91ndash100 2003

[25] T Masaki S Chiba H Tatsukawa et al ldquoAdiponectin protectsLPS-induced liver injury through modulation of TNF-120572 in KK-Ay obese micerdquo Hepatology vol 40 no 1 pp 177ndash184 2004

[26] J M Konter J L Parker E Baez et al ldquoAdiponectin attenuateslipopolysaccharide-induced acute lung injury through suppres-sion of endothelial cell activationrdquo Journal of Immunology vol188 no 2 pp 854ndash863 2012

[27] Y Watanabe R Shibata N Ouchi et al ldquoAdiponectin amelio-rates endotoxin-induced acute cardiac injuryrdquo BioMed ResearchInternational vol 2014 Article ID 382035 6 pages 2014

[28] Y Uji H Yamamoto H Tsuchihashi et al ldquoAdiponectindeficiency is associated with severe polymicrobial sepsis highinflammatory cytokine levels and high mortalityrdquo Surgery vol145 no 5 pp 550ndash557 2009

[29] W Jiang and D S Pisetsky ldquoThe role of IFN-alpha and nitricoxide in the release of HMGB1 by RAW 2647 cells stimulatedwith polyinosinic-polycytidylic acid or lipopolysacchariderdquoJournal of Immunology vol 177 no 5 pp 3337ndash3343 2006

[30] K Tsoyi H J Jang I T Nizamutdinova et al ldquoMetformininhibits HMGB1 release in LPS-treated RAW 2647 cells andincreases survival rate of endotoxaemic micerdquo British Journalof Pharmacology vol 162 no 7 pp 1498ndash1508 2011

[31] C K Zetterstrom W Jiang H Wahamaa et al ldquoPivotalAdvance inhibition ofHMGB1nuclear translocation as amech-anism for the anti-rheumatic effects of gold sodium thiomalaterdquoJournal of Leukocyte Biology vol 83 no 1 pp 31ndash38 2008

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Mediators of Inflammation 9

[32] A Chorny and M Delgado ldquoNeuropeptides rescue mice fromlethal sepsis by down-regulating secretion of the late-actinginflammatory mediator high mobility group box 1rdquo AmericanJournal of Pathology vol 172 no 5 pp 1297ndash1307 2008

[33] T Kadowaki and T Yamauchi ldquoAdiponectin and adiponectinreceptorsrdquo Endocrine Reviews vol 26 no 3 pp 439ndash451 2005

[34] T Yamauchi J Kamon Y Ito et al ldquoCloning of adiponectinreceptors that mediate antidiabetic metabolic effectsrdquo Naturevol 423 pp 762ndash769 2003

[35] T Kadowaki T Yamauchi N Kubota K Hara K Ueki andK Tobe ldquoAdiponectin and adiponectin receptors in insulinresistance diabetes and the metabolic syndromerdquo Journal ofClinical Investigation vol 116 no 7 pp 1784ndash1792 2006

[36] G Ashabi L Khalaj F Khodagholi M Goudarzvand and ASarkaki ldquoPre-treatment with metformin activates Nrf2 antiox-idant pathways and inhibits inflammatory responses throughinduction of AMPK after transient global cerebral ischemiardquoMetabolic Brain Disease vol 30 no 3 pp 747ndash754 2015

[37] J Lee and S Kim ldquoUpregulation of heme oxygenase-1 expres-sion by dehydrodiconiferyl alcohol (DHCA) through theAMPK-Nrf2 dependent pathwayrdquo Toxicology and Applied Phar-macology vol 281 no 1 pp 87ndash100 2014

[38] K Tsoyi T Y Lee Y S Lee et al ldquoHeme-oxygenase-1induction and carbon monoxide-releasing molecule inhibitlipopolysaccharide (LPS)-induced high-mobility group box 1release in vitro and improve survival of mice in LPS- and cecalligation and puncture-induced sepsis model in vivordquoMolecularPharmacology vol 76 no 1 pp 173ndash182 2009

[39] H-G Chen K-L Xie H-Z Han et al ldquoHeme oxygenase-1mediates the anti-inflammatory effect of molecular hydrogenin LPS-stimulated RAW 2647 macrophagesrdquo InternationalJournal of Surgery vol 11 no 10 pp 1060ndash1066 2013

[40] CN Lumeng ldquoInnate immune activation in obesityrdquoMolecularAspects of Medicine vol 34 no 1 pp 12ndash29 2013

[41] D M Rocha A P Caldas L L Oliveira J Bressan and HH Hermsdorff ldquoSaturated fatty acids trigger TLR4-mediatedinflammatory responserdquo Atherosclerosis vol 244 pp 211ndash2152016

[42] P Mandal S Roychowdhury P-H Park B T Pratt T Rogerand L E Nagy ldquoAdiponectin and heme oxygenase-1 suppressTLR4MyD88-independent signaling in rat Kupffer cells and inmice after chronic ethanol exposurerdquo The Journal of Immunol-ogy vol 185 no 8 pp 4928ndash4937 2010

[43] M J Yoon G Y Lee J-J Chung et al ldquoAdiponectin increasesfatty acid oxidation in skeletal muscle cells by sequential acti-vation of AMP-activated protein kinase p38 mitogen-activatedprotein kinase and peroxisome proliferator-activated receptor120572rdquo Diabetes vol 55 no 9 pp 2562ndash2570 2006

[44] E J Folco V Z Rocha M Lopez-Ilasaca and P LibbyldquoAdiponectin inhibits pro-inflammatory signaling in humanmacrophages independent of interleukin-10rdquo The Journal ofBiological Chemistry vol 284 no 38 pp 25569ndash25575 2009

[45] M E Grossmann K J Nkhata N K Mizuno A Ray andM PCleary ldquoEffects of adiponectin on breast cancer cell growth andsignalingrdquo British Journal of Cancer vol 98 no 2 pp 370ndash3792008

[46] N T Pun A Subedi M J Kim and P-H Park ldquoGlobularadiponectin causes tolerance to LPS-induced TNF-120572 expressionvia autophagy induction in RAW 2647 macrophages involve-ment of SIRT1FoxO3A axisrdquo PLoS ONE vol 10 no 5 ArticleID e0124636 2015

[47] Y M Kim H J Kim and K C Chang ldquoGlycyrrhizin reducesHMGB1 secretion in lipopolysaccharide-activated RAW 2647cells and endotoxemic mice by p38Nrf2-dependent inductionof HO-1rdquo International Immunopharmacology vol 26 no 1 pp112ndash118 2015

[48] J Wang X Hu J Xie W Xu and H Jiang ldquoBeta-1-adrenergicreceptors mediate Nrf2-HO-1-HMGB1 axis regulation to atten-uate hypoxiareoxygenation-induced cardiomyocytes injury invitrordquo Cellular Physiology and Biochemistry vol 35 no 2 pp767ndash777 2015

[49] T D Walko III V Di Caro J Piganelli T R Billiar R SClark and R K Aneja ldquoPoly(ADP-ribose) polymerase 1-sirtuin1 functional interplay regulates LPS-mediated high mobilitygroup box 1 secretionrdquoMolecularMedicine vol 20 pp 612ndash6242014

[50] M Kumada S Kihara N Ouchi et al ldquoAdiponectin specifi-cally increased tissue inhibitor of metalloproteinase-1 throughinterleukin-10 expression in human macrophagesrdquo Circulationvol 109 no 17 pp 2046ndash2049 2004

[51] P-H Park M R McMullen H Huang V Thakur and LE Nagy ldquoShort-term treatment of RAW2647 macrophageswith adiponectin increases tumor necrosis factor-120572 (TNF-120572)expression via ERK12 activation and Egr-1 expression role ofTNF-120572 in adiponectin-stimulated interleukin-10 productionrdquoThe Journal of Biological Chemistry vol 282 no 30 pp 21695ndash21703 2007

[52] P Mandal P-H Park M R McMullen B T Pratt and LE Nagy ldquoThe anti-inflammatory effects of adiponectin aremediated via a heme oxygenase-1-dependent pathway in ratkupffer cellsrdquo Hepatology vol 51 no 4 pp 1420ndash1429 2010

[53] J F Ndisang ldquoRole of the heme oxygenase-adiponectin-atrialnatriuretic peptide axis in renal functionrdquo Current Pharmaceu-tical Design vol 21 no 30 pp 4380ndash4391 2015

[54] A Paine B Eiz-Vesper R Blasczyk and S ImmenschuhldquoSignaling to heme oxygenase-1 and its anti-inflammatorytherapeutic potentialrdquo Biochemical Pharmacology vol 80 no12 pp 1895ndash1903 2010

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom