Research Article Vitamin A Supplementation Alleviates...

Research ArticleVitamin A Supplementation Alleviates Extrahepatic CholestasisLiver Injury through Nrf2 Activation

Guiyang Wang Peng Xiu Fu Li Cheng Xin and Kewei Li

Department of Biliary-Pancreatic Surgery Ren Ji Hospital School of Medicine Shanghai Jiao Tong University Shanghai 200127 China

Correspondence should be addressed to Kewei Li keweipig126com

Received 19 January 2014 Revised 12 June 2014 Accepted 17 June 2014 Published 9 July 2014

Academic Editor Felipe Dal-Pizzol

Copyright copy 2014 Guiyang Wang et alThis 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

Aim To investigate the role of vitamin A in liver damage induced by bile duct ligation (BDL) in ratsMethods Thirty male Wistarrats were randomly divided into three groups SHAM group BDL group and BDL + VitA group The concentrations of retinoland retinyl palmitate in the liver were analyzed using HPLC and liver function was evaluated by the level of TBIL ALT AST andALP in serum Hepatic oxidative status was estimated by measuring T-SOD CAT GSH MDA and AOPP Nrf2 expression wasassessed using immunohistochemistry and western blotting and EMSA was performed to determine Nrf2 DNA-binding activityThe expression of the downstream factors such as Ho1 and Nqo1 was also examined using immunohistochemistry and westernblotting assays Results Vitamin A treatment restored levels of retinoids in liver improved liver function alleviated oxidative stressand facilitated the translocation of Nrf2 to the nucleus in the experimental obstructive jaundice Vitamin A was also found toincrease the expression of Nrf2 downstream proteins such as Ho1 and Nqo1 Conclusion Vitamin A was here found to amelioratecholestatic liver injury This effect may be related to the activation of Nrf2ARE pathway in bile duct ligation rats

1 Introduction

The mechanisms of hepatic damage caused by cholestasisare complicated Recent studies have shown that reactiveoxygen species (ROS) play amajor role in the pathogenesis ofcholestasis [1ndash3] Excessive ROS generated during cholestasiscan cause oxidative damage to biological macromoleculesand triggermass consumption of antioxidants and aggravatedoxidative damage [2 4] The administration of antioxidantshas been shown to exert beneficial effects in the preventionof cholestasis liver injury [5ndash9] In this way maintaining thebalance between oxidation and antioxidant systems could bean important therapeutic target for cholestasis

The nuclear factor-erythroid 2-related factor 2 (Nrf2)has been thought to exert an important role in the cellulardefense against oxidative stress [10 11] Heme oxygenase 1(Ho1) and NADPH-quinone oxidoreductase 1 (Nqo1) arephase II detoxification enzymes They are regulated by theNrf2 [12ndash14] Induction of Nrf2ARE-dependent antioxidant

enzymes and phase II detoxification is one of the majorcellular defense mechanisms against oxidative stresses [11 1516]

Vitamin A is a micronutrient essential to a variety ofphysiological processes In healthy vertebrates the majorityof vitamin A is stored in the liver mainly in the form ofretinyl palmitate [17] Disruption of vitamin A homeostasiswas associated with many diseases particularly liver diseases[18] It has been reported that the concentrations of vitaminA are lower in cholestatic patients even during early stagesof the disease and vitamin A supplementation was foundto provide protection against hepatic damage in a BDL ratmodel induced by bile duct ligation [19ndash22] Vitamin A hasalso been reported to play its protective role by inducing Nrf2upregulation in neuronal cells [23] However whether vita-min A has similar effects on liver tissue is largely unknownThe aim of this study was to explore the impact of vitamin Asupplementation on the prevention of cholestatic liver injuryin rats and determine whether vitamin A administration

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2014 Article ID 273692 10 pageshttpdxdoiorg1011552014273692

2 Oxidative Medicine and Cellular Longevity

activates Nrf2ARE pathway to exert protective effects onliver damage induced by bile duct ligation

2 Materials and Methods

21 Animals Male Wistar rats (5 weeks old weighing 190ndash220 g) were obtained from the Shanghai Slac ExperimentalAnimal Centre (Shanghai China) The rats were housed incages under controlled temperature (24plusmn2∘C) and humidity(50 plusmn 10) conditions with 12 h lightdark cycles and weregiven a standard diet and water ad libitum The experimentswere carried out according to the guidelines set forth by theEthics Committee of Renji Hospital (SYXY(hu)2011-0121)

22 Experimental Design and Sample Collection The ratswere randomly assigned to three groups of 10 animals eachSHAM-operated group (SHAM) bile duct ligated group(BDL) and BDL treated with vitamin A group (BDL + VitA)Rats in the SHAM-operated group were subjected to a 15 cmupper midline abdominal incision and the common bileduct was isolated from the surrounding tissues The ratsin the BDL group underwent the same procedure and thecommon bile duct was doubly ligated with a 4ndash0 silk sutureand transected between the two ligatures Vitamin A-treatedrats received daily administration of retinyl acetate (DonghaiPharmaceutical Co Ltd Shanghai China) namely esters ofretinol 100000 IUkg dissolved in bean oil by gavage butthose in SHAM group and BDL group were only given anequal volume of bean oil The dose of vitamin A given torats was selected based on information from previous reports[21] After two weeks rats were killed to collect blood andlivers Plasma was immediately separated by centrifugation(3000 rmin for 10min at room temperature) and stored atminus20∘CThe same portion of each liver was removed and fixedin 10neutral formalin and the remaining portionwas storedat minus80∘C for further analysis

23 Liver Retinol and Retinyl Palmitate Extraction andQuantification by HPLC The sample extraction processreferenced to previous methodology [24ndash26] Retinol andretinyl palmitate in liver homogenates were extracted withethanol and hexaneThen 150mg liver samples were homog-enized in 2mL dehydrated ethanol containing 01 buty-lated hydroxytoluene (BHT) After complete homogeniza-tion an equal volume (2mL) of n-hexane was added tothe homogenateethanol-mixture and vortexed for 1minAfter centrifugation the hexane layer was transferred to acentrifugal tube and evaporated under N

2at about 40∘C

in a water bath The extraction manipulation was repeatedtwice and the merged hexane-phases were evaporated untildryness The residue was redissolved in 100 methanol andwas filtered with strainer (022120583m) An aliquot of 20 uLwas injected into the HPLC systems which consisted of abinaryHPLCpump (Waters1525) and ultra-violet absorbancedetector (Waters2487) for analysis Retinoids were separated

on a reverse phase column (C18 Hypersil ODS2 46mm times200mm 5 um) running at 1mLmin with the mobile phase(100 methanol) and monitored at 325 nm Quantificationwas performed by relating the area of the peak to areasobtained by the analysis of knownquantities of the retinol andretinyl palmitate standards and expressed as 120583gg wet weightQualitative analysis was performed with retention times ofthe chromatographic peaks The retinoids standard curveswere established for quantification of the retinol and retinylpalmitate All extraction manipulations and quantification oftissues were performed under dim light

24 Measurement of TBIL ALT AST and ALP in Serum Theserum levels of total bilirubin (TBIL) alanine transaminase(ALT) aspartate transaminase (AST) and alkaline phos-phatase (ALP) were measured with an automatic analyzer(HITACHI7600-20)

25 Measurement of T-SOD GSH CAT and MDA in LiverHomogenates The content of total superoxide dismutase (T-SOD) glutathione (GSH) catalase (CAT) and malondialde-hyde (MDA) in liver homogenate was detected with reagentskits purchased from Jiancheng Biologic Company (NanjingChina) Manipulations were performed according to themanufacturerrsquos protocols

26 Measurement of Advanced Oxidation Protein ProductsThe concentrations of advanced oxidation protein products(AOPP) were measured by spectrophotometry as describedbyWitko-Sarsat et al [27] Liver tissue samples were homog-enized by 50mM ice-cold Tris-HCl (pH 74) and centrifugedat 5000 g for 10min Tissue supernatants were then dilutedin phosphate buffered saline at a ratio of 1 5 The samplewas mixed with potassium iodide and acetic acid and theabsorbance of the reaction mixture was immediately read at340 nm Chloramine was chosen as a standard and the AOPPconcentrations were expressed as 120583mol per mg protein

27 Histological Examination and ImmunohistochemicalStaining Liver tissues were fixed in 10 neutral bufferedformalin embeddedin paraffin and sectioned The paraffinslices were stained with hematoxylin and eosin (HE) afterthe treatment of deparaffinization and dehydration Forimmunohistochemical analysis hepatic sections were placedin 3 H

2O2for 10min followed by incubation with anti-

Nrf2 (Santa Cruz CA USA) antibody anti-Ho1 (AbcamCambridge UK) and anti-Nqo1 (Sigma St Louis USA) at37∘C for 2 h Yellow and brown staining in the cytoplasm andnucleus were considered positive Five microscopic fieldswere randomly chosen per slice and the number of cells perfield was counted

28Western Blotting Nuclear and cytoplasmic proteins wereextracted with nuclear and cytoplasmic extraction reagents

Oxidative Medicine and Cellular Longevity 3

SHAM

(a)

BDL

(b)

BDL + VitA

(c)

Figure 1 Pathomorphological changes in each group (HE original magnification times200) (a) liver tissue in the SHAM group (b) Bile ductproliferation necrosis and inflammatory cell infiltration were observed in the BDL group (c) Proliferation of bile ducts necrosis andinflammatory cell infiltration in the BDL + vitA group was milder than in the BDL group

kits (Pierce Rockford IL USA) according to the manufac-turerrsquos protocol for Nrf2 Ho1 and Nqo1 detection Proteinconcentrations were measured using BCA protein assayProteins were separated by 10 or 12 SDS-PAGE andthen transferred onto PVDF membranes The transferredmembranes were blocked with TBS-T (10mmolL Tris-HC1150mmolL NaC1 01 Tween-20) containing 5 skim milkfor 1 h at room temperature The membranes were washed inTBS-T (10min times 3) and then the membranes were incubatedovernight at 4∘C with anti-Ho1 (Abcam Cambridge UK)anti-Nqo1 (Sigma St Louis USA) anti-Nrf2 (Santa CruzCA USA) anti-120573-actin (Abmart Shanghai China) and anti-PCNA (Santa Cruz CA USA) in TBS-T The membraneswerewashed three times again inTBS-T themembraneswereincubated with horseradish peroxidase conjugated to anti-rabbit IgG (Weiao Biotech Ltd Shanghai China) overnightat 4∘C The expression of targeted proteins was determinedusing Odyssey machine and optical density of band wasanalyzed by using Image J software

29 Electrophoretic Mobility Shift Assay EMSA was per-formed to determine Nrf2 DNA-binding activity using acommercial kit (Pierce Rockford IL USA) Consensusoligonucleotide probes ofNrf2 (51015840-TGGGGAACCTGTGCT-GAGTCACTGGAG-31015840) were labeled with biotin Nuclearextracts were prepared using a kit (Pierce Rockford ILUSA) Protein concentrations were quantified using a BCAprotein assay Nuclear extract (20 120583g) was incubated with20 ng of biotin-end-labeled target DNA for 30min at roomtemperature in 1 times binding buffer Assays were loaded ontonative 6 polyacrylamide gels preelectrophoresed in 05 timesTris-borate-EDTA (TBE) for 60min and electrophoresed at100V before transfer to a positively charged nylonmembranein 05 times TBE at 100V for 30min The membrane was UVcross-linked and binding activity of Nrf2 to the probe wasdetermined using a chemiluminescent EMSA kit (BeyotimeChina)

210 Statistical Analysis Statistical analysis was performedusing SPSS version 170 software Data are presented as mean

plusmn standard deviation (SD) Analysis of variance Duncanrsquostest and Dunnettrsquos test were used to assess differencesbetween multiple groups 119875 lt 005 was considered statisti-cally significant

3 Results

31 General Observations In the SHAM group all ratssurvived throughout the experiment In the BDLgroup 1 diedfrom duodenal rupture caused by operation on day 2 and 1rat died of abdominal infection due to bile leakage on day 6after operation In BDL + vitA group 1 died from primaryperitonitis on day 8 after operation Jaundice occurred in 3days after surgery and dilatation of the remnant common bileduct proximal to the ligature was observed upon reoperationin bile duct ligated rats

32 Morphological Studies Cholangiocyte hyperplasiaoccurred in BDL rats Necrosis was scattered in the liveraccompanied by inflammatory cells infiltrated especiallyaround the portal areas (Figure 1(b)) However vitaminA supplementation distinctly improved these pathologicalchanges of the liver in BDL + vitA group relative to the BDLgroup (Figure 1(c))

33 Concentrations of Retinol and Retinyl Palmitate in LiverRetinol and retinyl palmitate were eluted for 519 plusmn 008minand 2961 plusmn 029min respectively Levels of retinol andretinyl palmitate in the liver tissue were lower in the BDLgroup (retinol 1365 plusmn 256 120583gg wet weight versus 201 plusmn105 120583gg wet weight 119875 lt 005 retinyl palmitate 11841 plusmn1288 120583gg wet weight versus 4553 plusmn 942 120583gg wet weight119875 lt 005 Figure 2) and were significantly increased aftervitA supplementation (retinol 761 plusmn 201 120583gg wet weightversus 201plusmn105 120583gg wet weight119875 lt 005 retinyl palmitate8328 plusmn 1264 120583gg wet weight versus 4553 plusmn 942 120583gg wetweight 119875 lt 005 Figure 2)

34 Serum Concentrations of TBIL ALT AST and ALP Theserum concentrations of TBIL ALT AST and ALP in BDL


(min)500 1000 1500 2000 2500 3000

(AU

)010

008

006

004

002

000

SHAM

Retin

yl p

alm

itate29

392

Retin

ol5167

(a)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0005

0010

0015

0020

0025

0030BDL

Retin

yl p

alm

itate

Retin

ol

29892

5188

(b)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0010

0020

0030

0040

0050

0060 BDL + VitA

Retin

yl p

alm

itate

Retin

ol

29595

5172

(c)

SHAM

Retinol

Retinyl palmitate

BDL BDL + VitA

a

a

b

b

150

100

50

0(120583

gg

wet

wei

ght)

(d)

Figure 2 HPLC analysis of liver retinol and retinyl palmitate (a)ndash(c) SHAM group (119899 = 10) BDL group (119899 = 8) BDL + vitA group (119899 = 9)Abscissa and ordinate represent the retention time (min) and absorbance (AU) of retinol and retinyl palmitate (d) Data are presented asmean plusmn SD a119875 lt 005 versus SHAM group b119875 lt 005 versus BDL group

group increased visibly compared with those in SHAMgroup(TBIL 26007 plusmn 2832 120583molL versus 392 plusmn 143 120583molL 119875 lt005 ALT 24086 plusmn 5241UL versus 6877 plusmn 1504UL 119875 lt005 AST 69168 plusmn 6545UL versus 13859 plusmn 3840UL 119875 lt005 ALP 102914 plusmn 10840UL versus 33150 plusmn 4071UL119875 lt 005 Figure 3) The serum concentrations of TBILALT AST and ALP were significantly lower in the BDL +vitA group (TBIL 21146 plusmn 1983 120583molL versus 26007 plusmn2832 120583molL 119875 lt 005 ALT 14754 plusmn 2297UL versus24086 plusmn 5241UL 119875 lt 005 AST 37071 plusmn 7530UL versus69168plusmn 6545UL 119875 lt 005 ALP 66960plusmn 6565UL versus102914 plusmn 10840ULUL 119875 lt 005 Figure 3)

35 Levels of T-SOD GSH CAT and MDA in LiverHomogenates T-SOD GSH and CAT were all less active inliver homogenates and the level of MDA was higher in theBDL group than in the SHAM group (119875 lt 005 Table 1)However T-SOD GSH and CAT were more active in liverhomogenates and the level of MDA was lower in the BDL +vitA group than in the BDL group (119875 lt 005 Table 1)

SHAM BDL BDL + VitA

1500

1000

500

0

Live

r fun

ctio

n pa

ram

eter

(UL

)

TBILALT

ASTALP

a

a

aa

b

b

bb

Figure 3 Levels of TBIL ALT AST and ALP in serum A SHAMgroup (119899 = 10) B BDL (119899 = 8) B BDL + vitA group (119899 = 9)Data are presented as mean plusmn SD a119875 lt 005 versus SHAM groupb119875 lt 005 versus BDL group


Table 1 Levels of SOD GSH CAT and MDA in hepatic tissues (mean plusmn SD)

Group 119899 T-SOD (Umgprot) GSH (mggprot) CAT (Umgprot) MDA (nmolmgprot)SHAM 10 22270 plusmn 939 492 plusmn 019 11331 plusmn 968 081 plusmn 011BDL 8 17649 plusmn 1079a 350 plusmn 014a 7146 plusmn 484a 308 plusmn 087a

BDL + vitA 9 20268 plusmn 582b 423 plusmn 032b 8239 plusmn 400b 207 plusmn 014ba119875 lt 005 versus SHAM group b119875 lt 005 versus BDL group

SHAM BDL BDL + VitA0

2

4

6

a

b

(120583m

olm

g pr

otei

n)

Figure 4 Levels of advanced oxidation protein products (AOPP) inliver tissue A SHAM group (119899 = 10) B BDL (119899 = 8) C BDL +vitA group (119899 = 9) All these values are expressed as mean plusmn SDa119875 lt 005 versus SHAM group b

119875 lt 005 versus BDL group

36 Changes in Levels of Advanced Oxidation Protein Productsin the Liver Tissue Liver AOPP levels (120583molmg protein)were significantly higher in the BDL group than in the SHAMgroup (468 plusmn 043 versus 149 plusmn 058 119875 lt 005 Figure 4) butthey decreased after treatment with vitamin A (348 plusmn 038versus 468 plusmn 043 119875 lt 005 Figure 4)

37 Expression of Nrf2 and Downstream Proteins Immuno-histochemical staining and western blotting assays showedthat vitamin A led to Nrf2 accumulation in nucleus in BDLrats treated with vitamin A after 2 weeks (Figure 5 andFigure 6(a)) Statistical analysis also showed there is a signifi-cant difference between Nrf2 levels in cytoplasm of the threegroups (119875 lt 005) EMSA showedNrf2-ARE-binding activityhas been enhanced after BDL and substantially increased aftertreatment with vitamin A (Figure 6(b)) The expression ofantioxidative proteins Ho1 and Nqo1 downstream of Nrf2was also examined Immunohistochemistry and western blotresults indicated that both Ho1 and Nqo1 proteins wereupregulated after BDL (119875 lt 005 Figures 7 8 and 9)Additionally administration of vitamin A for 2 weeks furtherincreased the expression of protein overexpression in theBDLgroup (119875 lt 005 Figures 7ndash9)

4 Discussion

Cholestasis is a reduction in bile flow induced by a varietyof factors It may lead to hepatocellular injury fibrosiscirrhosis and finally liver failure There are few effectivetherapies for this disease [2] Previous studies in BDLmodelsconfirmed that vitamin A administration is effective in therepair of liver injury Murakami et al [21] demonstrated thatenhanced expression of fibrotic markers such as keratinocytegrowth factor alpha-smooth muscle actin and glial fibrillaryacidic protein are diminished by vitamin A administration inBDL rats He et al [19] demonstrated that supplementationof retinoic acid with ursodeoxycholic acid can produceeffects superior to those of ursodeoxycholic acid or retinoicacid treatment alone in BDL rats and that it works byreducing bile duct proliferation necrosis and fibrosis andliver hydroxyproline content and other inflammatory andfibrotic markers Wang et al [20] suggested that vitaminA derivatives such as retinoic acid inhibited liver fibrosisinduced by BDL by reducing the expression of transforminggrowth factor-1205731 connective tissue growth factor and tissueinhibitors of metalloproteinase-1 diminishing the inhibi-tion of tissue inhibitors of metalloproteinase-1 on matrixmetalloproteinase-2 and matrix metalloproteinase-13 thenpromoting the activity of matrix metalloproteinase-2 andmatrix metalloproteinase-13 However most of the reportsfocused on the antifibrotic effect of vitaminA and few reportshave reported the effects of vitamin A on oxidative damagecaused by cholestasis in livers

In the present study the concentration of vitamin A andthe levels of SOD GSH CAT MDA and AOPP in hepatictissues were used to explore the effects of vitamin A on liveroxidative damage induced by BDL These data showed thatvitamin A treatment resulted in a significant decrease inMDA andAOPP and a significant increase in SOD GSH andCAT levels in the livers of BDL rats by replenishing vitamin Aand eventually improving liver functionThis confirmed thatvitamin A supplementation alleviated oxidative stress injuryin livers of BDL rats

The transcription factor Nrf2 is a key regulator of theinduction of detoxifying enzymes and antioxidative stressgenes [28] Under normal conditions Nrf2 is repressed inthe cytoplasm by the actin-binding protein Kelch-like ECHassociating protein-1 (Keap1) Upon oxidative stress Nrf2dissociates from Keap1 and translocates to the nucleus tobind to antioxidant response element (ARE) which is an


SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

Nrf2 (cytoplasm)Nrf2 (nucleus)

BDL BDL + VitA

aa

b

b

0

20

40

60

80

Figure 5 Immunohistochemical staining of liver Nrf2 expression (originalmagnification times400 arrows indicate nucleus-positive cells)MoreNrf2 was concentrated in the nuclei of the BDL group than in the SHAM group and this concentration morphology was more apparent aftertreatment with vitamin A

enhancer element that initiates the transcription of a batteryof genes encoding phase-II enzymes and antioxidant enzymes[12] It has beenreported that Nrf2 activation counteractscholestatic liver injury via stimulation of hepatic defensesystems After BDL Keap1 gene-knockdown mice whichrepresent sustained activation of Nrf2 in the liver showedpronounced increases in the concentrations of detoxifyingenzymes and antioxidative stress genes in the livers butthe number of hepatic parenchymal necrosis and the ROSremained significantly lower than in WT mice [29] For thisreason activation of Nrf2may be a suitable therapeutic targetfor prevention and amelioration of cholestatic liver injuryData collected here showed that vitaminA exerted protectionagainst oxidative stress induced by BDL at least partiallyattributable to translocation of Nrf2 from the cytoplasm tothe nucleus

Both Ho1 and Nqo1 are easily-induced phase-II enzymesregulated by the Keap1Nrf2ARE pathway and the antioxi-dant activity of Ho1 and Nqo1 has been confirmed [30 31]The present study showed that the expression and concen-trations Ho1 and Nqo1 were significantly higher in the BDLgroup than in the control group and were even higher aftervitamin A treatment (119875 lt 005) These results indicated thatvitamin A promoted the activation of Nrf2 and its target Ho1and Nqo1 in liver tissue of BDL rats This may be associatedwith the improvement of hepatic injury induced by oxidative

stress in BDL rats The protective effects of vitamin A mayinvolve multiple mechanisms and more detailed study isneeded to identify the true mechanism

5 Conclusions

In conclusion these results suggest that the concentrationof vitamin A is lower in BDL rats than in control ratsbut is restored after supplementation Vitamin A activatedNrf2ARE signaling and promoted antioxidant mechanismsin BDL rats This was characterized by improvements inliver tissue pathology and liver function lower levels ofperoxidation products and an enhanced antioxidant capac-ity This was consistent with the expression and changesin the concentrations of Nrf2 Ho1 and Nqo1 Vitamin Asupplementation may not only improve vitamin A status butalso exerts a role of Nrf2 activators and alleviates cholestasisliver injury in BDL rats This may open new perspectives fortherapeutic uses of vitamin A in patients with cholestasis

Disclosure

All the authors have no competing interests to declare


(cyt

opla

sm)

BDL + VitA

BDL + VitA BDL + VitABDL

SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)

Figure 6 (a) Western blot results of Nrf2 expression in both the cytoplasm and nucleus from all three groups Vitamin A significantlyincreased the level of Nrf2 in nucleus and decreased the level of Nrf2 in cytoplasm PCNA and 120573-actin were used as protein loading control(b) Gel shift assay showing Nrf2-ARE-binding activity was higher after BDL and much higher after treatment with vitamin A SHAM group(119899 = 10) BDL group (119899 = 8) BDL + vitA group (119899 = 9) Data are presented as mean plusmn SD a

119875 lt 005 versus SHAM group b119875 lt 005 versus

BDL group

Conflict of Interests

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

Authorsrsquo Contribution

Guiyang Wang Peng Xiu and Cheng Xin performed themajority of experiments and wrote the paper Kewei Li and


SHAM BDL BDL + VitA

SHAM(expression of Ho1 protein immunohistochemistry)

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80

Figure 7 Immunohistochemical staining of liver Ho1 expression (original magnification times400 arrows indicate cytoplasm-positive cells)MoreHo1 was expressed in the cytoplasm of the BDL group than in the SHAMgroup and thismorphology wasmore apparent after treatmentwith vitamin A Data are presented as mean plusmn SD a

119875 lt 005 versus SHAM group b119875 lt 005 versus BDL group

SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80

(expression of Ho1 protein immunohistochemistry)

Figure 8 Immunohistochemical staining of liver Nqo1 expression (original magnification times400 arrows indicate cytoplasm-positive cells)More Nqo1 was expressed in the cytopalsm of the BDL group than in the SHAM group and this morphology was more apparent aftertreatment with vitamin A Data are presented as mean plusmn SD a



SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1

BDL BDL + VitASHAM BDL BDL + VitA

a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin

Figure 9 Western blot results of Ho1 and Nqo1 protein expression in the SHAM group (119899 = 10) BDL group (119899 = 8) BDL + vitA group(119899 = 9) Both Ho1 and Nqo1 proteins were upregulated after BDL and vitamin A increased their expression in hepatic tissue further 120573-actinwas used as a loading control Data are presented as mean plusmn SD a


Fu Li provided vital reagents and analytical tools designedthe study revised the paper and provided financial supportfor this work

Acknowledgment

The authors would like to thank Dr Li Sun for technicalsupport

References

[1] M G Roma and E J Sanchez Pozzi ldquoOxidative stress a radicalway to stop making bilerdquo Annals of Hepatology vol 7 no 1 pp16ndash33 2008

[2] B L Copple H Jaeschke and C D Klaassen ldquoOxidative stressand the pathogenesis of cholestasisrdquo Seminars in Liver Diseasevol 30 no 2 pp 195ndash204 2010

[3] S Antherieu P B Azzi J Dumont et al ldquoOxidative stress playsa major role in chlorpromazine-induced cholestasis in humanHepaRG cellsrdquo Hepatology vol 57 no 4 pp 1518ndash1529 2013

[4] K Jung andM Kwak ldquoThe Nrf2 system as a potential target forthe development of indirect antioxidantsrdquoMolecules vol 15 no10 pp 7266ndash7291 2010

[5] V Baron and P Muriel ldquoRole of glutathione lipid peroxidationand antioxidants on acute bile-duct obstruction in the ratrdquoBiochimica et Biophysica Acta vol 1472 no 1-2 pp 173ndash1801999

[6] A R Soylu H Umit A Tezel et al ldquoAntioxidants vitamin E andC attenuate hepatic fibrosis in biliary-obstructed ratsrdquo WorldJournal of Gastroenterology vol 12 no 42 pp 6835ndash6841 2006

[7] Y L Tain C C Chen C T Lee et al ldquoMelatonin regulates L-Arginine transport and NADPH oxidase in young rats with bileduct ligation role of protein kinase Crdquo Pediatric Research vol73 no 4 part 1 pp 395ndash401 2013

[8] M Tokac G Taner S Aydin et al ldquoProtective effects ofcurcumin against oxidative stress parameters andDNA damagein the livers and kidneys of rats with biliary obstructionrdquo Foodand Chemical Toxicology vol 61 pp 28ndash35 2013

[9] M Galicia-Moreno L Favari and P Muriel ldquoAntifibroticand antioxidant effects of N-acetylcysteine in an experimentalcholestatic modelrdquo European Journal of Gastroenterology andHepatology vol 24 no 2 pp 179ndash185 2012

[10] T Nguyen P Nioi and C B Pickett ldquoThe Nrf2-antioxidantresponse element signaling pathway and its activation byoxidative stressrdquo Journal of Biological Chemistry vol 284 no20 pp 13291ndash13295 2009

[11] S K Niture J W Kaspar J Shen and A K Jaiswal ldquoNrf2 sig-naling and cell survivalrdquo Toxicology and Applied Pharmacologyvol 244 no 1 pp 37ndash42 2010

[12] D D Zhang ldquoMechanistic studies of the Nrf2-Keap1 signalingpathwayrdquo Drug Metabolism Reviews vol 38 no 4 pp 769ndash7892006

[13] C Chen D Pung V Leong et al ldquoInduction of detoxifyingenzymes by garlic organosulfur compounds through transcrip-tion factor Nrf2 effect of chemical structure and stress signalsrdquoFree Radical Biology andMedicine vol 37 no 10 pp 1578ndash15902004

[14] W Jeong M Jun and A T Kong ldquoNrf2 a potential moleculartarget for cancer chemoprevention by natural compoundsrdquoAntioxidants and Redox Signaling vol 8 no 1-2 pp 99ndash1062006

[15] B N Das Y Kim and Y Keum ldquoMechanisms of Nrf2keap1-dependent phase II cytoprotective and detoxifying gene expres-sion and potential cellular targets of chemopreventive isothio-cyanatesrdquo Oxidative Medicine and Cellular Longevity vol 2013Article ID 839409 7 pages 2013

[16] JW Kaspar S K Niture and A K Jaiswal ldquoNrf2INrf2 (Keap1)signaling in oxidative stressrdquo Free Radical Biology andMedicinevol 47 no 9 pp 1304ndash1309 2009

[17] W S Blaner S M OrsquoByrne N Wongsiriroj et al ldquoHepaticstellate cell lipid droplets a specialized lipid droplet for retinoidstoragerdquo Biochimica et Biophysica Acta vol 1791 no 6 pp 467ndash473 2009

[18] M Venu E Martin K Saeian and S Gawrieh ldquoHigh preva-lence of vitamin A deficiency and vitamin D deficiency inpatients evaluated for liver transplantationrdquo Liver Transplanta-tion vol 19 no 6 pp 627ndash633 2013


[19] H He A Mennone J L Boyer and S Cai ldquoCombination ofretinoic acid and ursodeoxycholic acid attenuates liver injuryin bile duct-ligated rats and human hepatic cellsrdquo Hepatologyvol 53 no 2 pp 548ndash557 2011

[20] H Wang Z Dan and H Jiang ldquoEffect of all-trans retinoicacid on liver fibrosis induced by common bile duct ligation inratsrdquo Journal of Huazhong University of Science and Technology-Medical Science vol 28 no 5 pp 553ndash557 2008

[21] K Murakami T Kaji R Shimono et al ldquoTherapeutic effectsof vitamin A on experimental cholestatic rats with hepaticfibrosisrdquo Pediatric Surgery International vol 27 no 8 pp 863ndash870 2011

[22] A Floreani A Baragiotta D Martines R Naccarato and ADrsquoOdorico ldquoPlasma antioxidant levels in chronic cholestaticliver diseasesrdquo Alimentary Pharmacology and Therapeutics vol14 no 3 pp 353ndash358 2000

[23] F Zhao T Wu A Lau et al ldquoNrf2 promotes neuronal celldifferentiationrdquo Free Radical Biology and Medicine vol 47 no6 pp 867ndash879 2009

[24] O Carvalho and C Goncalves ldquoRetinol and retinyl palmitatein foetal lung mice sexual dimorphismrdquo Critical Care Researchand Practice vol 2013 Article ID 760305 7 pages 2013

[25] M Rasmussen R Blomhoff P Helgerud L A Solberg T Bergand K R Norum ldquoRetinol and retinyl esters in parenchymaland nonparenchymal rat liver cell fractions after long-termadministration of ethanolrdquo Journal of Lipid Research vol 26 no9 pp 1112ndash1119 1985

[26] M W Schaffer S S Roy S Mukherjee et al ldquoQualitativeand quantitative analysis of retinol retinyl esters tocopherolsand selected carotenoids out of various internal organs fromdifferent species by HPLCrdquoAnalytical Methods vol 2 no 9 pp1320ndash1332 2010

[27] V Witko-Sarsat M Friedlander C Capeillere-Blandin et alldquoAdvanced oxidation protein products as a novel marker ofoxidative stress in uremiardquo Kidney International vol 49 no 5pp 1304ndash1313 1996

[28] Y S Keum ldquoRegulation of Nrf2-mediated phase II detoxifica-tion and anti-oxidant genesrdquo Biomolecules amp Therapeutics vol20 no 2 pp 144ndash151 2012

[29] K Okada J Shoda K Taguchi et al ldquoNrf2 counteractscholestatic liver injury via stimulation of hepatic defense sys-temsrdquo Biochemical and Biophysical Research Communicationsvol 389 no 3 pp 431ndash436 2009

[30] A T Dinkova-Kostova and P Talalay ldquoNAD(P)Hquinoneacceptor oxidoreductase 1 (NQO1) a multifunctional antioxi-dant enzyme and exceptionally versatile cytoprotectorrdquoArchivesof Biochemistry and Biophysics vol 501 no 1 pp 116ndash123 2010

[31] D Martin A I Rojo M Salinas et al ldquoRegulation of hemeoxygenase-1 expression through the phosphatidylinositol 3-kinase Akt pathway and the Nrf2 transcription factor inresponse to the antioxidant phytochemical carnosolrdquoThe Jour-nal of Biological Chemistry vol 279 no 10 pp 8919ndash8929 2004

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



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OphthalmologyJournal of


Diabetes ResearchJournal of



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Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


activates Nrf2ARE pathway to exert protective effects onliver damage induced by bile duct ligation

2 Materials and Methods

21 Animals Male Wistar rats (5 weeks old weighing 190ndash220 g) were obtained from the Shanghai Slac ExperimentalAnimal Centre (Shanghai China) The rats were housed incages under controlled temperature (24plusmn2∘C) and humidity(50 plusmn 10) conditions with 12 h lightdark cycles and weregiven a standard diet and water ad libitum The experimentswere carried out according to the guidelines set forth by theEthics Committee of Renji Hospital (SYXY(hu)2011-0121)

22 Experimental Design and Sample Collection The ratswere randomly assigned to three groups of 10 animals eachSHAM-operated group (SHAM) bile duct ligated group(BDL) and BDL treated with vitamin A group (BDL + VitA)Rats in the SHAM-operated group were subjected to a 15 cmupper midline abdominal incision and the common bileduct was isolated from the surrounding tissues The ratsin the BDL group underwent the same procedure and thecommon bile duct was doubly ligated with a 4ndash0 silk sutureand transected between the two ligatures Vitamin A-treatedrats received daily administration of retinyl acetate (DonghaiPharmaceutical Co Ltd Shanghai China) namely esters ofretinol 100000 IUkg dissolved in bean oil by gavage butthose in SHAM group and BDL group were only given anequal volume of bean oil The dose of vitamin A given torats was selected based on information from previous reports[21] After two weeks rats were killed to collect blood andlivers Plasma was immediately separated by centrifugation(3000 rmin for 10min at room temperature) and stored atminus20∘CThe same portion of each liver was removed and fixedin 10neutral formalin and the remaining portionwas storedat minus80∘C for further analysis

23 Liver Retinol and Retinyl Palmitate Extraction andQuantification by HPLC The sample extraction processreferenced to previous methodology [24ndash26] Retinol andretinyl palmitate in liver homogenates were extracted withethanol and hexaneThen 150mg liver samples were homog-enized in 2mL dehydrated ethanol containing 01 buty-lated hydroxytoluene (BHT) After complete homogeniza-tion an equal volume (2mL) of n-hexane was added tothe homogenateethanol-mixture and vortexed for 1minAfter centrifugation the hexane layer was transferred to acentrifugal tube and evaporated under N

2at about 40∘C

in a water bath The extraction manipulation was repeatedtwice and the merged hexane-phases were evaporated untildryness The residue was redissolved in 100 methanol andwas filtered with strainer (022120583m) An aliquot of 20 uLwas injected into the HPLC systems which consisted of abinaryHPLCpump (Waters1525) and ultra-violet absorbancedetector (Waters2487) for analysis Retinoids were separated

on a reverse phase column (C18 Hypersil ODS2 46mm times200mm 5 um) running at 1mLmin with the mobile phase(100 methanol) and monitored at 325 nm Quantificationwas performed by relating the area of the peak to areasobtained by the analysis of knownquantities of the retinol andretinyl palmitate standards and expressed as 120583gg wet weightQualitative analysis was performed with retention times ofthe chromatographic peaks The retinoids standard curveswere established for quantification of the retinol and retinylpalmitate All extraction manipulations and quantification oftissues were performed under dim light

24 Measurement of TBIL ALT AST and ALP in Serum Theserum levels of total bilirubin (TBIL) alanine transaminase(ALT) aspartate transaminase (AST) and alkaline phos-phatase (ALP) were measured with an automatic analyzer(HITACHI7600-20)

25 Measurement of T-SOD GSH CAT and MDA in LiverHomogenates The content of total superoxide dismutase (T-SOD) glutathione (GSH) catalase (CAT) and malondialde-hyde (MDA) in liver homogenate was detected with reagentskits purchased from Jiancheng Biologic Company (NanjingChina) Manipulations were performed according to themanufacturerrsquos protocols

26 Measurement of Advanced Oxidation Protein ProductsThe concentrations of advanced oxidation protein products(AOPP) were measured by spectrophotometry as describedbyWitko-Sarsat et al [27] Liver tissue samples were homog-enized by 50mM ice-cold Tris-HCl (pH 74) and centrifugedat 5000 g for 10min Tissue supernatants were then dilutedin phosphate buffered saline at a ratio of 1 5 The samplewas mixed with potassium iodide and acetic acid and theabsorbance of the reaction mixture was immediately read at340 nm Chloramine was chosen as a standard and the AOPPconcentrations were expressed as 120583mol per mg protein

27 Histological Examination and ImmunohistochemicalStaining Liver tissues were fixed in 10 neutral bufferedformalin embeddedin paraffin and sectioned The paraffinslices were stained with hematoxylin and eosin (HE) afterthe treatment of deparaffinization and dehydration Forimmunohistochemical analysis hepatic sections were placedin 3 H

2O2for 10min followed by incubation with anti-

Nrf2 (Santa Cruz CA USA) antibody anti-Ho1 (AbcamCambridge UK) and anti-Nqo1 (Sigma St Louis USA) at37∘C for 2 h Yellow and brown staining in the cytoplasm andnucleus were considered positive Five microscopic fieldswere randomly chosen per slice and the number of cells perfield was counted

28Western Blotting Nuclear and cytoplasmic proteins wereextracted with nuclear and cytoplasmic extraction reagents


SHAM

(a)

BDL

(b)

BDL + VitA

(c)






3 Results






(min)500 1000 1500 2000 2500 3000

(AU

)010

008

006

004

002

000

SHAM

Retin

yl p

alm

itate29

392

Retin

ol5167

(a)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0005

0010

0015

0020

0025

0030BDL

Retin

yl p

alm

itate

Retin

ol

29892

5188

(b)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0010

0020

0030

0040

0050

0060 BDL + VitA

Retin

yl p

alm

itate

Retin

ol

29595

5172

(c)

SHAM

Retinol

Retinyl palmitate

BDL BDL + VitA

a

a

b

b

150

100

50

0(120583

gg

wet

wei

ght)

(d)




SHAM BDL BDL + VitA

1500

1000

500

0

Live

r fun

ctio

n pa

ram

eter

(UL

)

TBILALT

ASTALP

a

a

aa

b

b

bb







2

4

6

a

b

(120583m

olm

g pr

otei

n)





4 Discussion





SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()


BDL BDL + VitA

aa

b

b

0

20

40

60

80





5 Conclusions


Disclosure



(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SHAM

(a)

BDL

(b)

BDL + VitA

(c)






3 Results






(min)500 1000 1500 2000 2500 3000

(AU

)010

008

006

004

002

000

SHAM

Retin

yl p

alm

itate29

392

Retin

ol5167

(a)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0005

0010

0015

0020

0025

0030BDL

Retin

yl p

alm

itate

Retin

ol

29892

5188

(b)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0010

0020

0030

0040

0050

0060 BDL + VitA

Retin

yl p

alm

itate

Retin

ol

29595

5172

(c)

SHAM

Retinol

Retinyl palmitate

BDL BDL + VitA

a

a

b

b

150

100

50

0(120583

gg

wet

wei

ght)

(d)




SHAM BDL BDL + VitA

1500

1000

500

0

Live

r fun

ctio

n pa

ram

eter

(UL

)

TBILALT

ASTALP

a

a

aa

b

b

bb







2

4

6

a

b

(120583m

olm

g pr

otei

n)





4 Discussion





SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()


BDL BDL + VitA

aa

b

b

0

20

40

60

80





5 Conclusions


Disclosure



(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(min)500 1000 1500 2000 2500 3000

(AU

)010

008

006

004

002

000

SHAM

Retin

yl p

alm

itate29

392

Retin

ol5167

(a)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0005

0010

0015

0020

0025

0030BDL

Retin

yl p

alm

itate

Retin

ol

29892

5188

(b)

(min)500 1000 3500 4000 4500 5000000 1500 2000 2500 3000

(AU

)

0000

0010

0020

0030

0040

0050

0060 BDL + VitA

Retin

yl p

alm

itate

Retin

ol

29595

5172

(c)

SHAM

Retinol

Retinyl palmitate

BDL BDL + VitA

a

a

b

b

150

100

50

0(120583

gg

wet

wei

ght)

(d)




SHAM BDL BDL + VitA

1500

1000

500

0

Live

r fun

ctio

n pa

ram

eter

(UL

)

TBILALT

ASTALP

a

a

aa

b

b

bb







2

4

6

a

b

(120583m

olm

g pr

otei

n)





4 Discussion





SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()


BDL BDL + VitA

aa

b

b

0

20

40

60

80





5 Conclusions


Disclosure



(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















2

4

6

a

b

(120583m

olm

g pr

otei

n)





4 Discussion





SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()


BDL BDL + VitA

aa

b

b

0

20

40

60

80





5 Conclusions


Disclosure



(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()


BDL BDL + VitA

aa

b

b

0

20

40

60

80





5 Conclusions


Disclosure



(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(cyt

opla

sm)

BDL + VitA


SHAM BDL BDL + VitA

SHAM BDL BDL + VitA

a

b

00

05

10

15

Nrf2

(cyt

opla

sm)120573

-act

in

b

SHAM BDL BDL + VitA

SHAM BDL

a

SHAM BDL BDL + VitA

0

2

4

6

8

10

Nrf2

(nuc

leus

)PC

NA

Nrf2(cytoplasm)

120573-Actin PCNA

Nrf2(nucleus)

(a)

SHA

M

BDL

BDL

+ Vi

tA

Nrf2

(b)



BDL group






SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SHAM BDL BDL + VitA


Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

100

80



SHAM BDL BDL + VitA

SHAM

Posit

ive c

ells

()

BDL BDL + VitA

a

b

0

20

40

60

80





SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


References






































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















SHAM

Rela

tive i

nten

sity

Nqo1Ho1

Nqo1

Ho1


a

a

b

b

0

1

2

4

3

5

120573-Actin

120573-Actin




Acknowledgment


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 Vitamin A Supplementation Alleviates...

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Transcript of Research Article Vitamin A Supplementation Alleviates...