A Novel Antioxidant Multitarget Iron Chelator M30 Protects Hepatocytes against Ethanol-Induced Injury

Research ArticleA Novel Antioxidant Multitarget Iron Chelator M30 ProtectsHepatocytes against Ethanol-Induced Injury

Jia Xiao123 Yi Lv2 Bin Lin3 George L Tipoe3 Moussa B H Youdim4

Feiyue Xing2 and Yingxia Liu1

1National Key Disciplines for Infectious Diseases Shenzhen Third Peoplersquos Hospital Shenzhen 518112 China2Department of Immunobiology Institute of Tissue Transplantation and Immunology Jinan University Guangzhou 510632 China3Department of Anatomy The University of Hong Kong Pokfulam Hong Kong4Eve Topf Centers of Excellence Technion Rappaport Family Faculty of Medicine and Department of Pharmacology31096 Haifa Israel

Correspondence should be addressed to Feiyue Xing tfyxingjnueducn and Yingxia Liu yingxialiuhotmailcom

Received 13 October 2014 Accepted 14 January 2015

Academic Editor Vittorio Calabrese

Copyright copy 2015 Jia Xiao et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The multitarget iron chelator M30 is a novel antioxidant and protective agent against oxidative stress in a spectrum of diseasesHowever there is no report regarding its role in liver diseases Since oxidative stress is one of the major pathological events duringthe progression of alcoholic liver diseases the protective effects andmechanisms ofM30 on ethanol-induced hepatocyte injurywereinvestigated in this study Rat hepatocyte line BRL-3A was pretreated with M30 prior to ethanol treatment Cell death apoptosisoxidative stress and inflammation were examined Specific antagonists and agonists were applied to determine the involvementsof hypoxia inducible factor-1 alpha (HIF-1120572) and its upstream adenylate cyclase (AC)cyclic AMP (cAMP)protein kinase A(PKA)HIF-1120572NOD-like receptor 3 (NLRP3) inflammasomepathwayWe found thatM30 significantly attenuated ethanol-inducedcellular death apoptosis production of reactive oxygen species (ROS) and secretion of inflammatory cytokines and inhibitedactivation of the ACcAMPPKAHIF-1120572NLRP3 inflammasome pathway Inhibition and activation of the ACcAMPPKAHIF-1120572pathway mimicked and abolished the effects of M30 respectively In conclusion inhibition of the ACcAMPPKAHIF-1120572NLRP3inflammasome pathway by M30 partially contributes to its attenuation of hepatocyte injury caused by ethanol exposure

1 Introduction

Alcoholic liver diseases (ALDs) including acute alcoholicliver injury liver failure alcoholic fatty liver disease (AFLD)and alcoholic steatohepatitis (ASH) annually result in anestimated 25 million deaths (4 of all mortality) world-wide [1] Alcoholism or alcohol use disorder is defined asoverconsumption of ethanol (men gt 30 gday and women gt20 gday) [2] The only definitive clinical treatment for ALDis liver transplantation Abstinence is critical but usuallycannot reverse advanced ALD It should be accompanied bysupportive therapy and nutritional management [3] To datealthough a spectrum of key signaling pathways and thera-peutic targets have been described in ALD the interactionbetween oxidative stress and inflammation is considered to be

a central event during the initiation and progression of ALD[4 5]

Inflammasomes are a group of large caspase-1-activatingprotein complexes in response to the evocation of innateimmunity and the production of proinflammatory cytokinesinterleukin-1120573 (IL-1120573) and IL-18 Inflammasome activationhas been shown to induce cell pyroptosis a process ofprogrammed cell death distinct from apoptosis [6] Inflam-masomes particularly NOD-like receptor 3 (NLRP3) inflam-masome are shown to be activated in a variety of acuteand chronic liver diseases including ALD [7] Our previousstudy found that Lycium barbarum polysaccharide atten-uated ethanol-induced hepatocyte injury partially throughregulating the thioredoxin-interacting protein- (TXNIP-)NLRP3 inflammasome pathway [7] However the upstream

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2015 Article ID 607271 11 pageshttpdxdoiorg1011552015607271

2 Oxidative Medicine and Cellular Longevity

regulators of NLRP3 during ALD progression await furtherinvestigation

Hypoxia-inducible factor-1 alpha (HIF-1120572) is activated byhypoxia and is a master regulator of cellular redox status anddownstream production of proinflammatory cytokines (egtumor necrosis factor-alpha (TNF-120572) and IL-6) [8] It couldbe activated by increased cellular oxygen levels primarily atthe protein level [9] Recent studies pointed out that underhypoxic or oxidative stress environments adenylate cyclase(AC) activates HIF-1120572 through cyclic AMP (cAMP)proteinkinase A (PKA) pathway [10] Since ethanol induces hypoxiaand elevates HIF-1120572 in the liver [11] it was speculated thatHIF-1120572 might be a novel therapeutic target for ethanol-induced injury and an upstream regulator of NLRP3 inflam-masome

In the current study we aimed to determine whether theACcAMPPKAHIF-1120572 pathway andNLRP3 inflammasomeare involved in the protective effect of a novel multitarget ironchelator (M30) against ethanol-induced hepatocyte injury invitro

2 Materials and Methods

21 Drugs and Chemicals Themultifunctional iron chelatorM30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxy-quinoline) was synthesized and kindly provided by VarinelInc (Philadelphia PA) [12 13] CAY10585 (HIF-1120572 inhibitor)was purchased from Cayman Chemical (Ann Arbor MI)Forskolin (AC agonist) SQ22536 (AC inhibitor) H89 (PKAinhibitor) and db-cAMP (stable cAMP analogue) werepurchased from Sigma-Aldrich (St Louis MO) Antibodiesagainst catalase (CAT) glutathione peroxidase 1 (GPx1)NLRP3 apoptosis-associated speck-like protein containinga CARD (ASC) caspase-1 and 120573-actin were ordered fromAbcam (Cambridge UK)

22 Cell Culture and Treatments Rat normal hepatocyte cellline BRL-3A was supplied by the Cell Bank of Type Cul-ture Collection of Chinese Academy of Sciences (ShanghaiChina) Cells were cultured in DMEM with 10 (vv) FBS at37∘C with 5 CO

2 Before drug treatment cells must reach

a confluence of 60ndash70 For the pretreatment with M30PBS dissolved M30 was added 2 hours before the ethanoltreatment For the treatments using agonists and inhibitorsof the ACcAMPPKAHIF-1120572 pathway drugs were addedalong with M30 or individually 2 hours before the ethanoltreatment at designated concentrations

23 MTT Assay The cell viability was evaluated by the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma-Aldrich St Louis MO) method After drugtreatment cells were washed by sterile PBS for 3 timesand then incubated with 5mgmL MTT for 3 hours andsubsequently dissolved in dimethyl sulfoxide (DMSO) Theabsorbance of cells was measured at 570 nm The percent ofcell viability was defined as the relative absorbance of treatedcells versus untreated cells

24 Detection of Alanine Aminotransferase (ALT) LevelsAfter treatments cell supernatant was collected and the levelof ALT was detected by an ALTGPT kit (EIAab ScienceWuhan China) according to manufacturerrsquos instruction

25 Quantification of Apoptotic Cells After treatmentHoechst 33342 (Sigma 5 120583gmL) and propidium iodide(Sigma 5 120583gmL) were added to each well to stain live cellsThe cell population was separated into 3 groups live cellsshowed only a low level of fluorescence apoptotic cellsshowed a higher level of blue fluorescence and dead cellsshowed low-blue and high-red fluorescence Stained cellswere observed and quantified by two independent cellbiologists without knowing the grouping The results wereexpressed as the percentage of apoptosis (PA) PA = apoptoticcell numbertotal cell number times 100 [14]

26 Caspase-37 Activity The activity of caspase-37 in celllysate was measured using the Cell Meter Caspase-37 Activ-ity Apoptosis Assay Kit (AAT Bio Sunnyvale CA) accordingto the user manual The results were read at 520 nm in amicroplate reader (Bio-Rad) and expressed as fold change ofthe control

27 Measurement of ROS Production Intracellular produc-tion of ROS was detected by a fluorescence probemdash2101584071015840-dichlorofluorescin diacetate (DCFH-DA Sigma-Aldrich) aspreviously described [7] Briefly after treatment cells werewashed three times with PBS and then incubated with 10 120583MDCFH-DA for 30min at 37∘C for green fluorescent light visu-alization Quantification of green fluorescence was analyzedby using ImageJ software (Version 148 National Institutesof Health Bethesda MD) Fluorescent quantification resultswere then normalized by the protein amount of each specificplate well (sim110ndash150 120583gwell of a 24-well plate)

28 GSHGSSG Ratio Measurement To determine the intra-cellular oxidative status the ratio of reduced glutathione(GSH) to oxidized glutathione (GSSG) in each cellular pro-tein sample was measured by using a GSHGSSG detectionassay kit from Abcam (Cambridge UK)

29 RNA Extraction and Quantitative PCR Total cellularRNA was extracted using illustra RNAspin mini kit (GEhealthcare UK) The preparation of the first-strand cDNAwas conducted following the instruction of the SuperScriptFirst-Strand Synthesis System (Invitrogen Carlsbad CA)The mRNA expression levels of target genes were mea-sured by Takara SYBR premix Taq quantitative PCR system(Takara Bio Inc Shiga Japan) and in MyiQ2 real-time PCRmachine (Bio-Rad Hercules CA) Parallel amplification ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) wasused as an internal control Relative quantification wascalculated using the 2minusΔΔCt method Primers and PCR condi-tions were described in previous literature [15] The relativeexpression of the specific gene to the internal control wasobtained and then expressed as percentage of the controlvalue All real-time PCR procedures including the design of

Oxidative Medicine and Cellular Longevity 3

EtOH (250 mM) minusminus

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+minus

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24h48h

(a)

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Ctrl EtOH M30 E + M30

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leve

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IL)

lowastlowastlowast

(b)

Figure 1 M30 improved hepatocytes injury (a) Cell viability after 24-hour and 48-hour treatments and (b) released ALT concentration inthe culture medium Data from each group (119899 = 4) were expressed as means plusmn SEM Statistical comparison between groups was done usingthe Kruskal-Wallis test followed by Dunnrsquos post hoc test to detect differences in all groups ldquolowast lowast lowastrdquo means significantly different from controlgroup (119875 lt 0001) ldquordquo means significantly different from ethanol exposure group (119875 lt 001) ldquordquo means significantly different fromethanol exposure group (119875 lt 0001) EtOH ethanol M30 vehicle M30 treatment E + M30 ethanol + M30 cotreatment

primers validation of PCR environment and quantificationmethods were performed following the MIQE guideline [16]

210 Western Blot Western blot analyses of cell lysates(cytosolic or nuclear fractions) were performed as previouslydescribed [17] The ratio of the optical density of the proteinproduct to the internal control (120573-actin) was obtained and thedata were expressed as ratio or percentage of the control

211 Enzyme-Linked Immunosorbent Assay (ELISA) AssayThe levels of secreted TNF-120572 IL-1120573 and IL-6 in culturemedium were measured using corresponding ELISA kitsfrom PeproTech (PeproTech Inc Rocky Hill NJ) accordingto user instructions ELISA assay kit for secreted IL-18was purchased from Invitrogen (Carlsbad CA) The levelof HIF-1120572 was measured using an EIISA kit from Abcam(Cambridge UK)

212 Statistical Analysis Data were expressed as means plusmnSEM Comparison between groups was examined using theKruskal-Wallis test followed by Dunnrsquos post hoc test A valueof 119875 lt 005 was considered to be statistically significant(Prism 50 Graphpad software Inc San Diego CA)

3 Results

31 M30 Inhibited Ethanol-Induced Hepatocyte Injury andApoptosis Based on previous literatures [18 19] we selected05 120583Mand 5 120583MM30 in the current study Cell viability assaysuggested that after 24-hour exposure to ethanol around

30 of BRL-3A cells became inviable M30 pretreatment atboth concentrations significantly increased the cell viability(Figure 1(a)) A 48-hour treatment with ethanol andor M30exhibited similar results indicating that the protective effectsof M30 on ethanol-damaged hepatocyte mainly occurredwithin the first 24 hours (Figure 1(a)) Therefore we selected05 120583M and 24 hours as the incubation concentration andduration for the following studies In line with the cellviability results pretreatment with 05 120583M M30 significantlyreduced ethanol exposure caused release of ALT from dam-aged hepatocyte into the culture medium (Figure 1(b))

To examine the effects of M30 on cellular apoptosis wefirstly measured the apoptotic ratio of BRL-3A after varioustreatmentsWe found that ethanol exposure greatly increasedapoptosis of the cell from sim2 to sim18 (sim9-fold) andpretreatment of M30 partially abolished this effect of ethanol(Figure 2(a)) Quantification of cellular caspase-37 activityantiapoptotic gene Bcl-2 and proapoptotic gene Bax1 furthersupported these observations (Figures 2(b) and 2(c))

32 M30 Inhibited Ethanol-Induced Hepatocyte OxidativeStress Since formation of ROS and oxidative stress are mainconsequences of ethanol metabolism in the hepatocytes wethen tested the production of ROS in the culture mediumand within the hepatocyte after ethanol andor M30 incu-bation Fluorescence staining results suggested that ethanolobviously increased the signal of ROS staining while M30reduced it (Figures 3(a) and 3(b)) This phenomenon wasconsistentwith the finding that ethanol exposure significantlyreduced the protein expression level of antioxidant enzymesCAT and GPx1 which was recovered by the pretreatment


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Bax1Bcl-2

lowastlowast

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A ex

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(ct

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(c)

Figure 2M30 improved cellular apoptosis (a) Apoptotic ratio after various treatments (b)Activity of caspase-37 of hepatocytes after varioustreatments (c) Cellular Bcl-2 and Bax1 mRNA change after various treatments Data from each group (119899 = 4) were expressed as means plusmnSEM Statistical comparison between groups was done using the Kruskal-Wallis test followed by Dunnrsquos post hoc test to detect differences inall groups ldquolowastrdquo means significantly different from control group (119875 lt 005) ldquolowastlowastrdquo means significantly different from control group (119875 lt 001)ldquolowastlowastlowastrdquomeans significantly different from control group (119875 lt 0001) ldquordquomeans significantly different from ethanol exposure group (119875 lt 005)ldquordquo means significantly different from ethanol exposure group (119875 lt 001) ldquordquo means significantly different from ethanol exposure group(119875 lt 0001) EtOH ethanol M30 vehicle M30 treatment E + M30 ethanol + M30 cotreatment

with M30 (Figure 3(c)) In addition the status change ofcellular oxidative stress was exhibited by the ratio changeof cellular GSHGSSG since this ratio is an indicator ofcellular health with reduced GSH constituting up to 98of cellular GSH under normal conditions [20] Ethanoltreatment significantly reduced the GSHGSSG ratio fromsim29 to sim12 Pretreatment with M30 either independentlyor in combination with ethanol significantly increased theratio to a level (sim47) even higher than that of the controlgroup strongly suggesting the antioxidant property of M30(Figure 3(d)) We also quantified the activity of HIF-1120572 oneof the major regulators of oxidative stress generation inalcoholic liver diseases [9] In agreement with other studiesethanol significantly induced the activity of HIF-1120572 which

was reversed by the pretreatment with M30 Vehicle-M30treatment did not influence the basal activity level of HIF-1120572(Figure 3(e))

33 M30 Attenuated Ethanol-Induced Hepatocyte Inflam-matory Responses and NLRP3 Inflammasome ActivationOverproduction of ROS and oxidative stress in the liverafter ethanol exposure often induce potent inflammatoryresponses which may significantly aggravate the pathogene-sis of ALD [21]Thus whether pretreatmentwithM30 attenu-ates ethanol-induced proinflammatory cytokines productionand NLRP3 inflammasome activation is another importantaim for the current study ELISA results of secreted TNF-120572 and IL-6 showed that ethanol incubation greatly induced


Control EtOH M30 EtOH + M30

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-1120572

activ

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lowastlowastlowast

(e)

Figure 3M30 improved cellular oxidative stress (a) Production of ROS staining in the cells after various treatments (magnification 200x bar= 100120583m) (b) Quantification of ROS staining (c) Expressional changes of antioxidants CAT and GPx1 after various treatments (d) Changeof GSHGSSG ratio treatments (e) Changes of transcriptional activity of HIF-1120572 in each group Data from each group (119899 = 4) were expressedas means plusmn SEM Statistical comparison between groups was done using the Kruskal-Wallis test followed by Dunnrsquos post hoc test to detectdifferences in all groups ldquolowastrdquo means significantly different from control group (119875 lt 005) ldquolowastlowastrdquo means significantly different from controlgroup (119875 lt 001) ldquolowast lowast lowastrdquo means significantly different from control group (119875 lt 0001) ldquordquo means significantly different from ethanolexposure group (119875 lt 001) ldquordquo means significantly different from ethanol exposure group (119875 lt 0001) Letters ldquoardquo labelled above the valuesof control M30 and ethanol + M30 groups mean there is no significant change among these groups (119875 gt 005) Letter ldquobrdquo means the valueof ethanol-treated group is statistically different from other three values (119875 lt 005) EtOH ethanol M30 vehicle M30 treatment E + M30ethanol + M30 cotreatment


the secretion of these two proinflammatory cytokines fromBRL-3A cells indicating an inflammatory status of the cellsM30 totally or partially abolished such effects (Figures 4(a)and 4(b)) This phenomenon was further confirmed bythe change of transcription activity of NF-120581B the masterregulator of both oxidative stress and inflammation in theliver (Figure 4(c)) To characterize the involvement of NLRP3inflammasome in ethanol-induced hepatocyte damage andM30-mediated protection the cellular protein levels ofNLRP3 ASC and caspase-1 as well as the secreted proteinlevels of both IL-1120573 and IL-18 were measured by Westernblot or ELISA in all groups Ethanol exposure significantlyupregulated all the protein levels of these key componentsof NLRP3 inflammasome indicating that NLRP3 inflamma-some was activated during ethanol incubation Pretreatmentwith M30 significantly reduced their levels (Figures 4(d)ndash4(f))

34 Inhibition of HIF-1120572 Reduced NLRP3 InflammasomeActivation The production of IL-1120573 is a central step in ALDprogression Thus delineating the upstream signals thatlead to the activation of NLRP3 inflammasome and IL-1120573secretion is important for the pathological study of thisdisease [22 23] Since recent several reports suggested thatthere were HIF-1120572 response elements in the human andmouse Il1b promoter [24] it was interesting to find whetherblockade of HIF-1120572 could reduce the production of IL-1120573 andother inflammasome products (IL-18 and caspase-1) in oursystem The HIF-1120572 inhibitor CAY10585 was able to signif-icantly decrease ethanol induced IL-1120573 production withoutaffecting its basal level (Figure 5(a)) Addition ofM30 furtherreduced the production of IL-1120573 to a control-comparablelevel indicating that HIF-1120572 was indeed a regulating targetof M30 but there should be other targets that M30 used toplay its protective roles against ethanol (Figure 5(a)) Changeof IL-18 and caspase-1 protein level further confirmed thisobservation (Figures 5(b) and 5(c))

35 M30 Modulates ACcAMPPKA Pathway and Its Down-stream HIF-1120572 Since PKA activates HIF-1120572 through cAMPresponse element-binding protein (CREB) [25] and AC isreported to be the target of other hepatoprotective agents[26] we then considered that the ACcAMPPKA pathwaymight be repressed by M30 to downregulate the activity ofHIF-1120572 To test this hypothesis AC activator forskolin andinhibitor SQ22536 were used to treat BRL-3A cells in thepresence or absence of ethanol and M30 We found thatvehicle forskolin slightly increased the basal secretion of IL-1120573 but not IL-18 When pretreated with M30 before ethanolforskolin significantly reduced the effect of M30 on IL-1120573and IL-18 secretion Similarly when AC was inhibited bySQ22536 the secretion of IL-1120573 and IL-18 induced by ethanolexposure was significantly decreased (Figures 6(a) and 6(b))Pretreatment with SQ22536 before ethanol potentiated theeffect of M30 on the reduction of IL-1120573 and IL-18 secretionCollectively these data suggested that M30 exerted its ben-eficial effects on hepatocytes partially through the action ofAC

We then used a stable analogue of cAMP (db-cAMP) andan inhibitor of PKA (H89) to see the change of IL-1120573 andIL-18 secretion in our system In line with the results fromAC overactivation and repression db-cAMP significantlyimpaired the effects ofM30 on both IL-1120573 and IL-18 secretionwhileH89 partiallymimicked the effects ofM30 (Figures 6(c)and 6(d)) indicating that the cAMPPKA pathway was alsoapplied by M30 when treating alcoholic hepatocyte injury

4 Discussion

M30 is one of the most effective drugs possessing iron-che-latingradical scavenging potency and inhibition of lipid per-oxidation features Cytochrome P450 isoenzyme inhibitionand voltage-dependent potassium channel blocking studiesshowed that M30 was not cytotoxic in several cell lines[12 13] M30 has been shown to inhibit oxidative stress andinflammation in several diseases including type 2 diabetesmellitus [27] Alzheimerrsquos disease [28] Parkinsonrsquos disease[29] and drug-induced brain cell injury [30] Howeverlittle is known about its effect on the liver In this studywe demonstrated that pretreatment with M30 significantlypreserved cell viability attenuated cell damage and improvedcellular oxidative stress and inflammation in an in vitroethanol-induced hepatocyte injury model Pharmacologicalactivation and inhibition by specific agonist and inhibitorsof signaling molecules further confirmed the partial involve-ment of the ACcAMPPKAHIF-1120572 and the NLRP3 inflam-masome pathways in the hepatoprotective actions of M30

A number of previous papers reported that ethanolevoked HIF-1120572 activation in the liver For example ethanoltreatment induced fatty liver was alleviated in mice withhepatocyte-specifically knockdown of HIF-1120572 while overex-pression of HIF-1120572 aggravated that damage [31] Wang et alalso found that blocking HIF-1120572 activation and subsequentaction had therapeutic implication against ethanolCYP2E1-induced oxidative stress steatosis and liver injury [11] Inour study after ethanol exposure the activity of HIF-1120572 wassignificantly increased along with exacerbated hepatocytedamage oxidative stress inflammation and NLRP3 inflam-masome activation when compared with those in the con-trol group M30 pretreatment significantly reduced HIF-1120572activity and other cellular injuries Correspondingly ethanol-induced NLRP3 inflammasome activation was decreased incells treated with HIF-1120572 inhibitor CAY10585 M30 additionfurther reduced the inflammasome activation Collectivelythese data suggested that HIF-1120572 might play important rolesin ethanol-induced hepatocyte injury and M30-mediatedhepatoprotection This conclusion was consistent with avery recent paper finding that moderate hypoxia potentiatesNLRP3 inflammasome activation and IL-1120573 production inactivated humanmacrophages [32] Opposite to our findingsseveral previous studies suggested that M30 could ameliorateneuronal oxidative stress through the stabilization and induc-tion of HIF-1120572 proteinThis may be attributed to the differentdownstream signalings in different organs (brain versus liver)[27ndash29] Indeed further studies are warranted to delineatethis discrepancy


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Figure 4 M30 improved cellular inflammation and NLRP3 inflammasome activation (a b) Changes of secreted levels of TNF-120572 and IL-6in each group after treatments (c) Change of transcriptional activity of NF-120581B p65 subunit after treatments (d) Expressional changes ofNLRP3 ASC and caspase-1 proteins after treatments (e f) Changes of secreted levels of IL-1120573 and IL-18 in each group after treatmentsData from each group (119899 = 4) were expressed as means plusmn SEM Statistical comparison between groups was done using the Kruskal-Wallistest followed by Dunnrsquos post hoc test to detect differences in all groups ldquolowastrdquo means significantly different from control group (119875 lt 005)ldquolowastlowastrdquo means significantly different from control group (119875 lt 001) ldquolowast lowast lowastrdquo means significantly different from control group (119875 lt 0001)ldquordquo means significantly different from ethanol exposure group (119875 lt 005) ldquordquo means significantly different from ethanol exposure group(119875 lt 001) ldquordquo means significantly different from ethanol exposure group (119875 lt 0001) Letters ldquoardquo labelled above the values of control M30and ethanol + M30 groups mean there is no significant change among these groups (119875 gt 005) Letter ldquobrdquo means the value of ethanol-treatedgroup is statistically different from other three values (119875 lt 005) For NLRP3 protein letter ldquocrdquo on ethanol + M30 group means significantchange when compared with other three groups (ie control ethanol and M30 119875 lt 005) EtOH ethanol M30 vehicle M30 treatment E +M30 ethanol + M30 cotreatment


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-1120573

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lowastlowast

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(c)

Figure 5 HIF-1120572 is critical for theNLRP3 inflammasome activation (andashc) Changes of secreted IL-1120573 IL-18 and cellular caspase-1 expressionsafter ethanol exposure M30 pretreatment andor HIF-1120572 inhibitor CAY10585 Data from each group (119899 = 4) were expressed as means plusmnSEM Statistical comparison between groups was done using the Kruskal-Wallis test followed by Dunnrsquos post hoc test to detect differences inall groups ldquolowastrdquo means significantly different from control group (119875 lt 005) ldquolowastlowastrdquo means significantly different from control group (119875 lt 001)ldquolowastlowastlowastrdquomeans significantly different from control group (119875 lt 0001) ldquordquomeans significantly different from ethanol exposure group (119875 lt 005)ldquordquo means significantly different from ethanol exposure group (119875 lt 001) ldquordquo means significantly different from ethanol exposure group(119875 lt 0001)

The direct link between the ACcAMPPKA pathway andthe activation of HIF-1120572 has been recently examined in sev-eral cell models such as epithelial-mesenchymal transition inlung cancer [33] pancreatic beta cell injury [34] and inflam-mation in macrophage [10] We demonstrated that activationand inhibition of the ACcAMPPKA pathway could respec-tively increase and decrease the production of inflammasomeproducts (IL-1120573 and IL-18) Indeed the signaling mediator

in the upstream of AC (particularly the membrane receptor)that immediately transduces ethanol-induced damage andM30-mediated protection is the key missing link for thecurrent study Based on the studies that adenosine 1A or2A receptor antagonist prevented and reversed liver injuryin mouse models of ALD [35 36] and that adenosine2A receptor directed the activation of NLRP3 inflamma-some through the ACcAMPPKAHIF-1120572 pathway [10]


050

100150200250300350400450

IL-1120573

secr

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secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)

Figure 6 Role of the ACcAMPPKA pathway in NLRP3 inflammasome activation (a-b) Changes of secreted IL-1120573 and IL-18 after ethanolexposure M30 pretreatment andor AC agonist forskolininhibitor SQ22536 (c-d) Changes of secreted IL-1120573 and IL-18 after ethanolexposureM30 pretreatment andor cAMP analogue db-cAMPPKA inhibitor H89 Data from each group (119899 = 4) were expressed asmeans plusmnSEM Statistical comparison between groups was done using the Kruskal-Wallis test followed by Dunnrsquos post hoc test to detect differences inall groups ldquolowastrdquo means significantly different from control group (119875 lt 005) ldquolowastlowastrdquo means significantly different from control group (119875 lt 001)ldquolowastlowastlowastrdquomeans significantly different from control group (119875 lt 0001) ldquordquomeans significantly different from ethanol exposure group (119875 lt 005)ldquordquo means significantly different from ethanol exposure group (119875 lt 001) ldquordquo means significantly different from ethanol exposure group(119875 lt 0001)

we highly speculated that adenosine receptors could be thekey immediate molecules modulating the beneficial effects ofM30 This hypothesis deserves future investigations

In conclusion pretreatment with multitarget iron chela-tor and antioxidant M30 attenuates ethanol-induced injuryin rat BRL-3A hepatic cells The hepatoprotective propertyof M30 is partially achieved by modulating the ACcAMPPKAHIF-1120572 pathwayThis study also provides a rationale fordeveloping hepatoprotective therapies that target HIF-1120572 orits updownstream mediators

Abbreviations

AC Adenylate cyclaseAFLD Alcoholic fatty liver diseaseALD Alcoholic liver diseaseALT Alanine aminotransferaseASC Apoptosis-associated speck-like protein

containing a CARD

ASH Alcoholic steatohepatitiscAMP Cyclic AMPCAT CatalaseDCFH-DA 2101584071015840-Dichlorofluorescin diacetateDMSO Dimethyl sulfoxideELISA Enzyme-linked immunosorbent assayGAPDH Glyceraldehyde-3-phosphate

dehydrogenaseGPx1 Glutathione peroxidase 1GSH GlutathioneGSSG Oxidized glutathioneHIF-1120572 Hypoxia inducible factor-1 alphaIL InterleukinNLRP NOD-like receptor proteinPKA Protein kinase AROS Reactive oxygen speciesTNF Tumor necrosis factorTXNIP Thioredoxin-interacting protein


Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Jia Xiao and Yi Lv contributed equally

Acknowledgments

This study was supported by grants from National NaturalScience Foundation Projects (81370971) Guangdong Nat-ural Science Funds for Distinguished Young Scholar(S2013050013880) the Key Discipline Project of ShenzhenNew Emerging Infectious Diseases (no 201161) and Shenz-hen Municipal Science and Technology Innovation Fund(CXZZ20130322170220544)

References

[1] WorldHealthOrganizationGlobal Status Report onAlcohol andHealth World Health Organization Geneva Switzerland 2011

[2] S Bellentani G Saccoccio G Costa et al ldquoDrinking habits ascofactors of risk for alcohol induced liver damageTheDionysosStudy Grouprdquo Gut vol 41 no 6 pp 845ndash858 1997

[3] M M Jaurigue and M S Cappell ldquoTherapy for alcoholic liverdiseaserdquo World Journal of Gastroenterology vol 20 no 9 pp2143ndash2158 2014

[4] B Gao and R Bataller ldquoAlcoholic liver disease pathogenesisand new therapeutic targetsrdquo Gastroenterology vol 141 no 5pp 1572ndash1585 2011

[5] A Dey and A I Cederbaum ldquoAlcohol and oxidative liverinjuryrdquo Hepatology vol 43 no 2 supplement 1 pp S63ndashS742006

[6] S L Fink and B T Cookson ldquoApoptosis pyroptosis and necro-sis mechanistic description of dead and dying eukaryotic cellsrdquoInfection and Immunity vol 73 no 4 pp 1907ndash1916 2005

[7] J Xiao Y Zhu Y Liu G L Tipoe F Xing andK-F So ldquoLyciumbarbarum polysaccharide attenuates alcoholic cellular injurythrough TXNIP-NLRP3 inflammasome pathwayrdquo InternationalJournal of Biological Macromolecules vol 69 pp 73ndash78 2014

[8] G L Semenza ldquoHypoxia-inducible factors in physiology andmedicinerdquo Cell vol 148 no 3 pp 399ndash408 2012

[9] A JMajmundarW JWong andM C Simon ldquoHypoxia-indu-cible factors and the response to hypoxic stressrdquoMolecular Cellvol 40 no 2 pp 294ndash309 2010

[10] X Ouyang A Ghani A Malik et al ldquoAdenosine is required forsustained inflammasome activation via theA

2119860

receptor and theHIF-1120572 pathwayrdquo Nature Communications vol 4 article 29092013

[11] X Wang D Wu L Yang L Gan and A I Cederbaum ldquoCytochrome P450 2E1 potentiates ethanol induction of hypoxia andHIF-1120572 in vivordquo Free Radical Biology and Medicine vol 63 pp175ndash186 2013

[12] H Zheng L M Weiner O Bar-Am et al ldquoDesign synthesisand evaluation of novel bifunctional iron-chelators as potentialagents for neuroprotection in Alzheimerrsquos Parkinsonrsquos andother neurodegenerative diseasesrdquo Bioorganic and MedicinalChemistry vol 13 no 3 pp 773ndash783 2005

[13] H Zheng S Gal L M Weiner et al ldquoNovel multifunctionalneuroprotective iron chelator-monoamine oxidase inhibitordrugs for neurodegenerative diseases in vitro studies onantioxidant activity prevention of lipid peroxide formation andmonoamine oxidase inhibitionrdquo Journal of Neurochemistry vol95 no 1 pp 68ndash78 2005

[14] J Xiao Z-C Zhou C Chen et al ldquoTumor necrosis factor-alphagene from mandarin fish Siniperca chuatsi molecular cloningcytotoxicity analysis and expression profilerdquo Molecular Immu-nology vol 44 no 14 pp 3615ndash3622 2007

[15] J Xiao F Xing J Huo et al ldquoLycium barbarum polysaccharidestherapeutically improve hepatic functions in non-alcoholicsteatohepatitis rats and cellular steatosis modelrdquo ScientificReports vol 4 article 5587 2014

[16] S A Bustin V Benes J A Garson et al ldquoTheMIQE guidelinesminimum information for publication of quantitative real-timePCRexperimentsrdquoClinical Chemistry vol 55 no 4 pp 611ndash6222009

[17] J Xiao Y P Ching E C Liong A A Nanji M L Fung and GL Tipoe ldquoGarlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver disease in vivoanimal modelrdquo European Journal of Nutrition vol 52 no 1 pp179ndash191 2013

[18] Y Avramovich-Tirosh T Amit O Bar-Am H Zheng M Frid-kin and M B H Youdim ldquoTherapeutic targets and potentialof the novel brain-permeable multifunctional iron chelator-monoamine oxidase inhibitor drug M-30 for the treatment ofAlzheimerrsquos diseaserdquo Journal of Neurochemistry vol 100 no 2pp 490ndash502 2007

[19] L Kupershmidt O Weinreb T Amit S Mandel M T Carriand M B H Youdim ldquoNeuroprotective and neuritogenicactivities of novel multimodal iron-chelating drugs in motor-neuron-like NSC-34 cells and transgenic mouse model ofamyotrophic lateral sclerosisrdquo The FASEB Journal vol 23 no11 pp 3766ndash3779 2009

[20] J B Owen and D A Butterfield ldquoMeasurement of oxidizedreduced glutathione ratiordquo Methods in Molecular Biology vol648 pp 269ndash277 2010

[21] H J Wang B Gao S Zakhari and L E Nagy ldquoInflammationin alcoholic liver diseaserdquo Annual Review of Nutrition vol 32pp 343ndash368 2012

[22] G Kakiyama P B Hylemon H Zhou et al ldquoColonic inflam-mation and secondary bile acids in alcoholic cirrhosisrdquo TheAmerican Journal of PhysiologymdashGastrointestinal and LiverPhysiology vol 306 no 11 pp G929ndashG937 2014

[23] Y Peng B A French B Tillman et al ldquoThe inflammasomein alcoholic hepatitis its relationship with Mallory-Denk bodyformationrdquo Experimental and Molecular Pathology vol 97 no2 pp 305ndash313 2014

[24] W Zhang J-M Petrovic D Callaghan et al ldquoEvidence thathypoxia-inducible factor-1 (HIF-1) mediates transcriptionalactivation of interleukin-1120573 (IL-1120573) in astrocyte culturesrdquo Jour-nal of Neuroimmunology vol 174 no 1-2 pp 63ndash73 2006

[25] I Kvietikova R H Wenger H H Marti and M GassmannldquoThe transcription factors ATF-1 and CREB-1 bind constitu-tively to the hypoxia-inducible factor-1 (HIF-1) DNA recogni-tion siterdquoNucleic Acids Research vol 23 no 22 pp 4542ndash45501995

[26] S Mandal V K Nelson S Mukhopadhyay et al ldquo14-Deox-yandrographolide targets adenylate cyclase and prevents eth-anol-induced liver injury through constitutive NOS dependent


reduced redox signaling in ratsrdquo Food and Chemical Toxicologyvol 59 pp 236ndash248 2013

[27] D Mechlovich T Amit O Bar-Am O Weinreb and M B HYoudim ldquoMolecular targets of the multifunctional iron-chelating drug M30 in the brains of mouse models of type 2diabetes mellitusrdquoThe British Journal of Pharmacology vol 171no 24 pp 5636ndash5649 2014

[28] E Sofic M Salkovic-Petrisic I Tahirovic et al ldquoBrain catalasein the streptozotocin-rat model of sporadic Alzheimerrsquos diseasetreated with the iron chelator-monoamine oxidase inhibitorM30rdquo Journal of Neural Transmission 2014

[29] M B H Youdim L Kupershmidt T Amit and O Wein-reb ldquoPromises of novel multi-target neuroprotective and neu-rorestorative drugs for Parkinsonrsquos diseaserdquo Parkinsonism andRelated Disorders vol 20 supplement 1 pp S132ndashS136 2014

[30] S Johnson S Tazik and D Lu ldquoThe new inhibitor of mono-amine oxidase M30 has a neuroprotective effect against dex-amethasone-induced brain cell apoptosisrdquo Frontiers in Neuro-science vol 4 article 180 2010

[31] B Nath I Levin T Csak et al ldquoHepatocyte-specific hypoxia-inducible factor-1120572 is a determinant of lipid accumulation andliver injury in alcohol-induced steatosis in micerdquo Hepatologyvol 53 no 5 pp 1526ndash1537 2011

[32] E J Folco G K Sukhova T Quillard and P Libby ldquoModeratehypoxia potentiates interleukin-1120573 production in activatedhuman macrophagesrdquo Circulation Research vol 115 no 10 pp875ndash883 2014

[33] D Shaikh Q Zhou T Chen J C F Ibe J U Raj and G ZhouldquoCAMP-dependent protein kinase is essential for hypoxia-mediated epithelial-mesenchymal transition migration andinvasion in lung cancer cellsrdquo Cellular Signalling vol 24 no 12pp 2396ndash2406 2012

[34] S Van de Velde M F Hogan and M Montminy ldquomTOR linksincretin signaling to HIF induction in pancreatic beta cellsrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 41 pp 16876ndash16882 2011

[35] P Yang Z Wang Y Zhan et al ldquoEndogenous A1 adenosinereceptor protects mice from acute ethanol-induced hepatotoxi-cityrdquo Toxicology vol 309 pp 100ndash106 2013

[36] D J Chiang S Roychowdhury K Bush et al ldquoAdenosine 2Areceptor antagonist prevented and reversed liver fibrosis in amouse model of ethanol-exacerbated liver fibrosisrdquo PLoS ONEvol 8 no 7 Article ID e69114 2013


regulators of NLRP3 during ALD progression await furtherinvestigation

Hypoxia-inducible factor-1 alpha (HIF-1120572) is activated byhypoxia and is a master regulator of cellular redox status anddownstream production of proinflammatory cytokines (egtumor necrosis factor-alpha (TNF-120572) and IL-6) [8] It couldbe activated by increased cellular oxygen levels primarily atthe protein level [9] Recent studies pointed out that underhypoxic or oxidative stress environments adenylate cyclase(AC) activates HIF-1120572 through cyclic AMP (cAMP)proteinkinase A (PKA) pathway [10] Since ethanol induces hypoxiaand elevates HIF-1120572 in the liver [11] it was speculated thatHIF-1120572 might be a novel therapeutic target for ethanol-induced injury and an upstream regulator of NLRP3 inflam-masome

In the current study we aimed to determine whether theACcAMPPKAHIF-1120572 pathway andNLRP3 inflammasomeare involved in the protective effect of a novel multitarget ironchelator (M30) against ethanol-induced hepatocyte injury invitro

2 Materials and Methods

21 Drugs and Chemicals Themultifunctional iron chelatorM30 (5-[N-methyl-N-propargylaminomethyl]-8-hydroxy-quinoline) was synthesized and kindly provided by VarinelInc (Philadelphia PA) [12 13] CAY10585 (HIF-1120572 inhibitor)was purchased from Cayman Chemical (Ann Arbor MI)Forskolin (AC agonist) SQ22536 (AC inhibitor) H89 (PKAinhibitor) and db-cAMP (stable cAMP analogue) werepurchased from Sigma-Aldrich (St Louis MO) Antibodiesagainst catalase (CAT) glutathione peroxidase 1 (GPx1)NLRP3 apoptosis-associated speck-like protein containinga CARD (ASC) caspase-1 and 120573-actin were ordered fromAbcam (Cambridge UK)

22 Cell Culture and Treatments Rat normal hepatocyte cellline BRL-3A was supplied by the Cell Bank of Type Cul-ture Collection of Chinese Academy of Sciences (ShanghaiChina) Cells were cultured in DMEM with 10 (vv) FBS at37∘C with 5 CO

2 Before drug treatment cells must reach

a confluence of 60ndash70 For the pretreatment with M30PBS dissolved M30 was added 2 hours before the ethanoltreatment For the treatments using agonists and inhibitorsof the ACcAMPPKAHIF-1120572 pathway drugs were addedalong with M30 or individually 2 hours before the ethanoltreatment at designated concentrations

23 MTT Assay The cell viability was evaluated by the 3-(45-dimethylthiazol-2-yl)-25-diphenyltetrazolium bromide(MTT Sigma-Aldrich St Louis MO) method After drugtreatment cells were washed by sterile PBS for 3 timesand then incubated with 5mgmL MTT for 3 hours andsubsequently dissolved in dimethyl sulfoxide (DMSO) Theabsorbance of cells was measured at 570 nm The percent ofcell viability was defined as the relative absorbance of treatedcells versus untreated cells

24 Detection of Alanine Aminotransferase (ALT) LevelsAfter treatments cell supernatant was collected and the levelof ALT was detected by an ALTGPT kit (EIAab ScienceWuhan China) according to manufacturerrsquos instruction

25 Quantification of Apoptotic Cells After treatmentHoechst 33342 (Sigma 5 120583gmL) and propidium iodide(Sigma 5 120583gmL) were added to each well to stain live cellsThe cell population was separated into 3 groups live cellsshowed only a low level of fluorescence apoptotic cellsshowed a higher level of blue fluorescence and dead cellsshowed low-blue and high-red fluorescence Stained cellswere observed and quantified by two independent cellbiologists without knowing the grouping The results wereexpressed as the percentage of apoptosis (PA) PA = apoptoticcell numbertotal cell number times 100 [14]

26 Caspase-37 Activity The activity of caspase-37 in celllysate was measured using the Cell Meter Caspase-37 Activ-ity Apoptosis Assay Kit (AAT Bio Sunnyvale CA) accordingto the user manual The results were read at 520 nm in amicroplate reader (Bio-Rad) and expressed as fold change ofthe control

27 Measurement of ROS Production Intracellular produc-tion of ROS was detected by a fluorescence probemdash2101584071015840-dichlorofluorescin diacetate (DCFH-DA Sigma-Aldrich) aspreviously described [7] Briefly after treatment cells werewashed three times with PBS and then incubated with 10 120583MDCFH-DA for 30min at 37∘C for green fluorescent light visu-alization Quantification of green fluorescence was analyzedby using ImageJ software (Version 148 National Institutesof Health Bethesda MD) Fluorescent quantification resultswere then normalized by the protein amount of each specificplate well (sim110ndash150 120583gwell of a 24-well plate)

28 GSHGSSG Ratio Measurement To determine the intra-cellular oxidative status the ratio of reduced glutathione(GSH) to oxidized glutathione (GSSG) in each cellular pro-tein sample was measured by using a GSHGSSG detectionassay kit from Abcam (Cambridge UK)

29 RNA Extraction and Quantitative PCR Total cellularRNA was extracted using illustra RNAspin mini kit (GEhealthcare UK) The preparation of the first-strand cDNAwas conducted following the instruction of the SuperScriptFirst-Strand Synthesis System (Invitrogen Carlsbad CA)The mRNA expression levels of target genes were mea-sured by Takara SYBR premix Taq quantitative PCR system(Takara Bio Inc Shiga Japan) and in MyiQ2 real-time PCRmachine (Bio-Rad Hercules CA) Parallel amplification ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) wasused as an internal control Relative quantification wascalculated using the 2minusΔΔCt method Primers and PCR condi-tions were described in previous literature [15] The relativeexpression of the specific gene to the internal control wasobtained and then expressed as percentage of the controlvalue All real-time PCR procedures including the design of



minus

+minus

minus

minusminus

+

minus+

minus

+minus

+

+

minus

+0

20

40

60

80

100

120C

ell v

iabi

lity

(ct

rl)

lowastlowastlowast

M30 (05 120583M)M30 (5120583M)

24h48h

(a)

0

5

10

15

20

25


ALT

leve

l (U

IL)

lowastlowastlowast

(b)






3 Results






0

5

10

15

20

25


Apop

totic

ratio

()

lowastlowastlowast

(a)

0

50

100

150

200

250

300


Casp

ase-

37

activ

ity (

ctrl)

lowastlowast

(b)

0

50

100

150

200

250

300

350

400


Bax1Bcl-2

lowastlowast

lowastlowast

lowast

lowastmRN

A ex

pres

sion

(ct

rl)

(c)







(a)

0

100

200

300

400

500

600


lowastlowastlowast

lowast

ROS

stai

ning

sign

al p

er m

g pr

otei

n (

ctrl)

(b)

CAT

120573-actin

GPx1




(c)

0

1

2

3

4

5

6


GSH

GSS

G ra

tio

lowastlowast

lowast

(d)

0

50

100

150

200

250

300

350

400

450


HIF

-1120572

activ

ity (

ctrl)

lowastlowastlowast

(e)







4 Discussion




0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860
































minus

+minus

minus

minusminus

+

minus+

minus

+minus

+

+

minus

+0

20

40

60

80

100

120C

ell v

iabi

lity

(ct

rl)

lowastlowastlowast

M30 (05 120583M)M30 (5120583M)

24h48h

(a)

0

5

10

15

20

25


ALT

leve

l (U

IL)

lowastlowastlowast

(b)






3 Results






0

5

10

15

20

25


Apop

totic

ratio

()

lowastlowastlowast

(a)

0

50

100

150

200

250

300


Casp

ase-

37

activ

ity (

ctrl)

lowastlowast

(b)

0

50

100

150

200

250

300

350

400


Bax1Bcl-2

lowastlowast

lowastlowast

lowast

lowastmRN

A ex

pres

sion

(ct

rl)

(c)







(a)

0

100

200

300

400

500

600


lowastlowastlowast

lowast

ROS

stai

ning

sign

al p

er m

g pr

otei

n (

ctrl)

(b)

CAT

120573-actin

GPx1




(c)

0

1

2

3

4

5

6


GSH

GSS

G ra

tio

lowastlowast

lowast

(d)

0

50

100

150

200

250

300

350

400

450


HIF

-1120572

activ

ity (

ctrl)

lowastlowastlowast

(e)







4 Discussion




0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860































0

5

10

15

20

25


Apop

totic

ratio

()

lowastlowastlowast

(a)

0

50

100

150

200

250

300


Casp

ase-

37

activ

ity (

ctrl)

lowastlowast

(b)

0

50

100

150

200

250

300

350

400


Bax1Bcl-2

lowastlowast

lowastlowast

lowast

lowastmRN

A ex

pres

sion

(ct

rl)

(c)







(a)

0

100

200

300

400

500

600


lowastlowastlowast

lowast

ROS

stai

ning

sign

al p

er m

g pr

otei

n (

ctrl)

(b)

CAT

120573-actin

GPx1




(c)

0

1

2

3

4

5

6


GSH

GSS

G ra

tio

lowastlowast

lowast

(d)

0

50

100

150

200

250

300

350

400

450


HIF

-1120572

activ

ity (

ctrl)

lowastlowastlowast

(e)







4 Discussion




0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860
































(a)

0

100

200

300

400

500

600


lowastlowastlowast

lowast

ROS

stai

ning

sign

al p

er m

g pr

otei

n (

ctrl)

(b)

CAT

120573-actin

GPx1




(c)

0

1

2

3

4

5

6


GSH

GSS

G ra

tio

lowastlowast

lowast

(d)

0

50

100

150

200

250

300

350

400

450


HIF

-1120572

activ

ity (

ctrl)

lowastlowastlowast

(e)







4 Discussion




0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860



































4 Discussion




0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860































0

50

100

150

200

250


TNF-120572

secr

etio

n (

ctrl)

lowastlowastlowast

(a)

0

50

100

150

200

250

300

350


IL-6

secr

etio

n (

ctrl)

lowastlowastlowast

(b)

0

20

40

60

80

100

120

140

160

180


NF-120581

B p6

5 ac

tivity

(ct

rl)

lowast

lowast

(c)

NLRP3

120573-actin

ASC

Caspase-1





(d)

0

50

100

150

200

250


IL1-120573

secr

etio

n (

ctrl)

lowastlowastlowast

lowast

(e)

0

20

40

60

80

100

120

140

160

180

200


IL-1

8 se

cret

ion

(ct

rl)

lowastlowast

lowast

(f)



0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860































0

50

100

150

200

250IL

-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

(a)

0

50

100

150

200

250

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowastlowast

lowast lowast

(b)

Caspase-1

120573-actin

0

50

100

150

200

250

Casp

ase-

1 pr

otei

n (

ctrl)

EtOH (250 mM)CAY (25 120583M)M30 (05 120583M)

minusminus

minus

+minus

minus

++

minus

minus+

minus

minus+

+

++

+

minus

+

+

lowastlowast

lowastlowast

(c)





050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860































050

100150200250300350400450

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast

lowast

(a)

0

50

100

150

200

250

300

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

Forskolin (20 120583M)SQ (50 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowast

lowast

(b)

0

50

100

150

200

250

300

350

IL-1120573

secr

etio

n (

ctrl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

(c)

050

100150200250300350400450500

IL-1

8 se

cret

ion

(ct

rl)

EtOH (250 mM)

db-cAMP (200 120583M)H89 (20 120583M)


minus

minus

+minus

minus

minus

minusminus

+

minus

++

minus

minus

minus+

+

minus

++

+

minus

minusminus

minus

+

+minus

minus

+

minus+

minus

+

++

minus

+

minus

+

minus

+

lowastlowastlowast

lowast lowast

(d)




Abbreviations


containing a CARD








Acknowledgments


References











2119860



































Acknowledgments


References











2119860










































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