Research Article Lutein Has a Protective Effect on...

Research ArticleLutein Has a Protective Effect on Hepatotoxicity Induced byArsenic via Nrf2 Signaling

Shugang Li1 Yusong Ding1 Qiang Niu1 Shangzhi Xu1 Lijuan Pang2 Rulin Ma1

Mingxia Jing1 Gangling Feng1 Jing Xia Tang1 Qian Zhang3 Xiaomei Ma4 Yizhong Yan1

Jingyu Zhang1 Meng Wei1 Hai Xia Wang1 Feng Li2 and Shuxia Guo1

1Department of Public Health and Key Laboratory of Xinjiang Endemic and Ethnic Diseases of the Ministry of EducationShihezi University School of Medicine Shihezi 832002 China2Department of Pathology and Key Laboratory of Xinjiang Endemic and Ethnic Diseases of the Ministry of EducationShihezi University School of Medicine Shihezi Xinjiang 832002 China3Department of Science and Technology The Affiliated Tumor Hospital of Xinjiang Medical University Urumqi 830011 China4Department of Pathology The Affiliated Tumor Hospital of Xinjiang Medical University Urumqi 830011 China

Correspondence should be addressed to Feng Li lifeng7855126com and Shuxia Guo pge888sinacom

Received 31 October 2014 Revised 24 December 2014 Accepted 24 December 2014

Academic Editor Chetna Singh

Copyright copy 2015 Shugang Li 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

Arsenic produces liver disease through the oxidative stress While lutein can alleviate cytotoxic and oxidative injury nuclear factorerythroid 2-related factor 2 (Nrf2) pathway plays a critical role in defending oxidative species However the mechanisms by whichlutein protects the liver against the effect of arsenic are not knownTherefore this study aims to investigate themechanisms involvedin the action of lutein using mice model in which hepatotoxicity was induced by arsenic We found that mice treatment with luteincould reverse changes in morphological and liver indexes and result in a significant improvement in hepatic function comparingwith arsenic trioxide group Lutein treatment improved the activities of antioxidant enzymes and attenuated increasing of ROSand MDA induced by arsenic trioxide Lutein could increase the mRNA and protein expression of Nrf2 signaling related genes(Nrf2 Nqo1 Ho-1 and Gst) These findings provide additional evidence that lutein may be useful for reducing reproductive injuryassociated with oxidative stress by the activation of Nrf2 signaling Our findings suggest a possible mechanism of antioxidant luteinin preventing the hepatotoxicity which implicate that a dietary lutein may be a potential treatment for liver diseases especially forarsenicosis therapy

1 Introduction

Arsenic has been recognized as a pollutant in drinkingwater due to its significant toxicity worldwide The toxicityof arsenic has aroused concern in public health in manycountries including China Using current drinking waterstandard of 10 120583gL in China the estimated number of peoplesuffering from arsenic-contaminated water is about 196million people in China [1] Recent studies have reported theprotective effect of vitamin E selenium zinc and so forth [2ndash4] against arsenic However limited data is available for therecommended nutritional intake (RNI) of those substancesTherefore it is necessary to find a safe nutrient to preventarsenic toxicity

At present the oxidative stress is themostwidely acceptedmechanism to explain the toxicity induced by arsenic Liveris the major metabolic organ of the arsenic with the high-est concentration of arsenic retention [5] Consequentlymedications with antioxidants are potentially to reduce thehepatotoxicity induced by arsenic Lutein (LU) is the mostcommon carotenoids in deep yellow vegetables and fruitsincluding cooked spinach lettuce string beans and squash[6] The biologic properties of lutein and its derivatives havebeen reported such as being antioxidant neuroprotectantand studies have indicated the protective role of LU in manydiseases including diabetic retinopathy uveitis light inducedretinopathy and ischemiareperfusion injury [7ndash11]

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 315205 10 pageshttpdxdoiorg1011552015315205

2 BioMed Research International

Nuclear factor erythroid 2-related factor 2 (Nrf2) playsa critical role in defending tissues against elevated oxidativespecies and toxic damage [12 13] In resting cells the activityof Nrf2 is tightly controlled by Kelch-like ECH-associatedprotein 1 (Keap1) in the cytoplasm [14] In response to specificstimuli keap1 dissociates then Nrf2 translocates into thenucleus and activates its target genes [15] This process acti-vates the expression of detoxifying enzymes and antioxidantproteins such asHmox-1 Nqo1 glutathione transferase (Gst)and glutathione (GSH) [16ndash19]Many studies have shown thatactivation of the Nrf2 pathway could alleviate numerous liverdisorders [20ndash22]

The molecular mechanism of luteinrsquos antioxidation func-tion has not been elucidated Therefore the present studyaims to assess if lutein can relieve the arsenic-inducedhepatotoxicity through activating Nrf2 pathway If thesehypotheses are true these findings will provide additionalevidence that lutein may be useful for reducing reproductiveinjury associated with oxidative stress by activating Nrf2

2 Materials and Methods

21 Chemicals Arsenic trioxide (ATO As2O3) was pur-

chased from Beijing Chemical Reagent Corp (BeijingChina) ATO was dissolved in 1N NaOH at 01M as astock solution LU was obtained from JF-Natural (TianjinChina) GSH superoxide dismutase (SOD) total antiox-idative capacity (T-AOC) malondialdehyde (MDA) bicin-choninic acid protein alanine aminotransferase (ALT) andaspartate aminotransferase (AST) assay kits were purchasedfrom Nanjing Jiancheng Bioengineering Institute (NanjingChina) All other chemicals were of analytical grade and wereobtained commercially

22 Animals and Treatment 40 healthy Kunming mice (18ndash22 g 20 male 20 female) were obtained from the laboratorycenter of Xinjiang Medical University (Urumqi China) Theresearch was approved by the Ethics Committee of ShiheziUniversity Throughout the experimental period mice werehoused in well-ventilated cages in a temperature-controlledroom at 22ndash25∘CThemicewere kept under sterile conditionson a 12 h lightdark cycle These mice were fed a standardlaboratory diet composed of 60 corn meal 15 beans 10bran 10 corn oil 3 casein 1 mineral mixture and 1vitamin mixture The mice had access to food and water adlibitum

After a two-week acclimation period the mice weredivided based on body weight into four groups controlATO-treated LU-treated andATO+LU-treated Each groupconsisted of 10 mice (5 males and 5 females) and all groupswere dosed intragastrically for five weeks The control groupreceived normal saline (NS) A previous report showed thatboth 1 and 5mgkg ATO could induce liver injury but didnot affect survival in mice [23] Therefore the ATO groupreceived 4mgkgATO in our studyThe LUgroupwas treatedwith 40mgkg LU a dose that was shown to reduce thetoxic effects of semicarbazide [24] The ATO + LU groupreceived 4mgkg ATO in the morning and 40mgkg LU inthe evening

23 Preparation of Plasma and Liver Function ParametersThe blood samples were collected into evacuated tubescontaining heparin solution as anticoagulant and then cen-trifuged at 3000 g for 10min The activities of ALT and ASTwere detected using commercial kits

24 Collection of Liver Tissues and Liver Somatic Index Atthe end of the treatment period the body weight of eachmouse was recorded and the mice were sacrificed by cervicaldislocation After washing and drying with filter paper thelivers were collected and weighed to the nearest milligramon an electronic balance (Shimadzu Model BL-220H TokyoJapan) The relative liver weight was calculated accordingto the following formula index weight = liver weightbodyweight times 100

25 Histopathological Examination of Liver Tissue Afterweighing 3 of liver sections were created One section wasused for preparation of tissue homogenates and one sectionwas frozen in liquid nitrogen and stored at minus80∘C for totalRNA extraction and immunoblot analysis The remainingsections were fixed in 10 neutral formalin for at least 24 hdehydrated in different grades of alcohol and embedded inparaffin Sections (5 120583m) of fixed liver tissue were cut usinga rotary microtome The sections were processed and passedthrough a graded alcohol series dyed with hematoxylin andeosin cleared in xylene and inspected microscopically asdescribed previously [19]

26 Preparation of Liver Homogenates Liver tissues wereplaced in a lysis buffer (119898V = 1 9) containing 20mM Tris(pH 75) 150mM NaCl 1 Triton X-100 and the pro-tein inhibitors sodium pyrophosphate 120573-glycerophosphateethylenediaminetetraacetic acid Na

3VO4 and leupeptin

(Beyotime Biotechnology Shanghai China) The liver sec-tions were homogenized with a Tissue Lyser (Qiagen Valen-cia CA USA) After the homogenate was centrifuged at2500timesg for 10min at 4∘C the supernatant was collected todetermine the activity of ALT AST SOD and T-AOC and thecontent of GSH and MDA

27 Measurement of Lipid Peroxidation MDA a marker oflipid peroxidation was measured with a commercial kitfollowing the manufacturerrsquos instructions Briefly the sam-ples were treated with thiobarbituric acid which producesa red compound with an absorption maximum at 532 nmin the presence of MDA The concentration of MDA wascalculated by comparing the absorbance to that produced bythe standard 1133-tetraethoxypropane

28 Measurement of Antioxidant Enzyme Assay GSH specif-ically deoxidizes dithiobisnitrobenzoic acid to form a yellowproduct 2-nitro-5-SH-benzoic acid which can be measuredby colorimetry at 532 nm SOD activity was measured usinga tetrazolium salt for detection of superoxide radicals gen-erated by xanthine oxidase and hypoxanthine One unit ofSOD is defined as the amount of enzyme needed to exhibit50 dismutation of the superoxide radical at 37∘C The

BioMed Research International 3

reaction product was measured at 450 nm T-AOC in thetissue was measured with a commercial analysis kit This kitused antioxidants in the samples to reduce Fe3+ to Fe2+ whichwas chelated with porphyrin to produce a purple complexthat was quantified by measuring the absorbance at 550 nmThe T-AOC of the samples was determined by comparisonwith the control standard Results were normalized to thetotal amount of protein as measured by bicinchoninic acidprotein assay

29 Detection of mRNA of Nrf2 Related Genes by RealTime PCR Liver tissue was snap frozen in liquid nitrogenand total RNA was extracted by Trizol extraction method(Invitrogen Grand Island NY USA) according to themanufacturerrsquos instructions Equal amounts of RNA (2 120583g)were reverse-transcribed into cDNA using the TranscriptorFirst-Strand cDNA Synthesis Kit (Roche Indianapolis INUSA) Primers were synthesized by Sigma-Aldrich (St LouisMO USA) for the following Mus musculus genes Hmox-1 184 bp amplicon F 51015840-CAGGTGATGCTGACAGAGGA-31015840 R 51015840-ACAGGAAGCTGAGAGTGAGG-31015840 Gst 199 bpamplicon F 51015840-ATCGTTCCCTTTCTCGGCAT-31015840 R 51015840-GCAGCCTGTAAGCCATTGAC-31015840 Nqo1 112 bp ampliconF 51015840-TGGCCGAACACAAGAAGCTG-31015840 R 51015840-GCTACG-AGCACTCTCTCAAACC-31015840 Nrf2 173 bp amplicon F 51015840-TTCCATTTACGGAGACCCAC-31015840 R 51015840-ATTCACGCA-TAGGAGCACTG-31015840 and beta-actin 240 bp amplicon F51015840-CACGATGGAGGGGCCGGACTCATC-31015840 R 51015840-TAA-AGACCTCTATGCCAACACAGT-31015840

The relative gene expression of targets was detected usinga comparative cycle threshold method [20] All samples weretested in triplicate Real time quantitative PCR (qPCR) wasperformed on a mixture containing 10 120583L of PCR Supermix(Bio-Rad Laboratories Hercules CA USA) 1 120583L of forwardand reverse primers (Sangon Beijing China) 1120583L of tem-plate DNA and 8 120583L of distilled water

The qPCR conditions were as follows 1 cycle of initialdenaturation (94∘C for 3min) 30 cycles of amplification(94∘C for 30 s 57∘C for 30 s and 72∘C for 25 s) 1 cycle ofmelting curve measurement (95∘C for 5 s 65∘C for 60 s anda gradual increase in temperature to 97∘C) and a coolingperiod (40∘C for 30 s) The data presented were relativemRNA levels normalized to 120573-actin

210 Detection of Protein Expression of Nrf2 Related Protein byWestern Blot Liver tissues were homogenized in one volumeof sample buffer (50mMTris-Cl 100mMDTT 10 glyceroland 2 SDS) and centrifuged at 14800timesg at 4∘C for 15min tomove debris The samples were subjected to SDS-PAGE andtransferred to polyvinylidene difluoride membranes Afterblocking with skim milk (5) the blots were probed withthe primary antibodies (Abcam Cambridge UK) for Nrf2(1 1000) Hmox-1 (1 1000) Nqo1 (1 1000) Gst (1 1000)and 120573-actin (1 1000) at 4∘Covernight Incubation with theprimary antibodies was followed by incubation with sec-ondary antibodies (conjugated to horseradish peroxidase)afterwashing in Tris-buffered saline andTween 20 Blots wereprocessed using an ECL kit (Santa Cruz Biotechnology Inc)

and exposed to film All experiments were repeated threetimes

211 Immunohistochemical Staining Examination of LiverTissue Paraffin-embedded liver samples were prepared forimmunohistochemical analysis to elucidate the subcellularlocalization of Nrf2 and its target gene products Hmox-1 Nqo1 and Gst EnVisions two-step immunohistochem-ical kit (Zhongshan Golden Bridge Beijing China) and331015840-diaminobenzidine tetrahydrochloride (DAB) enhancer(Dako System Glostrup Denmark) were used to detectspecific target proteins Briefly tissue sections were cutdried and deparaffinized Antigen retrieval was performedby boiling in sodium citrate buffer After blocking peroxidaseand nonspecific binding tissue sections were incubated withprimary and then secondary antibodies (Abcam CambridgeUK) The sections were visualized by incubating with DABand counterstained with hematoxylin

212 Statistical Analysis The results were expressed as themean plusmn standard deviation A general linear model was usedto analyze the interactions of the effects of the combination ofATO and LU in a two-factor two-level factorial design Themodel was as follows in which a significant 119875 value for b3indicated an interaction between the effects of ATO and LU

119910 = constant + b1 times ATO + b2 times LU + b3 times (ATO times LU) (1)

where ldquo119910rdquo is a measured parameter such as body weightliver weight relative liver weight liver function and oxidativestress indices Analysis of variance (ANOVA) was usedto detect the differences among the experimental groupscontrol ATO LU and ATO + LU ANOVA was followed bypairwise comparisons with Bonferronirsquosmultiple comparisontests The data were analyzed using SPSS software for Win-dows version 150 (SPSS Inc Chicago IL USA) and a119875 valuelt 005 was considered to be statistically significant

3 Results

31 LU Alleviated Liver Damage Induced by ATO in MiceIn our study we found that the final body weight wassignificantly lower in ATO group than in the control group(119875 lt 0001) The liver index of the ATO group was highercompared with the control group (119875 lt 0001) We testedthe activities of ALT and AST in liver tissue and found bothactivities of ALT and AST in ATO group were markedlyhigher than those in the control group (119875 lt 0001) Inaddition a significant interaction was found between theeffects of ATO and LU on body weight liver index andthe ALT and AST activities (119875 lt 0001 resp) ATO +LU treatment increased the body weight and decreased liverindex and activities of ALT and AST compared with the ATOgroup see Table 1

To confirm the protective effect of LU on ATO-inducedliver damage we examined liver histology in tissues fromthe treated mice LU did not cause noticeable morphologicalchanges in the liver (Figure 1(c)) while arsenic exposure


Table 1 Effect of arsenic trioxide (ATO) andor lutein (LU) administration on indices related to hepatotoxicity in mice

Control (NS) ATO (4mgkg) LU (40mgKg) ATO + LU (4mgkg + 40mgKg)Initial body weight (g) 1941 plusmn 116 1931 plusmn 169 2112 plusmn 143 2050 plusmn 159Final body weight (g) 3468 plusmn 203 3482 plusmn 119ac 2583 plusmn 196 3037 plusmn 191ab

Body weight gain (g) 1527 plusmn 265 1552 plusmn 171 471 plusmn 268 988 plusmn 193Liver weight (g) 138 plusmn 017 323 plusmn 015ab 105 plusmn 007a 116 plusmn 026ab

Liver index () 399 plusmn 061 929 plusmn 047b 41 plusmn 049a 388 plusmn 096b

AST (Ug prot) 2201 plusmn 495 3162 plusmn 728b 1777 plusmn 497a 1087 plusmn 494ab

ALT (Ug prot) 7834 plusmn 2140 13651 plusmn 2931b 6884 plusmn 1725a 5998 plusmn 1708b

Note the results were described as mean plusmn SD (119899 = 10) aIndicating significant difference from control (NS) group at119875 lt 001 bindicating significant differencefrom the ATO group (4mgkg) at 119875 lt 001 cindicating significant difference from the ATO + LU group (4mgkg + 40mgKg) at 119875 lt 001

Table 2 Effect of lutein (LU) on malondialdehyde (MDA) glutathione (GSH) superoxide dismutase (SOD) and total antioxidative capacity(T-AOC) of arsenic trioxide- (ATO-) treated mice

Experimental group MDAnmolmg prot

GSH120583molg prot

SODUg prot

T-AOCUg prot

Control (NS) 289 plusmn 045 061 plusmn 004 048 plusmn 018 3719 plusmn 579LU (40mgkg) 303 plusmn 046 077 plusmn 006 056 plusmn 017 4657 plusmn 221b

ATO (4mgkg) 473 plusmn 054ac 050 plusmn 005ac 054 plusmn 014ac 2728 plusmn 413ac

ATO + LU (4mgkg + 40mgkg) 358 plusmn 033ab 067 plusmn 002ab 055 plusmn 017ab 2938 plusmn 320ab


resulted in dim boundary of hepatocyte dismissed cellmembrane cytoplasmdisintegrating pieces and the accumu-lation of lipid droplets intracytoplasm the specific hepatocyteballoon degeneration performance (Figure 1(b)) All thesechanges were mitigated by LU (Figure 1(d))

32 LU Reduced the Oxidative Damage Induced by ATO Weused ATO as an exogenous oxidative stressor in the liver Wetested GSH SOD and T-AOC in serum to assess the effectsof ATO on endogenous liver antioxidant system Treatmentwith ATO caused a prominent decrease of GSH and T-AOClevel compared with control group (119875 lt 001) The contentof MDA was significantly higher in ATO group than incontrol groupThere was a statistically significant interactionbetween ATO and LU on the content of GSH and MDA aswell as the level of T-AOC (119875 lt 001 resp) As shown inTable 2 treatment with ATO + LU elevated the content ofGSH and the level of T-AOC in liver tissue comparedwith theATO group Additionally the ATO + LU group had a lowercontent of MDA than the ATO group Hence LU remittedoxidative stress induced by ATO

33 LU Activated the mRNA and Protein Expression Levelsof Nrf2 Pathway Related Genes We found that neither themRNA nor the protein expression of Nrf2 in liver wasenhanced in ATO group compared with the control groupSimilar to Nrf2 expression of its target genes Nqo1 andGst were not detectably changed in the ATO group LUitself induced Nrf2 expression increasingly (119875 lt 001) Thegroup treated with ATO + LU showed the most prominent

expression of Nrf2 Similar to Nrf2 expression of its targetgenes Ho-1 Nqo1 and Gst (119875 lt 001 resp) were induced inthe same two groups (LU and ATO + LU) with the highestlevel seen in ATO + LU group (Figures 2 and 3)

In the present study we also tested the subcellularlocalization of Nrf2 Ho-1 Nqo1 and Gst by immunohisto-chemical staining We found that both the control and ATOgroups showed a low level ofNrf2 in the nucleuswhile nuclearNrf2 accumulation was enhanced in the LU group and theATO + LU group (Figure 4)

4 Discussion

Our study shows that LU can relieve arsenic-induced liverdysfunction by activating Nrf2 pathway Kuitun district(about 4000Km away from Beijing) next to Shihezi city (inwhich our university located) is the first arsenic poisoningdistrict in China inner land founded in 1980 This studyprovides a good way to protect the hepatotoxicity induced byarsenic whichwill help us to prevent and control local arsenicpoisoning

Oxidative stress has been correlated with the progressionand severity of many liver diseases Therefore antioxidanttherapy may be a feasible therapy to reduce liver damageArsenic is widely accepted as an exogenous stressor andhepatotoxicant [24] The major metabolic pathway of arsenicis methylation in the liver [25] However the methylationof arsenic is not a detoxification process because trivalentmethylated arsenical intermediates are highly toxic [26] Inour study we used arsenic to induce oxidative stress and


Control 400x

(a)

ATO 400x

(b)

LU 400x

(c)

ATO + LU 400x

(d)

Figure 1 Morphological changes in mouse liver after arsenic trioxide (ATO) andor lutein (LU) Control group showed normal structureof hepatic cord hepatic sinusoid and hepatocyte The basic performance of the hepatocyte poisoning could be found in ATO treatmentgroup such as dim boundary of hepatocyte dismissed cell membrane cytoplasm disintegrating pieces and the accumulation of lipid dropletsintracytoplasm meanwhile the specific hepatocyte balloon degeneration performance can be observed At the same time inflammatory cellsinfiltration scattered in the liver tissue AOT + LU group displayed fatty degeneration of hepatocytes and congestion but not observed balloondegeneration and hepatocyte disintegration LU treatment group showed normal structure of liver cells as control group (400xmagnification)

liver injury in mice The toxic effects of arsenic are relatedto its ability to induce reactive oxygen species [25] After 35-day treatment with ATO we found that ATO inhibited theoverall growth in mice and liver injury as measured by theincreased level of tissue ALT and AST compared with controlgroup Further noticeable morphological changes found inthe liver further confirmed the adverse effects of ATO onthe liver Compared with the control group livers frommice treated with ATO showed significantly decreased GSHcontent and T-AOC level and increased MDA content Theseresults strongly suggested that ATO disrupted the balancebetween oxidant and antioxidant agents and demonstratedATO-induced liver oxidative damage

One large scale randomized placebo-controlled trialsponsored by NIH lutein demonstrated that LU was effectivein decreasing the risk of developing advanced age-relatedmacular degeneration (AMD) To the best of our knowledgethere have been no reports on the effects of lutein on neuronsin the brain in a rodent stroke model [27] In the presentstudy we found significant interactions between LU andATO treatments on body weight liver index ALT AST T-AOC levels GSH and MDA contents We observed thatthese parameters in the ATO + LU group fell in between

the ATO and control group This indicates that LU canalleviate the oxidative stress and liver injury induced by ATOCompared to the ATO group pathological improvement inATO + LU group provided evidence that LU antagonizes thehepatotoxicity of ATO

Previous studies have shown that LU could reduce oxida-tive damage and improve the liver function However fewstudies addressed the exact mechanisms of these protectiveeffects and the involvement of Nrf2 pathway Activation ofNrf2 pathway can enhance the expression of detoxifyingenzymes and antioxidants [15 17] and attenuate hepaticfibrosis [18] nonalcoholic steatohepatitis [20] NAFLD [28]and drug-induced liver injury [21] In our study we foundthat LU can induce the expression of Nrf2 compared withcontrol group In addition the expression of Nrf2 wassignificantly highest in ATO + LU group We also measuredthe target genes of Nrf2 such asHmox-1Nqo1 andGst in thepresent study Hmox-1 possesses cytoprotective properties bypromoting the oxidative cleavage of the prooxidant hemeto carbon monoxide and bilirubin [28] Nqo1 is a phase IIenzyme and could reduce oxidative stress through reducingsuperoxide maintaining endogenous antioxidants and cat-alyzing themetabolism of xenobiotics [28] Gst is responsible


1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)

Figure 2 (a) Real time PCR analysis of treatment of arsenic trioxide (ATO) andor lutein (LU) Nuclear factor erythroid 2-related factor 2(Nrf2 molecular weight 173 bp) NAD(P)H dehydrogenase quinone 1 (Nqo1 molecular weight 112 bp) heme oxygenase-1 (Ho-1 molecularweight 184 bp) and glutathione transferase (Gst molecular weight 199 bp) mRNA expression levels were measured by real time PCR (b)Quantitative mRNA analysis of treatment of arsenic trioxide (ATO) andor lutein (LU) 119910-axis indicates mRNA expression of test markerversus 120573-actin Each bar represents the mean plusmn SD The gene expression of Nrf2 Nqo1 Ho-1 and Gst of LU treat group was significantlyhigher than those of control group The group treated with ATO + LU showed the most prominent mRNA expression of Nrf2 related genesSignificant differences relative to the control and ATO groups are indicated as follows lowast versus control group 119875 lt 001 versus ATO group119875 lt 001


Control ATO LU ATO + LU

Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)

Figure 3 (a) Western blot analysis protein levels of treatment of arsenic trioxide (ATO) andor lutein (LU) Nrf2 (molecular weight 57 kDa)Nqo1 (molecular weight 31 kDa) Ho-1 (molecular weight 32 kDa) and Gst (molecular weight 26 kDa) protein expression wasmeasured in thelivers of mice treated with arsenic trioxide (ATO) andor lutein (LU) by western blot (b) Quantitative protein analysis of treatment of arsenictrioxide (ATO) andor lutein (LU) Nrf2 Nqo1 Ho-1 and Gst protein expression levels were measured 119910 axis represents protein expressionof test protein relative to 120573-actin Each bar represents the mean plusmn SD The protein expression of Nrf2 Nqo1 Ho-1 and Gst of LU treat groupwas significantly higher than those of control group The group treated with ATO + LU showed the most prominent protein expression ofNrf2 related genes Significant differences relative to the control and arsenic trioxide (ATO) groups are indicated as follows lowast versus controlgroup 119875 lt 001 versus ATO group 119875 lt 001

for catalyzing the conjugation ofGSH to reactive electrophiles[29] Similar to Nrf2 expressions of Hmox-1 Nqo1 andGst were all induced in the two LU-treated groups with thehighest levels seen inATO+LU group at both themRNA andprotein levels In addition LU also increased the liver contentof GSH and the level of T-AOC decreased the liver contentMDA and alleviated the pathological alterations induced byATO Based on the alteration of oxidative stress parametersmitigation of pathological changes and enhanced expressionof Nrf2 pathway components we concluded that LU stimu-lated the Nrf2 pathway thus alleviating oxidative stress andreducing oxidative damage in the liver

Numerous studies have shown that arsenic was an Nrf2inducer in several cell types including human hepatocytes[30ndash32] It is very interesting that the expressions of eitherNrf2 or its downstream genes Noq1 and Gst were enhancedin the ATO group in our study Recent studies have shownthat expression levels of Nrf2 and its target genes initiallyincreased and then gradually fell over the duration of arsenicexposure [33] Acute exposure to arsenic or other exogenousstressors may activate the Nrf2 pathway to maintain cellularredox homeostasis and limit oxidative damage but theprotective compensatory reaction may be overpowered asexposure time may be prolonged It has been shown thatNrf2 is repressed by Keap1 in the cytosol and degraded bythe 26S proteasome in nonstressed cells Oxidative stress

activates Nrf2 by permitting its dissociation from Keap1and translocation into the nucleus where it binds to theantioxidant response element and leads to the expression ofthe target genes Given that subcellular localization is a majordetermining factor of the function of Nrf2 we studied itssubcellular location by immunohistochemical staining Wefound prominent nuclear accumulation of Nrf2 protein inboth the LU and ATO + LU groups Meanwhile Hmox-1Nqo1 and GST also showed accumulation in the nucleus

In summary we demonstrated that LU could alleviatearsenic-induced oxidative stress and liver injury throughactivating the Nrf2 pathway As oxidative damage is animportant toxic mechanism of arsenic and is associated withthe pathological processes of diverse liver diseases we believeLU could be useful in treating many liver disorders throughreducing oxidative stress by activating Nrf2 pathway We alsofound the possible role of the Nrf2 pathway in prolongedoxidative stress Our findings could help provide a betterunderstanding of the mechanism of LU This study alsoprovided more evidence that a dietary Nrf2 activator mightbe a plausible treatment for liver diseases Further studies toclarify these findings should include validation of the role ofthe Nrf2 pathway by studying the effects of LU on liver injuryin mice lacking the Nrf2 gene and cell experiments such asthe dissociation of Keap1Nrf2 complex the translocation ofNrf2 and the accumulation of Nrf2 in nucleus


Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)

Figure 4 Representative immunohistochemical slides Immunohistochemical slideswere stained forNrf2Nqo1Ho-1 andGst in liver tissueswith arsenic trioxide (ATO) andor lutein (LU) treatment (200x magnification) Nrf2 protein expression located in nucleus and cytoplasmtreated with ATO + LU Nqo1 Ho-1 and Gst protein expression located in cytoplasm nucleus or membrane treated with ATO + LU Theprotein expression level of Nrf2 Nqo1 Ho-1 and Gst of ATO + LU group was higher than other groups

Conflict of Interests

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

Authorsrsquo Contribution

Shugang Li and Feng Li conceived and designed theexperiments Qiang Niu Yusong Ding Shangzhi XuJingxia Tang Rulin Ma Mingxia Jing Gangling Feng andLijuan Pang performed the experiment Shuxia Guo MengWei and Hai Xia Wang analyzed the data Qian ZhangXiaomei Ma Yizhong Yan and Jingyu Zhang contributedreagentsmaterialsanalysis tools Shugang Li and Yusong

Ding wrote the paper Shugang Li Shuxia Guo and Feng Licontributed to paper revisions Shugang Li and Yusong Dingcontributed equally to this work

Acknowledgments

This work was supported by Xinjiang Production and Con-struction Corps Grant (2014BA039) and Shihezi UniversityGrant (RCZX201112)

References

[1] L Rodrıguez-LadoG SunMBerg et al ldquoGroundwater arseniccontamination throughout Chinardquo Science vol 341 no 6148pp 866ndash868 2013


[2] Q Wang H Jia and K-T Liu ldquoExperimental study of arsenicand the antagonistic action of vitamin E on lipid peroxidationin the liver of micerdquo Journal of Xinjiang Medical University vol27 no 4 pp 354ndash356 2004

[3] S E Afton B Catron and J A Caruso ldquoElucidating theselenium and arsenic metabolic pathways following exposureto the non hyperaccumulating chlorophytum comosum spiderplantrdquo Journal of Experimental Botany vol 60 no 4 pp 1289ndash1299 2013

[4] N Nan and Z Ping ldquoAnatagonism of zinc on testicular toxicityinduced by arsenic exposure in male ratsrdquo Journal of ShanghaiJiaotong Universtiy (Medical Science) vol 33 no 12 pp 1637ndash1639 2013

[5] L Benramdane M Accominotti L Fanton D Malicier and J-J Vallon ldquoArsenic speciation in human organs following fatalarsenic trioxide poisoningmdasha case reportrdquo Clinical Chemistryvol 45 no 2 pp 301ndash306 1999

[6] C-J Chang J-F Lin H-H Chang G-A Lee and C-F HungldquoLutein protects against methotrexate-induced and reactiveoxygen species-mediated apoptotic cell injury of IEC-6 cellsrdquoPLoS ONE vol 8 no 9 Article ID e72553 2013

[7] M Sasaki Y Ozawa T Kurihara et al ldquoNeurodegenerativeinfluence of oxidative stress in the retina of a murine model ofdiabetesrdquo Diabetologia vol 53 no 5 pp 971ndash979 2010

[8] M Sasaki Y Ozawa T Kurihara et al ldquoNeuroprotectiveeffect of an antioxidant lutein during retinal inflammationrdquoInvestigative Ophthalmology and Visual Science vol 50 no 3pp 1433ndash1439 2009

[9] M Wang C Zhang and X Lin ldquoProtective effect of luteinagainst blue light-induced retinal damage in ratrdquo Journal ofHygiene Research vol 37 no 4 pp 409ndash412 2008

[10] M Sasaki K Yuki and T Kurihara ldquoBiological role of luteinin the light-induced retinal degenerationrdquoThe Journal of Nutri-tional Biochemistry vol 23 no 3 pp 423ndash429 2013

[11] S Y Li Z J Fu H Ma et al ldquoEffect of lutein on retinalneurons and oxidative stress in a model of acute retinalischemiareperfusionrdquo Investigative Ophthalmology and VisualScience vol 50 no 2 pp 836ndash843 2009

[12] Y Mitsuishi H Motohashi and M Yamamoto ldquoTheKeap1ndashNrf2 system in cancers stress response and anabolicmetabolismrdquo Frontiers in Oncology vol 2 no 2 pp 200ndash2042012

[13] A Lau S A Whitman M C Jaramillo and D D ZhangldquoArsenic-mediated activation of the Nrf2-Keap1 antioxidantpathwayrdquo Journal of Biochemical and Molecular Toxicology vol27 no 2 pp 99ndash105 2013

[14] Y Hirotsu F Katsuoka R Funayama et al ldquoNrf2-MafGheterodimers contribute globally to antioxidant and metabolicnetworksrdquo Nucleic Acids Research vol 40 no 20 pp 10228ndash10239 2012

[15] D Liu X Duan D Dong et al ldquoActivation of the Nrf2pathway by inorganic arsenic in human hepatocytes and therole of transcriptional repressor bach1rdquoOxidative Medicine andCellular Longevity vol 2013 Article ID 984546 10 pages 2013

[16] Q Zhang J Pi C G Woods and M E Andersen ldquoA systemsbiology perspective on Nrf2-mediated antioxidant responserdquoToxicology and Applied Pharmacology vol 244 no 1 pp 84ndash972010

[17] B Li X Li B Zhu et al ldquoSodium arsenite induced reactiveoxygen species generation nuclear factor (erythroid-2 related)

factor 2 activation heme oxygenase-1 expression and glu-tathione elevation in Chang human hepatocytesrdquo Environmen-tal Toxicology vol 28 no 7 pp 401ndash410 2013

[18] C J Oh J-Y Kim A-K Min et al ldquoSulforaphane attenuateshepatic fibrosis via NF-E2-related factor 2-mediated inhibitionof transforming growth factor-120573Smad signalingrdquo Free RadicalBiology amp Medicine vol 52 no 5 pp 671ndash682 2012

[19] K CWu J Y Cui andCDKlaassen ldquoBeneficial role ofNrf2 inregulating NADPH generation and consumptionrdquo ToxicologicalSciences vol 123 no 2 pp 590ndash600 2011

[20] J Lamle S Marhenke J Borlak et al ldquoNuclear factor-eythroid2-related factor 2 prevents alcohol-induced fulminant liverinjuryrdquo Gastroenterology vol 134 no 4 pp 1159ndash1168 2008

[21] S A Reisman D B Buckley Y Tanaka and C D KlaassenldquoCDDO-Im protects from acetaminophen hepatotoxicitythrough induction of Nrf2-dependent genesrdquo Toxicology andApplied Pharmacology vol 236 no 1 pp 109ndash114 2009

[22] KKudohHUchinamiMYoshioka E Seki andYYamamotoldquoNrf2 activation protects the liver from ischemiareperfusioninjury in micerdquo Annals of Surgery vol 260 no 2 pp 118ndash1272014

[23] Q Wang Y Li S Li et al ldquoExperimental study of arsenic andthe antagonistic action of vitam in E on lipid peroxidation in theorgans of micrdquo Chinese Journal of Control of Endemic Diseasevol 24 no 1 pp 9ndash12 2009

[24] Z Huizhu W Jinghong B Jing et al ldquoProtective effect oflutein on acute alcoholic liver injury inmicerdquoPharmacology andClinics of Chinese Materia Medica vol 8 no 2 pp 68ndash71 2012

[25] Q Liu H Zhang L Smeester et al ldquoThe NRF2-mediatedoxidative stress response pathway is associated with tumor cellresistance to arsenic trioxide across the NCI-60 panelrdquo BMCMedical Genomics vol 3 article 37 2010

[26] K Islam A Haque R Karim et al ldquoDose-response relationshipbetween arsenic exposure and the serum enzymes for liverfunction tests in the individuals exposed to arsenic a crosssectional study in Bangladeshrdquo Environmental Health vol 10no 1 article 64 2011

[27] T T Y Woo S-Y Li W W K Lai D Wong and A C YLo ldquoNeuroprotective effects of lutein in a rat model of retinaldetachmentrdquo Graefersquos Archive for Clinical and ExperimentalOphthalmology vol 251 no 1 pp 41ndash51 2013

[28] A M Bataille and J E Manautou ldquoNrf2 a potential target fornew therapeutics in liver diseaserdquo Clinical Pharmacology andTherapeutics vol 92 no 3 pp 340ndash348 2012

[29] R N Hardwick C D Fisher M J Canet A D Lake and NJ Cherrington ldquoDiversity in antioxidant response enzymes inprogressive stages of human nonalcoholic fatty liver diseaserdquoDrug Metabolism and Disposition vol 38 no 2 pp 293ndash3012010

[30] X-J Wang Z Sun W Chen K E Eblin J A Gandolfi and DD Zhang ldquoNrf2 protects human bladder urothelial cells fromarsenite andmonomethylarsonous acid toxicityrdquoToxicology andApplied Pharmacology vol 225 no 2 pp 206ndash213 2007

[31] D Meng X Wang Q Chang et al ldquoArsenic promotes angio-genesis in vitro via a heme oxygenase-1-dependentmechanismrdquoToxicology and Applied Pharmacology vol 244 no 3 pp 291ndash299 2010

[32] Y Abiko Y Shinkai D Sumi and Y Kumagai ldquoReduction ofarsenic-induced cytotoxicity through Nrf2HO-1 signaling inHepG2 cellsrdquo Journal of Toxicological Sciences vol 35 no 3 pp419ndash423 2010


[33] A Ranganathan M B Sheshappa and V Baskaran ldquoQual-ity characteristics and lutein bioavailability from maize andvegetable-based health foodrdquo Journal of Dietary Supplementsvol 11 no 2 pp 131ndash144 2014

Submit your manuscripts athttpwwwhindawicom

PainResearch and TreatmentHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom

Volume 2014

ToxinsJournal of

VaccinesJournal of

Hindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

AntibioticsInternational Journal of

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Drug DeliveryJournal of



Advances in Pharmacological Sciences

Tropical MedicineJournal of


Medicinal ChemistryInternational Journal of


AddictionJournal of



BioMed Research International

Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

ScientificaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of


Pharmaceutics


MEDIATORSINFLAMMATION

of


Nuclear factor erythroid 2-related factor 2 (Nrf2) playsa critical role in defending tissues against elevated oxidativespecies and toxic damage [12 13] In resting cells the activityof Nrf2 is tightly controlled by Kelch-like ECH-associatedprotein 1 (Keap1) in the cytoplasm [14] In response to specificstimuli keap1 dissociates then Nrf2 translocates into thenucleus and activates its target genes [15] This process acti-vates the expression of detoxifying enzymes and antioxidantproteins such asHmox-1 Nqo1 glutathione transferase (Gst)and glutathione (GSH) [16ndash19]Many studies have shown thatactivation of the Nrf2 pathway could alleviate numerous liverdisorders [20ndash22]

The molecular mechanism of luteinrsquos antioxidation func-tion has not been elucidated Therefore the present studyaims to assess if lutein can relieve the arsenic-inducedhepatotoxicity through activating Nrf2 pathway If thesehypotheses are true these findings will provide additionalevidence that lutein may be useful for reducing reproductiveinjury associated with oxidative stress by activating Nrf2

2 Materials and Methods

21 Chemicals Arsenic trioxide (ATO As2O3) was pur-

chased from Beijing Chemical Reagent Corp (BeijingChina) ATO was dissolved in 1N NaOH at 01M as astock solution LU was obtained from JF-Natural (TianjinChina) GSH superoxide dismutase (SOD) total antiox-idative capacity (T-AOC) malondialdehyde (MDA) bicin-choninic acid protein alanine aminotransferase (ALT) andaspartate aminotransferase (AST) assay kits were purchasedfrom Nanjing Jiancheng Bioengineering Institute (NanjingChina) All other chemicals were of analytical grade and wereobtained commercially

22 Animals and Treatment 40 healthy Kunming mice (18ndash22 g 20 male 20 female) were obtained from the laboratorycenter of Xinjiang Medical University (Urumqi China) Theresearch was approved by the Ethics Committee of ShiheziUniversity Throughout the experimental period mice werehoused in well-ventilated cages in a temperature-controlledroom at 22ndash25∘CThemicewere kept under sterile conditionson a 12 h lightdark cycle These mice were fed a standardlaboratory diet composed of 60 corn meal 15 beans 10bran 10 corn oil 3 casein 1 mineral mixture and 1vitamin mixture The mice had access to food and water adlibitum

After a two-week acclimation period the mice weredivided based on body weight into four groups controlATO-treated LU-treated andATO+LU-treated Each groupconsisted of 10 mice (5 males and 5 females) and all groupswere dosed intragastrically for five weeks The control groupreceived normal saline (NS) A previous report showed thatboth 1 and 5mgkg ATO could induce liver injury but didnot affect survival in mice [23] Therefore the ATO groupreceived 4mgkgATO in our studyThe LUgroupwas treatedwith 40mgkg LU a dose that was shown to reduce thetoxic effects of semicarbazide [24] The ATO + LU groupreceived 4mgkg ATO in the morning and 40mgkg LU inthe evening

23 Preparation of Plasma and Liver Function ParametersThe blood samples were collected into evacuated tubescontaining heparin solution as anticoagulant and then cen-trifuged at 3000 g for 10min The activities of ALT and ASTwere detected using commercial kits

24 Collection of Liver Tissues and Liver Somatic Index Atthe end of the treatment period the body weight of eachmouse was recorded and the mice were sacrificed by cervicaldislocation After washing and drying with filter paper thelivers were collected and weighed to the nearest milligramon an electronic balance (Shimadzu Model BL-220H TokyoJapan) The relative liver weight was calculated accordingto the following formula index weight = liver weightbodyweight times 100

25 Histopathological Examination of Liver Tissue Afterweighing 3 of liver sections were created One section wasused for preparation of tissue homogenates and one sectionwas frozen in liquid nitrogen and stored at minus80∘C for totalRNA extraction and immunoblot analysis The remainingsections were fixed in 10 neutral formalin for at least 24 hdehydrated in different grades of alcohol and embedded inparaffin Sections (5 120583m) of fixed liver tissue were cut usinga rotary microtome The sections were processed and passedthrough a graded alcohol series dyed with hematoxylin andeosin cleared in xylene and inspected microscopically asdescribed previously [19]

26 Preparation of Liver Homogenates Liver tissues wereplaced in a lysis buffer (119898V = 1 9) containing 20mM Tris(pH 75) 150mM NaCl 1 Triton X-100 and the pro-tein inhibitors sodium pyrophosphate 120573-glycerophosphateethylenediaminetetraacetic acid Na

3VO4 and leupeptin

(Beyotime Biotechnology Shanghai China) The liver sec-tions were homogenized with a Tissue Lyser (Qiagen Valen-cia CA USA) After the homogenate was centrifuged at2500timesg for 10min at 4∘C the supernatant was collected todetermine the activity of ALT AST SOD and T-AOC and thecontent of GSH and MDA

27 Measurement of Lipid Peroxidation MDA a marker oflipid peroxidation was measured with a commercial kitfollowing the manufacturerrsquos instructions Briefly the sam-ples were treated with thiobarbituric acid which producesa red compound with an absorption maximum at 532 nmin the presence of MDA The concentration of MDA wascalculated by comparing the absorbance to that produced bythe standard 1133-tetraethoxypropane

28 Measurement of Antioxidant Enzyme Assay GSH specif-ically deoxidizes dithiobisnitrobenzoic acid to form a yellowproduct 2-nitro-5-SH-benzoic acid which can be measuredby colorimetry at 532 nm SOD activity was measured usinga tetrazolium salt for detection of superoxide radicals gen-erated by xanthine oxidase and hypoxanthine One unit ofSOD is defined as the amount of enzyme needed to exhibit50 dismutation of the superoxide radical at 37∘C The












3 Results













GSH120583molg prot

SODUg prot

T-AOCUg prot










4 Discussion




Control 400x

(a)

ATO 400x

(b)

LU 400x

(c)

ATO + LU 400x

(d)







1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)




Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of












3 Results













GSH120583molg prot

SODUg prot

T-AOCUg prot










4 Discussion




Control 400x

(a)

ATO 400x

(b)

LU 400x

(c)

ATO + LU 400x

(d)







1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)




Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of











GSH120583molg prot

SODUg prot

T-AOCUg prot










4 Discussion




Control 400x

(a)

ATO 400x

(b)

LU 400x

(c)

ATO + LU 400x

(d)







1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)




Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control 400x

(a)

ATO 400x

(b)

LU 400x

(c)

ATO + LU 400x

(d)







1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)




Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


1 2 3 4

1 2 3 4

2000 bp1000 bp750bp500bp250bp100 bp

Nrf2 (173bp) Nqo1 (112 bp)

Ho-1 (184 bp)

(1) ATO + LU(2) LU

(3) ATO(4) Control

120573-actin (240bp)

2000 bp1000 bp750bp500bp

250bp100 bp

2000 bp

1000 bp750bp500bp

250bp100 bp

2000 bp1000 bp750bp500bp250bp100 bp

1 2 3 4

1 2 3 4

Gst (199bp)

2000 bp1000 bp750bp500bp

250bp100 bp

(a)

00

05

10

15

20

25

Nrf2 Nqo1 Ho-1 Gst

mRN

A ex

pres

sion

ControlATO

LUATO + LU

lowast

lowast

lowast

lowastlowast

lowast

lowast

lowast

lowast

(b)




Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of



Nrf257kDa

Nqo131kDa

Ho-132kDa

Gst26kDa

120573-actin42kDa

(a)

000

020

040

060

080

Gst Ho-1 Nqo1 Nrf2

Prot

ein

expr

essio

nControlATO

LUATO + LU

lowast

lowast

lowastlowast

lowast

lowast

lowastlowast

lowast

(b)







Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of


Control ATO

ATO + LULUNrf2

(a)

Nqo1

Control ATO

ATO + LULU

(b)

Ho-1

Control ATO

ATO + LULU

(c)

Gst

Control ATO

ATO + LULU

(d)







Acknowledgments


References









































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of








































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Research Article Lutein Has a Protective Effect on...

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