Research Article The Protective Effect of Low-Dose Ethanol...

Research ArticleThe Protective Effect of Low-Dose Ethanol on MyocardialFibrosis through Downregulating the JNK Signaling Pathway inDiabetic Rats

Ying Yu1 Xian-Jie Jia2 Wei-ping Zhang1 Ting-ting Fang1 Jie Hu1

Shan-Feng Ma1 and Qin Gao1

1Department of Physiology Bengbu Medical College 2600 Dong Hai Avenue Bengbu 233030 China2Department of Epidemiology and Statistics Bengbu Medical College 2600 Dong Hai Avenue Bengbu 233030 China

Correspondence should be addressed to Qin Gao bbmcgq126com

Received 6 April 2016 Revised 30 June 2016 Accepted 3 July 2016

Academic Editor Nikolaos Papanas

Copyright copy 2016 Ying Yu et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Objective To investigate the effects of low dose ethanol feeding in diabetic rats and analyze its underlying mechanisms MethodsMale Sprague-Dawley rats were divided into 4 groups control (Con) diabetes at 4 weeks (DM4W) diabetes at 8 weeks(DM8W) and EtOH + DM8W After 8 weeks hemodynamic parameters were recorded and heart weightbody weight (HB) andhydroxyproline (Hp) content in myocardium were measured Morphology of collagen in myocardial tissue was observed withMassonrsquos trichrome staining method and collagen volume fraction (CVF) was analysed The mRNA expression of ALDH2 wasassessed with Real-Time PCR The protein expressions of p-JNK and JNK were evaluated using western blot Results In contrastto Con group there was no difference in hemodynamic parameters in DM4W group but mean arterial pressure and heart ratewere decreased in DM8W group and the ratios of HB Hp and CVF were markedly increased ALDH2 mRNA expression wasdecreased while the ratio of p-JNKJNKwere increased Compared with DM8W group the above indexes were improved in EtOH+ DM8W group Conclusion With low dose ethanol intervention enhanced ALDH2 expression can antagonize the happening ofmyocardial fibrosis in diabetic rats which may be relevant with downregulating the JNK pathway

1 Introduction

Diabetes mellitus (DM) is one of the major public healthproblems around the world [1] According to the compileddata of the World Health Organization (WHO) approxi-mately 150 million people have diabetes mellitus worldwideand this number of diabetic patients may be doubled by theyear 2025 [2]

Alongside established lifestyle factors such as smokingadiposity and diet ethanol consumption is thought to playa role in the development of diabetes Ethanol is a verycommonly used chemical substance and also has dose-relatedeffect on cardiac events Possible cardioprotective effectsof ethanol consumption continue to be hotly debated inthe medical literatures and popular media [3] Systematicreviews and meta-analyses have addressed that there is acurvilinear relationship between ethanol consumption and

cardiovascular disease with a protective effect of moder-ate ethanol consumption and a detrimental effect of largeamounts intake [3ndash7] Importantly epidemiological studiessuggest that light tomoderate alcohol consumption decreasesthe risk of cardiovascular events that is 1-2 drinks perd or 3ndash9 drinks per wk (one beer one glass of wine orone glass of spirit was approximated to one standard drinkdefined as 15 cL or 12 g of pure ethanol [7]) Moderatedrinking can activate metabolism of ethanol by acetaldehydedehydrogenase-2 generation [3] to reduce the incidence ofdiabetic cardiomyopathy [8 9]

Diabetic cardiomyopathy is defined as the ventriculardysfunction that occurs in diabetic patients independent ofanother cause such as coronary artery disease or hyperten-sion Diabetic cardiomyopathy is a common complication ofdiabetes which has become a major cause of diabetes-relatedmorbidity and mortality [10] Mitochondrial acetaldehyde

Hindawi Publishing CorporationJournal of Diabetes ResearchVolume 2016 Article ID 3834283 7 pageshttpdxdoiorg10115520163834283

2 Journal of Diabetes Research

dehydrogenase-2 (ALDH2) is a key enzyme which plays animportant role in the metabolism of acetaldehyde and othertoxic aldehydes [11] Preclinical studies in rats also suggestthat ALDH2 activity may affect diabetic pathology [12]Furthermore in transgenic mice overexpression of ALDH2has been shown to be protective against streptozotocin-induced diabetic cardiomyopathy [13] Our previous resultshad indicated that ALDH2 expression was further decreasedaccompanying the development of diabetes Meanwhile weinvestigated the effects of low-dose ethanol feeding in diabeticrats which could promote myocardial protection throughactivation of ALDH2 expression And more we also foundthat upregulation of ALDH2 played a protective effect inmyocardial ischemia and reperfusion injury and diabetescardiomyopathy ALDH2 may be an endogenous cardiacprotective factor in myocardial injury [2 8 14]

Several biological mechanisms have been proposed toexplain that myocardial fibrosis is one of the main pathologi-cal changes of diabetic cardiomyopathy Studies have foundC-JUN N-terminal kinase (JNK) signaling pathway playeda key role in the process of myocardial fibrosis [15ndash17]Evidence had shown that activation of the JNK pathway wasinvolved in the progression of diabetes induced myocardialfibrosis and that such a pathway could be a therapeutictarget for diabetic heart injury and cardiomyopathy [1819] However whether the JNK pathway is involved in thecardioprotective effect of low-dose ethanol on diabetic ratshas not been fully elucidated

So in this study we mimic diabetes model by intraperi-toneal injection of streptozotocin (STZ) in combinationwith low-dose ethanol the purpose of the present studyis as follows (1) to investigate the mechanism of low-dose ethanol which alleviates myocardial fibrosis in diabeticcardiomyopathy (2) to clarify whether low-dose ethanolmediated protection is associated with downregulating theJNK signaling pathway This study might shed some light onlow-dose ethanol as an effective therapeutic in the treatmentof diabetic cardiomyopathy

2 Methods

21 Animals Adult male Sprague-Dawley rats (200 to 250 g)were obtained fromBengbuMedical College Animal Admin-istration Center All animal studies were approved by theAnimal Ethics Committee of Bengbu Medical College andperformed in accordance with the ethical standards Therats were fed normal chow and had free access to distilledwater

22 Chemicals and Reagents Streptozotocin (STZ) was pur-chased from Sigma (USA) TRIzol was purchased fromInvitrogen (USA) hydroxyproline (Hp) was purchased fromNanjing Jiancheng Bioengineering Institute (China) Ethanol(EtOH) was purchased from Bengbu New Chemical ReagentFactory (China) 120573-actin antibodies were purchased fromSanta Cruz Biotechnology (USA) rabbit c-Jun N-terminalkinase (JNK) and phosphorylated JNK (p-JNK) were pur-chased from Anbo Biotechnology (USA) Chemilumines-cence reaction (ECL) system was purchased from Millipore

Billerica (USA) All primers were purchased from ShanghaiSangon Biotech (China)

23 Induction of Diabetes and Experimental Protocol Aspreviously described by our laboratory STZ at 55mgkgfreshly dissolved in 01molL sodium citrate buffer (pH 45)was injected intraperitoneally to induce diabetic models inovernight fasted rats [8] All rats were randomly dividedinto four groups normal control group (Con) diabetes at 4weeks group (DM4W) diabetes at 8 weeks group (DM8W)and ethanol + diabetes at 8 weeks group (EtOH + DM8W)respectively (119899 = 6) In Con group rats were fed withstandard rat chow and received an intraperitoneal injection ofthe same volume of citrate buffer for 8 weeks In EtOH + DMgroup DM rats were fed with 25 EtOH in their drinkingwater for oneweek to initiate drinking then it was changed to5 EtOH continuous access through the remaining 7 weeks

24 Hemodynamics Male SD rats were anaesthetized byuse of chloral hydrate (100mgkg) through intraperitonealinjectionThroughout the experiment systolic pressure (SP)diastolic pressure (DP) mean arterial pressure (MAP) andheart rate (HR) were determined by invasive hemodynamicevaluation methods for 30min At the end of the experi-mental period hearts were excised rapidly placed in ice-coldKrebs-Henseleit (K-H) buffer and weighed and the ratio ofheart weightbody weight (HB) was calculated

25 Detection of Hydroxyproline Content Heart tissue(100mg) was homogenized in ice-cold K-H buffer Thesupernatant was collected after centrifugation for 20min(2000 rpm) The protein concentration was measured bythe Bradford method Hydroxyproline (Hp) content wasdetected according to the instruction manual

26 The Content of Collagen Detection by Masson-StainingLeft ventricular tissue obtained from all groups was stainedwith Massonrsquos trichrome for the quantification of collagenThe histological sections were taken in 10 neutral formalin-fixed dehydrated paraffin embedded sections Then thesamples underwent the dehydration of gradient ethanolneutral resin embedding and Masson trichrome stainingThe collagen fibers were stained blue and cardiomyocyteswere stained redMyocardial collagenwas quantified at a finalmagnification of 200xwith a polarizedmicroscope connectedto a video camera Myocardial collagen volume fraction(CVF) was analyzed using Image Pro analysis softwareexpressed as the mean percentage of collagen area to the totalarea of eachmicroscopic field Five visions under microscopeof each sample were randomly chosen and the average ofthem was taken for analysis [17]

27 Detection of ALDH2 mRNA by Real-Time PCR TotalRNA was extracted from the left anterior myocardium usingTRIzol according to the manufacturerrsquos instructions TotalRNA (3 120583g) was reversely transcribed to cDNA and PCRwas performed by a routine method The primers used wereas follows for ALDH2 forward 5-GTG TTC GGA GAC

Journal of Diabetes Research 3

Table 1 Hemodynamic data in rats

Group SP (mmHg) DP (mmHg) MAP (mmHg) HR (beatsmin)Con 11109 plusmn 605 9547 plusmn 775 10068 plusmn 715 46263 plusmn 6520DM4W 10530 plusmn 474 9276 plusmn 490 9694 plusmn 485 43300 plusmn 1439

DM8W 8324 plusmn 932lowastlowast 6297 plusmn 1056lowastlowast 6972 plusmn 940lowastlowast 37706 plusmn 3904lowastlowast

EtOH + DM8W 10374 plusmn 438 8045 plusmn 590 8821 plusmn 537 41144 plusmn 2227lowast

Values are means plusmn SD (119899 = 6)Con normal control group DM4W diabetes at 4 weeks group DM8W diabetes at 8 weeks group and EtOH + DM8W ethanol + diabetes at 8 weeks grouplowast

119875 lt 005 lowastlowast119875 lt 001 compared with Con 119875 lt 005 119875 lt 001 compared with DM8W

Table 2 Changes of heart weightbody weight (HB) hydroxyproline (Hp) content and collagen volume fraction (CVF) in different groups

Group HB (mgg) Hp (umolmg) CVF ()Con 376 plusmn 015 027 plusmn 005 1250 plusmn 098DM4W 388 plusmn 020 035 plusmn 007 1574 plusmn 153lowast

DM8W 478 plusmn 010lowastlowast 043 plusmn 004lowastlowast 2575 plusmn 198lowastlowast

EtOH + DM8W 396 plusmn 017 034 plusmn 005 1443 plusmn 265

Values are means plusmn SD (119899 = 6)lowast

119875 lt 005 and lowastlowast119875 lt 001 compared with Con 119875 lt 005 and 119875 lt 001 compared with DM8W

GTCAAAGA-31015840 and reverse 51015840-GCAGAGCTTGGGACAGGT AA-31015840 and the product size was 187 bp for 120573-actinforward 51015840-GAG ACC TTC AAC ACC CCA GCC-31015840 andreverse 51015840-GGC CAT CTC TTG CTC GAA GTC-31015840 andthe product size was 312 bp [8] The PCR condition was asfollows predenaturing at 95∘C for 3min and then 40 cycles(50 s denaturation at 95∘C 50 s annealing at 625∘C and 60 sextension at 72∘C) followed by a final step at 72∘C for 10minReal-Time PCR was performed to determine the ALDH2mRNA level

28 Detection of JNK and p-JNK Protein Expressions by West-ern Blot Myocardium tissues (100mg) from each groupwerecollected and homogenized in a lysis buffer Homogenateswere sonicated and centrifuged at 12000timesg for 30min at 4∘CThe protein concentration was determined using the bicin-choninic acid (BCA) Protein Assay kit Total protein (80120583g)was separated by SDS-polyacrylamide gel electrophoresis(PAGE) and transferred electrophoretically to a polyvinyli-dene difluoride (PVDF) filter membrane [20]

The membranes were blocked with 5 nonfat milk inTris-buffered saline Tween (TBST) for 2 h and then theywere incubated at 4∘C overnight with the corresponding pri-mary rabbit JNK antibody (1 1000) rabbit p-JNK antibody(1 1000) and mouse 120573-actin antibody (1 500) All mem-branes were incubated for 1 h with corresponding secondaryantibody HRP-linked anti-mouse IgG or HRP-linked anti-rabbit IgG Autoradiographs were scanned and the banddensity was determined with Image J software

29 Statistical Analysis Data were expressed as mean plusmn SDOne-way analysis of variance (ANOVA) followed by Student-Newman-Keuls (SNK) was used for multiple comparisons119875 lt 005 was considered as statistically significant

3 Results

31 Hemodynamics In contrast to Con group there wasno statistical difference about hemodynamic parameters in

DM4W groups but systolic pressure (SP) diastolic pressure(DP) mean arterial pressure (MAP) and heart rate (HR)were decreased significantly in DM8W groups Comparedwith DM8W group SP DP MAP and HR were ratherincreased significantly in EtOH + DM8W group (Table 1)

32 The Ratio of Heart Weight to Body Weight and Hydrox-yproline Content in Myocardial Tissue Compared with Congroup there was no significant difference about the ratio ofheart weight to body weight (HB) and hydroxyproline (Hp)content in myocardial tissue in diabetic rat in DM4W grouphowever with extended duration HB ratio and myocardialHp content were increased in DM8W group and the differ-ence was statistically significant (Table 2) Compared withDM8W group HB ratio and myocardial Hp content weresignificantly deceased in EtOH + DM8W group (119875 lt 005)

33TheContent of CollagenDetection byMasson-Staining InMasson trichrome staining the collagen fibers were stainedblue and cardiomyocyteswere stained redThe collagen tissuewas appropriately arranged among cardiomyocytes in controlgroup However collagen tissue was increased markedly anddisrupted in some area in diabetic group

In DM4W group myocardial cells were approximatelyarranged well collagen fibers were sparsely distributedand interstitial collagen was dyed a little blue Comparedwith control group myocardial cells were in a disorderedarrangement the interstitial collagen were edematous andcollagen fibers were unevenly distributed and increasedmarkedly in DM8W group Compared with DM8W groupmyocardial cells were arranged neatly and collagen fiberswere significantly reduced in EtOH + DM8W group

Quantitative Analysis Results The content of collagen indiabetic group was higher than that of control group As dis-played in Table 2 compared to control group the contents ofcollagen volume fraction (CVF) were significantly increasedin DM4W group (119875 lt 005) and with the development


(a) (b)

(c) (d)

Figure 1 The result of collagen detection by Masson-staining (200) (a) Control (b) DM4W (c) DM8W and (d) EtOH + DM8W

p-JN

KJN

K120573-actin

54kD46kD

54kD46kD

43kD

lowastlowast

lowast

Con DM8W EtOH + DM8WDM4W


00

05

10

15

20

p-JN

KJN

K

Figure 2 The result of myocardial JNK and phospho-JNK protein lowast119875 lt 005 and lowastlowast119875 lt 001 compared with Con 119875 lt 005 and 119875 lt 001compared with DM8W

of diabetes CVF were further increased in DM8W groups(119875 lt 001) But in contrast to DM8W group the contentsof CVF were markedly decreased in EtOH + DM8W group(119875 lt 001 Figure 1 Table 2)

34 Changes of Myocardial ALDH2 mRNA Expression Real-Time PCR revealed that compared with control group theexpression of ALDH2 mRNA level was reduced in DM4W

(119875 lt 005) With the development of diabetes ALDH2mRNA were further decreased in DM8W groups (119875 lt001) In contrast to DM8W group the expression of ALDH2mRNA was increased in EtOH + DM8W group (119875 lt 001Table 3)

35 Changes of Myocardial JNK and Phospho-JNK ProteinLevels On western blot analysis compared with control


Table 3 The result of ALDH2 mRNA and p-JNKJNK proteinexpression

Group ALDH2 mRNA p-JNKJNKCon 085 plusmn 016 088 plusmn 025DM4W 056 plusmn 017lowast 120 plusmn 022lowast

DM8W 021 plusmn 00lowastlowast 171 plusmn 014lowastlowast

EtOH + DM8W 061 plusmn 018 115 plusmn 007


119875 lt 005 and lowastlowast119875 lt 001 compared with Con 119875 lt 005 and 119875 lt 001

compared with DM8W

group the ratio of p-JNKJNK protein expression wasincreased in DM4W (119875 lt 005) and with the developmentof diabetes the ratio of p-JNKJNK was further increased inDM8W groups (119875 lt 001) Compared with DM8W groupthe ratio in EtOH + DM8W group was decreased (119875 lt 001Figure 2 Table 3)

4 Discussion

Diabetes mellitus is becoming an epidemic health threat andrepresents one of the most prevalent chronic noncommu-nicable disorders [21] Cardiovascular disease is a seriouscomplication of diabetes and is responsible for 80 of thedeaths among diabetics [22] Diabetic cardiomyopathy is oneof the most common complications of diabetes its majorpathological characteristics are hypertrophy or hyperplasiaof cardiac myocytes Excessive deposition of myocardialinterstitial collagen and myocardial fibrosis often leads tocardiac hypertrophy and decrease of heart function whichplay a vital role in the occurrence and development of diabeticcardiomyopathy [23]

In the present study our results demonstrated thatwith the progression of diabetes systolic pressure diastolicpressure mean arterial pressure and heart rate were declinedsignificantly in DM8W group compared with Con groupbut the ratio of heart weightbody weight hydroxyprolinecontent and CVF were markedly increased Meanwhile themRNA expression of ALDH2 was decreased and the ratio ofp-JNKJNK was increased When the DM rats were treatedwith ethanol at low concentration hemodynamic parameterswere improved and hydroxyproline content and CVF weredecreased accompanied with the increase of myocardialALDH2 mRNA expression and decrease of p-JNKJNKprotein expression The results suggested that with low-dose ethanol intervention enhanced ALDH2 expression canantagonize the happening of myocardial fibrosis in diabeticcardiomyopathy whichmay be relevant with downregulatingthe JNK pathway

Mitochondrial aldehyde dehydrogenase 2 (ALDH2) isa kind of aldehyde oxidase that is involved in ethanolmetabolism which is closely related to human alcoholdrinking behavior [24] Heavier alcohol consumption isassociatedwith increased risk of developing diabetesmellitushypertension and cardiovascular and cerebrovascular dis-ease [25] Nevertheless there are increasing reports showinga critical role for light to moderate alcohol ingestion could

protect against myocardial injuries Researches indicated aU-shaped relationship between alcohol consumption andcardiovascular disease mortality [6 26] Statistics manifestedthe mortality of drinking alcohol up to or over 6 cups(355mLcup) per day is 16 times as high as control whilelight-to-moderate drinking of 1sim2 cups alcohol per day [fewerthan 2 cups or 1 ounce (283495 g)] reduced mortality fromcardiovascular disease [27] In addition clinical data showedthat moderate drinking can protect metabolic syndrome(cardiovascular disease and diabetes) via influencing variousfactors to reduce the diabetes prevalence such as glycosy-lated hemoglobin high density lipoprotein cholesterol andfibrinogen [28] After continuously feeding C57BL6 micewith 18 of ethanol for 12 weeks the myocardial mechanicswere restored and the expression and activation of PKC andAkt were increased which established that EtOH feedingcauses cardiac expression of activated PKC-120576 [29] Sun et alobserved that ischemic injury could result in downregulationof mitochondrial ALDH2 in mice hearts inducing an eleva-tion of aldehyde 4-hydroxy-2-nonenal (4-HNE) leading tocardiomyocyte apoptosis through downregulation of HSP70and activation of JNK and p53 ALDH2 detoxifies 4-HNE amediator of programmed cell death events by transmittinga mitochondrial ALDH2 signal to elicit a cytosolic responsethrough the JNKp53 pathway However using transgenetechnology to increase the expression of ALDH2 can con-tribute to antagonizing heart failure and decreasing heartfunction [30] Furthermore our previous research found thatthere was a close relationship between ALDH2 and oxidativedamage With the progression of diabetes the myocardialantioxidant ability of diabetic rats was decreased whichexacerbated the progression of myocardial fibrosis [8 13 31]In this study we found that the ratio of heart weightbodyweight hydroxyproline content and CVF were markedlyincreased in diabetic 4 and 8 weeks rats which suggesteddiabetic induced myocardial fibrosis When the diabetic ratwas treated with low doses of ethanol to induce ALDH2activity the hemodynamic parameters were increased andhydroxyproline content and CVF were decreased whichindicated that increasing ALDH2 expression can attenuatethe happening of myocardial fibrosis and the destroying ofmyocardial injuries

Mitogen-activated protein kinases (MAPKs) play animportant role in the signal transduction pathways fromthe membrane to intracellular compartments including thenucleus They regulate the functions of many gene productsand therefore affect cell growth differentiation and apoptosis[32] Oxidative stress inflammation endoplasmic reticulumstress and autophagy defect can activate MAPKs signalingpathway in the progression of diabetes complications Li etal observed that production of a large number of ROS wasthought to be an important contributing factor concomitantwith activation of JNK p38 MAPK and TGF-120573 in thedevelopment and the progression of diabetic cardiomyopathy[16]

C-Jun NH2-terminal kinase (JNK) is one of the majormembers of MAPKs and JNK activation is also implicatedin cardiac fibrosis [33 34] JNK signaling pathway plays akey role in the growth of cardiac fibroblasts induced by


high glucose Studies of diabetic rats had demonstrated thatmyocardial fibrosis was developed meanwhile JNK mRNAexpression level and activity were upregulated [17] Further-more disruption of the JNK protein kinase decreased theoccurrence and development of diabetic myocardial fibrosis[18 35] It is worthwhile to note that in our experiment wefound that with the progression of diabetes the expressionof myocardial ALDH2 at mRNA level was decreased andthe ratio of p-JNKJNK at protein level was increasedwhich suggested ALDH2 and JNK signaling pathway bothparticipated in the occurrence and development of diabeticcardiomyopathy Moreover further enhancing activation ofALDH2 expression of low doses of ethanol for 8 weeksin diabetic rats accompanied with the high-expression ofALDH2 p-JNKJNK were decreased in contrast to diabetesrats which suggested activation of ALDH2 expression mightbe associated with downregulating the JNK signaling path-way to relieve myocardial fibrosis and myocardial injuries

However there were some experimental limitations inour study We only observed that the protection of low-dose ethanol was relevant with downregulating the JNKpathway in diabetic cardiomyopathy To better understandthe mechanisms involved we will adopt the activation orinhibition of JNK to investigate the downstream signalingmolecules in the follow-up experiments To address this issuewe will use JNK inhibitor to observe whether inhibiting JNKpathway can play the cardiovascular role in diabetes ratsMoreover we further explore whether enhanced activation ofJNK signaling pathway can antagonize the cardioprotectiveeffect of activation of ALDH2 by low-dose ethanol Throughthese experiments we want to verify low-dose ethanol couldattenuate myocardial fibrosis via downregulating the JNKpathway in diabetic cardiomyopathy

In summary our results demonstrate that with theprogression of diabetes ALDH2 expression was decreasedaccompanied with the happening of myocardium fibrosisTreatment with ethanol at low concentration can protectthe heart by upregulating ALDH2 and downregulating theJNK signaling pathway against myocardial fibrosis in diabeticcardiomyopathy

Competing Interests

The authors have no potential conflict of interests to declare

Authorsrsquo Contributions

Ying Yu and Xian-Jie Jia are co-first authors and contributedequally to this paper

Acknowledgments

This work was supported by the National Natural Sci-ence Foundation of China (81000074) and the NaturalScience Foundation of Anhui Province (1508085QH1501508085MH169) China

References

[1] V Mohan Y K Seedat and R Pradeepa ldquoThe rising burdenof diabetes and hypertension in southeast Asian and Africanregions need for effective strategies for prevention and controlin primary health care settingsrdquo International Journal of Hyper-tension vol 2013 Article ID 409083 14 pages 2013

[2] J-F Hu G-J Zhang L Wang et al ldquoEthanol at low concen-tration attenuates diabetes induced lung injury in rats modelrdquoJournal of Diabetes Research vol 2014 Article ID 107152 6pages 2014

[3] P E Ronksley S E Brien B J Turner K J Mukamal andW A Ghali ldquoAssociation of alcohol consumption with selectedcardiovascular disease outcomes a systematic review andmeta-analysisrdquoTheBritishMedical Journal vol 342 no 3 article d6712011

[4] S E Brien P E Ronksley B J Turner K J Mukamal and WA Ghali ldquoEffect of alcohol consumption on biological markersassociatedwith risk of coronary heart disease systematic reviewand meta-analysis of interventional studiesrdquo BMJ vol 342article d636 2011

[5] Y-L Zheng F Lian Q Shi et al ldquoAlcohol intake and associatedrisk of major cardiovascular outcomes in women comparedwith men a systematic review and meta-analysis of prospectiveobservational studiesrdquo BMC Public Health vol 15 article 7732015

[6] M Roerecke and J Rehm ldquoAlcohol consumption drinkingpatterns and ischemic heart disease a narrative review ofmeta-analyses and a systematic review and meta-analysis ofthe impact of heavy drinking occasions on risk for moderatedrinkersrdquo BMCMedicine vol 12 article 182 2014

[7] L L Husemoen T Joslashrgensen K Borch-Johnsen T HansenO Pedersen and A Linneberg ldquoThe association of alcohol andalcohol metabolizing gene variants with diabetes and coronaryheart disease risk factors in a white populationrdquo PLoS ONE vol5 no 8 article e11735 2010

[8] Q Gao H-J Wang X-M Wang et al ldquoActivation of ALDH2with ethanol attenuates diabetes induced myocardial injury inratsrdquo Food and Chemical Toxicology vol 56 pp 419ndash424 2013

[9] MVHolmes C E Dale L Zuccolo et al ldquoAssociation betweenalcohol and cardiovascular disease mendelian randomisationanalysis based on individual participant datardquo British MedicalJournal vol 349 Article ID g4164 2014

[10] Z Xie C He and M-H Zou ldquoAMP-activated protein kinasemodulates cardiac autophagy in diabetic cardiomyopathyrdquoAutophagy vol 7 no 10 pp 1254ndash1255 2011

[11] J Ren ldquoAcetaldehyde and alcoholic cardiomyopathy lessonsfrom the ADH and ALDH2 transgenic modelsrdquo NovartisFoundation Symposium vol 285 pp 69ndash199 2007

[12] J Wang H Wang P Hao et al ldquoInhibition of aldehydedehydrogenase 2 by oxidative stress is associated with cardiacdysfunction in diabetic ratsrdquoMolecular Medicine vol 17 no 3-4 pp 172ndash179 2011

[13] Y Zhang S A Babcock NHu J RMaris HWang and J RenldquoMitochondrial aldehyde dehydrogenase (ALDH2) protectsagainst streptozotocin-induced diabetic cardiomyopathy roleof GSK3120573 and mitochondrial functionrdquo BMCMedicine vol 10article 40 2012

[14] H-J Wang P-F Kang W-J Wu et al ldquoChanges in cardiacmitochondrial aldehyde dehydrogenase 2 activity in relationto oxidative stress and inflammatory injury in diabetic ratsrdquoMolecular Medicine Reports vol 8 no 2 pp 686ndash690 2013


[15] Y Pan Y Wang Y Zhao et al ldquoInhibition of JNK phospho-rylation by a novel curcumin analog prevents high glucose-induced inflammation and apoptosis in cardiomyocytes and thedevelopment of diabetic cardiomyopathyrdquo Diabetes vol 63 no10 pp 3497ndash3511 2014

[16] C-J Li L Lv H Li and D-M Yu ldquoCardiac fibrosis anddysfunction in experimental diabetic cardiomyopathy are ame-liorated by alpha-lipoic acidrdquo Cardiovascular Diabetology vol11 article 73 2012

[17] H Zhou Y-J Li MWang et al ldquoInvolvement of RhoAROCKin myocardial fibrosis in a rat model of type 2 diabetesrdquo ActaPharmacologica Sinica vol 32 no 8 pp 999ndash1008 2011

[18] H Kaneto T-AMatsuoka N Katakami et al ldquoOxidative stressand the JNK pathway are involved in the development of type1 and type 2 diabetesrdquo Current Molecular Medicine vol 7 no 7pp 674ndash686 2007

[19] H Kaneto G Xu N Fujii S Kim S Bonner-Weir and GC Weir ldquoInvolvement of c-Jun N-terminal kinase in oxidativestress-mediated suppression of insulin gene expressionrdquo Journalof Biological Chemistry vol 277 no 33 pp 30010ndash30018 2002

[20] Y Yu X-J Jia Q-F Zong et al ldquoRemote ischemic postcondi-tioning protects the heart by upregulating ALDH2 expressionlevels through the PI3KAkt signaling pathwayrdquo MolecularMedicine Reports vol 10 no 1 pp 536ndash542 2014

[21] J Ren and J R Sowers ldquoApplication of a novel curcumin analogin the management of diabetic cardiomyopathyrdquo Diabetes vol63 no 10 pp 3166ndash3168 2014

[22] U Bildirici D Ural E Acar A Agacdiken and E Ural ldquoDiag-nostic value of poor R-wave progression in electrocardiogramsfor diabetic cardiomyopathy in type 2 diabetic patientsrdquoClinicalCardiology vol 33 no 9 pp 559ndash564 2010

[23] Y Kayama U Raaz A Jagger et al ldquoDiabetic cardiovasculardisease induced by oxidative stressrdquo International Journal ofMolecular Sciences vol 16 no 10 pp 25234ndash25263 2015

[24] I Baik N H Cho S H Kim B-GHan andC Shin ldquoGenome-wide association studies identify genetic loci related to alcoholconsumption in Korean menrdquoThe American Journal of ClinicalNutrition vol 93 no 4 pp 809ndash816 2011

[25] T Flensborg-Madsen J Knop E L Mortensen U Beckerand M Groslashnbaeligk ldquoAmount of alcohol consumption and riskof developing alcoholism in men and womenrdquo Alcohol andAlcoholism vol 42 no 5 pp 442ndash447 2007

[26] T Flensborg-Madsen U Becker M Groslashnbaeligk J Knop L Sherand E L Mortensen ldquoAlcohol consumption and later risk ofhospitalization with psychiatric disorders Prospective CohortStudyrdquo Psychiatry Research vol 187 no 1-2 pp 214ndash219 2011

[27] S Deutscher H E Rockette and V Krishnaswami ldquoEvalua-tion of habitual excessive alcohol consumption on myocardialinfarction risk in coronary disease patientsrdquo American HeartJournal vol 108 no 4 part 1 pp 988ndash995 1984

[28] V G Athyros E N Liberopoulos D P Mikhailidis et alldquoAssociation of drinking pattern and alcohol beverage typewith the prevalence of metabolic syndrome diabetes coronaryheart disease stroke and peripheral arterial disease in aMediterranean cohortrdquo Angiology vol 58 no 6 pp 689ndash6972008

[29] H-Z Zhou J S Karliner and M O Gray ldquoModerate alcoholconsumption induces sustained cardiac protection by activatingPKC-120576 and Aktrdquo American Journal of PhysiologymdashHeart andCirculatory Physiology vol 283 no 1 pp H165ndashH174 2002

[30] A Sun Y Zou P Wang et al ldquoMitochondrial aldehydedehydrogenase 2 plays protective roles in heart failure after

myocardial infarction via suppression of the cytosolic JNKp53pathway in micerdquo Journal of the American Heart Associationvol 3 no 5 Article ID e000779 2014

[31] Y Yu P-F Kang H-H Li et al ldquoEffects of activation of ALDH2by ethanol on the expression of JNK in kidney of diabetic ratsrdquoZhongguo Ying Yong Sheng Li Xue Za Zhi vol 30 no 3 pp 270ndash273 2014

[32] G Pearson F Robinson T Beers Gibson et al ldquoMitogen-activated protein (MAP) kinase pathways regulation and phys-iological functionsrdquo Endocrine Reviews vol 22 no 2 pp 153ndash183 2001

[33] T Omura M Yoshiyama R Matsumoto et al ldquoRole of c-JunNH2-terminal kinase in G-protein-coupled receptor agonist-induced cardiac plasminogen activator inhibitor-1 expressionrdquoJournal of Molecular and Cellular Cardiology vol 38 no 4 pp583ndash592 2005

[34] G-H Hao X-L Niu D-F Gao J Wei and N-P Wang ldquoAgo-nists at PPAR-120574 suppress angiotensin II-induced production ofplasminogen activator inhibitor-1 and extracellularmatrix in ratcardiac fibroblastsrdquoBritish Journal of Pharmacology vol 153 no7 pp 1409ndash1419 2008

[35] H Kaneto and T-A Matsuoka ldquoInvolvement of oxidativestress in suppression of insulin biosynthesis under diabeticconditionsrdquo International Journal of Molecular Sciences vol 13no 10 pp 13680ndash13690 2012

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


dehydrogenase-2 (ALDH2) is a key enzyme which plays animportant role in the metabolism of acetaldehyde and othertoxic aldehydes [11] Preclinical studies in rats also suggestthat ALDH2 activity may affect diabetic pathology [12]Furthermore in transgenic mice overexpression of ALDH2has been shown to be protective against streptozotocin-induced diabetic cardiomyopathy [13] Our previous resultshad indicated that ALDH2 expression was further decreasedaccompanying the development of diabetes Meanwhile weinvestigated the effects of low-dose ethanol feeding in diabeticrats which could promote myocardial protection throughactivation of ALDH2 expression And more we also foundthat upregulation of ALDH2 played a protective effect inmyocardial ischemia and reperfusion injury and diabetescardiomyopathy ALDH2 may be an endogenous cardiacprotective factor in myocardial injury [2 8 14]

Several biological mechanisms have been proposed toexplain that myocardial fibrosis is one of the main pathologi-cal changes of diabetic cardiomyopathy Studies have foundC-JUN N-terminal kinase (JNK) signaling pathway playeda key role in the process of myocardial fibrosis [15ndash17]Evidence had shown that activation of the JNK pathway wasinvolved in the progression of diabetes induced myocardialfibrosis and that such a pathway could be a therapeutictarget for diabetic heart injury and cardiomyopathy [1819] However whether the JNK pathway is involved in thecardioprotective effect of low-dose ethanol on diabetic ratshas not been fully elucidated

So in this study we mimic diabetes model by intraperi-toneal injection of streptozotocin (STZ) in combinationwith low-dose ethanol the purpose of the present studyis as follows (1) to investigate the mechanism of low-dose ethanol which alleviates myocardial fibrosis in diabeticcardiomyopathy (2) to clarify whether low-dose ethanolmediated protection is associated with downregulating theJNK signaling pathway This study might shed some light onlow-dose ethanol as an effective therapeutic in the treatmentof diabetic cardiomyopathy

2 Methods

21 Animals Adult male Sprague-Dawley rats (200 to 250 g)were obtained fromBengbuMedical College Animal Admin-istration Center All animal studies were approved by theAnimal Ethics Committee of Bengbu Medical College andperformed in accordance with the ethical standards Therats were fed normal chow and had free access to distilledwater

22 Chemicals and Reagents Streptozotocin (STZ) was pur-chased from Sigma (USA) TRIzol was purchased fromInvitrogen (USA) hydroxyproline (Hp) was purchased fromNanjing Jiancheng Bioengineering Institute (China) Ethanol(EtOH) was purchased from Bengbu New Chemical ReagentFactory (China) 120573-actin antibodies were purchased fromSanta Cruz Biotechnology (USA) rabbit c-Jun N-terminalkinase (JNK) and phosphorylated JNK (p-JNK) were pur-chased from Anbo Biotechnology (USA) Chemilumines-cence reaction (ECL) system was purchased from Millipore

Billerica (USA) All primers were purchased from ShanghaiSangon Biotech (China)

23 Induction of Diabetes and Experimental Protocol Aspreviously described by our laboratory STZ at 55mgkgfreshly dissolved in 01molL sodium citrate buffer (pH 45)was injected intraperitoneally to induce diabetic models inovernight fasted rats [8] All rats were randomly dividedinto four groups normal control group (Con) diabetes at 4weeks group (DM4W) diabetes at 8 weeks group (DM8W)and ethanol + diabetes at 8 weeks group (EtOH + DM8W)respectively (119899 = 6) In Con group rats were fed withstandard rat chow and received an intraperitoneal injection ofthe same volume of citrate buffer for 8 weeks In EtOH + DMgroup DM rats were fed with 25 EtOH in their drinkingwater for oneweek to initiate drinking then it was changed to5 EtOH continuous access through the remaining 7 weeks

24 Hemodynamics Male SD rats were anaesthetized byuse of chloral hydrate (100mgkg) through intraperitonealinjectionThroughout the experiment systolic pressure (SP)diastolic pressure (DP) mean arterial pressure (MAP) andheart rate (HR) were determined by invasive hemodynamicevaluation methods for 30min At the end of the experi-mental period hearts were excised rapidly placed in ice-coldKrebs-Henseleit (K-H) buffer and weighed and the ratio ofheart weightbody weight (HB) was calculated

25 Detection of Hydroxyproline Content Heart tissue(100mg) was homogenized in ice-cold K-H buffer Thesupernatant was collected after centrifugation for 20min(2000 rpm) The protein concentration was measured bythe Bradford method Hydroxyproline (Hp) content wasdetected according to the instruction manual

26 The Content of Collagen Detection by Masson-StainingLeft ventricular tissue obtained from all groups was stainedwith Massonrsquos trichrome for the quantification of collagenThe histological sections were taken in 10 neutral formalin-fixed dehydrated paraffin embedded sections Then thesamples underwent the dehydration of gradient ethanolneutral resin embedding and Masson trichrome stainingThe collagen fibers were stained blue and cardiomyocyteswere stained redMyocardial collagenwas quantified at a finalmagnification of 200xwith a polarizedmicroscope connectedto a video camera Myocardial collagen volume fraction(CVF) was analyzed using Image Pro analysis softwareexpressed as the mean percentage of collagen area to the totalarea of eachmicroscopic field Five visions under microscopeof each sample were randomly chosen and the average ofthem was taken for analysis [17]

27 Detection of ALDH2 mRNA by Real-Time PCR TotalRNA was extracted from the left anterior myocardium usingTRIzol according to the manufacturerrsquos instructions TotalRNA (3 120583g) was reversely transcribed to cDNA and PCRwas performed by a routine method The primers used wereas follows for ALDH2 forward 5-GTG TTC GGA GAC


















3 Results








(a) (b)

(c) (d)


p-JN

KJN

K120573-actin

54kD46kD

54kD46kD

43kD

lowastlowast

lowast



00

05

10

15

20

p-JN

KJN

K













compared with DM8W


4 Discussion











Competing Interests




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































3 Results








(a) (b)

(c) (d)


p-JN

KJN

K120573-actin

54kD46kD

54kD46kD

43kD

lowastlowast

lowast



00

05

10

15

20

p-JN

KJN

K













compared with DM8W


4 Discussion











Competing Interests




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (b)

(c) (d)


p-JN

KJN

K120573-actin

54kD46kD

54kD46kD

43kD

lowastlowast

lowast



00

05

10

15

20

p-JN

KJN

K













compared with DM8W


4 Discussion











Competing Interests




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























compared with DM8W


4 Discussion











Competing Interests




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















Competing Interests




Acknowledgments


References











































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Research Article The Protective Effect of Low-Dose Ethanol...

Documents

Transcript of Research Article The Protective Effect of Low-Dose Ethanol...