Research Article Rutin Attenuates Hepatotoxicity in High...

Research ArticleRutin Attenuates Hepatotoxicity inHigh-Cholesterol-Diet-Fed Rats

Shakir D AlSharari1 Salim S Al-Rejaie1 Hatem M Abuohashish12

Mohamed M Ahmed1 and Mohamed M Hafez1

1Department of Pharmacology and Toxicology College of Pharmacy King Saud University PO Box 2457 Riyadh 11451 Saudi Arabia2Department of Biomedical Dental Sciences College of Dentistry University of Dammam Dammam 31441 Saudi Arabia

Correspondence should be addressed to Mohamed M Hafez mohhafez 2000yahoocom

Received 7 December 2015 Revised 29 February 2016 Accepted 10 March 2016

Academic Editor Reiko Matsui

Copyright copy 2016 Shakir D AlSharari et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Background and Objective High-cholesterol diet (HCD) intends to increase the oxidative stress in liver tissues inducinghepatotoxicity Rutin is a natural flavonoid (vitamin p) which is known to have antioxidative propertiesThe aim of the present studywas to investigate the potential effects of Rutin on hypercholesterolemia-induced hepatotoxicity in rats Materials and MethodsMale Wistar rats were divided into four groups G-I control G-II Rutin G-III HCD and G-IV Rutin + HCD The liver functionsand lipid profile were used to evaluate the HCD-induced hepatotoxicity Quantitative real time-PCR was carried out to evaluatethe expression levels of genes in TGF-120573Smad signaling pathway Results Rutin in combination with HCD showed a significantprotective effect against hepatotoxicity HCD caused significant increase in the mRNA expression of transforming growth factorbeta (TGF-120573)MothersAgainstDecapentaplegicHomolog 2 (Smad-2)MothersAgainstDecapentaplegicHomolog 4 (Smad-4) Bcl-2-binding component 3 (Bbc3) caspase-3 P53 and Interleukin-6 (IL-6) and decrease in the expression levels of Cyclin dependedkinase inhibitor (P21) and Interleukin-3 (IL-3) in hepatic cells Conclusion TGF-120573Smad signaling pathway is involved in HCD-induced hepatotoxicity and Rutin inhibits the hepatotoxicity via suppressing this pathwayTherefore Rutin might be considered asa protective agent for hepatotoxicity

1 Introduction

Nonalcoholic steatohepatitis (NASH) is a form of chronicliver disease and a part of nonalcoholic fatty liver disease(NAFLD) which may lead to cirrhosis and hepatocellularcarcinoma (HCC) [1ndash7] The prevalence of NAFLD hassignificantly increased worldwide with the increase of obesity[4 8] Investigating the mechanisms of NAFLD may helpin finding the strategies against this disease [9] Oxidativestress induced by high-cholesterol diet can mediate a varietyof cellular responses leading to diverse outcomes such asapoptosis [10 11] which is involved in NASH causation [12]

Apoptosis occurs in many human liver disorders [13 14]and can trigger cell repair inflammation regeneration andfibrosis [15] Liver fibrosis may result in cirrhosis and end-stage liver disease [16 17] The uncontrolled hepatocyte apo-ptosis may be a central mechanism triggering liver fibro-genesis [18] The hepatocyte is specific genetic disruption

of the antiapoptotic member of the Bcl-2 family Bcl-xLresulting in hepatocyte apoptosis and liver fibrotic responses[19] Engulfment of apoptotic bodies by hepatic stellate cells(HSCs) stimulates fibrogenic activity [20] and theDNA fromapoptotic hepatocytes can act as an important mediator ofHSC activation and differentiation [21]

The TGF-120573 cytokine is involved in cell survival prolifera-tion differentiation and angiogenesis [13 14]TGF-120573 bindingto its receptor causes recruitment and phosphorylation ofotherTGF-120573 receptors that could activate the SmadpathwaysThe initiation of TGF-120573Smad signaling pathway started bythe formation of heteromeric receptor complexes [22 23]that lead to phosphorylation of Smad-2 and Smad-3 and thenthe formation of a complex with Smad4The phosphorylatedSmad-2 and Smad-3 associate with Smad-4 and then enterthe nucleus to regulate gene transcription [17] Further-more Smad proteins are mediators for the TGF-120573-induced

Hindawi Publishing CorporationOxidative Medicine and Cellular LongevityVolume 2016 Article ID 5436745 11 pageshttpdxdoiorg10115520165436745

2 Oxidative Medicine and Cellular Longevity

apoptosis [24] Transforming growth factor-1205731 is formedmainly by fibroblast and contributes to fibrosis developmenthypertrophy and apoptosis [25] The role of TGF-120573Smadsignaling in hepatocytes in the development of NASH isnot well understood and its role in metabolic disease is stilllimited In liver TGF-120573 signaling participates in fibrogenicresponse through hepatic stellate cell activation [26] Inchronic liver diseases HSCs are primary target for activeTGF-120573 and thus it plays a role in progression of fibrosis inadvanced NAFLD Procollagen-120572

1and procollagen-120572

2 tis-

sue inhibitor of metalloproteinase-1 and metalloproteinase-2(TIMP-1 and TIMP-2) and plasminogen activator inhibitor-1 (PAI-1) are identified as direct TGF-120573 target genes inHSCs [27ndash29] However the transcriptional activation ofmyofibroblast markers 120572-smooth muscle actin (SMA) andconnective tissue growth factor (CTGF) is induced in a TGF-120573-independent manner Instead TGF-120573 signaling is requiredfor 120572-SMA organization and stress-fiber formation [30]

Heme oxygenase-1 (HO-1) has protective activity againstacute and chronic liver injury [31] and is regulated by Nrf2transcription factor [32] Studies found that during hepaticinjury induced by oxidative stress HO-1 and Nrf2 weredownregulated and were associated withNF-120581B upregulation[33 34] The Rutin administration results in Nrf2 HO-1and NF-120581B overexpression Rutin acts as HO-1 inducer inliver ischemia-reperfusion injury rat model [35] The nucleartranslocation of HO-1 could regulate the genes responsiblefor cytoprotection against oxidative stress [36] The releaseof ROS is known to activate inhibitory kappa-B kinasewhich causes phosphorylation of I120581B Release of free NF-120581Benhances the inflammatory cytokines [37] and suppresses theantioxidant genes by downregulating Nrf-2HO-1 pathwayRutin is capable of inhibiting NF-120581B and activating the Nrf-2pathway Sirtuin 1 (Sirt1) a member of sirtuin proteins familydownregulation is associated with high insulin resistance andloss in mitochondrial biogenesis cells [38] High-cholesteroldiet decreases genes expression levels that are involvedin muscle mitochondrial biogenesis and function such asSirt1 peroxisome proliferator-activated receptor coactivator-1 (PGC-1) and mitochondrial transcription factor A (Tfam)[39] Rutin increases the expression levels of Sirt1 PGC-1and Tfam in skeletal muscle and brain of mice which leadto increase in muscle mitochondrial biogenesis and function[40] Therefore the antiobesity property of Rutin mightbe associated with Rutin-mediated muscle mitochondrialchanges

Flavonoids are polyphenolic compounds found in plantsand have an important role in detoxification of free radicals[41] Rutin is a flavonoid glycoside that possessed differ-ent protective effects [42 43] against lipid peroxidationand oxidative-stress-mediated diseases [44] Therefore thepresent study was aimed to investigate the preventive effectof Rutin against HCD-induced hepatotoxicity in rats throughstudying genes expression in the TGF-120573Smad pathways

2 Materials and Methods

21 Animals FortymaleWistar albino rats weighing between80 and 180 g were obtained from the Animal Care Center

College of Pharmacy King Saud University Riyadh SaudiArabia The animals were acclimatized to laboratory con-dition ten days prior to the experiment They were fedon Purina rat chow diet (manufactured by Grain Silos ampFlour Mills Organization Riyadh Saudi Arabia) and wateron a free access basis and were maintained under standardconditions of temperature (22 plusmn 1∘C) humidity (50ndash55)and 12 h lightdark cycles All methods including euthanasiaprocedure were conducted in accordance with guide forcare and use of laboratory animals Institute for LaboratoryAnimal Research National Institute of Health (NIH publi-cation number 80-23 1996) and they have been approvedby Research Ethics Committee of Excremental Animal CareCenter College of Pharmacy King Saud University RiyadhSaudi Arabia

22 Diets Diets were prepared in pellet form by adding 02Rutin (RT) (powder Sigma USA) or 1 cholesterol + 05cholic acid (HCD) or 02 RT + 1 cholesterol + 05 cholicacid (RT + HCD) in rat chow powder Rat chow was used asnormal diets and was prepared weekly and shade dried Foodintake gday was calculated daily for each group to the end ofexperiments

23 Experimental Design The animals were randomlydivided into 4 groups of 10 rats in each as follows Group Icontrol Group II RT Group III HCD and Group IV RT +HCD The experimental diets were fed on a free access basisfor 6 consecutive weeks At end of the experiment rats wereweighed and sacrificed by decapitation and the trunk bloodwas collected in heparinized tubes Rats livers were rapidlyexcised weighed and kept at minus80∘C till analysis Plasmasamples were collected after centrifugation and stored atminus20∘C till analysis

24 Bioassay Measurements Plasma levels of ALT AST TGTC HDL and LDL were estimated by using commerciallyavailable diagnostic kits (Human Wiesbaden Germany)

25 Measurement of Caspase-3 Activity in the Liver TissuesThe caspase-3 activity was measured in liver tissues by usingcolorimetric assay ab39401 (Abcam Cambridge MA 02139-1517 USA) kit according to the manufacturer instructions Inbrief 02mg of liver tissues was not homogenized completelyin 50120583L cell lysis buffer on ice for 10 minutes After centrifu-gation the protein concentrationswere adjusted to 50ndash200120583gprotein per reaction Fifty microlitres of caspase reactionmixcontaining 10mMDTTwas added to eachwell Two hundredmicromolars of DEVD-p-NA substrate was added and thenafter 120min incubation at 37∘C the plate was measured at405 nm Fold increase in caspase-3 activity can be determinedby comparing sample (treated) results with the level of theuntreated control

26 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Gene expression levels of TGF-120573 Smad-2 Smad-4 caspase-3 P21 P53 IL-3 and IL-6 were detected inliver tissues by quantitative real-time PCR Total RNA were

Oxidative Medicine and Cellular Longevity 3

Table 1 Primer sequence used in this study

Gene name Forward primer Reverse primerTGF-120573 51015840-TGCCTGACGGTCAGGTCA-31015840 51015840-CAGGAAGGAAGGCTGGAG-31015840

Smad-2 51015840-CCATCCCCGAGAACACTAACTT-31015840 51015840-TGGTGGTCGCTAGTTTCTCCAT-31015840

Smad-4 51015840-ACCAACTTCCCCAACATTCCT-31015840 51015840-ACTATGGCTGCCTGCCAGAA-31015840

Caspase-3 51015840-AAATTCAAGGGACGGGTCATG-31015840 51015840-GAGCTTGTGCGTACAGTT-31015840

Bbc3 51015840-AGCCAAACCTGACCACTAGC-31015840 51015840-CCAGATGAAGGTGAGGCAGG-31015840

P53 51015840-CACCATGAGCGTTGCTCTGAT-31015840 51015840-GATTTCCTTCCACCCGGATAA-31015840

P21 51015840-GAGAACTGGGGAGGGCTTTC-31015840 51015840-TCCTGAGCCTGTTTCGTGTC-31015840

IL-3 51015840-TTGTATTCCTGCAGCTGCGA-31015840 51015840-GGGCTGAGGTGGTCTAGAGA-31015840

IL-6 51015840-ATCTGCCCTTCAGGAACAGC-31015840 51015840-AGCCTCCGACTTGTGAAGTG-31015840

120573-actin 51015840-TGCCTGACGGTCAGGTCA-31015840 51015840-CAGGAAGGAAGGCTGGAAG-31015840

extracted from liver tissues using RNA Mini Kit (BiolineTaunton USA) according to the manufacturerrsquos protocolThe quantity and integrity of total RNA were characterizedusing a UV spectrophotometer (Nanodrop 8000 ThermoScientific USA) and ethidium bromide stained agarosegel The isolated RNA has an A 260280 ratio of 19ndash20First-strand cDNA was synthesized from 1120583g of total RNAby reverse transcription with a SuperScript first-strandsynthesis system kit (Invitrogen CA USA) according tothe manufacturerrsquos instructions Quantitative real-time PCRusing ΔΔCT method was done according to our previousstudy [10]120573-actin gene is used as internal control All primersused in this study were listed in Table 1

27 Statistical Analysis Differences between obtained values(meanplusmn SEM 119899 = 10) were carried out by oneway analysis ofvariance followed by theTukey-Kramermultiple comparisonThe differences were considered statistically significant at119875 lt005

3 Results

As in our previous study [10] the liver enzymes ALTand AST plasma levels were used as biochemical markersfor early acute hepatotoxicity Rats fed with HCD for 6weeks had significant increase in ALT (Figure 1(a)) and AST(Figure 1(b)) levels (119875 lt 0001) compared to control andHCD + Rutin groups Rutin supplementation alone showedno significant changes in biochemical markers Howeveradministration of Rutin in combination with HCD resultedin reversal of hepatic damage biomarker induced by HCD tothe values as in control group These reversal changes weresignificant compared to HCD group

The effect of HCD on the body and liver weights as well asthe food intake during the experiment was shown in Figure 2HCDsignificantly increased the liverweight (Figure 2(a)) andthe body weight (Figure 2(b)) compared to control and Rutingroups On the other hand there was no significant differenceobserved in food intake during experiment among all studiedgroups (Figure 2(c)) The supplementation of 02 Rutin or1 cholesterol + 05 cholic acid (HCD) or 02 RT + 1cholesterol + 05 cholic acid (RT + HCD) to the food didnot influence the food intake among all groups

The effects of HCD on the lipid parameters including TGTC HDL and LDL levels were shown in Figure 3 High-cholesterol diet significantly increased plasma levels of TG(Figure 3(a)) TC (Figure 3(b)) and LDL (Figure 3(c)) by48 89 and 67 respectively and significantly decreasedthe HDL (Figure 3(d)) levels by 17 compared to controlgroup Rutin supplementation in combination withHCD sig-nificantly increase TG TC and LDL levels and insignificantlydecreased plasma levels of HDL compared to control groupOn the other hand there were no significant differencesobserved in the plasma lipids levels (TG TC HDL and LDL)in RT group compared to control group

The expression levels of TGF-120573 Smad-2 Samd-4 P21caspase-3 and P53 genes were studied in rats hepatic cellsto investigate whether HCD stimulates the TGF-1205731 signalingcascade that leads to liver fibrosis and apoptosisThe effects ofHCD Rutin and their combination on TGF-1205731 Smad-2 andSmad-4 expression levels were shown in Figure 4 In HCDgroup the expression levels of TGF-120573 (Figure 4(a)) Smad-2 (Figure 4(b)) and Smad-4 (Figure 4(c)) were significantlyincreased by 8- 85- and 53-fold compared to the controlgroup On the other hand administration of Rutin in com-bination with HCD induced a significant repair of the HCD-induced alteration in the gene expression of TGF-120573 Smad-2and Smad-4 compared to the HCD group expression levels

Figure 5 shows the effect of HCD Rutin and theircombination on the expression level of caspase-3 (a) P53 (b)P21 (c) and Bbc3 (d) and on the caspase-3 activity (e) inrat liver tissues High-cholesterol diet resulted in significantincreases in expression levels of caspase-3 P53 and Bbc3 by62- 53- and 68-fold respectively compared to the controlgroup Interestingly administration of Rutin in combinationwith HCD resulted in a complete reversal change of caspase-3 P53 and Bbc3 induced by HCD to the normal expressionlevels as in control groupThe activity of caspase-3 cleavage inliver tissues of control rats which feed on rat chow was hardlydetectable while a significant increase by 289 and 192 inthe levels of cleavage caspase-3 was observed in the HCD-fed rat compared to control and Rutin group respectivelyThe Rutin in combination with HCD significantly decreasedthe levels of cleavage caspase-3 by 62 compared to HCDgroup On the other hand HCD suppressed the expressionlevel of P21 by 285-fold while administration of Rutin


0

10

20

30

40 lowast$A

LT (U

L)

HCD + RutinHCDRutinControl(a)

lowast$

0

20

40

60

AST

(UL

)

HCD + RutinHCDRutinControl(b)

Figure 1 The effect of HCD Rutin and their combination on the plasma levels of ALT (a) and AST (b) in rats Data are presented as mean plusmnSEM (119899 = 10) lowast and $ indicate significant change from control Rutin and HCD + Rutin respectively at 119875 lt 005 using ANOVA followedby Tukey-Kramer as a post-ANOVA test

0

1

2

3

4

5lowast

Rutin + HCD HCDRutinControl

g liv

er100

g bo

dy w

eigh

t

(a)

0

50

100

150

200

Body

wei

ght i

ncre

ased

(g) lowast

HCDRutinControl Rutin + HCD (b)

0

10

20

30

Food

inta

ke (g

day

)

Rutin + HCD HCDRutinControl(c)

Figure 2 The effect of HCD Rutin and their combination on the liver weight (a) body weight (b) and food intake (c) in rats Data areexpressed as mean plusmn SEM (119899 = 10) lowast and indicate significant change from control Rutin respectively at 119875 lt 005 using ANOVA followedby Tukey-Kramer as a post-ANOVA test

in combination with HCD resulted in a complete reversalchange of P21 induced by HCD to values as in control group

Figure 6 shows the effects of HCD Rutin and theircombination on the expression level of IL-3 (a) and IL-6 (b) inrat liver tissues The expression level of IL-6 was significantlyincreased by 58-fold in rats fed with HCD compared to

control group On the other hand rats fed with HCD haddecreases in the expression levels of IL-3 by 3-fold comparedto control group Interestingly supplementation of Rutinin combination with HCD resulted in a complete reversalchange of IL-6 and IL-3 induced byHCD to the normal valuesas in control group


0

50

100

150Tr

igly

cerid

es (m

gdL

)

lowastlowast


0

50

100

150

Tota

l cho

leste

rol (

mg

dL)

lowast


0

20

40

60

80

100

LDL

(mg

dL)

HCD + RutinHCDRutinControl

lowastlowast

(c)

0

10

20

30

40

HD

L (m

gdL

)


lowast

(d)

Figure 3 The effect of HCD Rutin and their combination on the plasma levels of triglyceride (a) total cholesterol (b) high-densitylipoprotein (c) and low-density lipoprotein (d) in rats Data are presented as mean plusmn SEM (119899 = 10) lowast and indicate significant changefrom control and Rutin groups respectively at 119875 lt 005 using ANOVA followed by Tukey-Kramer as a post-ANOVA test

4 Discussion

Obesity hypertriglyceridemia andor hypercholesterolemiaare the common causes for many diseases such as cardiovas-cular [45] and liver diseases [46 47] Rat fedwithHCDcan beused as model of the human obesity syndrome [48] In liverdisease cell repair inflammation regeneration and fibrosismay all be triggered by apoptosis [13 14] Nonalcoholicfatty liver disease can end in cirrhosis and hepatocellularcarcinoma [6] The present study examined the hepatopro-tective effect of Rutin against hepatotoxicity induced byHCD in rat model The HCD caused hepatotoxicity throughincreasing liver enzymes ALT and AST In agreement withearlier studies the elevated ALT andAST levels are attributedto hepatic damage that may contribute to oxidative stressunbalance [45 49] Earlier it is reported that Rutin haspotential to reduce the oxidative stress in liver kidney andbrain tissues of rats [50] As a result of Rutin supplementationALT and AST levels were lowered leading to decrease in thehepatic damage caused by HCD feedings The present studyshowed that Rutin can protect hepatocyte against the toxicityinduced by HCD

High-cholesterol diet leads to dyslipidemic syndromeand hyperlipidemia that is characterized by increase in TG

and decrease in HDL-cholesterol [51] In the current studyhigh-cholesterol diet led to TG increase andHDL-cholesteroldecrease The accumulation of excess triglycerides in hep-atocytes forms steatotic droplets in NAFLD [52 53] Thehyperlipidemia hypertriglyceridemia and the decreasing inHDL-cholesterol increase the risk of NAFLD [54] In thepresent study Rutin supplement ameliorated the effect ofHCD by lowering TC TG and LDL levels Similarly Rutindecreased the lipid in hypercholesterolemic rats by reducingthe activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase[55] Also Rutin has a strong ability to chelate multivalentmetal ions especially zinc calcium and iron [56]

The immunoregulatory cytokine such as IL-6 is a mul-tifunctional cytokine that regulates some biological processand may play essential role ranging from inflammation tohost defense toward tissue injury [57] Interleukin-6 withTNF120572 stimulates hepatic lipogenesis [58] impairs insulinsignaling [59] and can trigger different key steps in insulinsignaling pathway [60] In the current study HCD increasedIL-6 expression level in NAFLD compared to control groupSimilar study found that TNF-120572 and IL-6 levels were sig-nificantly increased in NAFLD patients [61] Interleukin-6 helps the hepatic survival by stimulating liver recoveryand gives hepatoprotection [62 63] Binding of IL-6 to its


0

5

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15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$

HCD + RutinHCDRutinControl(c)

Figure 4 The effect of HCD Rutin and their combination on the levels of TGF-120573 (a) Smad-2 (b) and Smad-4 (c) expression level in ratsliver tissues Data are presented as mean plusmn SEM (119899 = 10) lowast and $ indicate significant change from control Rutin and HCD plus Rutinrespectively at 119875 lt 005 using ANOVA followed by Tukey-Kramer as a post-ANOVA test

receptor (IL-6R) prompts STAT3 pathway activation throughbinding to glycoprotein 130 (gp130) IL-6 acts as both pro-and anti-inflammatory cytokines and may mediate liverdamage through different pathways Elevated levels of IL-6 are associated with disease states [64] The soluble formof IL-6R in addition to membrane-bound receptor bindsto IL-6 and prolongs its plasma half-life [65] The solubleIL-6R has roles in cellular proliferation differentiation andactivation of inflammatory responses [66 67] In the currentstudy downregulated IL-6 expression level as a result ofRutin administration may lead to the attenuation of the diet-induced hepatotoxicity Rutin possesses anti-inflammatoryeffect by downregulating IL-6 and TGF-1205731 which promotesextracellular matrix deposition and fibrosis [68] It has beenreported that Rutin produced anti-inflammatory effects byinhibiting proinflammatory cytokines in adjuvant-inducedarthritis in rats [69]

IL-3 cytokine is important for cell survival proliferationand differentiation [70 71] and participates in the responseto stress [72] IL-3 may be involved in alcohol induced liverinjury with unclear role [73] In the current study significantreduction in IL-3 was observed following HCD feedingThe downregulation of IL-3 in HCD group suggested thepotential involvement of IL-3 in HCD-induced liver injury

Recent study reported that the downregulation in IL-3 isassociatedwith liver injury [74] Similarly Sheng et al found adecrease in level of IL-3 in rat treated withDioscorea bulbifera[74]

TGF-1205731 gene downregulation can suppress the liver fibro-sis and apoptosis [75] In the present study HCD expresseshigh levels of hepatic TGF-1205731 Smad-2 and Smad-4 geneexpression These findings suggest the possible involvementof TGF-1205731 Smad-2 and Smad-4 genes in the regulationof hepatotoxicity process Phosphorylation of Smad-2 andSmad-3 byTGF-120573 leads to Smad-4 translocation into nucleusIn the nucleus Smad-4 functions to regulate transcriptionof target genes including P21 and proapoptotic Bcl-2 family[76] TGF-1205731Smad-2 signaling is responsible for insulinregulation and pancreatic 120573-cell function [77] The effectsof Smad genes on obesity and type 2 diabetes are not wellunderstood In the current study the HCD increased Smad-2 and Smad-4 expression levels The role of Smad-2 in liverfibrosis is less well characterized but in comparison withSmad-3 seems to regulate a distinct set of target genes [78 79]The combination of Rutin with HCD has downregulated theSmad-2 and Smad-4 gene expression levels These resultsindicated that Rutin exerted a negative regulatory effecton hepatotoxicity and fibrogenesis TGF-1205731Smad pathway


0

2

4

6

8caspase-3

relat

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n lowast$


0

2

4

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8

P53

relat

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n


lowast

(b)

00

05

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25

P21

relat

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0

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4

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10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)

Figure 5 The effect of HCD Rutin and their combination on the expression levels of caspase-3 (a) P53 (b) P21 (c) Bbc3 (d) and cleavagecaspase-3 (e) in rat liver tissues Data are presented as mean plusmn SEM (119899 = 10) lowast and $ indicate significant change from control Rutin andHCD plus Rutin respectively at 119875 lt 005 using ANOVA followed by Tukey-Kramer as a post-ANOVA test

and IL-6 are extensively studied as mutagenic and fibroticfactors and appear to be critical for matrix-producing cellsactivation TGF-1205731 is a fibrosis mediator which can enhanceextracellular matrix deposition by activating stellate cells toturn on fibrosis gene expression and by inhibiting collagenaseactivity via Smad-2-3 signaling [26] Thus they might beused in liver fibrosis treatment

Tumor suppressor gene P53 plays a central role in fattyliver disease [80ndash82] and its activation may be important in

fatty liver pathogenesis that facilitates apoptosis oxidativestress steatosis and injury The regulatory control of P53appears to fail in fatty liver disease [83] as a direct down-stream target of P53 [84] In the current study HCD increasesP53 expression level This increase in P53 expression levelin HCD may suggest that P53 activation may be involved inliver injuryThis finding was in agreement with another studythat found a positive correlation between steatosis and P53expression in human liver samples [80]


00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)

Figure 6The effect of HCD Rutin and their combination on the expression levels of Interleukin-3 (a) and Interleukin-6 (b) in rat liver tissuesData are presented as mean plusmn SEM (119899 = 10) lowast and $ indicate significant change from control Rutin and HCD plus Rutin respectively at119875 lt 005 using ANOVA followed by Tukey-Kramer as a post-ANOVA test

Increases in the P53 expression induce CTGF result inliver fibrosis in rodent and human [85] In the current studythe increase in the expression levels of P53 is associatedwith increase in the expression of Bbc3 Bbc3 interacts withantiapoptotic Bcl-2 family members such as Bcl-xL Bcl-2Mcl-1 Bcl-w and A1 inhibiting their interaction with Baxand Bak which leads to release of apoptosis-inducing factorand mitochondrial apoptotic proteins cytochrome c leadingto caspase activation and cell death [86]

The stage of liver steatosis increased the expression ofP53 in hepatocytes Levels of P21 often determine the cellularresponse to different stress induced by drug and others In thecurrent study HCD induce reduction in the expression levelof P21 Reduction of endogenous P21 expression attenuatesthe growth arrest and promotes cell death Similarly anotherstudy reported that the reduction in the expression levelof P21 in MCF7 cells by antisense P21 RNA attenuates thegrowth arrest and promotes cell death [87] Different types ofcellular stress lead to induction ofP21 expression by bothP53-dependent and -independent mechanisms High expressionofP21-induced growth arrest can protect cells from apoptosisHowever P21 possesses proapoptotic functions and may playan active role in apoptosis induced by activation of membersof the TNF family of death receptors Activated TNF120572CD95interacts with one or several cell surface receptors to triggercaspase activation and cytochrome c release In the cur-rent study HCD induce activation of caspase-3 Caspase-3-mediated cleavage of P21 and subsequent upregulation ofcyclin ACdk2 activity is considered an important mecha-nism for death associated with cyclin ACdk2 activation indifferent cell types

During hepatic injury induced by oxidative stress theHO-1 and Nrf2 expression levels were downregulated andwere associated with upregulation in NF-120581B [33 34] Over-expression of these transcription factors acts to promoteapoptosis The Rutin administration increased Nrf2 andHO-1 expression levels and suppressed the expression levels ofNF-120581B In addition to oxidative stress high-cholesterol diet

decreases the expression levels of genes involved in musclemitochondrial biogenesis and function such as Sirt1 [39]Themechanisms of liver apoptosis in NAFLD induced by HCDneed more study to understand the mechanism

5 Conclusion

In conclusion our findings demonstrated that Rutin couldlower and attenuate hepatotoxicity induced by HCD in ratmodel Signaling molecules in the TGF-120573Smad signalingpathway were restored to nearly their normal levels Consid-ering its excellent safety profile we propose that Rutin couldbe used in people at high risk of developing hepatotoxicityinduced by HCD

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

The authors extend their appreciation to the Deanship ofScientific Research at King Saud University for funding thiswork through Research Project no NFG-14-02-13

References

[1] J M Clark ldquoThe epidemiology of nonalcoholic fatty liverdisease in adultsrdquo Journal of Clinical Gastroenterology vol 40no 1 pp S5ndashS10 2006

[2] J P Ong andZM Younossi ldquoEpidemiology and natural historyof NAFLD and NASHrdquo Clinics in Liver Disease vol 11 no 1 pp1ndash16 2007

[3] M Lazo and J M Clark ldquoThe epidemiology of nonalcoholicfatty liver disease a global perspectiverdquo Seminars in LiverDisease vol 28 no 4 pp 339ndash350 2008

[4] J-G Fan andGC Farrell ldquoEpidemiology of non-alcoholic fattyliver disease in Chinardquo Journal of Hepatology vol 50 no 1 pp204ndash210 2009


[5] A St George A Bauman A Johnston G Farrell T Chey andJ George ldquoIndependent effects of physical activity in patientswith nonalcoholic fatty liver diseaserdquo Hepatology vol 50 no 1pp 68ndash76 2009

[6] L Gan S Chitturi and G C Farrell ldquoMechanisms and impli-cations of age-related changes in the liver nonalcoholic fattyliver disease in the elderlyrdquo Current Gerontology and GeriatricsResearch vol 2011 Article ID 831536 12 pages 2011

[7] J Manchanayake S Chitturi C Nolan and G C FarrellldquoPostprandial hyperinsulinemia is universal in non-diabeticpatients with nonalcoholic fatty liver diseaserdquo Journal of Gas-troenterology and Hepatology vol 26 no 3 pp 510ndash516 2011

[8] J-G Fan ldquoEpidemiology of alcoholic and nonalcoholic fattyliver disease in Chinardquo Journal of Gastroenterology and Hepa-tology vol 28 supplement 1 pp 11ndash17 2013

[9] G C Farrell and C Z Larter ldquoNonalcoholic fatty liver diseasefrom steatosis to cirrhosisrdquo Hepatology vol 43 no 2 supple-ment 1 pp S99ndashS112 2006

[10] S S Al-Rejaie A M Aleisa M M Sayed-Ahmed et alldquoProtective effect of rutin on the antioxidant genes expressionin hypercholestrolemic male Westar ratrdquo BMC Complementaryand Alternative Medicine vol 13 article no 136 2013

[11] B Wang J Sun Y Ma G Wu Y Shi and G Le ldquoIncreasedoxidative stress and the apoptosis of regulatory T cells in obesemice but not resistant mice in response to a high-fat dietrdquoCellular Immunology vol 288 no 1-2 pp 39ndash46 2014

[12] A E Feldstein A Canbay P Angulo et al ldquoHepatocyte apo-ptosis and Fas expression are prominent features of humannonalcoholic steatohepatitisrdquo Gastroenterology vol 125 no 2pp 437ndash443 2003

[13] P S Ribeiro H Cortez-Pinto S Sola et al ldquoHepatocyteapoptosis expression of death receptors and activation of NF-120581B in the liver of nonalcoholic and alcoholic steatohepatitispatientsrdquo American Journal of Gastroenterology vol 99 no 9pp 1708ndash1717 2004

[14] A E Feldstein and G J Gores ldquoApoptosis in alcoholic andnonalcoholic steatohepatitisrdquo Frontiers in Bioscience vol 10 no3 pp 3093ndash3099 2005

[15] M E Guicciardi and G J Gores ldquoApoptosis a mechanism ofacute and chronic liver injuryrdquoGut vol 54 no 7 pp 1024ndash10332005

[16] H Kayadibi E Sertoglu and M Uyanik ldquoWhich diagnostictool for the prediction of intermediate liver fibrosis stagesrdquoEuropean Journal of Gastroenterology and Hepatology vol 27no 2 p 182 2015

[17] C F Subasi U E Aykut and Y Yilmaz ldquoComparison ofnoninvasive scores for the detection of advanced fibrosis inpatients with nonalcoholic fatty liver diseaserdquo European Journalof Gastroenterology and Hepatology vol 27 no 2 pp 137ndash1412015

[18] A Canbay S Friedman and G J Gores ldquoApoptosis the nexusof liver injury and fibrosisrdquo Hepatology vol 39 no 2 pp 273ndash278 2004

[19] T Takehara T Tatsumi T Suzuki et al ldquoHepatocyte-specificdisruption of Bcl-x L leads to continuous hepatocyte apoptosisand liver fibrotic responsesrdquoGastroenterology vol 127 no 4 pp1189ndash1197 2004

[20] A Canbay P Taimr N Torok H Higuchi S Friedman and GJ Gores ldquoApoptotic body engulfment by a human stellate cellline is profibrogenicrdquo Laboratory Investigation vol 83 no 5 pp655ndash663 2003

[21] AWatanabe A Hashmi D A Gomes et al ldquoApoptotic hepato-cyte DNA inhibits hepatic stellate cell chemotaxis via toll-likereceptor 9rdquo Hepatology vol 46 no 5 pp 1509ndash1518 2007

[22] Y Shi and J Massague ldquoMechanisms of TGF-120573 signaling fromcell membrane to the nucleusrdquo Cell vol 113 no 6 pp 685ndash7002003

[23] G Euler-Taimor and J Heger ldquoThe complex pattern ofSMAD signaling in the cardiovascular systemrdquo CardiovascularResearch vol 69 no 1 pp 15ndash25 2006

[24] D Schneiders J Heger P Best H M Piper and G TaimorldquoSMAD proteins are involved in apoptosis induction in ventric-ular cardiomyocytesrdquoCardiovascular Research vol 67 no 1 pp87ndash96 2005

[25] B Wang J Hao S C Jones M-S Yee J C Roth and IM C Dixon ldquoDecreased Smad 7 expression contributes tocardiac fibrosis in the infarcted rat heartrdquo American Journal ofPhysiologymdashHeart and Circulatory Physiology vol 282 no 5pp H1685ndashH1696 2002

[26] W Huang L Li X Tian et al ldquoAstragalus and Paeoniaeradix rubra extract inhibits liver fibrosis by modulating thetransforming growth factor120573Smad pathway in ratsrdquoMolecularMedicine Reports vol 11 no 2 pp 805ndash814 2015

[27] H Herbst T Wege S Milani et al ldquoTissue inhibitor ofmetalloproteinase-1 and -2 RNA expression in rat and humanliver fibrosisrdquoTheAmerican Journal of Pathology vol 150 no 5pp 1647ndash1659 1997

[28] N Nieto J A Dominguez-Rosales L Fontana et al ldquoRathepatic stellate cells contribute to the acute-phase responsewith increased expression of 1205721 (I) and 1205721 (IV) colla-gens tissue inhibitor of metalloproteinase-1 and matrix-metalloproteinase-2 messenger RNAsrdquo Hepatology vol 33 no3 pp 597ndash607 2001

[29] S Kurita K Takeuchi Y Hayashi et al ldquoSignificance ofserumZn-1205722-glycoprotein for the regulation of blood pressurerdquoHypertension Research vol 38 no 4 pp 244ndash251 2015

[30] S Dooley J Hamzavi L Ciuclan et al ldquoHepatocyte-specificSmad7 expression attenuates TGF-120573-mediated fibrogenesis andprotects against liver damagerdquo Gastroenterology vol 135 no 2pp 642ndash659 2008

[31] G Sass R Barikbin and G Tiegs ldquoThe multiple functions ofheme oxygenase-1 in the liverrdquo Zeitschrift fur Gastroenterologievol 50 no 1 pp 34ndash40 2012

[32] Y-C Yang C-K Lii A-H Lin et al ldquoInduction of glutathionesynthesis and heme oxygenase 1 by the flavonoids butein andphloretin is mediated through the ERKNrf2 pathway andprotects against oxidative stressrdquo Free Radical Biology andMedicine vol 51 no 11 pp 2073ndash2081 2011

[33] Y H Choi ldquoThe cytoprotective effect of isorhamnetin againstoxidative stress is mediated by the upregulation of the Nrf2-dependent HO-1 expression in C2C12myoblasts through scav-enging reactive oxygen species and ERK inactivationrdquo GeneralPhysiology and Biophysics 2016

[34] Y H Choi ldquoThe cytoprotective effects of ethanol extractof Ecklonia cava against oxidative stress are associated withupregulation of Nrf2-mediated HO-1 and NQO-1 expressionthrough activation of the MAPK pathwayrdquo General Physiologyand Biophysics vol 35 no 1 pp 45ndash53 2016

[35] R Acquaviva R Lanteri G Li Destri et al ldquoBeneficial effectsof rutin and L-arginine coadministration in a rat model of liverischemia-reperfusion injuryrdquo American Journal of PhysiologymdashGastrointestinal and Liver Physiology vol 296 no 3 pp G664ndashG670 2009


[36] Q Lin SWeis G Yang et al ldquoHeme oxygenase-1 protein local-izes to the nucleus and activates transcription factors importantin oxidative stressrdquoThe Journal of Biological Chemistry vol 282no 28 pp 20621ndash20633 2007

[37] T Lawrence ldquoThenuclear factorNF-kappaB pathway in inflam-mationrdquo Cold Spring Harbor Perspectives in Biology vol 1 no 6Article ID a001651 2009

[38] J T Rodgers C Lerin W Haas S P Gygi B M Spiegelmanand P Puigserver ldquoNutrient control of glucose homeostasisthrough a complex of PGC-1120572 and SIRT1rdquo Nature vol 434 no7029 pp 113ndash118 2005

[39] R Bergeron J M Ren K S Cadman et al ldquoChronic activationof AMP kinase results in NRF-1 activation and mitochondrialbiogenesisrdquo American Journal of Physiology-Endocrinology andMetabolism vol 281 no 6 pp E1340ndashE1346 2001

[40] S Seo M-S Lee E Chang et al ldquoRutin increases musclemitochondrial biogenesis with AMPK activation in high-fatdiet-induced obese ratsrdquo Nutrients vol 7 no 9 pp 8152ndash81692015

[41] J D Potter ldquoCancer prevention epidemiology and experimentrdquoCancer Letters vol 114 no 1-2 pp 7ndash9 1997

[42] P N Aleksandrov T V Speranskaia G Bobkov Iu V AZagorevskii andDA Zykov ldquoEffect of rutin and esculamine onmodels of aseptic inflammationrdquo Farmakologiia i Toksikologiiavol 49 no 1 pp 84ndash86 1986

[43] E E Deschner J Ruperto G Wong and H L NewmarkldquoQuercetin and rutin as inhibitors of azoxymethanol-inducedcolonic neoplasiardquo Carcinogenesis vol 12 no 7 pp 1193ndash11961991

[44] A Lopez-Revuelta J I Sanchez-Gallego A Hernandez-Hernandez J Sanchez-Yague and M Llanillo ldquoMembranecholesterol contents influence the protective effects of quercetinand rutin in erythrocytes damaged by oxidative stressrdquoChemico-Biological Interactions vol 161 no 1 pp 79ndash91 2006

[45] S M Artham C J Lavie R V Milani and H O VenturaldquoThe obesity paradox impact of obesity on the prevalence andprognosis of cardiovascular diseasesrdquo Postgraduate Medicinevol 120 no 2 pp 34ndash41 2008

[46] S Bellentani G Saccoccio F Masutti et al ldquoPrevalence of andrisk factors for hepatic steatosis in northern Italyrdquo Annals ofInternal Medicine vol 132 no 2 pp 112ndash117 2000

[47] P Vitaglione F Morisco G Mazzone et al ldquoCoffee reducesliver damage in a rat model of steatohepatitis the underlyingmechanisms and the role of polyphenols and melanoidinsrdquoHepatology vol 52 no 5 pp 1652ndash1661 2010

[48] S K Das V Balakrishnan S Mukherjee and DM VasudevanldquoEvaluation of blood oxidative stress-related parameters inalcoholic liver disease and non-alcoholic fatty liver diseaserdquoScandinavian Journal of Clinical and Laboratory Investigationvol 68 no 4 pp 323ndash334 2008

[49] H-Y Song Z-M Mao L-L Yang et al ldquoDangfei liganningcapsules attenuate the susceptibility of rat nonalcoholic fattyliver to carbon tetrachloride toxicityrdquo Journal of TraditionalChinese Medicine vol 31 no 4 pp 327ndash333 2011

[50] N Kamalakkannan and P S M Prince ldquoAntihyperglycaemicand antioxidant effect of rutin a polyphenolic flavonoid instreptozotocin-induced diabetic wistar ratsrdquo Basic and ClinicalPharmacology and Toxicology vol 98 no 1 pp 97ndash103 2006

[51] J Halcox and A Misra ldquoType 2 diabetes mellitus metabolicsyndrome and mixed dyslipidemia how similar how differentand how to treatrdquo Metabolic Syndrome and Related Disordersvol 13 no 1 pp 1ndash21 2015

[52] Y-W Fu and H-B He ldquoApoptosis of periodontium cells instreptozototocin- and ligature-induced experimental diabeticperiodontitis in ratsrdquo Acta Odontologica Scandinavica vol 71no 5 pp 1206ndash1215 2013

[53] K N Otwombe F Laher T Tutu-Gxashe G Gray and LMohapi ldquoThe effect of a maturing antiretroviral program onearlymortality for patients with advanced immune-suppressionin Soweto South Africardquo PLoS ONE vol 8 no 11 Article IDe81538 2013

[54] A Dasgupta J Zheng N I Perrone-Bizzozero and O ABizzozero ldquoIncreased carbonylation protein aggregation andapoptosis in the spinal cord of mice with experimental autoim-mune encephalomyelitisrdquo ASN neuro vol 5 no 1 Article IDe00111 2013

[55] S Arumugam S Mito R A Thandavarayan et al ldquoMulberryleaf diet protects against progression of experimental autoim-munemyocarditis to dilated cardiomyopathy via modulation ofoxidative stress and MAPK-mediated apoptosisrdquo Cardiovascu-lar Therapeutics vol 31 no 6 pp 352ndash362 2013

[56] F Bayatli D Akkus E Kilic R Saraymen and M F SonmezldquoThe protective effects of grape seed extract on MDA AOPPapoptosis and eNOS expression in testicular torsion an exper-imental studyrdquoWorld Journal of Urology vol 31 no 3 pp 615ndash622 2013

[57] D A Papanicolaou R L Wilder S C Manolagas and GP Chrousos ldquoThe pathophysiologic roles of interleukin-6 inhuman diseaserdquo Annals of Internal Medicine vol 128 no 2 pp127ndash137 1998

[58] A J Thompson A J Muir M S Sulkowski et al ldquoInterleukin-28B polymorphism improves viral kinetics and is the strongestpretreatment predictor of sustained virologic response in geno-type 1 Hepatitis C virusrdquo Gastroenterology vol 139 no 1 pp120ndash129e18 2010

[59] R Valentino V DrsquoEsposito F Passaretti et al ldquoBisphenol-Aimpairs insulin action and up-regulates inflammatory pathwaysin human subcutaneous adipocytes and 3T3-L1 cellsrdquo PLoSONE vol 8 no 12 Article ID e82099 2013

[60] X B Cui Y Z Chen and F Li ldquoRecent advances on relationshipbetween phospholipase C epsilon-1 gene and tumorrdquo ZhonghuaBing Li Xue Za Zhi vol 41 no 3 pp 213ndash216 2012

[61] S K Das and V Balakrishnan ldquoRole of cytokines in thepathogenesis of non-alcoholic fatty liver diseaserdquo Indian Journalof Clinical Biochemistry vol 26 no 2 pp 202ndash209 2011

[62] M-D Jiang H-D Ma X-F Zhong F-W Xie andW-Z ZengldquoEffects of Erk signal transduction on the cell cycle of rat hepaticstellate cells stimulated by acetaldehyderdquo Zhonghua Gan ZangBing Za Zhi vol 11 no 11 pp 650ndash653 2003

[63] B Saile C Eisenbach H El-Armouche K Neubauer and GRamadori ldquoAntiapoptotic effect of interferon-120572 on hepatic stel-late cells (HSC) a novel pathway of IFN-120572 signal transductionvia Janus kinase 2 (JAK 2) and caspase-8rdquo European Journal ofCell Biology vol 82 no 1 pp 31ndash41 2003

[64] M A Ganaie T H Khan N A Siddiqui and M N AnsarildquoAmeliorative effect of methanol extract of Rumex vesicarius onCCl4-induced liver damage in Wistar albino ratsrdquo Pharmaceu-

tical Biology vol 53 no 8 pp 1163ndash1167 2015[65] M Peters K-H Meyer zum Buschenfelde and S Rose-John

ldquoThe function of the soluble IL-6 receptor in vivordquo ImmunologyLetters vol 54 no 2-3 pp 177ndash184 1996

[66] D Novick H Engelmann D Wallach and M RubinsteinldquoSoluble cytokine receptors are present in normal humanurinerdquo


Journal of Experimental Medicine vol 170 no 4 pp 1409ndash14141989

[67] M Honda S Yamamoto M Cheng et al ldquoHuman soluble IL-6receptor its detection and enhanced release by HIV infectionrdquoJournal of Immunology vol 148 no 7 pp 2175ndash2180 1992

[68] S S Edranov and P A Motavkin ldquoApoptosis as a mechanismof maxillary sinus mucosa injury in rats after experimentaltransection of the maxillary nerverdquo Bulletin of ExperimentalBiology and Medicine vol 153 no 4 pp 526ndash529 2012

[69] T Kauss D Moynet J Rambert et al ldquoRutoside decreaseshuman macrophage-derived inflammatory mediators andimproves clinical signs in adjuvant-induced arthritisrdquo ArthritisResearch andTherapy vol 10 no 1 article R19 2008

[70] M Eder G Geissler and A Ganser ldquoIL-3 in the clinicrdquo StemCells vol 15 no 5 pp 327ndash333 1997

[71] D E Johnson ldquoRegulation of survival pathways by IL-3 andinduction of apoptosis following IL-3 withdrawalrdquo Frontiers inBioscience vol 3 pp d313ndashd324 1998

[72] H Bessler M Bergman and H Salman ldquoInterleukin-3 andstressrdquo Biomedicine and Pharmacotherapy vol 54 no 6 pp299ndash304 2000

[73] X-G Zhang P Xu Q Liu et al ldquoEffect of tea polyphenol oncytokine gene expression in rats with alcoholic liver diseaserdquoHepatobiliary and Pancreatic Diseases International vol 5 no2 pp 268ndash272 2006

[74] Y Sheng Y Ma Z Deng Z Wang and L Ji ldquoCytokinesas potential biomarkers of liver toxicity induced by Dioscoreabulbifera Lrdquo Bioscience Trends vol 8 no 1 pp 32ndash37 2014

[75] R-M Liu and K A Gaston Pravia ldquoOxidative stress and glu-tathione in TGF-120573-mediated fibrogenesisrdquo Free Radical Biologyand Medicine vol 48 no 1 pp 1ndash15 2010

[76] J Schmutzhard R Glueckert C Pritz et al ldquoSepsis otopathyexperimental sepsis leads to significant hearing impairment dueto apoptosis and glutamate excitotoxicity in murine cochleardquoDisease Models amp Mechanisms vol 6 no 3 pp 745ndash754 2013

[77] J Zhao M Zhang and Y Chen ldquoExperimental research on themechanisms of human multiple myeloma LP-1 cell apoptosisinduced by oridoninrdquo Zhongguo Zhong Xi Yi Jie He Za Zhi vol32 no 12 pp 1642ndash1646 2012

[78] S Oksuz E Ulkur O Oncul G T Kose Z Kucukodac andM Urhan ldquoThe effect of subcutaneous mesenchymal stem cellinjection on statis zone and apoptosis in an experimental burnmodelrdquo Plastic and Reconstructive Surgery vol 131 no 3 pp463ndash471 2013

[79] S Velayuthaprabhu HMatsubayashi T Sugi et al ldquoExpressionof apoptosis in placenta of experimental antiphospholipid syn-drome mouserdquo American Journal of Reproductive Immunologyvol 69 no 5 pp 486ndash494 2013

[80] A Panasiuk J Dzieciol B Panasiuk and D ProkopowiczldquoExpression of p53 Bax and Bcl-2 proteins in hepatocytes innon-alcoholic fatty liver diseaserdquo World Journal of Gastroen-terology vol 12 no 38 pp 6198ndash6202 2006

[81] C S Lieber M A Leo X Wang and L M DeCarlildquoAlcohol alters hepatic FoxO1 p53 and mitochondrial SIRT5deacetylation functionrdquo Biochemical and Biophysical ResearchCommunications vol 373 no 2 pp 246ndash252 2008

[82] N Edebali I O Tekin B Acıkgoz et al ldquoApoptosis andnecrosis in the circumventricular organs after experimentalsubarachnoid hemorrhage as detected with annexin V andcaspase 3 immunostainingrdquo Neurological Research vol 36 no12 pp 1114ndash1120 2014

[83] J Lee A Padhye A Sharma et al ldquoA pathway involvingfarnesoid X receptor and small heterodimer partner positivelyregulates hepatic sirtuin 1 levels via MicroRNA-34a inhibitionrdquoJournal of Biological Chemistry vol 285 no 17 pp 12604ndash126112010

[84] H Hermeking ldquop53 enters the MicroRNA worldrdquo Cancer Cellvol 12 no 5 pp 414ndash418 2007

[85] T Kodama T Takehara H Hikita et al ldquoIncreases in p53expression induce CTGF synthesis by mouse and humanhepatocytes and result in liver fibrosis in micerdquo Journal ofClinical Investigation vol 121 no 8 pp 3343ndash3356 2011

[86] J Vavrova and M Rezacova ldquoImportance of proapoptoticprotein PUMA in cell radioresistancerdquo Folia Biologica vol 60no 2 pp 53ndash56 2014

[87] A L Gartel and A L Tyner ldquoThe role of the cyclin-dependentkinase inhibitor p21 in apoptosisrdquo Molecular Cancer Therapeu-tics vol 1 no 8 pp 639ndash649 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


apoptosis [24] Transforming growth factor-1205731 is formedmainly by fibroblast and contributes to fibrosis developmenthypertrophy and apoptosis [25] The role of TGF-120573Smadsignaling in hepatocytes in the development of NASH isnot well understood and its role in metabolic disease is stilllimited In liver TGF-120573 signaling participates in fibrogenicresponse through hepatic stellate cell activation [26] Inchronic liver diseases HSCs are primary target for activeTGF-120573 and thus it plays a role in progression of fibrosis inadvanced NAFLD Procollagen-120572

1and procollagen-120572

2 tis-

sue inhibitor of metalloproteinase-1 and metalloproteinase-2(TIMP-1 and TIMP-2) and plasminogen activator inhibitor-1 (PAI-1) are identified as direct TGF-120573 target genes inHSCs [27ndash29] However the transcriptional activation ofmyofibroblast markers 120572-smooth muscle actin (SMA) andconnective tissue growth factor (CTGF) is induced in a TGF-120573-independent manner Instead TGF-120573 signaling is requiredfor 120572-SMA organization and stress-fiber formation [30]

Heme oxygenase-1 (HO-1) has protective activity againstacute and chronic liver injury [31] and is regulated by Nrf2transcription factor [32] Studies found that during hepaticinjury induced by oxidative stress HO-1 and Nrf2 weredownregulated and were associated withNF-120581B upregulation[33 34] The Rutin administration results in Nrf2 HO-1and NF-120581B overexpression Rutin acts as HO-1 inducer inliver ischemia-reperfusion injury rat model [35] The nucleartranslocation of HO-1 could regulate the genes responsiblefor cytoprotection against oxidative stress [36] The releaseof ROS is known to activate inhibitory kappa-B kinasewhich causes phosphorylation of I120581B Release of free NF-120581Benhances the inflammatory cytokines [37] and suppresses theantioxidant genes by downregulating Nrf-2HO-1 pathwayRutin is capable of inhibiting NF-120581B and activating the Nrf-2pathway Sirtuin 1 (Sirt1) a member of sirtuin proteins familydownregulation is associated with high insulin resistance andloss in mitochondrial biogenesis cells [38] High-cholesteroldiet decreases genes expression levels that are involvedin muscle mitochondrial biogenesis and function such asSirt1 peroxisome proliferator-activated receptor coactivator-1 (PGC-1) and mitochondrial transcription factor A (Tfam)[39] Rutin increases the expression levels of Sirt1 PGC-1and Tfam in skeletal muscle and brain of mice which leadto increase in muscle mitochondrial biogenesis and function[40] Therefore the antiobesity property of Rutin mightbe associated with Rutin-mediated muscle mitochondrialchanges

Flavonoids are polyphenolic compounds found in plantsand have an important role in detoxification of free radicals[41] Rutin is a flavonoid glycoside that possessed differ-ent protective effects [42 43] against lipid peroxidationand oxidative-stress-mediated diseases [44] Therefore thepresent study was aimed to investigate the preventive effectof Rutin against HCD-induced hepatotoxicity in rats throughstudying genes expression in the TGF-120573Smad pathways

2 Materials and Methods

21 Animals FortymaleWistar albino rats weighing between80 and 180 g were obtained from the Animal Care Center

College of Pharmacy King Saud University Riyadh SaudiArabia The animals were acclimatized to laboratory con-dition ten days prior to the experiment They were fedon Purina rat chow diet (manufactured by Grain Silos ampFlour Mills Organization Riyadh Saudi Arabia) and wateron a free access basis and were maintained under standardconditions of temperature (22 plusmn 1∘C) humidity (50ndash55)and 12 h lightdark cycles All methods including euthanasiaprocedure were conducted in accordance with guide forcare and use of laboratory animals Institute for LaboratoryAnimal Research National Institute of Health (NIH publi-cation number 80-23 1996) and they have been approvedby Research Ethics Committee of Excremental Animal CareCenter College of Pharmacy King Saud University RiyadhSaudi Arabia

22 Diets Diets were prepared in pellet form by adding 02Rutin (RT) (powder Sigma USA) or 1 cholesterol + 05cholic acid (HCD) or 02 RT + 1 cholesterol + 05 cholicacid (RT + HCD) in rat chow powder Rat chow was used asnormal diets and was prepared weekly and shade dried Foodintake gday was calculated daily for each group to the end ofexperiments

23 Experimental Design The animals were randomlydivided into 4 groups of 10 rats in each as follows Group Icontrol Group II RT Group III HCD and Group IV RT +HCD The experimental diets were fed on a free access basisfor 6 consecutive weeks At end of the experiment rats wereweighed and sacrificed by decapitation and the trunk bloodwas collected in heparinized tubes Rats livers were rapidlyexcised weighed and kept at minus80∘C till analysis Plasmasamples were collected after centrifugation and stored atminus20∘C till analysis

24 Bioassay Measurements Plasma levels of ALT AST TGTC HDL and LDL were estimated by using commerciallyavailable diagnostic kits (Human Wiesbaden Germany)

25 Measurement of Caspase-3 Activity in the Liver TissuesThe caspase-3 activity was measured in liver tissues by usingcolorimetric assay ab39401 (Abcam Cambridge MA 02139-1517 USA) kit according to the manufacturer instructions Inbrief 02mg of liver tissues was not homogenized completelyin 50120583L cell lysis buffer on ice for 10 minutes After centrifu-gation the protein concentrationswere adjusted to 50ndash200120583gprotein per reaction Fifty microlitres of caspase reactionmixcontaining 10mMDTTwas added to eachwell Two hundredmicromolars of DEVD-p-NA substrate was added and thenafter 120min incubation at 37∘C the plate was measured at405 nm Fold increase in caspase-3 activity can be determinedby comparing sample (treated) results with the level of theuntreated control

26 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Gene expression levels of TGF-120573 Smad-2 Smad-4 caspase-3 P21 P53 IL-3 and IL-6 were detected inliver tissues by quantitative real-time PCR Total RNA were















3 Results







0

10

20

30

40 lowast$A

LT (U

L)


lowast$

0

20

40

60

AST

(UL

)



0

1

2

3

4

5lowast


g liv

er100

g bo

dy w

eigh

t

(a)

0

50

100

150

200

Body

wei

ght i

ncre

ased

(g) lowast


0

10

20

30

Food

inta

ke (g

day

)







0

50

100

150Tr

igly

cerid

es (m

gdL

)

lowastlowast


0

50

100

150

Tota

l cho

leste

rol (

mg

dL)

lowast


0

20

40

60

80

100

LDL

(mg

dL)


lowastlowast

(c)

0

10

20

30

40

HD

L (m

gdL

)


lowast

(d)


4 Discussion






0

5

10

15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$








0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






























3 Results







0

10

20

30

40 lowast$A

LT (U

L)


lowast$

0

20

40

60

AST

(UL

)



0

1

2

3

4

5lowast


g liv

er100

g bo

dy w

eigh

t

(a)

0

50

100

150

200

Body

wei

ght i

ncre

ased

(g) lowast


0

10

20

30

Food

inta

ke (g

day

)







0

50

100

150Tr

igly

cerid

es (m

gdL

)

lowastlowast


0

50

100

150

Tota

l cho

leste

rol (

mg

dL)

lowast


0

20

40

60

80

100

LDL

(mg

dL)


lowastlowast

(c)

0

10

20

30

40

HD

L (m

gdL

)


lowast

(d)


4 Discussion






0

5

10

15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$








0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

10

20

30

40 lowast$A

LT (U

L)


lowast$

0

20

40

60

AST

(UL

)



0

1

2

3

4

5lowast


g liv

er100

g bo

dy w

eigh

t

(a)

0

50

100

150

200

Body

wei

ght i

ncre

ased

(g) lowast


0

10

20

30

Food

inta

ke (g

day

)







0

50

100

150Tr

igly

cerid

es (m

gdL

)

lowastlowast


0

50

100

150

Tota

l cho

leste

rol (

mg

dL)

lowast


0

20

40

60

80

100

LDL

(mg

dL)


lowastlowast

(c)

0

10

20

30

40

HD

L (m

gdL

)


lowast

(d)


4 Discussion






0

5

10

15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$








0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150Tr

igly

cerid

es (m

gdL

)

lowastlowast


0

50

100

150

Tota

l cho

leste

rol (

mg

dL)

lowast


0

20

40

60

80

100

LDL

(mg

dL)


lowastlowast

(c)

0

10

20

30

40

HD

L (m

gdL

)


lowast

(d)


4 Discussion






0

5

10

15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$








0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

5

10

15

lowast$


TGF-120573

relat

ive e

xpre

ssio

n

(a)

0

5

10

15

Smad2

relat

ive e

xpre

ssio

n

lowast$


0

2

4

6

8

Smad4

relat

ive e

xpre

ssio

n lowast$








0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

2

4

6

8caspase-3

relat

ive e

xpre

ssio

n lowast$


0

2

4

6

8

P53

relat

ive e

xpre

ssio

n


lowast

(b)

00

05

10

15

20

25

P21

relat

ive e

xpre

ssio

n


0

2

4

6

8

10

Bbc3

relat

ive g

ene e

xpre

ssio

n


lowast

(d)

00

02

04

06

08

10

lowast$


OD405

nm

(e)






00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















00

05

10

15

20Interle

ukin-3

relat

ive e

xpre

ssio

n


0

2

4

6

Interle

ukin-6

relat

ive e

xpre

ssio

n


lowast$

(b)






5 Conclusion


Competing Interests


Acknowledgments


References


































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of













































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of












































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















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