resveratrol ameliorates experimental alcoholic liver...

Research ArticleResveratrol Ameliorates Experimental Alcoholic Liver Disease byModulating Oxidative Stress

He Peiyuan1 Hou Zhiping2 Song Chengjun2 Wang Chunqing1

Li Bingqing1 andMustapha Umar Imam3

1Department of Gastroenterology The Affiliated Hospital of Chengde Medical University Chengde Hebei 067000 China2Department of Pathology Chengde Medical University Chengde Hebei 067000 China3Precision Nutrition Innovation Center School of Public Health Zhengzhou University Zhengzhou 450001 China

Correspondence should be addressed to Hou Zhiping zhipinghou163com and Mustapha Umar Imam mustyimamgmailcom

Received 30 June 2017 Revised 8 November 2017 Accepted 28 November 2017 Published 31 December 2017

Academic Editor Eman Al-Sayed

Copyright copy 2017 He Peiyuan 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

The aim of this study was to investigate the hepatoprotective effects of resveratrol in alcoholic liver disease (ALD) Alcoholwas administered to healthy female rats starting from 6 (vv) and gradually increased to 20 (vv) by the fifth week After 16weeks of intervention liver enzymes (aspartate aminotransferase [AST] and alanine aminotransferase [ALT]) were analyzed usinga chemistry analyzer while hepatic antioxidant enzymes oxidative stress markers and caspase 3 activity were assessed usingELISA kits Furthermore hepatic CYP2E1 protein levels and mRNA levels of antioxidant and inflammation-related genes weredetermined using western blotting and RT-PCR respectively The results showed that resveratrol significantly attenuated alcohol-induced elevation of liver enzymes and improved hepatic antioxidant enzymes Resveratrol also attenuated alcohol-inducedCYP2E1increase oxidative stress and apoptosis (caspase 3 activity)Moreover genes associatedwith oxidative stress and inflammationwereregulated by resveratrol supplementation Taken together the results suggested that resveratrol alleviated ALD through regulationof oxidative stress apoptosis and inflammation which was mediated at the transcriptional level The data suggests that resveratrolis a promising natural therapeutic agent against chronic ALD

1 Introduction

Alcoholic liver disease (ALD) often results from bingeoverconsumption of alcohol Epidemiological investigationshave shown that in the developed countries of Europe andAmerica liver diseases account for a significant cause ofmorbidity and mortality [1 2] ALD is becoming a globalproblemwith increasing cases in the developing countries [3]ALD encompasses fatty liver alcoholic hepatitis and chronichepatitis with liver fibrosis which are caused by the toxiceffects of the byproducts of alcoholmetabolism [4 5] Clinicaltesting has demonstrated that ALD patients normally haveprotein andor combined protein-calorie malnutrition [6]On the other hand rats fed with corn oil and ethanol showedaccelerated liver injury because of the polyunsaturated fattyacids intake [7] Accordingly risk factors for the developmentof ALD including excessive alcohol intake high fat diet andcalorie malnutrition have been adopted in the rat model

Oxidative stress and inflammation are central to thedevelopment and progression of ALD [8 9] often leadingto apoptotic cell death as a result of accumulating metabolicend-products of alcohol Oxidative stress through an imbal-ance of prooxidant and antioxidant proteins results in oxida-tive damage including lipid peroxidation [5] Inflammationon the other hand is often a secondary factor that furtherpromotes hepatic damage [10] Despite the huge burden ofthe problem at present there is a lack of effective therapeuticsto protect against ALD Moreover available pharmaceuticalagents are costly and may have side effects For this reasoninterest has heightened in natural products as sources of costeffective and safer alternative therapies for ALD

Resveratrol is a polyphenol existing in natural productsespecially in grape seed It is a nutraceutical withwide rangingpotential therapeutic actions including antioxidant [11] anti-inflammation [12] cardioprotective [13] and anticancer [14]activities in vitro and in vivo Additionally results of animal

HindawiEvidence-Based Complementary and Alternative MedicineVolume 2017 Article ID 4287890 10 pageshttpsdoiorg10115520174287890

2 Evidence-Based Complementary and Alternative Medicine

experimentation and clinical trial in nonalcoholic fatty liverdisease setting have suggested that resveratrol could reducehepatic fat accumulation partly through antioxidant andanti-inflammatory actions [15 16] Furthermore nonalco-holic liver disease shares overlapping mechanisms with ALDsuggesting that resveratrol may also be effective on ALDMoreover resveratrol has been shown to attenuate subacuteALD via regulation of inflammation and hepatosteatosis [1718] There is still need however to determine the effect ofresveratrol on chronic ALD and the underlying mechanismsinvolved Thus in this study we explored the effects ofresveratrol supplementation against liver damage caused bychronic alcohol intake with limited high fat diet

2 Materials and Methods

21 Materials Rat superoxide dismutase (SOD) and catalase(CAT) enzyme-linked immunosorbent assay (ELISA) kitswere purchased from Cell Biolabs (INC USA) Glutathioneperoxidase (GPx) and caspase 3 colorimetric assay kits werepurchased from Beyotime (Jiangsu China) Rat chow waspurchased fromSpecialty Feeds (Glen ForrestWAAustralia)and other solvents of analytical grade were purchased fromMerck (Darmstadt Germany)

22 Animals Diets and Experimental Design Thirty-sixfemale Wistar rats weighing approximately 200 g were usedfor the animal experimentation at the Tianjin Medical Uni-versity (Tianjin China) All protocols for animal experi-mentation and maintenance were conducted in accordancewith ChengdeMedical University Animal Ethics CommitteeThe animals were kept in individual cages under controlledtemperature (25 plusmn 2∘C) and humidity and a 12 h lightdarkcycle Animals were acclimatized for 2 weeks on standardrat chow ad libitum and free access to water After that ratswere randomly distributed into three groups of nine animalseach control group maintained on high fat diet alcoholicliver fibrosis group maintained on high fat diet and freeaccess to alcohol resveratrol group maintained on high fatdiet alcohol and resveratrol (250mgkg BWday) [19 20]The high fat diet fed to the alcohol and resveratrol groupswas based on our previous publication [21] and contained85 standard rat chow plus 15 corn oil The ethanol wasintroduced as 6 (vv) ethanol-containing water for oneweek and gradually increased to 9 (vv) for three days12 (vv) for two weeks and finally 20 (vv) from the fifthweek onwards Body weights were monitored weekly andfood intake was monitored daily The alcohol and resveratrolgroups were fed with isocaloric high fat diet for 16 weeksBefore sacrifice the rats were fasted for 12 hours and bloodwas collected and stored at minus80∘C At sacrifice portions ofthe liver tissue were snap-frozen in liquid nitrogen or fixed in10 neutral formalin for further analyses

23 Serum Biochemical Assays Serum aspartate aminotrans-ferase (AST) and alanine aminotransferase (ALT) analyseswere performed at several times during the study using bloodcollected via cardiac puncture after a 12 h fast Analyses wereperformed using analytical kits (Biosino Bio-Technology

and Science Inc Beijing China) based on manufacturerrsquosinstructions on the Hitachi-High-Tech instrument (HitachiHigh-Technologies Co Ltd Shanghai China)

24 Morphological Observation For light microscopy thedissected livers were fixed in 10 (vv) formalin saline andsubsequently processed accordingly Samples were finallystained with hematoxylin and eosin To evaluate the liverchanges samples were observed at times200 magnification andthe scale and degrees of liver injury were assessed by twopathologists For transmission electron microscopy 1mmcubes of rat liver were fixed for 4 h at 0∘C in 25 glutaralde-hyde in 01M sodium cacodylate buffer (pH 7sdot4) washedovernight and postfixed in 2 (wv) osmium tetroxideThe tissue was then stained with 2 (wv) uranyl acetateethanol dehydrated and finally embedded in resin Sectionswere poststained with lead citrate and observed under thetransmission electron microscope (Hitachi H-7650)

25 SOD CAT and GPx Antioxidant Activities SOD CATand GPx activities of the liver were analyzed using commer-cial ELISA kits based on manufacturerrsquos instructions Finallythe absorbances were read at the appropriate wavelengthson the Synergy H1 Hybrid Multimode Microplate Reader(BioTek Winooski VT US) and the results were calculatedfrom the corresponding standard curves (SOD 119910 = 0319119909 minus0126 1199032 = 0998 CAT 119910 = 0279119909 minus 0300 1199032 = 0995 GPx119910 = 0246119909 minus 0107 1199032 = 0996)

26 Thiobarbituric Acid Reactive Substances (TBARS) AssayLiver samples were homogenized in PBS (50mg50 120583l PBS)and added to a mixture of 025N HCl 15 TCA and 0375TBA The samples were then incubated at 100∘C for 10minafter which they were centrifuged at 3000 rpm for 15minFinally the absorbance of supernatants was read at 540 nmusing the Synergy H1 Hybrid Multimode Microplate Reader(BioTek Winooski VT US) Tetramethoxypropane (TMP)was used as the standard (119910 = 0115119909 minus 0266 1199032 = 0983)

27 CYP2E1 Western Blotting Liver samples were homog-enized in radio-immune precipitation assay buffer (RIPA)with protease inhibitors The homogenate was centrifuged at10000119892 for 20min at 4∘C Then 30mg protein determinedby the bicinchoninic acid (BCA) protein assay kit wasloaded per well onto a 10 resolving gel and 4 stackinggel Proteins were transferred to polyvinylidene difluoride(PVDF) membranes and incubated with primary antibodyCYP2E1 (1 500 dilution Abnova Taipei Taiwan) at 4∘Covernight The horseradish peroxidase- (HRP-) conjugatedsecondary antibody (Abnova Taipei Taiwan)was then addedfor 1 h at room temperature The membrane was strippedonce for 20min with stripping buffer and then reprobedwith firstsecondary antibody as described above for 120573-actin(Sigma St Louis MO USA) which was used as a loadingcontrol Bands were visualized using 331015840-diaminobenzidine(DAB) kit (Nacalai Tesque Inc Kyoto Japan) The relativeintensities of the immunoreactive bands were captured usinga Molecular Imager ChemiDoc XRS + System (Bio-RadHercules CA) and quantified with Quantity One Analysis

Evidence-Based Complementary and Alternative Medicine 3

Table 1 Body weights and food intake in alcoholic liver disease rat model after 16 weeks of intervention

Rat groups Control group Alcohol group Resveratrol groupBody weight at beginning (g) 18889 plusmn 1224 18545 plusmn 321 19021 plusmn 631Body weight at end of 4th week (g) 20734 plusmn 1355 20495 plusmn 1450 20285 plusmn 1218Body weight at end of 8th week (g) 23240 plusmn 1745 22191 plusmn 1870 22265 plusmn 1438Body weight at end of 12th week (g) 25586 plusmn 2674 22974 plusmn 2387a 23211 plusmn 48a

Body weight at end of 16th week (g) 2986 plusmn 3591 21785 plusmn 2516ab 25497 plusmn 2499a

Total food intake (Kcalkgday) 15583 plusmn 856 6456 plusmn 443 7221 plusmn 566Total alcohol intake (gkgday) NA 823 plusmn 214 789 plusmn 331Values are mean plusmn SD 119899 = 9 Control group received standard rat chow for 16 weeks alcohol group gained high fat diet and alcohol liquid resveratrol groupreceived high fat diet and alcohol liquid with 250mgkg BWday resveratrol a119875 lt 005 versus normal control group b119875 lt 051 versus alcohol group

AA A A

A A B A A B

1st day 28th day 56th day 84th day 112th day

ControlAlcoholResveratrol

0102030405060708090

100

ALT

(UL

)

(a)


A A A AB A B A

1st day 28th day 56th day 84th day 112th day0

50100150200250300350

AST

(UL

)

(b)

Figure 1 Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of the rats during chronic alcoholadministration for 16 weeks with resveratrol Control group received standard rat chow for 16 weeks alcohol group received high fat diet andalcohol resveratrol group received high fat diet and alcohol with 250mgkg BWday resveratrol Data represent mean plusmn SD of 5 individualvalues A119875 lt 005 versus alcohol group B119875 lt 005 versus control group

Software Version 464 (Bio-Rad Hercules CA) The resultswere expressed as the ratio of protein to 120573-actin

28 Liver Caspase 3 Activity Caspase 3 Activity Assay Kitwas used to quantify hepatic caspase 3 activity according tomanufacturerrsquos instructions (Beyotime Jiangsu China) Theabsorbances were read at 405 nm on the Synergy H1 HybridMultimode Microplate Reader (BioTek Winooski VT US)

29 Quantitative RT-PCR The total RNAwas extracted fromrat hepatic tissue using the total RNA extraction kit (San-gon Biotech Shanghai China) while reverse transcriptionwas performed using the TaqMan Reverse TranscriptionKit according to the manufacturerrsquos instructions Primersequences (Table 2) were designed on the National Center forBiotechnology Information (NCBI) website and purchasedfrom Sangon Biotech (Shanghai China) Quantitative PCRwas performed using SYBR Green PCRMaster Mix (QiagenInc Valencia CA) in a Thermal Cycler (Bioer TechnologyGermany) Furthermore the results were normalized to beta-actin expression and analyzed based on the manufacturerrsquosinstructions

210 Statistical Analysis Data were analyzed by one-wayANOVA with Tukeyrsquos HSD test 119879 test Pearson correlationand linear regression analysis using the Statistical Analysis

System (SAS Institute Cary NC USA) Results are expressedas the mean and SEM 119875 le 005 was taken as the level ofstatistical significance

3 Result

31 Ethanol Intake Food Intake and Body Weight As can berecalled ALD often results from alcohol overconsumptionAlcohol metabolism byproducts can propagate liver dis-ease through steatohepatitis fibrosis cirrhosis liver failureandor hepatocellular carcinoma [10] At the beginning ofthe intervention no significant differences in weights wereobserved among all the groups (Table 1) At the end of 12weeks of intervention however the control group was sig-nificantly different from the alcohol and resveratrol groupswhile after 16 weeks the body weights of the resveratrolgroup were increased and significantly different compared tothe alcohol group although they were still lower than thoseof the control group In the ALD-inducing period there wasno significant difference in food and alcohol intake betweenthe alcohol and resveratrol groupsThe total food and alcoholintake are shown in Table 1

32 Liver Enzymes There were no significant differences inthe ALT and AST among all groups at the beginning ofthe study (Figure 1 ALT 119875 = 04543 AST 119875 = 02694)


Table 2 Names accession number and primer sequences used in the study

Gene name Accession number Forward sequence Reverse sequenceKan(r)lowastlowast

Beta-actinlowast NM 031144 AGGTGACACTATAGAATAGGCATCCTGACCCTGAAGTA

GTACGACTCACTATAGGGAAGACGCAGGATGGCATGAG

SOD1 NM 017050 AGGTGACACTATAGAATATCAATATGGGGACAATACAC

GTACGACTCACTATAGGGATACTTTCTTCATTTCCACCTT

SOD2 NM 017051 AGGTGACACTATAGAATATGTATGAAAGTGCTCAAGAT

GTACGACTCACTATAGGGAGCCCTCTTGTGAGTATAAGT

SOD3 NM 012880 AGGTGACACTATAGAATATCGAACTACTTTATGCCC

GTACGACTCACTATAGGGAGAAGACAAACGAGGTCTCTA

CAT NM 012520 AGGTGACACTATAGAATAACTGCAAGTTCCATTACAAG

GTACGACTCACTATAGGGAGTTCAACTTCAGCAAAATAAT

GPx NM 001039848 AGGTGACACTATAGAATAGTGCCAGAGCCGGGG

GTACGACTCACTATAGGGAGGTCTTGCCTCACTGGGA

CYP2E1 NM 031543 AGGTGACACTATAGAATAAGAGGGAATTGGATC

GTACGACTCACTATAGGGATGCCTGGGATCAGGTTA

FasL NM 010177 AGGTGACACTATAGAATAGTGCCCCGGAATTGGG

GTACGACTCACTATAGGGAGGGCTCAATTCCCAA

TNF-120572 NM 001024447 AGGTGACACTATAGAATAGCCAAGGGGTTGGGAA

GTACGACTCACTATAGGGAGGGACTTTGGGGGAAA

lowastHousekeeping gene Underlined sequences are left and right universal left and right sequences (tags) lowastlowastInternal control supplied by Beckman Coulter Inc(Miami FL USA) as part of the GeXP kit RT conditions were 48∘C for 1 min 37∘C for 5 min 42∘C for 60min 95∘C for 5min and then held at 4∘C PCRconditions were initial denaturation at 95∘C for 10min followed by two-step cycles of 94∘C for 30 sec and 55∘C for 30 sec ending in a single extension cycleof 68∘C for 1min IDE insulin degrading enzyme LRP low density lipoprotein receptor-related protein 1 SOD superoxide dismutase CAT catalase PSENpresenilin APP amyloid precursor protein Gapdh glyceraldehyde-3-phosphate dehydrogenase Kan(r) kanamycin resistance

At the end of the fourth week of intervention howeverthe alcohol group showed elevated ALT and AST levels incomparison with the other two groups (ALT 119875 lt 00001AST 119875 = 00004) which progressively increased until theend of the intervention period suggesting ongoing damage tothe liver The liver enzymes (AST and ALT) in the resveratrolsupplementation group were significantly lower than in thealcohol group (119875 lt 005) after the fourth week

33 Morphological Changes Morphological changes in theliver are shown in Figure 2 The control group showedliver cells in normal state with clear cellular boundaryrounded and centrally located nuclear membrane centrallobular vein and radially arranged and clear sinusoids withno dilatation or congestion (Figure 2(a)(A)) The alcoholgroup however had ballooning of hepatocytes at the acinarzone III with signs of necrosis (Figure 2(a)(B) shown asarrow) dark double-nucleus hepatocytes and lymphocyteand neutrophil infiltration There were also signs of acinarperivascular fibrosis evidenced by the large numbers of bun-dles of collagen fibers and a wide range of bridging fibrosis(Figure 2(a)(B) shown as arrow) In some areas steatosis andinflammation were also visible (Figure 2(a)(B)) Interestinglythe resveratrol group had no bundles of collagen fibers andshowed attenuation of the alcohol-induced changes althoughrounded fat droplets were still visible in the liver tissue(Figure 2(a)(C))

No fatty infiltration and bundles of collagen or otherabnormal changes were seen in the control group under TEM(Figure 2(b)(A)) In the alcohol group the Disse space was

filled with bundles of collagen while the microvilli werereduced and the mitochondria were spherical There werealso fewer and shorter mitochondrial cristae and the sur-faces of the endoplasmic reticulum were smooth and cysticBundles of collagen between intercellular spaces and roundedlipid droplets were also visible (Figure 2(b)(B)) Notwith-standing the resveratrol group significantly attenuatedalcohol-induced hepatocellular damage (Figure 2(b)(C))Accordingly no deformation of mitochondrial and collagenstructures was observed although few rounded lipid dropletswere observed

34 Hepatic Antioxidant Capacity The levels of SOD enzymeactivity were assessed in hepatic homogenate As shownin Figure 3 at the end of the intervention the alcoholgroup had significantly increased SOD level (119875 lt 00001Figure 3(a)) which was attenuated by resveratrol supplemen-tation although it was not as low as that of the control group(119875 = 00067) The alcohol group had significantly lowerlevels of CAT andGPx activities in comparisonwith the othergroups (CAT 119875 lt 00001 Figure 3(b) GPx 119875 lt 00001 Fig-ure 3(c)) Interestingly the resveratrol-treated group had sig-nificantly higher activities of both enzymes (CAT119875 lt 00001GPx 119875 = 00001) Additionally the ratio of SODCAT+GPxin the resveratrol group was lower than that of the alcoholgroup (119875 lt 00001) although it was not as low as thatof the control group (119875 lt 00001)

35 Hepatic Oxidative Damage The hepatic malondialde-hyde (MDA) level was estimated as a measure of oxidative


Normal group Alcohol group Resveratrol

(B)(A) (C)

(a)


(B)(A) (C)

(b)

Figure 2 Histological study in the different groups ((A) control (B) alcohol and (C) resveratrol) (a) Hematoxylin and eosin staining (lightmicroscope) of liver tissue times200 (b) Transmission electron microscopy of liver tissue times15000 Groups are the same as Figure 1

damage to the liver As described in Figure 4 the alcoholgroup had higher levels of MDA (119875 lt 00001) in comparisonwith the other groups which were not significantly differentbetween each other (119875 = 00613)

36 CYP2E1 Protein Expression The alcohol group hadsignificantly higher CYP2E1 protein expression in the livercompared to the control and resveratrol groups (119875 lt00001 Figure 5) Conversely the resveratrol group had lowerCYP2E1 protein expression in comparison with the alcoholgroup (119875 lt 00001)

37 Hepatic Caspase 3 Activity Cleaved caspase 3 was upreg-ulated in the alcohol group in comparison with the othergroups (119875 lt 00001 Figure 6) Resveratrol supplementationdecreased cleaved caspase 3 activity in comparison with thealcohol group (119875 lt 0001) although it was not as low as thatof the control group

38 mRNA Levels of Hepatic Antioxidant and Inflammation-Related Genes Figure 7(a) shows the effects of the inter-ventions on the expression of antioxidant genes (SOD1SOD2 SOD3 CAT and GPx) In the present study alcoholupregulated SOD genes (SOD1 119875 lt 00001 SOD2 119875 lt00001 SOD3 119875 lt 00001) and decreased the expression

of the CAT and GPx genes (CAT 119875 lt 00001 GPx 119875 lt00001) Interestingly resveratrol supplementation attenu-ated the alcohol-inducedmRNA changes (SOD1 119875 = 00004SOD2 119875 lt 00001 SOD3 119875 lt 00001 CAT 119875 = 00003GPx 119875 lt 00001) Additionally significant differences wereobserved inmRNA levels of SOD andGPx genes between thecontrol and resveratrol groups (119875 = 00003 and 119875 = 00073resp) Moreover the mRNA levels of inflammation-relatedgenes (CYP2E1 FasL and TNF-120572) were significantly higherin the alcohol group in comparison with the other groups(CAP2E1 119875 lt 00001 FasL 119875 lt 00001 TNF-120572 119875 lt00001) although the levels in the control group were signif-icantly lower than in the resveratrol group except for TNF-120572mRNA expression (119875 = 01286)

4 Discussion

In the present study ALD was established using chronicalcohol administrationwith high fat diet consumption whichis known to induce hepatic steatosis inflammation andhepatocyte ballooning similar to the features of human ALD[22] Accordingly signs of incipient fibrosis worsening ofliver function and antioxidant status and increased inflam-matory markers and caspase 3 expression were indicative ofsuccessful induction of ALD in the alcohol group


Control ResveratrolAlcoholGroups

A

A B

0

2

4

6

8

10

12

14

16SO

D ac

tivity

(Um

g pr

otei

n)

(a)

A

A B

Control Resveratrol

CAT

activ

ity (U

mg

prot

ein)

AlcoholGroups

0

1

2

3

4

5

6

7

8

9

10

(b)


A A

0

1

2

3

4

5

6

GPx

activ

ity (U

mg

prot

ein)

(c)


002040608

112141618

SOD

[CA

４+

Px]

(d)

Figure 3 Antioxidant enzymes following chronic alcohol administration for 16 weeks with or without resveratrol supplementation (a) liversuperoxide dismutase (SOD) levels (b) liver catalase (CAT) levels (c) glutathione peroxidase (GPx) activity (d) SOD[CAT + GPx] levelGroups are the same as Figure 1 Data represent mean plusmn SD of 5 individual values A119875 lt 005 versus alcohol group B119875 lt 005 versus controlgroup

aa


0

05

1

15

2

25

MD

A co

nten

t (nm

olL

)

Figure 4 Hepatic malondialdehyde (MDA) at the end of chronic alcohol administration for 16 weeks with resveratrol Groups are the sameas Figure 1 Data represent mean plusmn SD of 5 individual values a119875 lt 005 versus alcohol group


CYP2E1

Control Alcohol Resveratrol

-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)

Figure 5 Hepatic CYP2E1 protein expression of rats at the end ofchronic alcohol administration after 16 weeks (a) Representativewestern blot assay and (b) relative optical density in hepatichomogenate Groups are the same as Figure 1 Data representmeanplusmnSD of 5 individual values A119875 lt 005 versus alcohol group B119875 lt 005versus control group

a

a b

Control Alcohol ResveratrolGroups

0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)

Figure 6 Hepatic caspase 3 protein expression of rats at the end ofchronic alcohol administration after 16 weeks Groups are the sameas Figure 1 Data represent mean plusmn SD of 5 individual values a119875 lt005 versus alcohol group b119875 lt 005 versus control group

The effects of resveratrol onALDwere assessed includingmechanistic bases for such effects In the past the protec-tive role of resveratrol against liver damage has been pre-dominantly attributed to antioxidant and anti-inflammatoryactivities However both inflammation and oxidative stressare largely secondary effects of liver damage Therefore wesought to establish whether resveratrol exerted any directeffect on the major enzymes in the alcohol metabolismpathway The elimination of alcohol is primarily dependent

on the oxidation of alcohol and its metabolic byproducts byCYP2E1 which produces toxic byproducts like hydroxyethylsuperoxide anion and hydroxyl radicals [23 24] Interest-ingly we observed that resveratrol could attenuate CYP2E1activity and protein levels via western blotting assay Thepresent findings appear to be consistent with the benefitsof resveratrol on suppression of CYP2E1 observed in otherliver damage disease models because it is also involved in themetabolism of other toxins in addition to alcohol [25]

On the basis of observations that ethanol administrationyields increased amounts of free radicals and hydrogen per-oxide one would logically predict a compensatory increasein enzyme activity to overcome oxidative stress Moreoveroxidative stress is a well-established factor in the progressionof liver damage due to alcohol intake and a high fat dietIn ALD the byproducts of dysregulated alcohol and lipidmetabolism result in the excessive production of free radicalswhich leads to oxidative stress [26 27] ALDhas been demon-strated to be more severe in mice lacking specific antioxidantenzymes [28 29] underpinning the central role of oxidativedamage in ALD and the importance of antioxidants inattenuating such effects Additionally chronic alcohol admin-istration typically reduces the antioxidant enzyme activitiesas indicated in the present study by the reduced SOD CATand GPx activities Insufficient antioxidant activity allowsfree radicals to accumulate and bind to unsaturated fattyacids in cell membranes which causes lipid peroxidation[30] Kessova et al [31] reported that the induction ofsteatosis after chronic alcohol administration is more severein CuZn SOD knockout mice The increased CAT and GPxactivities with lower SOD activity in the resveratrol group incomparisonwith the alcohol control group is suggestive of anantioxidative mechanism in attenuation of alcohol-inducedoxidative damage by resveratrol This is further supportedby the reduced oxidative stress (MDA) and improved liverfunction (AST and ALT) in the resveratrol group There aredivergent views on the significance of the absolute values ofantioxidant enzyme activities as a reflection of their effects[32 33] in which case SODCAT + GPx ratio has been used[34 35] The data again suggested that resveratrol modulatedantioxidant enzymes which may have been the basis forthe improved attenuation of oxidative stress and ultimatelyALD Additionally increased caspase 3 activation in thealcohol group may have been mediated through increasedoxidative stress Moreover oxidative stress and apoptosis arereported to share common pathways and the end result ofoxidative stress in cells is often apoptotic death [36 37] Thisis supported by the potentiation of antioxidant enzymes andattenuation of both MDA and caspase 3 by resveratrol in thisstudy

It is well documented that transcriptional regulation ofmetabolic processes precedes any biochemical changes andin the present study transcriptomic regulation of antioxidantand inflammation-related genes were evaluated The datashowed that the effects of resveratrolmay have beenmediatedat the transcriptional level Accordingly the effects of resver-atrol may be long lasting since transcriptional regulation isreported to produce longer lasting effects than biochemicalchanges alone [38 39] Such nutrigenomic regulation may


AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)

Figure 7 Hepatic mRNA levels of SOD1 SOD2 SOD3 CAT GPx1 CYP2E1 FasL and TNF-120572 genes at the end of chronic alcoholadministration after 16 weeks Groups are the same as Figure 1 Data represent mean plusmn SD of 5 individual values A119875 lt 005 versus alcoholgroup B119875 lt 005 versus control group

mean that resveratrolmay be amore effective alternative thanpalliative pharmaceuticals presently in use for ALD

The findings from this study mirror those of previousstudies that have shown the beneficial effects of resveratrolon ALD [15 16] However our data adds to the existingknowledge on how resveratrol protects the liver againstalcohol damage Moreover the duration of our study (16weeks) mimics that of chronic alcohol exposure more thanthe previous studies which lasted between 4 and 6 weeksSimilarly while the previous studies have shown that resver-atrol attenuates ALD through regulating inflammation andhepatic lipid metabolism our data shows that multiplemechanisms including the modulation of oxidative stressinflammation and apoptosis may be involved in the longterm effect of resveratrol against ALD Another study [19]had demonstrated that resveratrol could attenuate alcohol-induced oxidative stress similar to what was observed in thisstudy However the dose used in that study (5 gkg bodyweight) calls the reliability of the findings into questionCrowell et al [20] have demonstrated that doses of resveratrolabove 300mgkg in rodents are toxic suggesting that theresults from the study byKasdallah-Grissa et al [19]may havebeen influenced by toxicity from resveratrol

Taken together the present findings indicate that resvera-trol may have alleviated ALD through alleviation of oxidativestress apoptosis and inflammation which were mediatedat the transcriptional level Moreover resveratrol affectedmultiple processes and pathways suggesting its effectivenessin alleviating ALD

5 Conclusions

These results demonstrated that resveratrol (250mgkg BWday)effectively attenuated alcohol-induced hepatotoxicity infemaleWistar ratsThe hepatoprotective effects of resveratrolwere mediated by modulating multiple pathways includingoxidative stress inflammation and apoptosis The evidenceobtained in this study suggested the effectiveness of res-veratrol as a natural therapy for ALD

Abbreviations

ALD Alcoholic liver diseaseSOD Superoxide dismutaseCAT CatalaseGPx Glutathione peroxidaseFasL Fas ligandCYP2E1 Cytochrome P450 2E1TNF Tumor necrosis factorALT Alanine aminotransferaseAST Aspartate aminotransferase

Conflicts of Interest

The authors declare no conflicts of interest

Authorsrsquo Contributions

Study design was performed by He Peiyuan Hou Zhipingand Li Bingqing Primer design for gene expression studywas performed by He Peiyuan and Hou Zhiping Conductof experimental parts was performed by He Peiyuan WangChunqing Hou Zhiping Song Chengjun and Li BingqingData analyses and preparation ofmanuscript were performedby Hou Zhiping He Peiyuan and Mustapha Umar ImamReview of manuscript and final approval for submissionwere performed by Hou Zhiping Li Bingqing andMustaphaUmar Imam

Acknowledgments

The present work was supported by grant from Healthand Family Planning Commission of Hebei (no 20110179)and Hebei Province Clinical Talent Project (361008) Theauthors thank all staff of Morphology Experimental Centerof Chengde Medical University for their help during thestudy


References

[1] Y Xie B Feng Y Gao and L Wei ldquoCharacteristics of alcoholicliver disease and predictive factors for mortality of patientswith alcoholic cirrhosisrdquo Hepatobiliary amp Pancreatic DiseasesInternational vol 12 no 6 pp 594ndash601 2013

[2] F Nassir and J A Ibdah ldquoRole of mitochondria in alcoholicliver diseaserdquo World Journal of Gastroenterology vol 20 no 9pp 2136ndash2142 2014

[3] R Williams ldquoGlobal challenges in liver diseaserdquo Hepatologyvol 44 no 3 pp 521ndash526 2006

[4] R S OrsquoShea S Dasarathy and A J McCullough ldquoAlcoholicliver diseaserdquo Hepatology vol 51 no 1 pp 307ndash328 2010

[5] H-I Lee R A McGregor M-S Choi et al ldquoLow doses ofcurcumin protect alcohol-induced liver damage by modulationof the alcohol metabolic pathway CYP2E1 and AMPKrdquo LifeSciences vol 93 no 18-19 pp 693ndash699 2013

[6] C L Mendenhall S Anderson R E Weesner S J Goldbergand K A Crolic ldquoProtein-calorie malnutrition associated withalcoholic hepatitis Veterans administration cooperative studygroup on alcoholic hepatitisrdquoAmerican Journal ofMedicine vol76 no 2 pp 211ndash222 1984

[7] R Polavarapu D R Spitz J E Sim et al ldquoIncreased lipid perox-idation and impaired antioxidant enzyme function is associatedwith pathological liver injury in experimental alcoholic liverdisease in rats fed diets high in corn oil and fish oilrdquoHepatologyvol 27 no 5 pp 1317ndash1323 1998

[8] E Grasselli A D Compalati A Voci et al ldquoAltered oxidativestressantioxidant status in blood of alcoholic subjects is associ-ated with alcoholic liver diseaserdquoDrug and Alcohol Dependencevol 143 no 1 pp 112ndash119 2014

[9] P Zhang X Qiang M Zhang et al ldquoDemethyleneberberine anatural mitochondria-targeted antioxidant inhibits mitochon-drial dysfunction oxidative stress and steatosis in alcoholicliver disease mouse modelrdquo The Journal of Pharmacology andExperimental Therapeutics vol 352 no 1 article no A19 pp139ndash147 2015

[10] D Seth P S Haber W-K Syn A M Diehl and C PDay ldquoPathogenesis of alcohol-induced liver disease classicalconcepts and recent advancesrdquo Journal of Gastroenterology andHepatology vol 26 no 7 pp 1089ndash1105 2011

[11] A Bishayee A S Darvesh T Politis and R McGory ldquoResver-atrol and liver disease frombench to bedside and communityrdquoLiver International vol 30 no 8 pp 1103ndash1114 2010

[12] S Das and D K Das ldquoAnti-inflammatory responses of resver-atrolrdquo Inflammation amp Allergy-Drug Targets vol 6 no 3 pp168ndash173 2007

[13] G Petrovski N Gurusamy and D K Das ldquoResveratrol incardiovascular health and diseaserdquo Annals of the New YorkAcademy of Sciences vol 1215 no 1 pp 22ndash33 2011

[14] B B Aggarwal A Bhardwaj R S Aggarwal N P Seeram SShishodia and Y Takada ldquoRole of resveratrol in prevention andtherapy of cancer preclinical and clinical studiesrdquo AnticancerReseach vol 24 no 5 pp 2783ndash2840 2004

[15] G Musso ldquoObeticholic acid and resveratrol in nonalcoholicfatty liver disease All that is gold does not glitter not all thosewho wander are lostrdquo Hepatology vol 61 no 6 pp 2104ndash21062015

[16] J Shang L-L Chen F-X Xiao H Sun H-C Ding and HXiao ldquoResveratrol improves non-alcoholic fatty liver disease byactivating AMP-activated protein kinaserdquo Acta PharmacologicaSinica vol 29 no 6 pp 698ndash706 2008

[17] J M Ajmo X Liang C Q Rogers B Pennock and M YouldquoResveratrol alleviates alcoholic fatty liver in micerdquo AmericanJournal of Physiology-Gastrointestinal and Liver Physiology vol295 no 4 pp G833ndashG842 2008

[18] L Bujanda M Garcıa-Barcina V Gutierrez-de Juan et alldquoEffect of resveratrol on alcohol-induced mortality and liverlesions in micerdquo BMC Gastroenterology vol 6 article 35 2006

[19] A Kasdallah-Grissa BMornagui E Aouani et al ldquoResveratrola red wine polyphenol attenuates ethanol-induced oxidativestress in rat liverrdquo Life Sciences vol 80 no 11 pp 1033ndash10392007

[20] J A Crowell P J Korytko R L Morrissey T D Booth and BS Levine ldquoResveratrol-associated renal toxicityrdquo ToxicologicalSciences vol 82 no 2 pp 614ndash619 2004

[21] P-Y He Z-P Hou S-M Gao M-J Wang L-X Ma and B-Q Li ldquoEstablishment of a rat model of alcoholic liver fibrosisand detection of hepatic expression of smad7 TGF-120573rdquo WorldChinese Journal of Digestology vol 22 no 19 pp 2692ndash27022014

[22] S Ceccarelli V Nobili and A Alisi ldquoToll-like receptor-mediated signaling cascade as a regulator of the inflammationnetwork during alcoholic liver diseaserdquo World Journal of Gas-troenterology vol 20 no 44 pp 16443ndash16451 2014

[23] T-M Leung and N Nieto ldquoCYP2E1 and oxidant stress inalcoholic and non-alcoholic fatty liver diseaserdquo Journal ofHepatology vol 58 no 2 pp 395ndash398 2013

[24] M G Neuman S Malnick Y Maor et al ldquoAlcoholic liverdisease Clinical and translational researchrdquo Experimental andMolecular Pathology vol 99 no 3 pp 596ndash610 2015

[25] G Aljomah S S Baker W Liu et al ldquoInduction of CYP2E1 innon-alcoholic fatty liver diseasesrdquo Experimental and MolecularPathology vol 99 no 3 pp 677ndash681 2015

[26] K-H Han N Hashimoto and M Fukushima ldquoRelationshipsamong alcoholic liver disease antioxidants and antioxidantenzymesrdquo World Journal of Gastroenterology vol 22 no 1 pp37ndash49 2016

[27] B-J Park Y-J Lee andH-R Lee ldquoChronic liver inflammationClinical implications beyond alcoholic liver diseaserdquo WorldJournal of Gastroenterology vol 20 no 9 pp 2168ndash2175 2014

[28] G Liu Y Zhang C Liu et al ldquoLuteolin alleviates alcoholic liverdisease induced by chronic and binge ethanol feeding in micerdquoJournal of Nutrition vol 144 no 7 pp 1009ndash1015 2014

[29] W Okiyama N Tanaka T Nakajima et al ldquoPolyenephos-phatidylcholine prevents alcoholic liver disease in PPAR120572-null mice through attenuation of increases in oxidative stressrdquoJournal of Hepatology vol 50 no 6 pp 1236ndash1246 2009

[30] P Velu A Vijayalakshmi P Iyappan and D Indumathi ldquoEval-uation of antioxidant and stabilizing lipid peroxidation natureof Solanum xanthocarpum leaves in experimentally diethylni-trosamine induced hepatocellular carcinogenesisrdquo Biomedicineamp Pharmacotherapy vol 84 pp 430ndash437 2016

[31] I G Kessova Y-S Ho S Thung and A I CederbaumldquoAlcohol-Induced Liver Injury in Mice Lacking Cu Zn-Superoxide DismutaserdquoHepatology vol 38 no 5 pp 1136ndash11452003

[32] E Ceni T Mello and A Galli ldquoPathogenesis of alcoholicliver disease role of oxidative metabolismrdquo World Journal ofGastroenterology vol 20 no 47 pp 17756ndash17772 2014

[33] O Tsedensodnom A M Vacaru D L Howarth C Yin andK C Sadler ldquoEthanol metabolism and oxidative stress arerequired for unfolded protein response activation and steatosis


in zebrafish with alcoholic liver diseaserdquo Disease Models ampMechanisms vol 6 no 5 pp 1213ndash1226 2013

[34] F A D R Lıvero G G Martins J E Queiroz Telles etal ldquoHydroethanolic extract of Baccharis trimera amelioratesalcoholic fatty liver disease in micerdquo Chemico-Biological Inter-actions vol 260 pp 22ndash32 2016

[35] W Zhang R Hong and T Tian ldquoSilymarinrsquos protective effectsand possible mechanisms on alcoholic fatty liver for ratsrdquoBiomolecules ampTherapeutics vol 21 no 4 pp 264ndash269 2013

[36] L Xie Z Wang C Li K Yang and Y Liang ldquoProtective effectof nicotinamide adenine dinucleotide (NAD+) against spinalcord ischemiandashreperfusion injury via reducing oxidative stress-induced neuronal apoptosisrdquo Journal of Clinical Neurosciencevol 36 pp 114ndash119 2017

[37] N D Singh A K Sharma P Dwivedi et al ldquoEffect of feedinggraded doses of citrinin on apoptosis and oxidative stress inmale Wistar rats through the F1 generationrdquo Toxicology ampIndustrial Health vol 32 no 3 pp 385ndash397 2016

[38] S Guo Q Yao Z Ke H Chen J Wu and C Liu ldquoResvera-trol attenuates high glucose-induced oxidative stress and car-diomyocyte apoptosis through AMPKrdquoMolecular and CellularEndocrinology vol 412 pp 85ndash94 2015

[39] D Babu G Leclercq V Goossens et al ldquoAntioxidant potentialof CORM-A1 and resveratrol during TNF-acycloheximide-induced oxidative stress and apoptosis in murine intestinalepithelial MODE-K cellsrdquo Toxicology and Applied Pharmacol-ogy vol 288 no 2 pp 161ndash178 2015

Submit your manuscripts athttpswwwhindawicom

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


experimentation and clinical trial in nonalcoholic fatty liverdisease setting have suggested that resveratrol could reducehepatic fat accumulation partly through antioxidant andanti-inflammatory actions [15 16] Furthermore nonalco-holic liver disease shares overlapping mechanisms with ALDsuggesting that resveratrol may also be effective on ALDMoreover resveratrol has been shown to attenuate subacuteALD via regulation of inflammation and hepatosteatosis [1718] There is still need however to determine the effect ofresveratrol on chronic ALD and the underlying mechanismsinvolved Thus in this study we explored the effects ofresveratrol supplementation against liver damage caused bychronic alcohol intake with limited high fat diet

2 Materials and Methods

21 Materials Rat superoxide dismutase (SOD) and catalase(CAT) enzyme-linked immunosorbent assay (ELISA) kitswere purchased from Cell Biolabs (INC USA) Glutathioneperoxidase (GPx) and caspase 3 colorimetric assay kits werepurchased from Beyotime (Jiangsu China) Rat chow waspurchased fromSpecialty Feeds (Glen ForrestWAAustralia)and other solvents of analytical grade were purchased fromMerck (Darmstadt Germany)

22 Animals Diets and Experimental Design Thirty-sixfemale Wistar rats weighing approximately 200 g were usedfor the animal experimentation at the Tianjin Medical Uni-versity (Tianjin China) All protocols for animal experi-mentation and maintenance were conducted in accordancewith ChengdeMedical University Animal Ethics CommitteeThe animals were kept in individual cages under controlledtemperature (25 plusmn 2∘C) and humidity and a 12 h lightdarkcycle Animals were acclimatized for 2 weeks on standardrat chow ad libitum and free access to water After that ratswere randomly distributed into three groups of nine animalseach control group maintained on high fat diet alcoholicliver fibrosis group maintained on high fat diet and freeaccess to alcohol resveratrol group maintained on high fatdiet alcohol and resveratrol (250mgkg BWday) [19 20]The high fat diet fed to the alcohol and resveratrol groupswas based on our previous publication [21] and contained85 standard rat chow plus 15 corn oil The ethanol wasintroduced as 6 (vv) ethanol-containing water for oneweek and gradually increased to 9 (vv) for three days12 (vv) for two weeks and finally 20 (vv) from the fifthweek onwards Body weights were monitored weekly andfood intake was monitored daily The alcohol and resveratrolgroups were fed with isocaloric high fat diet for 16 weeksBefore sacrifice the rats were fasted for 12 hours and bloodwas collected and stored at minus80∘C At sacrifice portions ofthe liver tissue were snap-frozen in liquid nitrogen or fixed in10 neutral formalin for further analyses

23 Serum Biochemical Assays Serum aspartate aminotrans-ferase (AST) and alanine aminotransferase (ALT) analyseswere performed at several times during the study using bloodcollected via cardiac puncture after a 12 h fast Analyses wereperformed using analytical kits (Biosino Bio-Technology

and Science Inc Beijing China) based on manufacturerrsquosinstructions on the Hitachi-High-Tech instrument (HitachiHigh-Technologies Co Ltd Shanghai China)

24 Morphological Observation For light microscopy thedissected livers were fixed in 10 (vv) formalin saline andsubsequently processed accordingly Samples were finallystained with hematoxylin and eosin To evaluate the liverchanges samples were observed at times200 magnification andthe scale and degrees of liver injury were assessed by twopathologists For transmission electron microscopy 1mmcubes of rat liver were fixed for 4 h at 0∘C in 25 glutaralde-hyde in 01M sodium cacodylate buffer (pH 7sdot4) washedovernight and postfixed in 2 (wv) osmium tetroxideThe tissue was then stained with 2 (wv) uranyl acetateethanol dehydrated and finally embedded in resin Sectionswere poststained with lead citrate and observed under thetransmission electron microscope (Hitachi H-7650)

25 SOD CAT and GPx Antioxidant Activities SOD CATand GPx activities of the liver were analyzed using commer-cial ELISA kits based on manufacturerrsquos instructions Finallythe absorbances were read at the appropriate wavelengthson the Synergy H1 Hybrid Multimode Microplate Reader(BioTek Winooski VT US) and the results were calculatedfrom the corresponding standard curves (SOD 119910 = 0319119909 minus0126 1199032 = 0998 CAT 119910 = 0279119909 minus 0300 1199032 = 0995 GPx119910 = 0246119909 minus 0107 1199032 = 0996)

26 Thiobarbituric Acid Reactive Substances (TBARS) AssayLiver samples were homogenized in PBS (50mg50 120583l PBS)and added to a mixture of 025N HCl 15 TCA and 0375TBA The samples were then incubated at 100∘C for 10minafter which they were centrifuged at 3000 rpm for 15minFinally the absorbance of supernatants was read at 540 nmusing the Synergy H1 Hybrid Multimode Microplate Reader(BioTek Winooski VT US) Tetramethoxypropane (TMP)was used as the standard (119910 = 0115119909 minus 0266 1199032 = 0983)

27 CYP2E1 Western Blotting Liver samples were homog-enized in radio-immune precipitation assay buffer (RIPA)with protease inhibitors The homogenate was centrifuged at10000119892 for 20min at 4∘C Then 30mg protein determinedby the bicinchoninic acid (BCA) protein assay kit wasloaded per well onto a 10 resolving gel and 4 stackinggel Proteins were transferred to polyvinylidene difluoride(PVDF) membranes and incubated with primary antibodyCYP2E1 (1 500 dilution Abnova Taipei Taiwan) at 4∘Covernight The horseradish peroxidase- (HRP-) conjugatedsecondary antibody (Abnova Taipei Taiwan)was then addedfor 1 h at room temperature The membrane was strippedonce for 20min with stripping buffer and then reprobedwith firstsecondary antibody as described above for 120573-actin(Sigma St Louis MO USA) which was used as a loadingcontrol Bands were visualized using 331015840-diaminobenzidine(DAB) kit (Nacalai Tesque Inc Kyoto Japan) The relativeintensities of the immunoreactive bands were captured usinga Molecular Imager ChemiDoc XRS + System (Bio-RadHercules CA) and quantified with Quantity One Analysis






AA A A

A A B A A B



0102030405060708090

100

ALT

(UL

)

(a)


A A A AB A B A


50100150200250300350

AST

(UL

)

(b)







3 Result

































(B)(A) (C)

(a)


(B)(A) (C)

(b)







4 Discussion




A

A B

0

2

4

6

8

10

12

14

16SO

D ac

tivity

(Um

g pr

otei

n)

(a)

A

A B

Control Resveratrol

CAT

activ

ity (U

mg

prot

ein)

AlcoholGroups

0

1

2

3

4

5

6

7

8

9

10

(b)


A A

0

1

2

3

4

5

6

GPx

activ

ity (U

mg

prot

ein)

(c)


002040608

112141618

SOD

[CA

４+

Px]

(d)


aa


0

05

1

15

2

25

MD

A co

nten

t (nm

olL

)



CYP2E1


-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)


a

a b


0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)







AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















AA A A

A A B A A B



0102030405060708090

100

ALT

(UL

)

(a)


A A A AB A B A


50100150200250300350

AST

(UL

)

(b)







3 Result

































(B)(A) (C)

(a)


(B)(A) (C)

(b)







4 Discussion




A

A B

0

2

4

6

8

10

12

14

16SO

D ac

tivity

(Um

g pr

otei

n)

(a)

A

A B

Control Resveratrol

CAT

activ

ity (U

mg

prot

ein)

AlcoholGroups

0

1

2

3

4

5

6

7

8

9

10

(b)


A A

0

1

2

3

4

5

6

GPx

activ

ity (U

mg

prot

ein)

(c)


002040608

112141618

SOD

[CA

４+

Px]

(d)


aa


0

05

1

15

2

25

MD

A co

nten

t (nm

olL

)



CYP2E1


-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)


a

a b


0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)







AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of














































(B)(A) (C)

(a)


(B)(A) (C)

(b)







4 Discussion




A

A B

0

2

4

6

8

10

12

14

16SO

D ac

tivity

(Um

g pr

otei

n)

(a)

A

A B

Control Resveratrol

CAT

activ

ity (U

mg

prot

ein)

AlcoholGroups

0

1

2

3

4

5

6

7

8

9

10

(b)


A A

0

1

2

3

4

5

6

GPx

activ

ity (U

mg

prot

ein)

(c)


002040608

112141618

SOD

[CA

４+

Px]

(d)


aa


0

05

1

15

2

25

MD

A co

nten

t (nm

olL

)



CYP2E1


-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)


a

a b


0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)







AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















A

A B

0

2

4

6

8

10

12

14

16SO

D ac

tivity

(Um

g pr

otei

n)

(a)

A

A B

Control Resveratrol

CAT

activ

ity (U

mg

prot

ein)

AlcoholGroups

0

1

2

3

4

5

6

7

8

9

10

(b)


A A

0

1

2

3

4

5

6

GPx

activ

ity (U

mg

prot

ein)

(c)


002040608

112141618

SOD

[CA

４+

Px]

(d)


aa


0

05

1

15

2

25

MD

A co

nten

t (nm

olL

)



CYP2E1


-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)


a

a b


0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)







AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CYP2E1


-Actin

(a)

AA B


0

05

1

15

2

25

Rela

tive o

ptic

al d

ensit

y

(b)


a

a b


0

200

400

600

800

1000

1200

1400

Casp

ase 3

activ

ity (p

gm

l)







AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















AA B

A AA AA A B

A A B


0123456

Relat

ive e

xpre

ssio

n

SOD1SOD2SOD3

CATGPx1

(a)

AA B

AA BA B


0123456789

Relat

ive e

xpre

ssio

n

CAP2E1FasLTNF-

(b)





5 Conclusions


Abbreviations






Acknowledgments



References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















References















































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















resveratrol ameliorates experimental alcoholic liver...

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