Research ArticleLigusticum wallichii Extract Inhibitedthe Expression of IL-1120573 after AMI in Rats

Zhuo Yuan1 Junping Zhang1 and Cui Yang2

1 First Teaching Hospital of Tianjin University of Traditional Chinese Medicine 314 An Shan Xi RoadNan Kai District Tianjin 300193 China

2 Tianjin University of Traditional Chinese Medicine 312 An Shan Xi Road Nan Kai District Tianjin 300193 China

Correspondence should be addressed to Junping Zhang tjzhtcm163com

Received 31 January 2014 Revised 30 July 2014 Accepted 31 July 2014 Published 17 August 2014

Academic Editor Tabinda Ashfaq

Copyright copy 2014 Zhuo Yuan et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This study investigated the effects of Ligusticumwallichii on IL-1120573 expression inmyocardium and central nervous system after AMIAMI rat was administrated with Ligusticumwallichii extract A series of assays were used to detect the effects of Ligusticumwallichiiextract on infarct size left ventricular ejection fraction expression of TLR-4 NF-120581B and IL-1120573 in myocardium IL-1120573 expression inserum and hypothalamus and NPY expression in hypothalamus We observed that Ligusticum wallichii extract improved the leftventricular ejection fraction and reduced infarct area enlargement after AMI by inhibiting the expression of IL-1120573 in myocardiumserum and hypothalamus Ligusticum wallichii extract reduced the expression of IL-1120573 in myocardium by regulating TLR4-NF-120581Bsignaling pathway and inhibited IL-1120573 in hypothalamus by regulating NPY mRNA expression

1 Introduction

Myocardial infarction (MI) is a major cause of death globallyworldwide studies have indicated that MI is characterized byan intense inflammatory response within the myocardiumIL-1120573 is considered a key inflammatory mediator after acutemyocardial infarction IL-1120573 has been demonstrated to besignificantly related to infarction and left ventricular functionafter MI [1 2] and inhibited IL-1120573 expression could preventheart failure after MI [3]

There are substantial evidences implicating that centralnervous system- (CNS-) mediated mechanism involved theregulation of cardiac function after MI Recent studies showthat following myocardial infarction elicited by coronaryartery occlusion there is an increase in IL-1120573 levels in thehypothalamus within 24 h after myocardial infarct It hasalso elevated at the time when heart failure is establishedapproximately 6ndash8 weeks after a myocardial infarct in therat [4] The IL-1120573 elevated both in the circulation and in thehypothalamus after MI but the relationship between them isnot clear

Ligusticum wallichii (ChuanXiong) is a Chinese medici-nal herb that has been used orally with other herbs for heart[5] and brain diseases [6 7] for thousands of years Previousstudies have indicated the possible link between the plant andheart disease L wallichii could improve blood fluidity [8]and inhibits endothelial cell damage [9] and vascular smoothmuscle cell proliferation [10] Current research shows thatproinflammatory cytokines are modulators of cardiovascularfunction by a variety ofmechanisms L wallichiiwas found tobe related to anti-inflammatory activity by reducing serumTNF-120572 IL-6 and IL-8 levels [11] L wallichii also inhib-ited production of TNF-120572 and IL-1120573 activated by cerebralischemia [12 13]

An emerging area in cardiovascular research is theapparent significance of inflammation mechanisms Manyrecent studies have suggested that anti-inflammation radicalsmay be important participants in a wide array of cardiacconditions and several clinical trials evaluating the use ofanti-inflammation as therapeutics either have already beenconducted or are underway The present study was designed

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 620359 8 pageshttpdxdoiorg1011552014620359

2 Evidence-Based Complementary and Alternative Medicine

to investigate the effects of Ligusticum wallichii extract onanti-inflammation activities in AMI rats

2 Materials and Methods

21 Materials The reagents used in the study were purchasedas follows pentobarbital sodium from Sigma AmericaELISA kits for IL-1120573 from Jiancheng Biotech Nanjing ChinaNBT from Light Biotech Beijing China mice anti-rat toll-like receptor 4 (TLR4) and mice anti-rat TNF receptorassociated with factor-6 (TRAF-6) from Boster BiotechWuhan China mice anti-rat NF-120581B p65 from Santa CruzAmerica IL-1120573monoclonal antibodies from Sigma AmericaSP kits and DAB kits from ZhongShanJinqiao BiotechBeijing China prestained protein ladder 10ndash170 kDa fromThermo Scientific America Ligusticum wallichii from FirstTeaching Hospital of Tianjin university of TCM lot numberY20050124 aspirin from Bayer National License MedicalNumber J20080078

22 Preparation of Ligusticum wallichii Extract A batchof 63 g of the Ligusticum wallichii was soaked in 1000mLdeionized water for 05 h and continuously extracted usingdeionized water at boiling point for 1 h the extracts werecollected The herbs were then soaked in 800mL deionizedwater extracted for another 1 h Then all these extracts weremixed filtered concentrated dried and weighed

23 Animals AMI Induction and Treatment Sprague-Dawley rats (180 plusmn 50 g 8ndash10 weeks) were obtained fromthe experimental animal center of Military Medical ScienceAcademy Tianjin China They were housed in groups (4 percage) with free access to a regular diet and clean drinkingwater All the experimental procedures described belowadhered strictly to the guidelines set forth by the NationalScience and Technology Commission of China and approvedby the institutional ethics committee

For AMI induction rats were anesthetized with anintraperitoneal dose of 02mL 2 pentobarbital under intu-bated mechanical ventilation at 80 breaths per minuteThoracotomy was performed through the fourth intercostalspace and the left anterior descending coronary artery wasidentified and ligated with 60 prolene suture in the middleportion A few minutes after ligation pallor and akinesiawere seen in the anterior wall and apical left ventricular areaThe interface between the pale and normal areas was definedas ldquoinfarction border zonerdquo The presence of infarction zoneand ST segment elevation were considered as criteria ofsuccessful induction of AMI A group of animals (119899 = 10)were left untreated controls (sham group) The animals thatwere successfully induced to develop AMI were randomizedto AMI group and received PBS (10mgKgd 119899 = 10)Ligusticum wallichii group (10mgKgd 119899 = 10) and aspiringroup (10mgKgd 119899 = 10) respectively

24 Left Ventricular Ejection Fraction Evaluation Echocar-diographic examinationswere performed 7 days after surgery

with the Philips Sonos 5500 ultrasound system (HP Ger-many) The frequency of the probe was set at 175MHzsampling frequency in M-mode at 1000s and scanningspeed 50ndash100mms The probes were placed on precordiumand the detection was carried out from the section ofventricular bands Left ventricular anterior wall (LVAW)thickness left ventricular end-diastolic diameters (LVEDD)and left ventricular end-systolic diameters (LVESD) weremeasured Based on these measurements left ventricularend-diastolic volume (LVEDV) left ventricular end-systolicvolume (LVESV) and left ventricular ejection fraction(LVEF) were calculated as follows

LVEDV = 70 times LVEDD3

(24 + LVEDD)

LVESV = 70 times LVESD3

(24 + LVESD)

LVEF = LVEDV minus LVESVLVEDV

times 100

(1)

25 Infarct Area Size Assessment Areas identified as infarctnonischemic based on NBT staining were measured by com-puterized video planimetry and from these measurementsinfarct size (MIS) was calculated as a percentage of the regionat risk [14] MIS = ischemia area weightventricular weight times100

26 Expression of TLR4 TRAF-6 NF-120581B and IL-1120573 inMyocardium by Immunohistochemical Staining Sectionswere deparaffinized by a standard method cut fixed in 100acetone and stored at minus20∘C The mouse anti-rat TLR4TNF receptor associated to factor-6 (TRAF-6) NF-120581B andIL-1120573 polyclonal antibody were used for the identificationof TLR4 TRAF-6 NF-120581B and IL-1120573 positive cells Sectionswere washed twice in Tris HCl and pH 76 and briefly inbuffer containing 1 polymerized bovine albumin

The sections were incubated for 1 hour at room temper-ature with the primary antibodies diluted 1 100 for TLR41 150 for TRAF-6 1 25 for NF-120581B and 1 200 for IL-1120573And then the secondary antibody is biotinylated and thelabel is peroxidase conjugated streptavidin Control sectionswere treated with the same procedure except they wereincubated without the specific primary antibodies And thendiaminobenzidine (DAB) staining was performed Positivesignals were quantified using the Image Pro-Plus 51 softwareThe number of cells positive for TLR4 TRAF-6 NF-120581B orIL-1120573 was counted in 10 high power fields (times400)

27 Measurement of IL-1120573 in Serum by ELISA The cytokinemeasurements were performed according to the manufac-turerrsquos instructions for the ELISA kit (BD Biosciences USA)

28 Measurement of IL-1120573 and NPY in Myocardium andHypothalamus by Real-Time Quantitative RT-PCR AnalysisTotal RNA was extracted using a commercial RNApreppure kit (number DP430 Tiangen China) Total RNA con-centration was determined from spectrophotometric optical

Evidence-Based Complementary and Alternative Medicine 3

density measurement (260 and 280 nm) For each sampletested the ratio between the spectrophotometric readings at260 nm and 280 nm (OD260OD280) was used to provide anestimate of the purity of the nucleic acid and the ratio in allsamples ranged between 18 and 20

Total RNA was reverse transcribed at 46∘C for 60min70∘C for 5min and 5∘C for 5min using the PrimeScriptRT master mix kit (number DRR036 TaKaRa Japan) andthen cDNA was stored at minus20∘C Quantitative PCR reactionswere carried out with the SYBR Premix Ex TaqTM IIkit (number DRR081 TaKaRa Japan) in a thermocycler(Applied Biosystems 7500 Real-Time PCR System USA)Amplification conditions included 95∘C for 10 s followed by39 cycles of 95∘C for 5 s and 60∘C for 20 s The forward IL-1120573primer was 51015840-TGTGATGTTCCCATTAGAC-31015840 and thereverse IL-1120573 primer was 51015840-AATACCACTTGTTGGCTTA-31015840 (131 bp product) The forward GAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHPwas 51015840-GGATGCAGGGATGATGTTCT-31015840 (254 bp product)The forward NPY primer was 51015840-CTGACCCTCGCTCTA-TCC-31015840 and the reverse NPY primer was 51015840-GGTCTT-CAAGCCTTGTTCT-31015840 (247 bp product) The forwardGAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHP was 51015840-GGATGCAGGGATGAT-GTTCT-31015840 (254 bp product)

Amplification specificity was verified by the meltingcurve following the manufacturerrsquos instructions and 15agarose gel electrophoresis The data were normalized to theCt value of the internal housekeeping gene beta-actin andthe relative mRNA level in the untreated group was used ascalibrator Fold change of the target gene mRNA expressionwas calculated using the 2-ΔΔCT method

29 Statistical Analysis Values of all experiments were rep-resented as mean plusmn SD Statistical significance was assessedby one-way ANOVA followed by LSD post hoc multiplecomparisons The level of significance was set at 119875 lt 005

3 Result

31 Ligusticum wallichii Extract Improved the Left VentricularEjection Fraction (LVEF) The left ventricular ejection frac-tion was assessed by echocardiograph (Figure 1) The LVEFwas significantly decreased in theAMI groupwhen comparedwith the sham group (119875 lt 001) However that alternationwas reduced (119875 lt 005) by feeding LW and aspirin Butthere were no remarkable differences between LW group andaspirin group

32 Ligusticum wallichii Extract Reduced Infarct Area SizeThe infarct area was assessed by NBT staining (Figure 2)NBT stain showed that the color of myocardium in thecontrol group was dark blue but the infarct area of AMImodel group was hoar One week after infarction a largeinfarcted area with a collapsed and pale left ventricular wallwas seen under the ligated silk Compared to the shamgroup larger infarcted area was detected accompanyingglobal enlargement of the heart in the AMI group Ligusticum

0

20

40

60

80

100

Sham AMI Aspirin LW

LVEF

()

lowastlowast

lowastlowast

Figure 1 Left ventricular ejection fraction was assessed by echocar-diograph The LVEF was significantly decreased in the AMI groupwhen compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii (10mgKgd body weight) and aspirin (10mgKgd bodyweight) could improve the LVEF of AMI rat versus AMI group(lowast119875 lt 005) But there were no remarkable differences between LWgroup and aspirin group

wallichii and aspirin treatment reduced infarct area enlarge-ment

33 Ligusticum wallichii Extract Inhibited the Expressionof IL-1120573 in Myocardium Serum and Hypothalamus IL-1120573immunohistochemical staining could be detected to the cellsof rat myocardium after AMI (Figures 3(a) and 3(b)) TheIL-1120573 protein was predominantly expressed in the cyto-plasm Few cells positive for IL-1120573-like immunoreaction wereobserved in the sham group Intensive IL-1120573-like immunos-taining was present in myocardium after AMI Significantchanges of IL-1120573 protein expression were observed in the LWgroup and aspirin group

The serum IL-1120573 level was assessed by ELISA kit(Figure 3(c)) The serum IL-1120573 level was elevated 1W afterAMI LW and aspirin inhibited the IL-1120573 overexpression inserum but there were no remarkable differences between LWgroup and aspirin group

The hypothalamus IL-1120573mRNA expression was assessedby quantitative reverse transcription-PCR (Figure 3(d)) Thehypothalamus IL-1120573mRNA expression was elevated 1W afterAMI LW and aspirin inhibited IL-1120573 mRNA expressionin hypothalamus but there were no remarkable differencesbetween LW group and aspirin group

34 Ligusticum wallichii Extract Inhibited the Expression ofTLR4 TRAF-6 and NF-120581B in Myocardium TLR4 (Figures4(a) and 4(b)) and TRAF-6 (Figures 4(c) 4(d)) immuno-histochemical staining could be detected to the cells of ratmyocardium after AMI The TLR4 and TRAF-6 proteinwas predominantly expressed in the cytoplasm Few cellspositive for TLR4-like and immunoreaction were observedin the sham group Intensive TLR4-like and TRAF-6-likeimmunostaining was present in myocardium after AMISignificant changes of TLR4 and TRAF-6 protein expressionwere observed in the Ligusticum wallichii group and aspiringroup

4 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LW group

(a)

0

5

10

15

20

25

30

Sham AMI Aspirin LW

Myo

card

ium

infa

rct s

ize (

)

lowast lowast

(b)

Figure 2 Representative images for infarcted hearts at one week time point after AMI (a) NBT staining of infarcted area The color ofmyocardium in the control group was dark blue but the infarct area was hoar (b) Ligusticum wallichii (10mgKgd body weight) and aspirin(10mgKgd body weight) reduced the MIS versus AMI group (lowast119875 lt 005) But there were no remarkable differences between LW group andaspirin group

NF-120581B immunohistochemical staining could be detectedto the cells of rat myocardium after AMI (Figures 4(e) and4(f)) In sham group the NF-120581B protein was predominantlyexpressed in the cytoplasm However after AMI expressionwas observed both in the cytoplasm and nucleus Few cellspositive for NF-120581B-like immunoreaction were observed inthe sham group Intensive NF-120581B-like immunostaining waspresent in myocardium after AMI Significant changes ofNF-120581B protein expression were observed in the Ligusticumwallichii group and aspirin group

35 Ligusticumwallichii Extract Inhibited NPYmRNAExpres-sion in Hypothalamus The hypothalamus NPY mRNAexpressionwas assessed by quantitative reverse transcription-PCR (Figure 5) The hypothalamus NPY mRNA expressionwas elevated 1W after AMI LW and aspirin inhibited NPYmRNA expression in hypothalamus but there were noremarkable differences between LW group and aspirin group

4 Discussion

The objective of our study was to investigate the effectsof Ligusticum wallichii extract on anti-inflammation activ-ities after AMI We found that Ligusticum wallichii extract(10mgKgd body weight) could improve the left ventricu-lar ejection fraction and reduced infarct area enlargementLigusticum wallichii extract also inhibited the expression ofIL-1120573 in myocardium serum and hypothalamus Previousstudies have indicated that MI is characterized by an intenseinflammatory response IL-1120573 is considered a key inflamma-tory mediator after acute myocardial infarction IL-1120573 hasbeen demonstrated to be significantly related to infarctionand left ventricular function afterMI [2] Recent studies showthat following myocardial infarction elicited by coronary

artery occlusion there is an increase in IL-1120573 levels in thehypothalamus within 24 h after myocardial infarct [15] Andinhibited brain IL-1120573 synthesis could reduce infarction andimprove left ventricular function [16] this suggests thatinhibiting IL-1120573 expression in brain could improve heartfunction

Our results identify that Ligusticum wallichii extract(10mgKgd body weight) could inhibit the expression ofTLR4 and NF-120581B in myocardium after AMI Toll-like recep-tors (TLRs) play an important role in the regulation ofinnate immune and inflammatory responses by recognitionof pathogen associated molecular patterns (PAMPs) that arenot present in the host [17] TLR4 is a member of the TLRsthat have natural pattern recognition The function of TLR4is tomediate transmembrane signaling transduction inwhichTLR4 could serve as a bridge that links innate immunityand vascular inflammation TLR4 widely recognizes specificpathogen-associated molecular patterns such as couplessignal transduction pathways to activate inflammatory cellswhich results in a series of inflammatory responses and leadsto the synthesis and release of cytokines and inflammatorymediators In TLR4-deficient mice this vascular proinflam-matory gene cannot be expressed regardless of the extent ofobesity dyslipidemia or high fat intake [18]

Nuclear factor (NF)-120581B is downstream of the signalingpathway activating IL-1120573 NF-120581B pathway plays an importantrole in TLR4-mediated inflammatory regulation [19] It isan essential transcription factor that regulates inflammatoryresponses through modulation of the expression of variousproinflammatory mediators including cytokines and NONF-120581B is also a primary regulator of genes that are involvedin the production of proinflammatory cytokines and enzymesinvolved in the process of inflammation [20 21] TLR4 wasupregulated in response to IL-1120573 IL-1120573 activates NF-120581B

Evidence-Based Complementary and Alternative Medicine 5

Sham group AMI group

Aspirin group LW group

(a)

0

2

4

6

8

10

12

Sham AMI Aspirin LW

posit

ive a

rea r

atio

()

lowast lowast

IL-1120573

in m

yoca

rdiu

m

lowastlowast

(b)

0

500

1000

1500

2000

Sham AMI Aspirin LW

IL-1120573

in se

rum

(120583g

mL)

lowastlowast

lowastlowast

(c)

0

2

4

6

8

Sham AMI model Aspirin LW

Expr

essio

n of

IL-

1120573 m

RNA

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

(d)

Figure 3 Ligusticum wallichii extract inhibited the expression of IL-1120573 (a) Representative micrographs were taken at a magnification oftimes400 The brown areas were IL-1120573 positive cell the brown areas were significantly increased in the AMI model group Ligusticum wallichiiand aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred less in the cytoplasm The number ofIL-1120573-like immunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001)Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited IL-1120573 protein expression versus AMIgroup (lowast119875 lt 005) but therewere no remarkable differences betweenLWgroup and aspirin group (c) Serum IL-1120573 levelwas assessed byELISAkitThe serum IL-1120573 level was significantly upregulated 1w after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticum wallichii(10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited the IL-1120573 overexpression after AMI versus AMImodel (lowast119875 lt005) but there were no remarkable differences between LW group and aspirin group (d) Effect of Ligusticum wallichii on the IL-1120573mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCRwas performedThe IL-1120573mRNA expression was significantly upregulated1w after AMI versus sham group (lowastlowast119875 lt 001) Ligusticum wallichii and aspirin inhibited the hypothalamus IL-1120573 mRNA expression versusAMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group

resulting in transcriptional activation of a wide variety ofgenes such as inflammatory mediators

Tumor necrosis factor (TNF) receptor associated factors(TRAFs) play important roles in intracellular signal transduc-tion of many receptor families such as the IL-1 receptors (IL-1R) [22]They could lead to activation of transcription factorssuch as NF-120581B and inflammatory responses RemarkablyTRAF6 is uniquely pleiotropic in participating in the signaltransduction ofmany receptor systemswhile TRAF2 TRAF3and TRAF5 appear to signal only within the TNF receptorsuperfamily [23] TRAF-6 could be active by TLR4 andthen activate the inhibitor of 120581B (I120581B) kinase (IKK) leadingultimately to activation of NF-120581B [24] Levels of TRAF-6 arerelated to inflammation in CAD patients [25]

Neuropeptide Y (NPY) is one of the most abundantneuropeptides present in the human peripheral and centralnervous systems [26] It acts as a neurotransmitter regulating

various autonomic and endocrine functions [27] SpecificallyNPY-containing neurons are present in the paraventricu-lar nucleus (PVN) of the hypothalamus the ventrolateralmedulla (VLM) the NTS and the sympathetic fibres thatinnervate blood vessels [28] NPY played a significant rolein central cardiovascular regulation [29 30] More recentlygenome wide association studies have linked NPY to humancoronary artery disease (CAD) SNPs in the NPY genecorrelated to CAD in humans and evenmore so in early onsetpatients [31]

In recent years NPY has been also described to playa pivotal role in the immune system NPY can increasethe IL-1120573 secretion [32] And NPY receptors are presenton the surface of various leukocyte subgroups modulatingthe release of different cytokines NPY Y1 receptor signalingcould preventNF-120581B activation triggered by IL-1120573 [33] In ourstudy Ligusticum wallichii extract inhibited the expression of

6 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

Submit your manuscripts athttpwwwhindawicom

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Disease Markers

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PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom

2 Evidence-Based Complementary and Alternative Medicine

to investigate the effects of Ligusticum wallichii extract onanti-inflammation activities in AMI rats

2 Materials and Methods

21 Materials The reagents used in the study were purchasedas follows pentobarbital sodium from Sigma AmericaELISA kits for IL-1120573 from Jiancheng Biotech Nanjing ChinaNBT from Light Biotech Beijing China mice anti-rat toll-like receptor 4 (TLR4) and mice anti-rat TNF receptorassociated with factor-6 (TRAF-6) from Boster BiotechWuhan China mice anti-rat NF-120581B p65 from Santa CruzAmerica IL-1120573monoclonal antibodies from Sigma AmericaSP kits and DAB kits from ZhongShanJinqiao BiotechBeijing China prestained protein ladder 10ndash170 kDa fromThermo Scientific America Ligusticum wallichii from FirstTeaching Hospital of Tianjin university of TCM lot numberY20050124 aspirin from Bayer National License MedicalNumber J20080078

22 Preparation of Ligusticum wallichii Extract A batchof 63 g of the Ligusticum wallichii was soaked in 1000mLdeionized water for 05 h and continuously extracted usingdeionized water at boiling point for 1 h the extracts werecollected The herbs were then soaked in 800mL deionizedwater extracted for another 1 h Then all these extracts weremixed filtered concentrated dried and weighed

23 Animals AMI Induction and Treatment Sprague-Dawley rats (180 plusmn 50 g 8ndash10 weeks) were obtained fromthe experimental animal center of Military Medical ScienceAcademy Tianjin China They were housed in groups (4 percage) with free access to a regular diet and clean drinkingwater All the experimental procedures described belowadhered strictly to the guidelines set forth by the NationalScience and Technology Commission of China and approvedby the institutional ethics committee

For AMI induction rats were anesthetized with anintraperitoneal dose of 02mL 2 pentobarbital under intu-bated mechanical ventilation at 80 breaths per minuteThoracotomy was performed through the fourth intercostalspace and the left anterior descending coronary artery wasidentified and ligated with 60 prolene suture in the middleportion A few minutes after ligation pallor and akinesiawere seen in the anterior wall and apical left ventricular areaThe interface between the pale and normal areas was definedas ldquoinfarction border zonerdquo The presence of infarction zoneand ST segment elevation were considered as criteria ofsuccessful induction of AMI A group of animals (119899 = 10)were left untreated controls (sham group) The animals thatwere successfully induced to develop AMI were randomizedto AMI group and received PBS (10mgKgd 119899 = 10)Ligusticum wallichii group (10mgKgd 119899 = 10) and aspiringroup (10mgKgd 119899 = 10) respectively

24 Left Ventricular Ejection Fraction Evaluation Echocar-diographic examinationswere performed 7 days after surgery

with the Philips Sonos 5500 ultrasound system (HP Ger-many) The frequency of the probe was set at 175MHzsampling frequency in M-mode at 1000s and scanningspeed 50ndash100mms The probes were placed on precordiumand the detection was carried out from the section ofventricular bands Left ventricular anterior wall (LVAW)thickness left ventricular end-diastolic diameters (LVEDD)and left ventricular end-systolic diameters (LVESD) weremeasured Based on these measurements left ventricularend-diastolic volume (LVEDV) left ventricular end-systolicvolume (LVESV) and left ventricular ejection fraction(LVEF) were calculated as follows

LVEDV = 70 times LVEDD3

(24 + LVEDD)

LVESV = 70 times LVESD3

(24 + LVESD)

LVEF = LVEDV minus LVESVLVEDV

times 100

(1)

25 Infarct Area Size Assessment Areas identified as infarctnonischemic based on NBT staining were measured by com-puterized video planimetry and from these measurementsinfarct size (MIS) was calculated as a percentage of the regionat risk [14] MIS = ischemia area weightventricular weight times100

26 Expression of TLR4 TRAF-6 NF-120581B and IL-1120573 inMyocardium by Immunohistochemical Staining Sectionswere deparaffinized by a standard method cut fixed in 100acetone and stored at minus20∘C The mouse anti-rat TLR4TNF receptor associated to factor-6 (TRAF-6) NF-120581B andIL-1120573 polyclonal antibody were used for the identificationof TLR4 TRAF-6 NF-120581B and IL-1120573 positive cells Sectionswere washed twice in Tris HCl and pH 76 and briefly inbuffer containing 1 polymerized bovine albumin

The sections were incubated for 1 hour at room temper-ature with the primary antibodies diluted 1 100 for TLR41 150 for TRAF-6 1 25 for NF-120581B and 1 200 for IL-1120573And then the secondary antibody is biotinylated and thelabel is peroxidase conjugated streptavidin Control sectionswere treated with the same procedure except they wereincubated without the specific primary antibodies And thendiaminobenzidine (DAB) staining was performed Positivesignals were quantified using the Image Pro-Plus 51 softwareThe number of cells positive for TLR4 TRAF-6 NF-120581B orIL-1120573 was counted in 10 high power fields (times400)

27 Measurement of IL-1120573 in Serum by ELISA The cytokinemeasurements were performed according to the manufac-turerrsquos instructions for the ELISA kit (BD Biosciences USA)

28 Measurement of IL-1120573 and NPY in Myocardium andHypothalamus by Real-Time Quantitative RT-PCR AnalysisTotal RNA was extracted using a commercial RNApreppure kit (number DP430 Tiangen China) Total RNA con-centration was determined from spectrophotometric optical

Evidence-Based Complementary and Alternative Medicine 3

density measurement (260 and 280 nm) For each sampletested the ratio between the spectrophotometric readings at260 nm and 280 nm (OD260OD280) was used to provide anestimate of the purity of the nucleic acid and the ratio in allsamples ranged between 18 and 20

Total RNA was reverse transcribed at 46∘C for 60min70∘C for 5min and 5∘C for 5min using the PrimeScriptRT master mix kit (number DRR036 TaKaRa Japan) andthen cDNA was stored at minus20∘C Quantitative PCR reactionswere carried out with the SYBR Premix Ex TaqTM IIkit (number DRR081 TaKaRa Japan) in a thermocycler(Applied Biosystems 7500 Real-Time PCR System USA)Amplification conditions included 95∘C for 10 s followed by39 cycles of 95∘C for 5 s and 60∘C for 20 s The forward IL-1120573primer was 51015840-TGTGATGTTCCCATTAGAC-31015840 and thereverse IL-1120573 primer was 51015840-AATACCACTTGTTGGCTTA-31015840 (131 bp product) The forward GAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHPwas 51015840-GGATGCAGGGATGATGTTCT-31015840 (254 bp product)The forward NPY primer was 51015840-CTGACCCTCGCTCTA-TCC-31015840 and the reverse NPY primer was 51015840-GGTCTT-CAAGCCTTGTTCT-31015840 (247 bp product) The forwardGAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHP was 51015840-GGATGCAGGGATGAT-GTTCT-31015840 (254 bp product)

Amplification specificity was verified by the meltingcurve following the manufacturerrsquos instructions and 15agarose gel electrophoresis The data were normalized to theCt value of the internal housekeeping gene beta-actin andthe relative mRNA level in the untreated group was used ascalibrator Fold change of the target gene mRNA expressionwas calculated using the 2-ΔΔCT method

29 Statistical Analysis Values of all experiments were rep-resented as mean plusmn SD Statistical significance was assessedby one-way ANOVA followed by LSD post hoc multiplecomparisons The level of significance was set at 119875 lt 005

3 Result

31 Ligusticum wallichii Extract Improved the Left VentricularEjection Fraction (LVEF) The left ventricular ejection frac-tion was assessed by echocardiograph (Figure 1) The LVEFwas significantly decreased in theAMI groupwhen comparedwith the sham group (119875 lt 001) However that alternationwas reduced (119875 lt 005) by feeding LW and aspirin Butthere were no remarkable differences between LW group andaspirin group

32 Ligusticum wallichii Extract Reduced Infarct Area SizeThe infarct area was assessed by NBT staining (Figure 2)NBT stain showed that the color of myocardium in thecontrol group was dark blue but the infarct area of AMImodel group was hoar One week after infarction a largeinfarcted area with a collapsed and pale left ventricular wallwas seen under the ligated silk Compared to the shamgroup larger infarcted area was detected accompanyingglobal enlargement of the heart in the AMI group Ligusticum

0

20

40

60

80

100

Sham AMI Aspirin LW

LVEF

()

lowastlowast

lowastlowast

Figure 1 Left ventricular ejection fraction was assessed by echocar-diograph The LVEF was significantly decreased in the AMI groupwhen compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii (10mgKgd body weight) and aspirin (10mgKgd bodyweight) could improve the LVEF of AMI rat versus AMI group(lowast119875 lt 005) But there were no remarkable differences between LWgroup and aspirin group

wallichii and aspirin treatment reduced infarct area enlarge-ment

33 Ligusticum wallichii Extract Inhibited the Expressionof IL-1120573 in Myocardium Serum and Hypothalamus IL-1120573immunohistochemical staining could be detected to the cellsof rat myocardium after AMI (Figures 3(a) and 3(b)) TheIL-1120573 protein was predominantly expressed in the cyto-plasm Few cells positive for IL-1120573-like immunoreaction wereobserved in the sham group Intensive IL-1120573-like immunos-taining was present in myocardium after AMI Significantchanges of IL-1120573 protein expression were observed in the LWgroup and aspirin group

The serum IL-1120573 level was assessed by ELISA kit(Figure 3(c)) The serum IL-1120573 level was elevated 1W afterAMI LW and aspirin inhibited the IL-1120573 overexpression inserum but there were no remarkable differences between LWgroup and aspirin group

The hypothalamus IL-1120573mRNA expression was assessedby quantitative reverse transcription-PCR (Figure 3(d)) Thehypothalamus IL-1120573mRNA expression was elevated 1W afterAMI LW and aspirin inhibited IL-1120573 mRNA expressionin hypothalamus but there were no remarkable differencesbetween LW group and aspirin group

34 Ligusticum wallichii Extract Inhibited the Expression ofTLR4 TRAF-6 and NF-120581B in Myocardium TLR4 (Figures4(a) and 4(b)) and TRAF-6 (Figures 4(c) 4(d)) immuno-histochemical staining could be detected to the cells of ratmyocardium after AMI The TLR4 and TRAF-6 proteinwas predominantly expressed in the cytoplasm Few cellspositive for TLR4-like and immunoreaction were observedin the sham group Intensive TLR4-like and TRAF-6-likeimmunostaining was present in myocardium after AMISignificant changes of TLR4 and TRAF-6 protein expressionwere observed in the Ligusticum wallichii group and aspiringroup

4 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LW group

(a)

0

5

10

15

20

25

30

Sham AMI Aspirin LW

Myo

card

ium

infa

rct s

ize (

)

lowast lowast

(b)

Figure 2 Representative images for infarcted hearts at one week time point after AMI (a) NBT staining of infarcted area The color ofmyocardium in the control group was dark blue but the infarct area was hoar (b) Ligusticum wallichii (10mgKgd body weight) and aspirin(10mgKgd body weight) reduced the MIS versus AMI group (lowast119875 lt 005) But there were no remarkable differences between LW group andaspirin group

NF-120581B immunohistochemical staining could be detectedto the cells of rat myocardium after AMI (Figures 4(e) and4(f)) In sham group the NF-120581B protein was predominantlyexpressed in the cytoplasm However after AMI expressionwas observed both in the cytoplasm and nucleus Few cellspositive for NF-120581B-like immunoreaction were observed inthe sham group Intensive NF-120581B-like immunostaining waspresent in myocardium after AMI Significant changes ofNF-120581B protein expression were observed in the Ligusticumwallichii group and aspirin group

35 Ligusticumwallichii Extract Inhibited NPYmRNAExpres-sion in Hypothalamus The hypothalamus NPY mRNAexpressionwas assessed by quantitative reverse transcription-PCR (Figure 5) The hypothalamus NPY mRNA expressionwas elevated 1W after AMI LW and aspirin inhibited NPYmRNA expression in hypothalamus but there were noremarkable differences between LW group and aspirin group

4 Discussion

The objective of our study was to investigate the effectsof Ligusticum wallichii extract on anti-inflammation activ-ities after AMI We found that Ligusticum wallichii extract(10mgKgd body weight) could improve the left ventricu-lar ejection fraction and reduced infarct area enlargementLigusticum wallichii extract also inhibited the expression ofIL-1120573 in myocardium serum and hypothalamus Previousstudies have indicated that MI is characterized by an intenseinflammatory response IL-1120573 is considered a key inflamma-tory mediator after acute myocardial infarction IL-1120573 hasbeen demonstrated to be significantly related to infarctionand left ventricular function afterMI [2] Recent studies showthat following myocardial infarction elicited by coronary

artery occlusion there is an increase in IL-1120573 levels in thehypothalamus within 24 h after myocardial infarct [15] Andinhibited brain IL-1120573 synthesis could reduce infarction andimprove left ventricular function [16] this suggests thatinhibiting IL-1120573 expression in brain could improve heartfunction

Our results identify that Ligusticum wallichii extract(10mgKgd body weight) could inhibit the expression ofTLR4 and NF-120581B in myocardium after AMI Toll-like recep-tors (TLRs) play an important role in the regulation ofinnate immune and inflammatory responses by recognitionof pathogen associated molecular patterns (PAMPs) that arenot present in the host [17] TLR4 is a member of the TLRsthat have natural pattern recognition The function of TLR4is tomediate transmembrane signaling transduction inwhichTLR4 could serve as a bridge that links innate immunityand vascular inflammation TLR4 widely recognizes specificpathogen-associated molecular patterns such as couplessignal transduction pathways to activate inflammatory cellswhich results in a series of inflammatory responses and leadsto the synthesis and release of cytokines and inflammatorymediators In TLR4-deficient mice this vascular proinflam-matory gene cannot be expressed regardless of the extent ofobesity dyslipidemia or high fat intake [18]

Nuclear factor (NF)-120581B is downstream of the signalingpathway activating IL-1120573 NF-120581B pathway plays an importantrole in TLR4-mediated inflammatory regulation [19] It isan essential transcription factor that regulates inflammatoryresponses through modulation of the expression of variousproinflammatory mediators including cytokines and NONF-120581B is also a primary regulator of genes that are involvedin the production of proinflammatory cytokines and enzymesinvolved in the process of inflammation [20 21] TLR4 wasupregulated in response to IL-1120573 IL-1120573 activates NF-120581B

Evidence-Based Complementary and Alternative Medicine 5

Sham group AMI group

Aspirin group LW group

(a)

0

2

4

6

8

10

12

Sham AMI Aspirin LW

posit

ive a

rea r

atio

()

lowast lowast

IL-1120573

in m

yoca

rdiu

m

lowastlowast

(b)

0

500

1000

1500

2000

Sham AMI Aspirin LW

IL-1120573

in se

rum

(120583g

mL)

lowastlowast

lowastlowast

(c)

0

2

4

6

8

Sham AMI model Aspirin LW

Expr

essio

n of

IL-

1120573 m

RNA

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

(d)

Figure 3 Ligusticum wallichii extract inhibited the expression of IL-1120573 (a) Representative micrographs were taken at a magnification oftimes400 The brown areas were IL-1120573 positive cell the brown areas were significantly increased in the AMI model group Ligusticum wallichiiand aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred less in the cytoplasm The number ofIL-1120573-like immunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001)Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited IL-1120573 protein expression versus AMIgroup (lowast119875 lt 005) but therewere no remarkable differences betweenLWgroup and aspirin group (c) Serum IL-1120573 levelwas assessed byELISAkitThe serum IL-1120573 level was significantly upregulated 1w after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticum wallichii(10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited the IL-1120573 overexpression after AMI versus AMImodel (lowast119875 lt005) but there were no remarkable differences between LW group and aspirin group (d) Effect of Ligusticum wallichii on the IL-1120573mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCRwas performedThe IL-1120573mRNA expression was significantly upregulated1w after AMI versus sham group (lowastlowast119875 lt 001) Ligusticum wallichii and aspirin inhibited the hypothalamus IL-1120573 mRNA expression versusAMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group

resulting in transcriptional activation of a wide variety ofgenes such as inflammatory mediators

Tumor necrosis factor (TNF) receptor associated factors(TRAFs) play important roles in intracellular signal transduc-tion of many receptor families such as the IL-1 receptors (IL-1R) [22]They could lead to activation of transcription factorssuch as NF-120581B and inflammatory responses RemarkablyTRAF6 is uniquely pleiotropic in participating in the signaltransduction ofmany receptor systemswhile TRAF2 TRAF3and TRAF5 appear to signal only within the TNF receptorsuperfamily [23] TRAF-6 could be active by TLR4 andthen activate the inhibitor of 120581B (I120581B) kinase (IKK) leadingultimately to activation of NF-120581B [24] Levels of TRAF-6 arerelated to inflammation in CAD patients [25]

Neuropeptide Y (NPY) is one of the most abundantneuropeptides present in the human peripheral and centralnervous systems [26] It acts as a neurotransmitter regulating

various autonomic and endocrine functions [27] SpecificallyNPY-containing neurons are present in the paraventricu-lar nucleus (PVN) of the hypothalamus the ventrolateralmedulla (VLM) the NTS and the sympathetic fibres thatinnervate blood vessels [28] NPY played a significant rolein central cardiovascular regulation [29 30] More recentlygenome wide association studies have linked NPY to humancoronary artery disease (CAD) SNPs in the NPY genecorrelated to CAD in humans and evenmore so in early onsetpatients [31]

In recent years NPY has been also described to playa pivotal role in the immune system NPY can increasethe IL-1120573 secretion [32] And NPY receptors are presenton the surface of various leukocyte subgroups modulatingthe release of different cytokines NPY Y1 receptor signalingcould preventNF-120581B activation triggered by IL-1120573 [33] In ourstudy Ligusticum wallichii extract inhibited the expression of

6 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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

Evidence-Based Complementary and Alternative Medicine 3

density measurement (260 and 280 nm) For each sampletested the ratio between the spectrophotometric readings at260 nm and 280 nm (OD260OD280) was used to provide anestimate of the purity of the nucleic acid and the ratio in allsamples ranged between 18 and 20

Total RNA was reverse transcribed at 46∘C for 60min70∘C for 5min and 5∘C for 5min using the PrimeScriptRT master mix kit (number DRR036 TaKaRa Japan) andthen cDNA was stored at minus20∘C Quantitative PCR reactionswere carried out with the SYBR Premix Ex TaqTM IIkit (number DRR081 TaKaRa Japan) in a thermocycler(Applied Biosystems 7500 Real-Time PCR System USA)Amplification conditions included 95∘C for 10 s followed by39 cycles of 95∘C for 5 s and 60∘C for 20 s The forward IL-1120573primer was 51015840-TGTGATGTTCCCATTAGAC-31015840 and thereverse IL-1120573 primer was 51015840-AATACCACTTGTTGGCTTA-31015840 (131 bp product) The forward GAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHPwas 51015840-GGATGCAGGGATGATGTTCT-31015840 (254 bp product)The forward NPY primer was 51015840-CTGACCCTCGCTCTA-TCC-31015840 and the reverse NPY primer was 51015840-GGTCTT-CAAGCCTTGTTCT-31015840 (247 bp product) The forwardGAPDHP primer was 51015840-CTGATGCCTCCATGTTTGTG-31015840 the reverse GAPDHP was 51015840-GGATGCAGGGATGAT-GTTCT-31015840 (254 bp product)

Amplification specificity was verified by the meltingcurve following the manufacturerrsquos instructions and 15agarose gel electrophoresis The data were normalized to theCt value of the internal housekeeping gene beta-actin andthe relative mRNA level in the untreated group was used ascalibrator Fold change of the target gene mRNA expressionwas calculated using the 2-ΔΔCT method

29 Statistical Analysis Values of all experiments were rep-resented as mean plusmn SD Statistical significance was assessedby one-way ANOVA followed by LSD post hoc multiplecomparisons The level of significance was set at 119875 lt 005

3 Result

31 Ligusticum wallichii Extract Improved the Left VentricularEjection Fraction (LVEF) The left ventricular ejection frac-tion was assessed by echocardiograph (Figure 1) The LVEFwas significantly decreased in theAMI groupwhen comparedwith the sham group (119875 lt 001) However that alternationwas reduced (119875 lt 005) by feeding LW and aspirin Butthere were no remarkable differences between LW group andaspirin group

32 Ligusticum wallichii Extract Reduced Infarct Area SizeThe infarct area was assessed by NBT staining (Figure 2)NBT stain showed that the color of myocardium in thecontrol group was dark blue but the infarct area of AMImodel group was hoar One week after infarction a largeinfarcted area with a collapsed and pale left ventricular wallwas seen under the ligated silk Compared to the shamgroup larger infarcted area was detected accompanyingglobal enlargement of the heart in the AMI group Ligusticum

0

20

40

60

80

100

Sham AMI Aspirin LW

LVEF

()

lowastlowast

lowastlowast

Figure 1 Left ventricular ejection fraction was assessed by echocar-diograph The LVEF was significantly decreased in the AMI groupwhen compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii (10mgKgd body weight) and aspirin (10mgKgd bodyweight) could improve the LVEF of AMI rat versus AMI group(lowast119875 lt 005) But there were no remarkable differences between LWgroup and aspirin group

wallichii and aspirin treatment reduced infarct area enlarge-ment

33 Ligusticum wallichii Extract Inhibited the Expressionof IL-1120573 in Myocardium Serum and Hypothalamus IL-1120573immunohistochemical staining could be detected to the cellsof rat myocardium after AMI (Figures 3(a) and 3(b)) TheIL-1120573 protein was predominantly expressed in the cyto-plasm Few cells positive for IL-1120573-like immunoreaction wereobserved in the sham group Intensive IL-1120573-like immunos-taining was present in myocardium after AMI Significantchanges of IL-1120573 protein expression were observed in the LWgroup and aspirin group

The serum IL-1120573 level was assessed by ELISA kit(Figure 3(c)) The serum IL-1120573 level was elevated 1W afterAMI LW and aspirin inhibited the IL-1120573 overexpression inserum but there were no remarkable differences between LWgroup and aspirin group

The hypothalamus IL-1120573mRNA expression was assessedby quantitative reverse transcription-PCR (Figure 3(d)) Thehypothalamus IL-1120573mRNA expression was elevated 1W afterAMI LW and aspirin inhibited IL-1120573 mRNA expressionin hypothalamus but there were no remarkable differencesbetween LW group and aspirin group

34 Ligusticum wallichii Extract Inhibited the Expression ofTLR4 TRAF-6 and NF-120581B in Myocardium TLR4 (Figures4(a) and 4(b)) and TRAF-6 (Figures 4(c) 4(d)) immuno-histochemical staining could be detected to the cells of ratmyocardium after AMI The TLR4 and TRAF-6 proteinwas predominantly expressed in the cytoplasm Few cellspositive for TLR4-like and immunoreaction were observedin the sham group Intensive TLR4-like and TRAF-6-likeimmunostaining was present in myocardium after AMISignificant changes of TLR4 and TRAF-6 protein expressionwere observed in the Ligusticum wallichii group and aspiringroup

4 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LW group

(a)

0

5

10

15

20

25

30

Sham AMI Aspirin LW

Myo

card

ium

infa

rct s

ize (

)

lowast lowast

(b)

Figure 2 Representative images for infarcted hearts at one week time point after AMI (a) NBT staining of infarcted area The color ofmyocardium in the control group was dark blue but the infarct area was hoar (b) Ligusticum wallichii (10mgKgd body weight) and aspirin(10mgKgd body weight) reduced the MIS versus AMI group (lowast119875 lt 005) But there were no remarkable differences between LW group andaspirin group

NF-120581B immunohistochemical staining could be detectedto the cells of rat myocardium after AMI (Figures 4(e) and4(f)) In sham group the NF-120581B protein was predominantlyexpressed in the cytoplasm However after AMI expressionwas observed both in the cytoplasm and nucleus Few cellspositive for NF-120581B-like immunoreaction were observed inthe sham group Intensive NF-120581B-like immunostaining waspresent in myocardium after AMI Significant changes ofNF-120581B protein expression were observed in the Ligusticumwallichii group and aspirin group

35 Ligusticumwallichii Extract Inhibited NPYmRNAExpres-sion in Hypothalamus The hypothalamus NPY mRNAexpressionwas assessed by quantitative reverse transcription-PCR (Figure 5) The hypothalamus NPY mRNA expressionwas elevated 1W after AMI LW and aspirin inhibited NPYmRNA expression in hypothalamus but there were noremarkable differences between LW group and aspirin group

4 Discussion

The objective of our study was to investigate the effectsof Ligusticum wallichii extract on anti-inflammation activ-ities after AMI We found that Ligusticum wallichii extract(10mgKgd body weight) could improve the left ventricu-lar ejection fraction and reduced infarct area enlargementLigusticum wallichii extract also inhibited the expression ofIL-1120573 in myocardium serum and hypothalamus Previousstudies have indicated that MI is characterized by an intenseinflammatory response IL-1120573 is considered a key inflamma-tory mediator after acute myocardial infarction IL-1120573 hasbeen demonstrated to be significantly related to infarctionand left ventricular function afterMI [2] Recent studies showthat following myocardial infarction elicited by coronary

artery occlusion there is an increase in IL-1120573 levels in thehypothalamus within 24 h after myocardial infarct [15] Andinhibited brain IL-1120573 synthesis could reduce infarction andimprove left ventricular function [16] this suggests thatinhibiting IL-1120573 expression in brain could improve heartfunction

Our results identify that Ligusticum wallichii extract(10mgKgd body weight) could inhibit the expression ofTLR4 and NF-120581B in myocardium after AMI Toll-like recep-tors (TLRs) play an important role in the regulation ofinnate immune and inflammatory responses by recognitionof pathogen associated molecular patterns (PAMPs) that arenot present in the host [17] TLR4 is a member of the TLRsthat have natural pattern recognition The function of TLR4is tomediate transmembrane signaling transduction inwhichTLR4 could serve as a bridge that links innate immunityand vascular inflammation TLR4 widely recognizes specificpathogen-associated molecular patterns such as couplessignal transduction pathways to activate inflammatory cellswhich results in a series of inflammatory responses and leadsto the synthesis and release of cytokines and inflammatorymediators In TLR4-deficient mice this vascular proinflam-matory gene cannot be expressed regardless of the extent ofobesity dyslipidemia or high fat intake [18]

Nuclear factor (NF)-120581B is downstream of the signalingpathway activating IL-1120573 NF-120581B pathway plays an importantrole in TLR4-mediated inflammatory regulation [19] It isan essential transcription factor that regulates inflammatoryresponses through modulation of the expression of variousproinflammatory mediators including cytokines and NONF-120581B is also a primary regulator of genes that are involvedin the production of proinflammatory cytokines and enzymesinvolved in the process of inflammation [20 21] TLR4 wasupregulated in response to IL-1120573 IL-1120573 activates NF-120581B

Evidence-Based Complementary and Alternative Medicine 5

Sham group AMI group

Aspirin group LW group

(a)

0

2

4

6

8

10

12

Sham AMI Aspirin LW

posit

ive a

rea r

atio

()

lowast lowast

IL-1120573

in m

yoca

rdiu

m

lowastlowast

(b)

0

500

1000

1500

2000

Sham AMI Aspirin LW

IL-1120573

in se

rum

(120583g

mL)

lowastlowast

lowastlowast

(c)

0

2

4

6

8

Sham AMI model Aspirin LW

Expr

essio

n of

IL-

1120573 m

RNA

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

(d)

Figure 3 Ligusticum wallichii extract inhibited the expression of IL-1120573 (a) Representative micrographs were taken at a magnification oftimes400 The brown areas were IL-1120573 positive cell the brown areas were significantly increased in the AMI model group Ligusticum wallichiiand aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred less in the cytoplasm The number ofIL-1120573-like immunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001)Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited IL-1120573 protein expression versus AMIgroup (lowast119875 lt 005) but therewere no remarkable differences betweenLWgroup and aspirin group (c) Serum IL-1120573 levelwas assessed byELISAkitThe serum IL-1120573 level was significantly upregulated 1w after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticum wallichii(10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited the IL-1120573 overexpression after AMI versus AMImodel (lowast119875 lt005) but there were no remarkable differences between LW group and aspirin group (d) Effect of Ligusticum wallichii on the IL-1120573mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCRwas performedThe IL-1120573mRNA expression was significantly upregulated1w after AMI versus sham group (lowastlowast119875 lt 001) Ligusticum wallichii and aspirin inhibited the hypothalamus IL-1120573 mRNA expression versusAMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group

resulting in transcriptional activation of a wide variety ofgenes such as inflammatory mediators

Tumor necrosis factor (TNF) receptor associated factors(TRAFs) play important roles in intracellular signal transduc-tion of many receptor families such as the IL-1 receptors (IL-1R) [22]They could lead to activation of transcription factorssuch as NF-120581B and inflammatory responses RemarkablyTRAF6 is uniquely pleiotropic in participating in the signaltransduction ofmany receptor systemswhile TRAF2 TRAF3and TRAF5 appear to signal only within the TNF receptorsuperfamily [23] TRAF-6 could be active by TLR4 andthen activate the inhibitor of 120581B (I120581B) kinase (IKK) leadingultimately to activation of NF-120581B [24] Levels of TRAF-6 arerelated to inflammation in CAD patients [25]

Neuropeptide Y (NPY) is one of the most abundantneuropeptides present in the human peripheral and centralnervous systems [26] It acts as a neurotransmitter regulating

various autonomic and endocrine functions [27] SpecificallyNPY-containing neurons are present in the paraventricu-lar nucleus (PVN) of the hypothalamus the ventrolateralmedulla (VLM) the NTS and the sympathetic fibres thatinnervate blood vessels [28] NPY played a significant rolein central cardiovascular regulation [29 30] More recentlygenome wide association studies have linked NPY to humancoronary artery disease (CAD) SNPs in the NPY genecorrelated to CAD in humans and evenmore so in early onsetpatients [31]

In recent years NPY has been also described to playa pivotal role in the immune system NPY can increasethe IL-1120573 secretion [32] And NPY receptors are presenton the surface of various leukocyte subgroups modulatingthe release of different cytokines NPY Y1 receptor signalingcould preventNF-120581B activation triggered by IL-1120573 [33] In ourstudy Ligusticum wallichii extract inhibited the expression of

6 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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

4 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LW group

(a)

0

5

10

15

20

25

30

Sham AMI Aspirin LW

Myo

card

ium

infa

rct s

ize (

)

lowast lowast

(b)

Figure 2 Representative images for infarcted hearts at one week time point after AMI (a) NBT staining of infarcted area The color ofmyocardium in the control group was dark blue but the infarct area was hoar (b) Ligusticum wallichii (10mgKgd body weight) and aspirin(10mgKgd body weight) reduced the MIS versus AMI group (lowast119875 lt 005) But there were no remarkable differences between LW group andaspirin group

NF-120581B immunohistochemical staining could be detectedto the cells of rat myocardium after AMI (Figures 4(e) and4(f)) In sham group the NF-120581B protein was predominantlyexpressed in the cytoplasm However after AMI expressionwas observed both in the cytoplasm and nucleus Few cellspositive for NF-120581B-like immunoreaction were observed inthe sham group Intensive NF-120581B-like immunostaining waspresent in myocardium after AMI Significant changes ofNF-120581B protein expression were observed in the Ligusticumwallichii group and aspirin group

35 Ligusticumwallichii Extract Inhibited NPYmRNAExpres-sion in Hypothalamus The hypothalamus NPY mRNAexpressionwas assessed by quantitative reverse transcription-PCR (Figure 5) The hypothalamus NPY mRNA expressionwas elevated 1W after AMI LW and aspirin inhibited NPYmRNA expression in hypothalamus but there were noremarkable differences between LW group and aspirin group

4 Discussion

The objective of our study was to investigate the effectsof Ligusticum wallichii extract on anti-inflammation activ-ities after AMI We found that Ligusticum wallichii extract(10mgKgd body weight) could improve the left ventricu-lar ejection fraction and reduced infarct area enlargementLigusticum wallichii extract also inhibited the expression ofIL-1120573 in myocardium serum and hypothalamus Previousstudies have indicated that MI is characterized by an intenseinflammatory response IL-1120573 is considered a key inflamma-tory mediator after acute myocardial infarction IL-1120573 hasbeen demonstrated to be significantly related to infarctionand left ventricular function afterMI [2] Recent studies showthat following myocardial infarction elicited by coronary

artery occlusion there is an increase in IL-1120573 levels in thehypothalamus within 24 h after myocardial infarct [15] Andinhibited brain IL-1120573 synthesis could reduce infarction andimprove left ventricular function [16] this suggests thatinhibiting IL-1120573 expression in brain could improve heartfunction

Our results identify that Ligusticum wallichii extract(10mgKgd body weight) could inhibit the expression ofTLR4 and NF-120581B in myocardium after AMI Toll-like recep-tors (TLRs) play an important role in the regulation ofinnate immune and inflammatory responses by recognitionof pathogen associated molecular patterns (PAMPs) that arenot present in the host [17] TLR4 is a member of the TLRsthat have natural pattern recognition The function of TLR4is tomediate transmembrane signaling transduction inwhichTLR4 could serve as a bridge that links innate immunityand vascular inflammation TLR4 widely recognizes specificpathogen-associated molecular patterns such as couplessignal transduction pathways to activate inflammatory cellswhich results in a series of inflammatory responses and leadsto the synthesis and release of cytokines and inflammatorymediators In TLR4-deficient mice this vascular proinflam-matory gene cannot be expressed regardless of the extent ofobesity dyslipidemia or high fat intake [18]

Nuclear factor (NF)-120581B is downstream of the signalingpathway activating IL-1120573 NF-120581B pathway plays an importantrole in TLR4-mediated inflammatory regulation [19] It isan essential transcription factor that regulates inflammatoryresponses through modulation of the expression of variousproinflammatory mediators including cytokines and NONF-120581B is also a primary regulator of genes that are involvedin the production of proinflammatory cytokines and enzymesinvolved in the process of inflammation [20 21] TLR4 wasupregulated in response to IL-1120573 IL-1120573 activates NF-120581B

Evidence-Based Complementary and Alternative Medicine 5

Sham group AMI group

Aspirin group LW group

(a)

0

2

4

6

8

10

12

Sham AMI Aspirin LW

posit

ive a

rea r

atio

()

lowast lowast

IL-1120573

in m

yoca

rdiu

m

lowastlowast

(b)

0

500

1000

1500

2000

Sham AMI Aspirin LW

IL-1120573

in se

rum

(120583g

mL)

lowastlowast

lowastlowast

(c)

0

2

4

6

8

Sham AMI model Aspirin LW

Expr

essio

n of

IL-

1120573 m

RNA

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

(d)

Figure 3 Ligusticum wallichii extract inhibited the expression of IL-1120573 (a) Representative micrographs were taken at a magnification oftimes400 The brown areas were IL-1120573 positive cell the brown areas were significantly increased in the AMI model group Ligusticum wallichiiand aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred less in the cytoplasm The number ofIL-1120573-like immunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001)Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited IL-1120573 protein expression versus AMIgroup (lowast119875 lt 005) but therewere no remarkable differences betweenLWgroup and aspirin group (c) Serum IL-1120573 levelwas assessed byELISAkitThe serum IL-1120573 level was significantly upregulated 1w after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticum wallichii(10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited the IL-1120573 overexpression after AMI versus AMImodel (lowast119875 lt005) but there were no remarkable differences between LW group and aspirin group (d) Effect of Ligusticum wallichii on the IL-1120573mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCRwas performedThe IL-1120573mRNA expression was significantly upregulated1w after AMI versus sham group (lowastlowast119875 lt 001) Ligusticum wallichii and aspirin inhibited the hypothalamus IL-1120573 mRNA expression versusAMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group

resulting in transcriptional activation of a wide variety ofgenes such as inflammatory mediators

Tumor necrosis factor (TNF) receptor associated factors(TRAFs) play important roles in intracellular signal transduc-tion of many receptor families such as the IL-1 receptors (IL-1R) [22]They could lead to activation of transcription factorssuch as NF-120581B and inflammatory responses RemarkablyTRAF6 is uniquely pleiotropic in participating in the signaltransduction ofmany receptor systemswhile TRAF2 TRAF3and TRAF5 appear to signal only within the TNF receptorsuperfamily [23] TRAF-6 could be active by TLR4 andthen activate the inhibitor of 120581B (I120581B) kinase (IKK) leadingultimately to activation of NF-120581B [24] Levels of TRAF-6 arerelated to inflammation in CAD patients [25]

Neuropeptide Y (NPY) is one of the most abundantneuropeptides present in the human peripheral and centralnervous systems [26] It acts as a neurotransmitter regulating

various autonomic and endocrine functions [27] SpecificallyNPY-containing neurons are present in the paraventricu-lar nucleus (PVN) of the hypothalamus the ventrolateralmedulla (VLM) the NTS and the sympathetic fibres thatinnervate blood vessels [28] NPY played a significant rolein central cardiovascular regulation [29 30] More recentlygenome wide association studies have linked NPY to humancoronary artery disease (CAD) SNPs in the NPY genecorrelated to CAD in humans and evenmore so in early onsetpatients [31]

In recent years NPY has been also described to playa pivotal role in the immune system NPY can increasethe IL-1120573 secretion [32] And NPY receptors are presenton the surface of various leukocyte subgroups modulatingthe release of different cytokines NPY Y1 receptor signalingcould preventNF-120581B activation triggered by IL-1120573 [33] In ourstudy Ligusticum wallichii extract inhibited the expression of

6 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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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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

Evidence-Based Complementary and Alternative Medicine 5

Sham group AMI group

Aspirin group LW group

(a)

0

2

4

6

8

10

12

Sham AMI Aspirin LW

posit

ive a

rea r

atio

()

lowast lowast

IL-1120573

in m

yoca

rdiu

m

lowastlowast

(b)

0

500

1000

1500

2000

Sham AMI Aspirin LW

IL-1120573

in se

rum

(120583g

mL)

lowastlowast

lowastlowast

(c)

0

2

4

6

8

Sham AMI model Aspirin LW

Expr

essio

n of

IL-

1120573 m

RNA

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

(d)

Figure 3 Ligusticum wallichii extract inhibited the expression of IL-1120573 (a) Representative micrographs were taken at a magnification oftimes400 The brown areas were IL-1120573 positive cell the brown areas were significantly increased in the AMI model group Ligusticum wallichiiand aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred less in the cytoplasm The number ofIL-1120573-like immunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001)Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited IL-1120573 protein expression versus AMIgroup (lowast119875 lt 005) but therewere no remarkable differences betweenLWgroup and aspirin group (c) Serum IL-1120573 levelwas assessed byELISAkitThe serum IL-1120573 level was significantly upregulated 1w after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticum wallichii(10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited the IL-1120573 overexpression after AMI versus AMImodel (lowast119875 lt005) but there were no remarkable differences between LW group and aspirin group (d) Effect of Ligusticum wallichii on the IL-1120573mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCRwas performedThe IL-1120573mRNA expression was significantly upregulated1w after AMI versus sham group (lowastlowast119875 lt 001) Ligusticum wallichii and aspirin inhibited the hypothalamus IL-1120573 mRNA expression versusAMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group

resulting in transcriptional activation of a wide variety ofgenes such as inflammatory mediators

Tumor necrosis factor (TNF) receptor associated factors(TRAFs) play important roles in intracellular signal transduc-tion of many receptor families such as the IL-1 receptors (IL-1R) [22]They could lead to activation of transcription factorssuch as NF-120581B and inflammatory responses RemarkablyTRAF6 is uniquely pleiotropic in participating in the signaltransduction ofmany receptor systemswhile TRAF2 TRAF3and TRAF5 appear to signal only within the TNF receptorsuperfamily [23] TRAF-6 could be active by TLR4 andthen activate the inhibitor of 120581B (I120581B) kinase (IKK) leadingultimately to activation of NF-120581B [24] Levels of TRAF-6 arerelated to inflammation in CAD patients [25]

Neuropeptide Y (NPY) is one of the most abundantneuropeptides present in the human peripheral and centralnervous systems [26] It acts as a neurotransmitter regulating

various autonomic and endocrine functions [27] SpecificallyNPY-containing neurons are present in the paraventricu-lar nucleus (PVN) of the hypothalamus the ventrolateralmedulla (VLM) the NTS and the sympathetic fibres thatinnervate blood vessels [28] NPY played a significant rolein central cardiovascular regulation [29 30] More recentlygenome wide association studies have linked NPY to humancoronary artery disease (CAD) SNPs in the NPY genecorrelated to CAD in humans and evenmore so in early onsetpatients [31]

In recent years NPY has been also described to playa pivotal role in the immune system NPY can increasethe IL-1120573 secretion [32] And NPY receptors are presenton the surface of various leukocyte subgroups modulatingthe release of different cytokines NPY Y1 receptor signalingcould preventNF-120581B activation triggered by IL-1120573 [33] In ourstudy Ligusticum wallichii extract inhibited the expression of

6 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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 Evidence-Based Complementary and Alternative Medicine

Sham group AMI group

Aspirin group LWgroup

(a)

0

10

20

30

40

50

Sham AMI Aspirin LW

TLR4

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowastlowast

lowastlowast

(b)

Sham group AMI group

LW groupAspirin group

(c)

012345678

Sham AMI Aspirin LW

TRA

F-6

in m

yoca

rdiu

mpo

sitiv

e are

a rat

io (

)

lowast lowast

lowastlowast

(d)

AMI group

LW group

Sham group

Aspirin group

(e)

Sham AMI Aspirin LW02468

101214

posit

ive a

rea r

atio

()

NF-120581

B in

myo

card

ium

lowast

lowast

lowastlowast

(f)

Figure 4 Ligusticum wallichii extract inhibited the expression of TLR4 TRAF-6 NF-120581B in myocardium (a) Representative micrographswere taken at a magnification of times400 The brown areas were TLR4 positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (b) In the sham group the immunoreactive staining occurred lessin the cytoplasmThe number of TLR4-like immunoreactive cells increased significantly in the myocardium after AMI when compared withthe sham group (lowastlowast119875 lt 001) Ligusticum wallichii (10mgKgd body weight) and aspirin (10mgKgd body weight) both inhibited TLR4protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differences between LW group and aspirin group (c) Thebrown areas were TRAF-6 positive cell the brown areas were significantly increased in the AMImodel group Ligusticumwallichii and aspirindecreased the brown area (d) In the sham group the immunoreactive staining occurred less in the cytoplasm The number of TRAF-6-likeimmunoreactive cells increased significantly in the myocardium after AMI when compared with the sham group (lowastlowast119875 lt 001) Ligusticumwallichii and aspirin both inhibited TRAF-6 protein expression versus AMI group (lowast119875 lt 005) but there were no remarkable differencesbetween LW group and aspirin group (e)The brown areas were NF-120581B positive cell the brown areas were significantly increased in the AMImodel group Ligusticum wallichii and aspirin decreased the brown area (f) In the sham group the immunoreactive staining occurred less inthe cytoplasm The number of NF-120581B-like immunoreactive cells increased significantly in the cytoplasm and nucleus after AMI Ligusticumwallichii and aspirin both inhibited NF-120581B protein expression versus AMI group (lowast119875 lt 005)

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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

Evidence-Based Complementary and Alternative Medicine 7

0

1

2

3

4

Sham AMI model Aspirin LW

Expr

essio

n of

NPY

mRN

A

(in h

ypot

hala

mus

)

lowastlowast

lowastlowast

Figure 5 Effect of Ligusticum wallichii on the NPY mRNAexpression in the hypothalamus of AMI rats Quantitative RT-PCR was performed The NPY mRNA expression was significantlyupregulated 1w after AMI versus sham group (lowastlowast119875 lt 001)Ligusticum wallichii and aspirin inhibited the hypothalamus NPYmRNA expression versus AMI group (lowast119875 lt 005) but there wereno remarkable differences between LW group and aspirin group

NPY in hypothalamus suggesting that it could reduce the IL-1120573 level in hypothalamus afterAMI by inhibitingNPYmRNAexpression

5 Conclusion

Ligusticumwallichii extract improved the left ventricular ejec-tion fraction and reduced infarct area enlargement after AMIby inhibiting the expression of IL-1120573 in myocardium serumand hypothalamus Ligusticum wallichii extract reduced theexpression of IL-1120573 in myocardium by regulating TLR4-NF-120581B signaling pathway and inhibited the expression of IL-1120573 inhypothalamus by regulating NPY mRNA expression

Conflict of Interests

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

References

[1] M Sun F Dawood W Wen et al ldquoExcessive tumor necrosisfactor activation after infarction contributes to susceptibility ofmyocardial rupture and left ventricular dysfunctionrdquo Circula-tion vol 110 no 20 pp 3221ndash3228 2004

[2] A Abbate F N Salloum E Vecile et al ldquoAnakinra a recom-binant human interleukin-1 receptor antagonist inhibits apop-tosis in experimental acute myocardial infarctionrdquo Circulationvol 117 no 20 pp 2670ndash2683 2008

[3] A Abbate F N Salloum B W van Tassell et al ldquoAlterationsin the interleukin-1interleukin-1 receptor antagonist balancemodulate cardiac remodeling following myocardial infarctionin the mouserdquo PLoS ONE vol 6 no 11 Article ID e27923 2011

[4] J Francis Y Chu A K Johnson R M Weiss and R B FelderldquoAcute myocardial infarction induces hypothalamic cytokinesynthesisrdquo The American Journal of PhysiologymdashHeart andCirculatory Physiology vol 286 no 6 pp H2264ndashH2271 2004

[5] S Z Li Compendium of Materia Medica (Bencao Gangmu)JiLin University Publishing House ChangChun China 2009

[6] J W Ho andM Jie ldquoPharmacological activity of cardiovascularagents from herbal medicinerdquo Cardiovascular and Hematolog-ical Agents in Medicinal Chemistry vol 5 no 4 pp 273ndash2772007

[7] H Xu D Shi and C Guan ldquoClinical application and pharma-cological actions of ligustrazinerdquo Zhongguo Zhong Xi Yi Jie HeZa Zhi vol 23 no 5 pp 376ndash379 2003

[8] F Yan and R Luo ldquoEffects of ligustrazine on blood vessels andblood componentsrdquo Zhong Yao Cai vol 25 no 2 pp 143ndash1452002

[9] G F Wang C G Shi M Z Sun et al ldquoTetramethylpyrazineattenuates atherosclerosis development and protects cells ox-LDLrdquo Cardiovascular Drugs andTherapy vol 27 no 3 pp 199ndash210 2013

[10] M J Liang L C He and G D Yang ldquoScreening analysis andin vitro vasodilatation of effective components fromLigusticumChuanxiongrdquo Life Sciences vol 78 no 2 pp 128ndash133 2005

[11] Q Zengyong M Jiangwei and L Huajin ldquoEffect of Ligusticumwallichii aqueous extract on oxidative injury and immunityactivity in myocardial ischemic reperfusion ratsrdquo InternationalJournal of Molecular Sciences vol 12 no 3 pp 1991ndash2006 2011

[12] G Zhang Z Yu and H Zhao ldquoProtective effect of paeonol onrepeated cerebral ischemia in ratsrdquo Zhong Yao Cai vol 20 no12 pp 626ndash628 1997

[13] H J Kadhim J Duchateau and G Sebire ldquoCytokines and braininjury invited reviewrdquo Journal of Intensive Care Medicine vol23 no 4 pp 236ndash249 2008

[14] Y T Xuan Y Guo Y Zhu O L Wang G Rokosh and R BollildquoEndothelial nitric oxide synthase plays an obligatory role in thelate phase of ischemic preconditioning by activating the proteinkinaseC120576-p4442mitogen-activated protein kinase-pSer-signaltransducers and activators of transcription13 pathwayrdquo Circu-lation vol 116 no 5 pp 535ndash544 2007

[15] A L Paul and Q Wu ldquoVascular dysfunction in brain hem-orrhage translational pathways to developing new treatmentsfrom old targetsrdquo Journal of Neurology amp Neurophysiology vol2011 p S1-e001 2011

[16] Y M Kang Z H Zhang B Xue R M Weiss and R BFelder ldquoInhibition of brain proinflammatory cytokine synthesisreduces hypothalamic excitation in rats with ischemia-inducedheart failurerdquo The American Journal of Physiology Heart andCirculatory Physiology vol 295 no 1 pp H227ndashH236 2008

[17] A Aderem and R J Ulevitch ldquoToll-like receptors in theinduction of the innate immune responserdquoNature vol 406 no6797 pp 782ndash787 2000

[18] D M L Tsukumo M A Carvalho-Filho J B C Carvalheira etal ldquoLoss-of-function mutation in toll-like receptor 4 preventsdiet-induced obesity and insulin resistancerdquo Diabetes vol 56no 8 pp 1986ndash1998 2007

[19] S Akira and K Takeda ldquoToll-like receptor signallingrdquo NatureReviews Immunology vol 4 no 7 pp 499ndash511 2004

[20] J W Lee M S Lee T H Kim et al ldquoInhibitory effect ofinflexinol on nitric oxide generation and iNOS expression viainhibition of NF-120581B activationrdquoMediators of Inflammation vol2007 Article ID 93148 pp 93148ndash93157 2007

[21] E T Baima J A Guzova S Mathialagan et al ldquoNovel insightsinto the cellular mechanisms of the anti-inflammatory effectsof NF-120581B essential modulator binding domain peptidesrdquo TheJournal of Biological Chemistry vol 285 no 18 pp 13498ndash135062010

8 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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 Evidence-Based Complementary and Alternative Medicine

[22] H Wu ldquoAssembly of post-receptor signaling complexes for thetumor necrosis factor receptor superfamilyrdquoAdvances in ProteinChemistry vol 68 pp 225ndash279 2004

[23] H Ye J R Arron B Lamothe et al ldquoDistinct molecularmechanism for initiating TRAF6 signallingrdquo Nature vol 418no 6896 pp 443ndash447 2002

[24] Q Yin S C Lin B Lamothe et al ldquoE2 interaction and dimer-ization in the crystal structure of TRAF6rdquoNature Structural andMolecular Biology vol 16 no 6 pp 658ndash666 2009

[25] P Ramkaran S Khan A Phulukdaree D Moodley and AA Chuturgoon ldquomiR-146a polymorphism influences levelsof miR-146a IRAK-1 and TRAF-6 in young patients withcoronary artery diseaserdquo Cell Biochemistry and Biophysics vol68 pp 259ndash266 2014

[26] ldquoNPY and cohorts in human disease Proceedings of the 8thInternational NPY Meeting April 22-26 2006 St PetersburgFlorida USArdquo Peptides vol 28 no 2 pp 197ndash483 2007

[27] M J Morris and J M Pavia ldquoIncreased endogenous nora-drenaline and neuropeptide Y release from the hypothalamusof streptozotocin diabetic ratsrdquo Brain Research vol 1006 no 1pp 100ndash106 2004

[28] M L Wolak M R de Joseph A D Cator A S Mokashi MS Brownfield and J H Urban ldquoComparative distribution ofneuropeptide Y Y1 and Y5 receptors in the rat brain by usingimmunohistochemistryrdquo Journal of Comparative Neurology vol464 no 3 pp 285ndash311 2003

[29] H N Huang P J Lu W C Lo C H Lin M Hsiao and CJ Tseng ldquoIn situ Akt phosphorylation in the nucleus tractussolitarii is involved in central control of blood pressure andheart raterdquo Circulation vol 110 no 16 pp 2476ndash2483 2004

[30] W Ho P Lu M Hsiao et al ldquoAdenosine modulates cardio-vascular functions through activation of extracellular signal-regulated kinases 1 and 2 and endothelial nitric oxide synthasein the nucleus tractus solitarii of ratsrdquo Circulation vol 117 no6 pp 773ndash780 2008

[31] S H Shah N J Freedman L Zhang et al ldquoNeuropeptide Ygene polymorphisms confer risk of early-onset atherosclerosisrdquoPLoS Genetics vol 5 no 1 Article ID e1000318 2009

[32] S Bedoui S von Horsten and T Gebhardt ldquoA role forneuropeptide Y (NPY) in phagocytosis implications for innateand adaptive immunityrdquo Peptides vol 28 no 2 pp 373ndash3762007

[33] J Choi and S Koh ldquoRole of brain inflammation in epileptoge-nesisrdquo Yonsei Medical Journal vol 49 no 1 pp 1ndash18 2008

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