research article ruscogenin ameliorates experimental...

Research ArticleRuscogenin Ameliorates ExperimentalNonalcoholic Steatohepatitis via SuppressingLipogenesis and Inflammatory Pathway

Hung-Jen Lu1 Thing-Fong Tzeng2 Shorong-Shii Liou2 Chia Ju Chang2

Cheng Yang2 Ming-Chang Wu1 and I-Min Liu2

1 Department of Food Science College of Agriculture National Pingtung University of Science and TechnologyNeipu Township Pingtung County Taiwan

2Department of Pharmacy and Graduate Institute of Pharmaceutical Technology Tajen University Yanpu TownshipPingtung County Taiwan

Correspondence should be addressed to Ming-Chang Wu mcwumailnpustedutw and I-Min Liu imltajenedutw

Received 12 April 2014 Accepted 11 June 2014 Published 20 July 2014

Academic Editor Anton M Jetten

Copyright copy 2014 Hung-Jen Lu et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The aim of the study was to investigate the protective effects of ruscogenin a major steroid sapogenin in Ophiopogon japonicuson experimental models of nonalcoholic steatohepatitis HepG2 cells were exposed to 300120583molL palmitic acid (PA) for 24 hwith the preincubation of ruscogenin for another 24 h Ruscogenin (100120583moll) had inhibitory effects on PA-induced triglycerideaccumulation and inflammatorymarkers inHepG2 cellsMale goldenhamsterswere randomly divided into five groups fed a normaldiet a high-fat diet (HFD) or a HFD supplemented with ruscogenin (03 10 or 30mgkgday) by gavage once daily for 8 weeksRuscogenin alleviated dyslipidemia liver steatosis and necroinflammation and reversed plasmamarkers of metabolic syndrome inHFD-fed hamsters Hepatic mRNA levels involved in fatty acid oxidation were increased in ruscogenin-treated HFD-fed hamstersConversely ruscogenin decreased expression of genes involved in hepatic lipogenesis Gene expression of inflammatory cytokineschemoattractive mediator nuclear transcription factor-(NF-) 120581B and 120572-smooth muscle actin were increased in the HFD groupwhich were attenuated by ruscogenin Ruscogenin may attenuate HFD-induced steatohepatitis through downregulation of NF-120581B-mediated inflammatory responses reducing hepatic lipogenic gene expression and upregulating proteins in 120573-oxidation pathway

1 Introduction

Nonalcoholic fatty liver disease (NAFLD) is a potentiallysevere condition that comprises a spectrum of pathologiescharacterized by vesicular fatty change in the liver in theabsence of excessive alcohol consumption [1] NAFLD isstrongly associated with obesity insulin resistance and type2 diabetes and is now well recognized as being part of themetabolic syndrome [2] The metabolic pathways leading tothe development of hepatic steatosis are multiple includingenhanced nonesterified fatty acid release from adipose tissue(lipolysis) increased de novo fatty acids (lipogenesis) anddecreased 120573-oxidation To date caloric restriction and aer-obic exercise are effective treatments for NAFLD but theyare difficult to achieve for most NAFLD patients Other than

lifestyle and diet modifications there is no universally proventreatment [3] Therefore the development of additionaltherapies for controlling lipid levels is warranted to attenuatehepatic steatosis

NAFLD is classified into simple steatosis and nonalco-holic steatohepatitis (NASH) In NASH not only steato-sis but also intralobular inflammation and hepatocellularballooning are present often accompanied by progressivefibrosis [1] A variety of liver cells such as hepatocyteshepatic macrophages and hepatic stellate cells (HSCs) areinvolved in the pathogenesis of NASH [4] Among theinflammatorymediators chemokines play pivotal roles in therecruitment of a variety of cells including immune cells to thesites of inflammation through interaction with chemokinereceptors [5] The interaction of cytokinesgrowth factors

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 652680 10 pageshttpdxdoiorg1011552014652680

2 BioMed Research International

HO

HO OO

H

H

HH

H

Figure 1 Structure of ruscogenin

with their receptors initiates different signaling pathwaysleading to the activation of multiple transcriptional factorssuch as nuclear transcription factor- (NF-) 120581B which alsohas a role in liver fibrogenesis [6] Therapies that limithepatic injury and the related occurrence of inflammationand fibrosis are particularly appealing for this condition

Ruscogenin ((1120573 3120573 25R)-Spirost-5-ene-13-diolFigure 1) is a major effective steroidal sapogen in thetraditional Chinese herb Radix Ophiopogon japonicus thathas been used to treat acute and chronic inflammatory andcardiovascular diseases [7 8] Ruscogenin has been found toexert significant anti-inflammatory activities in many aspectssuch as antielastase activity inhibiting leukocyte adhesionand migration and antithrombotic activity [9ndash11] Previousresearch has proved that the possible anti-inflammatorymechanism of ruscogenin was linked with the suppression ofintercellular adhesion molecule-1 expression in endothelialcells mainly through the inhibition of the NF-120581B signalingpathway [12] It was also found that ruscogenin significantlyattenuated lipopolysaccharide- (LPS-) induced acutelung injury model in mice which possibly linked withinhibition of NF-120581B activation [13] Ruscogenin has alsobeen documented to reduce cerebral ischemic injury viaNF-120581B mediated inflammatory pathway in the mousemodel of experimental stroke [14] Downregulation ofNF-120581B-mediated inflammatory responses induced byruscogenin may indicate its potential protection in NASHhowever there is no report about it until now Therefore theaim of the present study was to investigate the protectiveeffects of ruscogenin supplementation on liver steatosisand injury in hamsters fed a high-fat diet (HFD) Theseeffects of ruscogenin were further characterized in HepG2hepatocytes

2 Materials and Methods

21 Cell Cultures Human hepatoma HepG2 cells obtainedfrom Bioresource Collection and Research Center (BCRC60025) of the Food Industry Research and DevelopmentInstitute (Hsinchu Taiwan) were cultured in minimumessential medium containing 10 (volvol) fetal bovineserum 2mmolL l-glutamine 1mmolL sodium pyruvate100UmL penicillin 100 120583gmL streptomycin and 1mmolLsodium pyruvate at 37∘C in a humidified atmosphere con-taining 5 CO

2 The cells were grown to 70 confluence

and incubated in serum free medium overnight before

treatments After the starvation for 24 h cells were exposedto 300 120583molL palmitic acid (PA Sigma-Aldrich Co StLouis MO Cat no P0500) for 24 h with or without thepreincubation of 01 10 or 100 120583molL ruscogenin (ge98Chengdu Biopurify Phytochemicals Ltd Chengdu SichuanChina Cat no 472-11-7) for another 24 h Cell viability afterPA treatments was monitored by trypan blue exclusion Nochange in viability was observedwith the concentrations usedin this study Subconfluent monolayers of HepG2 cells werestained with Oil Red O (Sigma-Aldrich Co) to determine fataccumulation

22 Observation of PA-Induced Lipids Accumulation inHepG2Cells The PA-induced lipid accumulation in HepG2 cellswas evaluated by Oil Red O staining and the measure-ment of triglyceride (TG) content Briefly samples werefixed with 4 paraformaldehyde and then stained with OilRed O for 15min Then the samples were counterstainedwith hematoxylin for 5min Results were examined bylight microscopy Intracellular TG content was evaluatedafter lysis of the cells with cell lysis buffer (1 Triton X-100 150mmolL NaCl 10mmolL Tris pH 74 1mmolLEDTA 1mmolL EGTA 02mmolL phenylmethylsulfonylfluoride 02mmolL sodium orthovanadate and 05 NP-40) (Promega Madison WI USA) The concentration ofTG was determined by the Triglyceride Colorimetric AssayKit (Cat no 10010303 Cayman Chemical Company AnnArbor Michigan USA) according to the protocol providedby manufacturer and normalized by protein content

23 Measurement of PA-Induced Inflammatory Cytokines inHepG2 Cells The cell culture media were centrifuged at10000 g for 10min at 4∘C and the supernatants were storedat minus20∘C before analysis Secretory levels of inflammatorycytokines including monocyte chemoattractant protein-(MCP-) 1 (Cat no ab100721) tumor necrosis factor- (TNF-)120572(Cat no ab46070) interleukin- (IL-) 1120573 (Cat no ab100768)and IL-6 (Cat no ab100772) in cell-free culture supernatantswere determined by commercial enzyme-linked immunosor-bent assay (ELISA) kits purchased from Abcam Inc (Cam-bridge MA USA) The color generated was determined bymeasuring the OD value at 450 nm of spectrophotometricmicrotiter plate reader (Molecular Devices Corp SunnyvaleCAUSA)A standard curvewas runon each assay plate usingrecombinant proteins in serial dilutions

24 Animal Models Male golden Syrian hamsters 8-weekold and weighing 90 plusmn 10 g were obtained from the NationalLaboratoryAnimal Center (Taipei Taiwan)Theyweremain-tained in a temperature-controlled room (25 plusmn 1∘C) ona 12 h 12 h light-dark cycle (lights on at 06 00 h) in ouranimal center Food and water were provided ad libitum Aregular chow diet (RCD 10 kcal fat no D12450B ResearchDiets New Brunswick NJ) was used as the maintenanceand control diet A purified HFD with 45 kcal fat obtainedprimarily from lard (no D12451 Research Diets) was used toinduce a rapid increase in body weight and obesity [15] Allanimal procedures were performed according to the Guide

BioMed Research International 3

for the Care and Use of Laboratory Animals of the NationalInstitutes of Health as well as the guidelines of the Ani-mal Welfare Act These studies were conducted with theapproval of the InstitutionalAnimalCare andUseCommittee(IACUC) at Tajen University (approval number IACUC 101-29 approval date December 22 2012)

25 Treatment Protocols in Animals After being fed a HFDfor two weeks hamsters were dosed by oral gavage onceper day for eight weeks with ruscogenin doses of 03 10 or30mgkg in a volume of 15mLkg distilled waterThe dosageregimen was selected based on a previous report demon-strating that ruscogenin at the indicated dosage regimen waspotentially effective in inhibiting LPS-induced inflammationin mice [13] Another group of HFD- and RCD-fed hamsterswas treated similarly but the same volume of vehicle (distilledwater) was used to prepare the tested compound solutionduring the same treatment period The water consumptionfood intake and body weight were measured once dailyat the same time (09 00) on each day throughout theexperiment Food cups containing fresh food were weighedat the beginning and end of each 24 h period Food intake wascalculated by determining the difference in food cup weightsadjusting for any spillage that occurred Water intake wascalculated bymeasuring the difference inwater bottle weightsat the beginning and end of the daily change of water

Eight weeks after treatment with ruscogenin animalswereweighed and anesthetizedwith ketamine after fasting for12 hours Blood samples were taken from the inferior venacava for analysis After blood collection liver epididymaladipose and perirenal adipose tissues were removed rinsedwith a physiological saline solution and immediately storedat minus80∘C in liquid nitrogen until assayed The coefficient ofhepatic weight was also calculated as liver weight (g) dividedby body weight (100 g) Other hepatic tissues were fixed in10 neutralized formalin for histology

26 Determination of Metabolic Parameters and Insulin Sen-sitivity Blood samples were centrifuged at 2000timesg for 10minutes at 4∘C The plasma was then removed and placedinto aliquots for the respective analyses Kits for determin-ing plasma glucose (Cat no 10009582) concentration werepurchased from Cayman Chemical Company (Ann ArborMI) Commercial ELISA kits were used to quantify plasmainsulin concentration (LINCO Research Inc St CharlesMO Cat no EZRMI-13 K) Whole body insulin sensitivitywas estimated using the homeostasis model assessment ofinsulin resistance (HOMA-IR) with the following formula[fasting plasma glucose (mmol) times fasting plasma insulin(mUmL)]225 [16] Diagnostic kits for determination ofplasma levels of total cholesterol (TC Cat no 10007640)and TG (Cat no 10010303) were purchased from CaymanChemical Company The diagnostic kit for determination ofplasma levels of high density lipoprotein cholesterol (HDL-C) was purchased from Bio-Quant Diagnostics (Cat no BQ019CR SanDiego CAUSA) Lowdensity lipoprotein choles-terol (LDL-C) concentrations in plasma were determinedby commercial ELISA kit (antibodies-online Inc Atlanta

GA USA Cat no ABIN416222) Plasma free fatty acid(FFA) levels were determined using an FFA quantification kitobtained fromAbcam plc (Cat no ab65341 CambridgeMAUSA) All experimental assays were carried out according tothe manufacturerrsquos instruction All samples were analyzed intriplicate

27 Measurement of Hepatic Lipids Sections of fresh liversamples were collected for determining the lipid contentLiver (125 g) was homogenized with chloroformmethanol(1 2 375mL) Chloroform (125mL) and distilled water(125mL) were then added to the homogenate and mixedwell After centrifugation (1500timesg for 10min) the lower clearorganic phase of the solution was transferred into a newglass tube and then lyophilized The lyophilized powder wasdissolved in chloroformmethanol (1 2) and stored at minus20∘Cfor less than 3 days [17] Hepatic TC and TG levels in lipidextracts were analyzed using the same diagnostic kits thatwere used for plasma analysis

28 Histological Analysis of Liver At sacrifice livers wereperfused with phosphate buffered saline (PBS) solution viathe portal vein After removal of the liver a section ofapproximately 4mm2 was fixed in PAF and embedded inparaffin Paraffin-embedded sections (5 120583m) were stainedwith hematoxylin and eosin (HampE) to evaluate the degreeof hepatic steatosis Liver tissues were scored for hepaticsteatosis (0 none 1 1ndash25 2 26ndash50 3 51ndash75 and 476ndash100 hepatocytes affected) and necroinflammation (0no inflammation 1 mild lobularportal inflammation 2moderate lobularportal inflammation and 3 severe lob-ularportal inflammation) [18] Other frozen liver sectionswere stained with macrophage-specific monoclonal antibodyF480 (1 500 Cat no sc-377009 Santa Cruz BiotechnologySanta Cruz CA USA) followed by detection with biotiny-lated secondary antibody and streptavidin-horseradish per-oxidase to evaluate the degree of macrophage infiltration andfibrosis All slides were scanned at a total magnification of200x using Image Pro Plus 70 software (Media Cybernet-ics) under a light microscope (Olympus BX51 microscopeTokyo Japan)

29 Analysis of mRNA Expression of Hepatic Genes Foranalysis of gene expression total RNA was extracted from100mg frozen liver samples using Trizol reagent (InvitrogenBoston MA USA) RNA was quantified by A260 and itsintegrity verified by agarose gel electrophoresis using ethid-ium bromide for visualization For the reverse transcrip-tase reaction 1 120583g of total RNA per sample and 85 120583g120583Lrandom hexamer primers were heated at 65∘C for 5minand then quenched on ice This mixture was combinedwith 500 120583molL of each of dATP dTTP dCTP and dGTP10mmolL DTT 20mmolL Tris-HCl (pH 84) 50mmolLKCl 5mmolL MgCl

2 40 units of RNaseOUT recombinant

ribonuclease inhibitor (Invitrogen) and 100 units Super-Script III reverse transcriptase (Invitrogen) Samples weresubjected to DNase (PromegaMadisonWI USA) treatmentat 37∘C for 20min in a GeneAmp 9700 Thermal Cycler

4 BioMed Research International

(Applied Biosystems Foster City California USA) and thenheld at 4∘C After aliquots were taken for immediate use inPCR the remainder of the cDNAwas stored atminus20∘CmRNAexpression was measured by quantitative real-time reversetranscription polymerase chain reaction (RT-PCR) in a flu-orescent temperature Lightcycler 480 (Roche DiagnosticsMannheim Germany)The following primer sequences wereused 51015840-TCTCTTCCTCCACCACTATGCA-31015840(forward)and 51015840-GGCTGAGACAGCACGTGGAT-31015840 (reverse) forMCP-1 51015840-ATGGATCTCAAAGACAACCA-31015840 (forward)and 51015840-TCCTGGTATGAAATGGCAAA-31015840 (reverse) forTNF-120572 51015840-GGTCAAAGGTTTGGAAGCAG-31015840(forward)and 51015840-TGTGAAATGCCACCTTTTGA-31015840 (reverse) forIL-1120573 51015840-AAAAGTCCTGATCCAGTTC-31015840 (forward) and51015840-GAGATGAGTTGTCATGTCC-31015840 (reverse) for IL-651015840-TGCTGTCCCTCTATGCCTCT-31015840 (forward) and 51015840-GAAGGAATAGCCACGTCAG-31015840 (reverse) for 120572-smoothmuscle actin (120572-SMA) 51015840-GAAGAAAATGGTGGAGTC-TG-31015840 (forward) and 51015840-GGTTCACTAGTTTCCAAGTC-31015840 (reverse) for nuclear transcription factor-120581B (NF-120581B)p6551015840-CGTCCTGGCCTTCTAAACGTAG-31015840 (forward) and 51015840-CCTGTAGATCTCCTGCAGTAGCG-31015840 (reverse) for per-oxisome proliferator-activated receptor (PPAR) 120572 51015840-CAG-ACCTGACACAACACGG-31015840 (forward) and 51015840-CTTGAA-AAATTGCTTGCGTC-31015840 (reverse) for PPAR120574 coactivator-1120572 (PGC-1120572) 51015840-ATGGCAGAGGCTCACCAAGCTGTG-31015840 (forward) and 51015840-CCTCTGTGGTACACAACAATG-TGC-31015840 (reverse) for carnitine palmitoyltransferase (CPT)-1 51015840-ATGGTTGGACTGAAGCCTTCAG-31015840 (forward) and5 1015840-TCAAAACGGTGATTCCCGTAAC-31015840 (reverse) foruncoupling protein (UCP)2 51015840-GAGGAGGAGGGATTC-TGGTC-31015840 (forward) and 51015840-CACGTCTCCAACCTT-CCATT-31015840 (reverse) for UCP3 51015840-TCAACAACCAAG-ACAGTGACTTCCCTGGCC-31015840 (forward) and 51015840-GTT-CTCCTGCTTGAGCTTCTGGTTGCTGTG-31015840(reverse) forsterol regulatory element-binding protein (SREBP)-1c 51015840-TCGTGGGCTACAGCATGGT-31015840 (forward) and 51015840-GCC-CTCTGAAGTCGAAGAAGAA-31015840 (reverse) for fatty acidsynthase (FAS) 51015840-CTGTAGAAACCCGGACAGTAGAAC-31015840 (forward) and 51015840-GGTCAGCATACATCTCCATGTG-31015840(reverse) for acetyl-CoA carboxylase (ACC) 51015840-TGTGAT-GGTGGGAATGGGTCAG-31015840 (forward) and 51015840-TTTGAT-GTCACGCACGATTTCC-31015840 (reverse) for 120573-actin Primerswere designed using Primer Express Software version 20System (Applied Biosystems Foster City CA USA) ThePCR reaction was performed using the following cyclingprotocol 95∘C for 5min followed by 45 cycles of 95∘C for5 s 58∘C for 15 s and 72∘C for 20 s Dissociation curves wererun after amplification to identify the specific PCR productsThe mRNA expression levels were normalized to 120573-actinmRNA levels and calculated according to the delta-delta Ctmethod [19]

210 Statistical Analysis Data are expressed as the meanplusmn standard error mean (mean plusmn SEM) Statistical analysiswas performed with one-way analysis of variance (ANOVA)Dunnett range post hoc comparisons were used to determinethe source of significant differences where appropriate For

the histological study a nonparametric Kruskal-Wallis testwas performed and Mann-Whitneyrsquos 119880 test was used tocompare data within the groupsThe SigmaPlot (Version 110)programwas used for statistical analysis A119875 valuelt 005 wasconsidered statistically significant

3 Results

31 Effects of Ruscogenin on PA-Induced Lipids Accumula-tion and Inflammatory Cytokines in HepG2 Cells The lipidaccumulation was measured by Oil Red O staining Asshown in Figure 2(a) HepG2 cells treated for 24 h with PAexhibited significant lipid droplet accumulation comparedwith untreated cells Preincubation with ruscogenin signifi-cantly prevented PA-induced lipid deposition and the mosteffective inhibition of lipid accumulation occurred at a doseof 10 120583molL (Figure 2(a)) Consistently treatment with PAresulted in an obvious increase in TG content comparedwith untreated cells which was attenuated significantly bypretreatmentwith ruscogenin at a concentration of 10120583molL(Figure 2(b)) Ruscogenin alone or vehicle did not affect basallevels of lipid deposition (data not shown)

Figure 2(c) showed that PA exposure significantlyincreased the production of MCP-1 TNF-120572 IL-1120573 andIL-6 compared with the untreated cells Pretreatmentwith 10 120583molL ruscogenin for 24 h significantly alleviatedPA-induced overproduction of all these inflammatorycytokines

32 Effects of Ruscogenin on the Body Weight the Liverand Visceral Fat Weight and Feeding Behaviors of HamstersThe body weight visceral fat and relative liver weightsin HFD-fed hamsters were significantly higher than thosein the RCD-fed group (Table 1) High doses of ruscogenin(30mgkgday) significantly suppressed body weight gainThe relative weights of liver epididymal adipose tissue andperirenal adipose tissue were significantly lower in rusco-genin (30mgkgday) treated groups than those in the HFDgroup (Table 1) No significant differences in daily food orwater intake were observed between the groups over theexperimental period (Table 1)

33 Effects of Ruscogenin on Insulin Sensitivity and PlasmaLipids Levels of Hamsters HFD-fed hamsters were insulinresistant as reflected by hyperinsulinemia as well as sig-nificantly increased HOMA-IR values (Table 2) Treatmentof HFD-fed hamsters with ruscogenin (30mgkgday) hada beneficial effect on insulin resistance as evidenced by areduction in fasting plasma insulin levels and improvedlevels of HOMA-IR (Table 2)

The HFD caused elevated concentrations of plasma TCTG and LDL Oral administration of ruscogenin at a doseof 03 10 or 30mgkgday significantly reduced plasmaTC levels (237 311 and 397 reduction resp) thereduction of plasma TG activity induced by ruscogenin at30mgkgday was nearly 301 (Table 2) Ruscogenin at theoral dose of 03 10 or 30mgkgday significantly reducedtotal plasma LDL levels (329 426 and 543 reduction

BioMed Research International 5

Control PA PA + RUS 01 PA + RUS 100PA + RUS 10

(a)

d

20

0

40

60

80

100

00

3000

30001

30010

300100

bb c

b c

a d

TG (120583

mol

mg

prot

ein)

PA (120583molL)RUS (120583molL)

(b)

0

50

100

150

200

250

300

0

40050200

600

800

1000

1200

1400

d

d

d

b c

b cb c

b c

b cb c

a d

a d

a d

0

100

150

200

b

b

b250

PA (120583molL)RUS (120583molL)

PA (120583molL)RUS (120583molL)

PA (120583molL)RUS (120583molL)

0

0

300

0

300

01

300

10

300

100

0

0

300

0

300

01

300

10

300

100

0

0

300

0

300

01

300

10

300

100

0

0

300

0

300

01

300

10

300

100

MCP

-1(p

gm

L)

d

b c b c

b

0

20

40

60

80

100

120

140

b d

PA (120583molL)RUS (120583molL)

IL-6

(pg

mL)

Il-120573

(pg

mL)

TNF120572

(pg

mL)

(c)

Figure 2 Effects of ruscogenin (RUS) on PA-induced lipids accumulation and inflammatory cytokines overproduction in HepG2 cellsCells were exposed to PA (300120583molL) for 24 h with or without the preincubation of 01 (RUS 01) 10 (RUS 10) or 100 120583molL ruscogenin(RUS 100) (a) Representative Oil Red O staining of cells with different treatments is shown Cells were examined by light microscopy at amagnification of 400x (b) Intracellular TG content was measured by an ELISA assay TG concentration was normalized by protein content(c) Inflammatory cytokines in cell-free culture supernatants were determined by ELISA kits The results are presented as the mean plusmn SEM offour experiments a119875 lt 005 and b

119875 lt 001 compared to the control values of untreated cells (control) respectively c119875 lt 005 and d119875 lt 001

compared to the values of PA-treated cells (PA) respectively

resp) compared to that of vehicle-treated HFD-fed hamsters(Table 2)

The plasma concentration of HDL-C in HFD-fed ham-sters was reduced to 604 of the level observed in theRCD-fed group (Table 2) After 8 weeks of ruscogenin(30mgkgday) treatment plasma HDL-C concentrations inHFD-fed hamsters increased to 905of the level in theRCD-fed group (Table 2)

Plasma FFA levels in vehicle-treated HFD-fed hamsterswere about 20-fold of that observed in the RCD-fed group(Table 2) The plasma FFA levels decreased by 381 inHFD-fed hamsters treated with ruscogenin (30mgkgday)compared to their vehicle-treated counterparts (Table 2)

34 Effects of Ruscogenin on Hepatic Steatosis The hepaticTC level was significantly higher in HFD-fed hamsters than

6 BioMed Research International

Table 1 Effects of ruscogenin on body weight liver and visceral fat weight and feeding behaviors of hamsters

RCD-fed HFD-fed

Vehicle Vehicle Ruscogenin (mgkgday)03 10 30

Final body weight (BW) (g) 1271 plusmn 91d

1643 plusmn 71b

1584 plusmn 81b

1539 plusmn 63b 1473 plusmn 73ac

Epididymal WAT (g100 g BW) 16 plusmn 03d

25 plusmn 03b

21 plusmn 04b

19 plusmn 02d

18 plusmn 03d

Perirenal WAT (g100 g BW) 08 plusmn 02c

12 plusmn 02a

11 plusmn 03 11 plusmn 02 09 plusmn 02

Liver weight (g100 g BW) 48 plusmn 03c

57 plusmn 03a

55 plusmn 03a

52 plusmn 03 51 plusmn 03

Food intake (gday) 99 plusmn 13 110 plusmn 13 107 plusmn 18 109 plusmn 14 98 plusmn 15

Water intake (mLday) 126 plusmn 44 132 plusmn 62 130 plusmn 45 126 plusmn 59 131 plusmn 43

The vehicle (distilled water) used to prepare the tested medication solution was given at the same volume Values (mean plusmn SEM) were obtained from eachgroup of 8 animals after 8 weeks of the experimental period a119875 lt 005 and b

119875 lt 001 compared to the values of vehicle-treated RCD-fed hamsters in eachgroup respectively c119875 lt 005 and d

119875 lt 001 compared to the values of vehicle-treated HFD-fed hamsters in each group respectively

RCD-vehicle

HFD-RUS

HFD-vehicle

Figure 3 Representative images of HampE and stained livers fromRCD- or HFD-fed hamsters receiving 8 weeks of treatmentsPhotomicrographs (original magnification 400x) are of tissuesisolated from vehicle-treated RCD-fed hamsters (RCD-vehicle)vehicle-treated HFD-fed hamsters (HFD-vehicle) or ruscogenin(30mgkgday) treated HFD-fed hamsters (HFD-ruscogenin)Arrows and arrow head indicate fat droplets and necroinflammatoryfoci respectively The severity of hepatic steatosis and necroinflam-mation were scored in Table 3

in hamsters from the RCD-fed group which was reducedby 285 in HFD-fed hamsters treated with ruscogenin(30mgkgday Table 2) Similarly ruscogenin treatment(30mgkgday) also produced a significant reduction inhepatic TG concentration to 632 of that in vehicle-treatedHFD-fed hamsters (Table 2)

Representative histological photomicrographs of liverspecimens are shown in Figure 3 Hamsters fed a RCDhad normal liver histological findings however numerousmacrovascular fat droplets and mild necroinflammatory fociwere present in livers of those fed a HFD Treatment ofHFD-fed hamsters with ruscogenin (30mgkgday) reducedfat liver depots and less macrovesicular steatosis as revealed

in vehicle-treated counterparts (Figure 3) Ruscogenin treat-ment (30mgkgday) clearly reduced hepatic necroinflam-mation (Figure 3) Histological grading of liver sections con-firmed that ruscogenin treatment significantly amelioratedboth hepatic steatosis and necroinflammation (Table 3)

Livers from RCD-fed hamsters did not show anysignificant macrophage (F480-positive cells) infiltration(Figure 4) In contrast HFD-fed hamsters demonstratedprominent macrophage infiltration of the liver (Figure 4)Treatment of HFD-fed hamsters with ruscogenin(30mgkgday) for 8 weeks showed a marked reductionin macrophage influx by 293 when compared with theirvehicle-treated counterparts (Figure 4)

35 Effects of Ruscogenin on Inflammatory Cytokines inHamsters All inflammatory mediators were expressed atvery low levels in the livers of RCD-fed hamsters (Figure 5)In HFD-fed hamsters hepatic mRNA levels of MCP-1TNF-120572 IL-1120573 IL-6 and NF-120581B were significantly increasedcompared to RCD-fed group (Figure 5) These increaseswere approached to control values in hamsters treated withruscogenin In addition the hepatic fibrosis index of 120572-SMAin HFD-fed hamsters was significantly increased to 43-foldof that observed in the RCD-fed group and decreased (415decreases) by ruscogenin treatment (Figure 5)

36 Effects of Ruscogenin on Hepatic mRNA Expression of 120573-Oxidation-Related Genes and Lipogenic Genes The mRNAlevels of PPAR120572 in livers ofHFD-fed hamsterswere decreasedto 382 of those of the RCD-fed group (Figure 6(a))Administration of ruscogenin to HFD-fed hamsters for 8weeks significantly upregulated hepatic PPAR120572mRNA levelsto 17-fold that of vehicle-treated counterparts (Figure 6(a))Hepatic mRNA levels of PGC-1120572 CPT-1 UCP2 andUCP3 inHFD-fed hamsters were clearly lower than those of the RCD-fed group and were upregulated by ruscogenin treatment(1732 1443 1632 and 1983 increases resp) (Figure 6(a))

HFD feeding markedly increased the hepatic mRNAlevels of SREBP-1c in hamsters to 23-fold that of the RCD-fed group (Figure 6(b)) Ruscogenin suppressed the HFD-induced increase in hepatic mRNA levels of SREBP-1c by

BioMed Research International 7

RCD-vehicle

HFD-RUS

HFD-vehicle

RCD-vehicle HFD-vehicle HFD-RUS

25

20

15

10

5d

0

30 b

F480

stai

ning

tota

l are

a (

)

b c

Figure 4 Representative images of F480 staining in livers from RCD- or HFD-fed hamsters receiving 8 weeks of treatmentsPhotomicrographs are of tissues isolated from vehicle-treated RCD-fed hamsters (RCD-vehicle) vehicle-treated HFD-fed hamsters (HFD-vehicle) or ruscogenin (30mgkgday) treated HFD-fed hamsters (HFD-ruscogenin) Arrows indicate inflammatory foci Quantification ofhepatic macrophage accumulation is presented as the percentage of the brown stained area relative to the whole area of the photomicrograph(original magnification 400x) Values (mean plusmn SEM) were obtained from 5 animals in each group b119875 lt 001 compared to vehicle-treatedRCD-fed hamsters c119875 lt 005 and d

119875 lt 001 compared to the values of vehicle-treated HFD-fed hamsters in each group respectively

Table 2 Effects of ruscogenin on insulin sensitivity plasma lipid profile and free fatty acid and hepatic lipids of hamsters

RCD-fed HFD-fed

Vehicle Vehicle Ruscogenin (mgkgday)03 10 30

Plasma glucose (mgdL) 922 plusmn 38d 1564 plusmn 41b 1436 plusmn 41bc 1396 plusmn 39bc 1305 plusmn 29bc

Plasma insulin (mU) 217 plusmn 03d 408 plusmn 05b 376 plusmn 04bc 322 plusmn 03bc 290 plusmn 03ad

HOMA-IR 49 plusmn 02 157 plusmn 02b 133 plusmn 03bc 111 plusmn 03bc 95 plusmn 03bd

Plasma TC (mgdL) 1151 plusmn 31d 2578 plusmn 44b 1962 plusmn 51bc 1780 plusmn 39bc 1558 plusmn 42ad

Plasma TG (mgdL) 902 plusmn 21d 1570 plusmn 39b 1309 plusmn 41bc 1186 plusmn 31ac 1097 plusmn 33ad

Plasma LDL (mgdL) 490 plusmn 38d 1972 plusmn 54b 1329 plusmn 69bc 1137 plusmn 61bc 901 plusmn 54ad

Plasma HDL (mgdL) 481 plusmn 30c 299 plusmn 29a 371 plusmn 31ac 405 plusmn 41ac 437 plusmn 36c

Plasma FFAs (mgdL) 299 plusmn 33d 602 plusmn 44b 537 plusmn 42b 480 plusmn 37ac 372 plusmn 35ad

Hepatic TC (120583molg liver) 99 plusmn 08d 192 plusmn 14b 173 plusmn 11b 155 plusmn 09bc 137 plusmn 14ac

Hepatic TG (120583molg liver) 86 plusmn 08d 170 plusmn 17b 157 plusmn 18b 123 plusmn 19bc 110 plusmn 12ac

The vehicle (distilled water) used to prepare the tested medication solution was given at the same volume Values (mean plusmn SEM) were obtained from eachgroup of 8 animals after 8 weeks of the experimental period a119875 lt 005 and b

119875 lt 001 compared to the values of vehicle-treated RCD-fed hamsters in eachgroup respectively c119875 lt 005 and d

119875 lt 001 compared to the values of vehicle-treated HFD-fed hamsters in each group respectively

Table 3 Effects of ruscogenin on scores for hepatic steatosis and necroinflammation of hamsters

RCD-fed HFD-fedVehicle Vehicle Ruscogenin (30mgkgday)

Steatosis (scores 0ndash4) 0 plusmn 0d 31 plusmn 06b 17 plusmn 03bd

Necroinflammation (scores 0ndash3) 0 plusmn 0d 11 plusmn 03b 06 plusmn 02bc

The vehicle (distilled water) used to prepare the tested medication solution was given at the same volume Values (mean plusmn SEM) were obtained from eachgroup of 8 animals after 8 weeks of the experimental period a119875 lt 005 and b

119875 lt 001 compared to the values of vehicle-treated RCD-fed hamsters in eachgroup respectively c119875 lt 005 and d

119875 lt 001 compared to the values of vehicle-treated HFD-fed hamsters in each group respectively

8 BioMed Research International

273 relative to vehicle-treated counterparts (Figure 6(b))HFD caused a 19-fold induction of hepatic FAS mRNA anda 20-fold induction of hepatic ACC mRNA over those ofthe RCD-fed group (Figure 6(b)) The HFD-induced mRNAlevels of FAS and ACC in liver were significantly reversedafter ruscogenin treatment (decreased by 213 and 285resp) compared to those of vehicle-treated counterparts(Figure 6(b))

4 Discussion

As inflammation plays a pivotal role in NAFLD an importantpharmacological objective in treating this disorder is thedirect targeting of inflammatory activation [5] Because PAis known to induce a hyperlipidemic condition viainflamma-tory liver injury [20 21] it could be thought that PA treatmentsufficiently caused hepatic steatosis and inflammation Ourdata demonstrated that exposure of HepG2 cells to PAresults in lipid accumulation and the overproduction ofinflammatory cytokines such as MCP-1 TNF-120572 IL-1120573 andIL-6 This is in agreement with a previous study that theplasma concentrations of FFAs were increased in patientswith NAFLD and correlated with the development of moresevere liver disease [22] Our study clearly demonstratedthat preincubation with ruscogenin significantly attenuatesthe lipid accumulation and inflammatory cytokines overpro-duction in HepG2 cells exposed to excess PA These dataindicated that ruscogenin might be effective for preventingand reversing lipid accumulation and the inflammatoryresponse which may accelerate the lipid metabolism disorderandor more severe liver injuries

The HFD-induced animal model of NAFLD has beenwidely used to study its pathogenesis and to evaluate newtreatments [23] We then investigated whether ruscogenincould improve fatty liver changes by decreasing inflamma-tory cytokines in the HFD-induced NAFLD hamsters Withruscogenin treatment ofHFD-fed hamsters we observed thatthe increased plasma levels of TG TC LDL-C and FFAwere significantly suppressed whereas the decreased plasmaHDL-C levels were clearly elevated In addition the relativeliver weight of ruscogenin-treated hamsters was significantlylower than that of HFD-fed hamsters Morphologically thelivers of HFD-fed hamsters showed large abundant lipiddroplets and clear derangement compared to those of RCD-fed hamsters However the livers of HFD-fed hamstersreceiving ruscogenin had fewer lipid droplets and morenormal liver morphology suggesting that ruscogenin hadthe beneficial effects of preventing lipid accumulation andreversing disrupted liver structure

Hepatic macrophages are the key cells inducing liverinflammation and infiltration of hepatic macrophages wasincreased in hamsters onHFD Inflammation and subsequentchemoattraction of cell migration to the liver are critical forthe development and progression of NAFLD [24] Admin-istration of ruscogenin during NAFLD development signif-icantly attenuated reduced hepatic macrophage infiltrationas well as inflammatory mediators in HFD-fed hamstersMolecules that initiate hepatic fibrosis may also be activated

0

3

2

1

5

4

8

7

6

9

RCD-vehicleHFD-vehicleHFD-RUS

d

b

d

b

d

b

d

b

d

b

d

b

MCP-1 TNF120572 IL-1120573 IL-6 NF-120581B 120572-SMA

Relat

ive m

RNA

expr

essio

n(n

orm

aliz

ed b

y120573

-act

in)

a c

a c

a c a ca db c

Figure 5 Real-time PCR analysis of mRNA expression of NF-120581B-dependent proinflammatory markers in the livers of HFD-fed hamsters receiving 8 weeks of treatment with ruscogenin(30mgkgday) The mRNA expressions of the inflammatorycytokines and NF-120581B were normalized to an internal control (120573-actin) Animals not receiving any treatment were given the samevolume of vehicle used to dissolve ruscogenin Similar results wereobtained with an additional 4 replications Data were expressed asthemeanwith SEM (119899 = 5 per group) in each column a119875 lt 001 andb119875 lt 001 compared to vehicle-treated RCD-fed hamsters c119875 lt 005and d119875 lt 001 compared to the values of vehicle-treated HFD-fed

hamsters in each group respectively

by excessive FFAs through lipid peroxidation or cytokineproduction [5] In addition activated hepatic Kupffer cellsthe direct source of proinflammatory cytokine productionmay in turn activate HSC to synthesize collagen initiatingthe process of liver remodeling in the form of fibrosis andcirrhosis [5 6] Expression of 120572-SMA has been consideredone of the dominant features of HSC activation and hasbecome an important evaluation index for hepatic fibrosis[25] We observed that the expression of 120572-SMA induced byHFDwas significantly reduced by ruscogenin suggesting thatthe inhibition of inflammatory factor expression also effec-tively suppressed HSC activation blocking the occurrence ofhepatic fibrosis at the source

SinceNF-120581B is themaster regulator ofmolecules that takepart in cellular proliferation inflammation and apoptosisblockade of NF-120581B pathway has shown therapeutic efficacy[6] The previous studies have demonstrated that ruscogeninhad significant anti-inflammatory and antithrombotic activ-ities which might be related to the inhibition of ICAM-1 and NF-120581B pathways [12] The inhibition of NF-120581B byruscogenin might be a critical step for the prevention ofcascading inflammatory response in the liver In the presentstudy ruscogenin suppressed the mRNA expression of NF-120581B in liver of HFD-fed hamsters reflecting the effectiveblockage of HFD-induced NF-120581B activation These resultssuggest that ruscogenin inhibits the activation of NF-120581Bleading to downregulation of proinflammatory mediators

BioMed Research International 9

00

02

04

06

08

10

bb

b

b

b

12

d d dd d

CPT-1 UCP2 UCP3

RCD-vehicleHFD-vehicleHFD-RUS

Relat

ive m

RNA

expr

essio

n(n

orm

aliz

ed b

y120573

-act

in)

b c b cb c

b c

b c

PPAR120572 PCG-1120572

(a)

00

10

05

15

b

b

20

25

d

b

dd

SREBP-1c FAS ACC

RCD-vehicleHFD-vehicleHFD-RUS

Relat

ive m

RNA

expr

essio

n(n

orm

aliz

ed b

y120573

-act

in) b c b c

b c

(b)

Figure 6 The hepatic mRNA levels of 120573-oxidation-related genes (a) and lipogenic genes (b) in HFD-fed hamsters receiving 8 weeks oftreatment with ruscogenin (30mgkgday HFD-RUS) The mRNA expressions of the 120573-oxidation-related genes and lipogenic genes weremeasured by RT-PCR and normalized to an internal control (120573-actin) Animals not receiving any treatment were given the same volume ofvehicle used to dissolve ruscogenin Similar results were obtained with an additional 4 replications Data were expressed as the mean withSEM (119899 = 5 per group) in each column b119875 lt 001 compared to vehicle-treated RCD-fed hamsters (RCD-vehicle) c119875 lt 005 and d

119875 lt 001

compared to the values of vehicle-treated HFD-fed hamsters (HFD-vehicle) in each group respectively

and amelioration of fibrogenesis and therefore shows apromising effective in preventing NAFLD

Importantly insulin resistance is further associated withthe development of steatosis and liver fibrosis [26] Besidesthe direct effects of inflammatory cytokines on lipogenesisand fibrogenesis MCP-1 TNF-120572 and IL-1120573 IL-6 can induceinsulin resistance [24 26] Therefore patients with type 2diabetes are at a higher risk of NAFLD and other inflamma-tory processes We observed that the effects of ruscogeninimproved HOMA-IR in HFD-fed hamsters These resultsfurther suggest that ruscogenin not only suppresses therecruitment of macrophages but also inhibits the release ofinflammatory cytokines from hepatic macrophages prevent-ing hepatic steatosis fibrosis and insulin resistance Accord-ing to our results ruscogenin could positively influence fattyliver changes in type 2 diabetes and in insulin resistant state

Hepatic lipidmetabolism is mainly regulated by lipid reg-ulatory proteins such as 120573-oxidation-related and lipogenicproteins Expression of fatty acid 120573-oxidation proteins is theindicator of higher 120573-oxidation rates and attenuates hepaticlipid accumulation [27 28] In contrast lowered expression oflipogenic proteins and SREBP-1c an important lipid synthetictranscription factor mediates hypolipidemic effects of lipidlowering agents [29ndash31] To explore the possible mechanismswhereby ruscogenin decreases hepatic lipid accumulationwe investigated the expression levels of several genes relatedto lipid metabolism Hepatic mRNA levels involved in fattyacid oxidation (PPAR120572 PGC-1a CPT-1 UCP2 and UCP3)were significantly increased in ruscogenin-treated HFD-fedhamsters Conversely ruscogenin decreased the expressionof genes involved in lipogenesis (SREBP-1c ACC and FAS)

These results suggest that ruscogenin reduces hepatic lipidaccumulation in twoways downregulating lipogenic proteinsand upregulating proteins in 120573-oxidation pathway

5 Conclusion

We demonstrated that ruscogenin has a beneficial effectin inhibiting fat accumulation in liver improves insulinresistance inhibits inflammation and possesses a repressiveproperty on hepatic lipogenesis these effects are associatedwith the inhibition of NF-120581B and SREBP-1c and induction ofPPAR120572 Therefore ruscogenin could represent a promisingagent to reduce fatty liver or reverse hepatic disorders linkedto type 2 diabetes as a monotherapy or in combination withother existing drugs

Conflict of Interests

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

Acknowledgment

Thepresent studywas supported by aGrant from theNationalScience Council (NSC 102-2320-B-127-001-MY3) of Taiwan

References

[1] MA IbrahimMKelleni andAGeddawy ldquoNonalcoholic fattyliver disease current and potential therapiesrdquo Life Sciences vol92 no 2 pp 114ndash118 2013

10 BioMed Research International

[2] P Paschos and K Paletas ldquoNon alcoholic fatty liver disease andmetabolic syndromerdquoHippokratia vol 13 no 1 pp 9ndash19 2009

[3] M D Beaton ldquoCurrent treatment options for nonalcoholicfatty liver disease and nonalcoholic steatohepatitisrdquo CanadianJournal of Gastroenterology vol 26 no 6 pp 353ndash357 2012

[4] Z J Xu J G Fan X D Ding L Qiao and G L Wang ldquoChar-acterization of high-fat diet-induced non-alcoholic steatohep-atitis with fibrosis in ratsrdquo Digestive Diseases and Sciences vol55 no 4 pp 931ndash940 2010

[5] V Braunersreuther G L Viviani F Mach and F MontecuccoldquoRole of cytokines and chemokines in non-alcoholic fatty liverdiseaserdquo World Journal of Gastroenterology vol 18 no 8 pp727ndash735 2012

[6] F Oakley J Mann S Nailard et al ldquoNuclear factor-120581B1 (p50)limits the inflammatory and fibrogenic responses to chronicinjuryrdquo The American Journal of Pathology vol 166 no 3 pp695ndash708 2005

[7] J Kou Y Tian Y Tang J Yan and B Yu ldquoAntithrombotic activ-ities of aqueous extract from Radix Ophiopogon japonicus andits two constituentsrdquoBiological andPharmaceutical Bulletin vol29 no 6 pp 1267ndash1270 2006

[8] J Kou Y Sun Y Lin et al ldquoAnti-inflammatory activities ofaqueous extract from Radix Ophiopogon japonicus and its twoconstituentsrdquoBiological andPharmaceutical Bulletin vol 28 no7 pp 1234ndash1238 2005

[9] C Capra ldquoPharmacology and toxicology of some componentsof Ruscus aculeatusrdquo Fitoterapia vol 4 pp 99ndash113 1972

[10] E Bouskela F Z G A Cyrino and G Marcelon ldquoPossiblemechanisms for the inhibitory effect of Ruscus extract onincreased microvascular permeability induced by histamine inhamster cheek pouchrdquo Journal of Cardiovascular Pharmacologyvol 24 no 2 pp 281ndash285 1994

[11] R M Facino M Carini R Stefani G Aldini and L SaibeneldquoAnti-elastase and anti-hyaluronidase activities of saponins andsapogenins from Hedera helix Aesculus hippocastanum andRuscus aculeatus factors contributing to their efficacy in thetreatment of venous insufficiencyrdquo Archiv der Pharmazie vol328 no 10 pp 720ndash724 1995

[12] Y L Huang J P Kou L Ma J X Song and B Yu ldquoPossiblemechanism of the anti-inflammatory activity of ruscogeninrole of intercellular adhesionmolecule-1 and nuclear factor-120581BrdquoJournal of Pharmacological Sciences vol 108 no 2 pp 198ndash2052008

[13] Q Sun L Chen M Gao et al ldquoRuscogenin inhibitslipopolysaccharide-induced acute lung injury in mice Involve-ment of tissue factor inducible NO synthase and nuclear factor(NF)-120581Brdquo International Immunopharmacology vol 12 no 1 pp88ndash93 2012

[14] T Guan Q Liu Y Qian et al ldquoRuscogenin reduces cerebralischemic injury via NF-120581B-mediated inflammatory pathway inthe mouse model of experimental strokerdquo European Journal ofPharmacology vol 714 no 1ndash3 pp 303ndash311 2013

[15] M van Heek D S Compton C F France et al ldquoDiet-inducedobese mice develop peripheral but not central resistance toleptinrdquo Journal of Clinical Investigation vol 99 no 3 pp 385ndash390 1997

[16] D R Matthews J P Hosker A S Rudenski B A Naylor DF Treacher and R C Turner ldquoHomeostasis model assessmentinsulin resistance and 120573-cell function from fasting plasmaglucose and insulin concentrations in manrdquo Diabetologia vol28 no 7 pp 412ndash419 1985

[17] J Folch M Lees and G H Sloane-Stanley ldquoA simple methodfor the isolation and purification of total lipides from animaltissuesrdquo The Journal of Biological Chemistry vol 226 no 1 pp497ndash509 1957

[18] D E Kleiner E M Brunt M van Natta et al ldquoDesign andvalidation of a histological scoring system for nonalcoholic fattyliver diseaserdquo Hepatology vol 41 no 6 pp 1313ndash1321 2005

[19] K J Livak and T D Schmittgen ldquoAnalysis of relative geneexpression data using real-time quantitative PCR and the 2-ΔΔCT methodrdquoMethods vol 25 no 4 pp 402ndash408 2001

[20] S Joshi-Barve S S Barve K Amancherla et al ldquoPalmitic acidinduces production of proinflammatory cytokine interleukin-8from hepatocytesrdquoHepatology vol 46 no 3 pp 823ndash830 2007

[21] D Gao S Nong X Huang et al ldquoThe effects of palmitate onhepatic insulin resistance are mediated by NADPH oxidase 3-derived reactive oxygen species through JNK and p38 MAPKpathwaysrdquo The Journal of Biological Chemistry vol 285 no 39pp 29965ndash29973 2010

[22] V Nehra P Angulo A L Buchman and K D LindorldquoNutritional and metabolic considerations in the etiology ofnonalcoholic steatohepatitisrdquo Digestive Diseases and Sciencesvol 46 no 11 pp 2347ndash2352 2001

[23] J Bhathena A Kulamarva C Martoni et al ldquoDiet-inducedmetabolic hamster model of nonalcoholic fatty liver diseaserdquoDiabetes Metabolic Syndrome and Obesity vol 4 pp 195ndash2032011

[24] HKanda S Tateya Y Tamori et al ldquocontributes tomacrophageinfiltration into adipose tissue insulin resistance and hepaticsteatosis in obesityrdquoThe Journal of Clinical Investigation vol 116no 6 pp 1494ndash1505 2006

[25] M Malaguarnera M Di Rosa F Nicoletti and L Malaguarn-era ldquoMolecular mechanisms involved in NAFLD progressionrdquoJournal of Molecular Medicine vol 87 no 7 pp 679ndash695 2009

[26] I A Leclercq A Da Silva Morais B Schroyen N Van Hul andAGeerts ldquoInsulin resistance in hepatocytes and sinusoidal livercells mechanisms and consequencesrdquo Journal of Hepatologyvol 47 no 1 pp 142ndash156 2007

[27] J K Reddy and T Hashimoto ldquoPeroxisomal 120573-oxidation andperoxisome proliferatormdashactivated receptor 120572 an adaptivemetabolic systemrdquo Annual Review of Nutrition vol 21 pp 193ndash230 2001

[28] J Huang Y Jia T Fu et al ldquoSustained activation of PPAR120572 byendogenous ligands increases hepatic fatty acid oxidation andprevents obesity in obob micerdquoThe FASEB Journal vol 26 no2 pp 628ndash638 2012

[29] T F Osborne ldquoSterol regulatory element-binding proteins(SREBPS) key regulators of nutritional homeostasis and insulinactionrdquo Journal of Biological Chemistry vol 275 no 42 pp32379ndash32382 2000

[30] A P Jensen-Urstad and C F Semenkovich ldquoFatty acid synthaseand liver triglyceride metabolism housekeeper or messengerrdquoBiochimica et Biophysica Acta vol 1821 no 5 pp 747ndash753 2012

[31] J Mao F J DeMayo H Li et al ldquoLiver-specific deletion ofacetyl-CoA carboxylase 1 reduces hepatic triglyceride accumu-lation without affecting glucose homeostasisrdquo Proceedings of theNational Academy of Sciences of theUnited States of America vol103 no 22 pp 8552ndash8557 2006

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

MEDIATORSINFLAMMATION

of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Behavioural Neurology

EndocrinologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Disease Markers

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

OncologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Oxidative Medicine and Cellular Longevity

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Computational and Mathematical Methods in Medicine

OphthalmologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Diabetes ResearchJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Research and TreatmentAIDS

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Gastroenterology Research and Practice

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom