research article ergosterol is the active compound of...

Research ArticleErgosterol Is the Active Compound of Cultured MyceliumCordyceps sinensis on Antiliver Fibrosis

Yuan Peng1 Yanyan Tao12 Qinglan Wang3 Li Shen12 Tao Yang1

Zulong Liu1 and Chenghai Liu123

1 Institute of Liver Diseases Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine528 Zhangheng Road Pudong New Area Shanghai 201203 China

2 Shanghai Clinical Key Laboratory of Traditional Chinese Medicine Shanghai 201203 China3 E-Institute of TCM Internal Medicine Shanghai Municipal Education Commission Shanghai 201203 China

Correspondence should be addressed to Chenghai Liu chenghailiuhotmailcom

Received 9 July 2014 Accepted 9 September 2014 Published 15 October 2014

Academic Editor Jae Youl Cho

Copyright copy 2014 Yuan Peng 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

CulturedmyceliumCordyceps sinensis (CMCS) is a Chinese herbalmedicine which is widely used for a variety of diseases includingliver injury in clinicThe current study aims to investigate the protective effects of CMCS against liver fibrosis and to exploit its activeantifibrotic substances in vivo and in vitro For evaluating the antifibrotic effect of CMCS and ergosterol male C57BL6 mice wereinjected intraperitoneally with carbon tetrachloride (CCl

4) and treated with CMCS (120mgkgd) or ergosterol (50mgkgd) Four

weeks later serum liver function hepatic hydroxyproline (Hyp) content liver inflammation collagen deposition and expressionof alpha smooth muscle actin (120572-SMA) in liver tissue were evaluated Besides toxicological effects of active compounds of CMCSon hepatocytes and hepatic stellate cells (HSCs) were detected and expressions of permeability of the lysosomal membrane EdUF-actin and 120572-SMA of activated HSCs were analyzed to screen the antifibrotic substance in CMCS in vitro Our results showed thatCMCS could significantly alleviate levels of serum liver functions attenuate hepatic inflammation decrease collagen depositionand relieve levels of 120572-SMA in liver respectively Ergosterol the active compound in CMCS that was detected by HPLC played adose-dependent inhibition role on activated HSCs via upregulating expressions of permeability of the lysosomal membrane anddownregulating levels of EdU F-actin and 120572-SMA on activated HSCs in vitro Moreover ergosterol revealed the antifibrotic effectalike in vivo In conclusion CMCS is effective in alleviating liver fibrosis induced by CCl

4and ergosterol might be the efficacious

antifibrotic substance in CMCS in vivo and in vitro

1 Introduction

Liver fibrosis is a wound-healing response to liver injury thatinvolves a wide range of etiologic agents including virusesdrugs metabolic disorders and immune attacks [1] Liverfibrosis involves an increased production of extracellularmatrix (ECM) components particularly the deposition ofcollagens in liver [2] Activated hepatic stellate cells (HSCs)portal fibroblasts and myofibroblasts of bone marrow originhave been identified as the major collagen-producing cells inthe injured liver [3] Above all HSCs especially the activatedHSCs were identified as the main collagen-producing cellsin the liver [4] In carbon tetrachloride (CCl

4) model of liver

fibrosis quiescent HSCs are activated to become fibrogenic

myofibroblasts [5] which express 120572-smooth muscle actin(120572-SMA) Since liver fibrosis is closely linked with severepathological changes including the development of hepaticcarcinoma it is essential to elucidate the mechanisms ofrestraining the activation and elimination of HSCs which isconsidered as a target for the treatment of liver fibrosis [6]

Cordyceps sinensis (Berk) Sacc as a well-known tonicherb in TCM is a highly valued fungus in China in regulatingthe immune function In recent years cultured myceliumCordyceps sinensis (CMCS) was successfully developed andwidely used as the alternative of natural Cordyceps sinensis(Berk) Sacc [7] Accumulated evidences from both ani-mal and human studies indicated that Cordyceps sinensiswas capable of antifibrosis [8] and anti-inflammation [9]

Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2014 Article ID 537234 12 pageshttpdxdoiorg1011552014537234

2 Evidence-Based Complementary and Alternative Medicine

Whether the active ingredients of CMCS could inhibit HSCsactivation and liver fibrosis remained unknown

In this study we aimed to elucidate the effects of CMCSon CCl

4-induced liver fibrosis in vivoThen we evaluated the

effects of the active compounds of CMCS onHSCs activationand hepatic fibrosis in vitro and in vivo To clarify our aimseveral questions should be formulated Specifically theywere as follows (1) Could CMCS inhibit hepatic fibrosis (2)What were the ingredients of CMCS (3)Whichmight be theactive antifibrotic compound of CMCS that inhibited HSCsactivation and hepatic fibrosisThe results demonstrated thatboth CMCS and ergosterol an active ingredient of CMCSreduced liver injury andhepatic fibrosisThese findingsmightsuggest its potent effects on antifibrosis and exceed its role fordesigning better antifibrotic drugs

2 Material and Methods

21 Reagents CCl4of analytical reagent grade was obtained

from Sinopharm Group Co Ltd All the other chemicals andsolvents used were of analytical grade

22 Drug Preparation The powder of cultured myceliumCordyceps sinensis was purchased from Shanghai SundiseChinese Medicine Technology Development Co Ltd Theextraction process of CMCS and determination and quan-titation of active compounds in CMCS analyzed by HPLCwere all as previously described [10] Adenosine ergosterolvernine and uridine were all purchased from ShanghaiWinherb Medical Science Co Ltd The purity of thesestandards determined by HPLC was more than 98 as perthe manufacturerrsquos specification

23 Mice Male C57BL6 mice (10 weeks of age) wereobtained from Shanghai SLAC Laboratory Animal Co Ltd(Shanghai China) Mice were housed in a room undercontrolled temperature (22ndash25∘C) humidity (46ndash52) andlighting (12-hour artificial light and dark cycle) with freeaccess to tap water andmouse chowThe standard diet pelletscontained not less than 20 protein 5 fibers 35 fatsand 65 ash and vitamins mixture Our experiment wasconducted according to the local ethical guidelines (ShanghaiUniversity of TCM Shanghai China)

24 Induction of Hepatic Fibrosis For liver fibrosismicewereintraperitoneally injected with 10 CCl

4olive oil 2mLkg

body weight three times a week for 4 weeks [11] Followingpentobarbital sodium anesthesia all mice were sacrificed24 hours after the last injection Serum and liver sampleswere harvested For histology tissue specimens were fixed inbuffered formalin and embedded in paraffinwax For fluores-cence liver specimens were embedded in OCT compoundSerum and liver samples were kept frozen at minus70∘C untilassayed

25 Animal Groups and Experimental Design To evaluate theeffect of CMCS against liver fibrosis mice were randomlydivided into 4 groups (10 mice per group) normal controlgroup (Normal control) CMCS-treated group (CMCS con-trol) CCl

4-treated group (Model control) and CCl

4plus

CMCS-treated group (CMCS treatment) Mice in CMCScontrol and CMCS treatment were treated orally with CMCSat a daily dose of 120mgkg of body weight respectivelywhich was equivalent to the dosage of a 60 kg adult Mice inNormal control and Model control were treated with ddH

2O

by gavage To investigate the effect of ergosterol against liverfibrosis mice were randomly divided into 3 groups (10 miceper group) normal control group (Normal control) CCl

4-

treated group (Model control) and CCl4plus ergosterol-

treated group (Ergosterol treatment) Mice in Ergosteroltreatment were administered orally by gavage with ergosterolat a daily dose of 50mgkg of body weight

26 Measurements of Serum Liver Function and Hydroxypro-line Content The activities of serum alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST) and thelevels of serum albumin (Alb) and total bilirubin (TBil) werequantitated by following the instructions provided by themanufacturer (Nanjing JianCheng Bioengineering InstituteNanjing China) including use of standardization Levels ofhydroxyproline (Hyp) in tissue were measured by Jamallrsquosmethod [12]

27 Histopathological Assessment of Liver Injury Liver spec-imens were fixed in 10 formaldehyde solution and dehy-drated in a graded alcohol series embedded in paraffinblocks and cut into 4 120583m thick slices For histopathologicalexamination slices were stained by using standard procedureof hematoxylin and eosin (HampE) For investigation of thehepatic collagen deposition Sirius red-polarization stainingwas performed and a red color staining was consideredwherethe collagen deposition Images were analyzed with lightmicroscope (Olympus BX40 Japan)

28 Cell Culture The LX-2 cell line a spontaneously immor-talized human hepatic stellate cell was a gift from Dr ScottL Friedman (Mount Sinai School of Medicine New YorkNY) LX-2 cells were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM) supplemented with 10 fetal bovineserum (FBS) 100 unitsmL of penicillin and 100 unitsmLof streptomycin The human hepatic cell line HL-7702an immortal cell line derived from human adult hepatictissue was purchased from Institute of Biochemistry andCell Biology SIBS CAS (Shanghai China) HL-7702 cellswere incubated in RPMI 1640 medium containing 10 heat-inactivated FBS 100UmL penicillin 100 120583gmL strepto-mycin and 2mM glutamine All the cells were grown in ahumidified incubator at 37∘C in a 5 CO

2atmosphere

29 Drug Treatments Cells were treated with 6 concentra-tions (25 5 10 20 40 and 80120583M) of each compoundin duplicate for a period of 24 h The compounds wereinitially dissolved as concentrated stock solutions in dimethylsulfoxide ([DMSO] Sigma-Aldrich USA)

210 Cell Viability and Proliferation Assay ForHL-7702 cellscell viability and proliferation assays were performed by usingthe Cell Counting Kit-8 (CCK8 Beyotime BiotechnologyJiangsu China) according to the manufacturerrsquos protocol

Evidence-Based Complementary and Alternative Medicine 3

For LX-2 cells cell viability and proliferation assays weredetected by Thiazolyl Blue Tetrazolium Blue (MTT Sigma-Aldrich USA) according to the manufacturerrsquos protocolBesides cytotoxicity of ergosterol on LX-2 was tested byMultiparameter Cytotoxicity II Kit (ThermoFisher ScientificGermany) aiming to investigate the membrane physicalstate on lysosomal permeability and cell proliferation wasobserved by Cell-Light EdU Apollo 643 DNA In Vitro Kit(RiboBio Co LTD Guangzhou China) according to themanufacturerrsquos protocol Images of the permeability of thelysosomal membrane and EdU were taken by CellomicsArrayScan VTI HCS Reader (ThermoFisher Scientific USA)and data were analyzed by Cellomics Cell Health ProfilingBioApplication Software

211 Immunocytochemistry and Immunofluorescence for 120572-SMA For immunocytochemistry LX-2 cells cultured on 96wells were washed with cold PBS twice and fixed with coldmethanol acetone (1 1) for 10min on ice After extensivewashing with PBS three times cells were permeated with005 saponin for 15min The cells were then blocked with5 bovine serum albumin in PBS buffer for 30min atroom temperature before being incubated with the alphasmooth muscle actin (120572-SMA) (1 100) primary antibodies(A2547 Sigma USA) Cells were then stained with FITC-conjugated secondary antibodies After washing cells weredouble-stained with Hoechst 33258 (Beyotime Biotechnol-ogy Jiangsu China) to visualize the nuclei Imageswere takenby Cellomics ArrayScan VTI HCS Reader For immunoflu-orescence liver tissue embedded in OCT compound wascut into 9 micrometers thick and fixed in 4 formaldehyde(Dingguo Shanghai China) After washing with PBS forthree times sections were permeabilized with 01 Triton X-100 and blocked with 5 BSA in 01M PBS for 20 minutesat room temperature to reduce nonspecific binding andthen were incubated with primary antibodies against 120572-SMA (M0851 DAKO Japan) followed by the biotinylatedsecondary antibody To visualize the primary antibodies cellswere stained with FITC-conjugated secondary antibodiesAfterwashing cells were double stainedwith 410158406-diamidino-2-phenylindole (DAPI) to visualize the nuclei Images wereobtained using a confocal microscope and photographs weretaken with Olympus confocal software The semiquantifica-tion data for 120572-SMA level in the liver tissue were determinedwith the computer-assisted image analysis system

212 F-Actin Cytoskeleton Staining LX-2 cells were grown ina 96-well chamber and treated with 5 ngmL transforminggrowth factor-beta1 (TGF-1205731) and drugs After 24 hours cellswere fixed in 4 formaldehyde and permeabilized with 02triton X-100 F-actin was stained with rhodamine phalloidin(1 100) (Molecular Probes Inc Eugene Oregon USA) andthe nucleus with DAPI according to the manufacturerrsquosprotocol Cells were visualized by Cellomics ArrayScan VTIHCSReader and data were analyzed by Cellomics Cell HealthProfiling BioApplication Software

213 Statistical Analysis All data were analyzed by usingPASW Statistics 18 software Differences between the groups

were assessed by nonparametric one-way analysis of variance(ANOVA) followed by the least significant difference (LSD)post hoc tests Values in the text are means plusmn standarddeviation (SD) Differences with 119875 lt 005 were consideredto be statistically significant

3 Results

31 CMCS Attenuated CCl4-Induced Liver Fibrosis in Mice

In order to evaluate the effect of CMCS on CCl4-induced

liver fibrosis levels of serum liver functions were assayedby kits Levels of serum ALT AST and TBil and higherlevels of serum Alb were lower in Normal control comparedwith those in Model control (Figures 1(a) 1(b) 1(c) and1(d)) The levels of ALT AST and TBil were significantlyreduced inCMCS-treatedmice Besides CMCS alleviated theratios of liverbody weight (BW) () and spleenBW (Ğ) inCCl4-induced hepatic fibrosis mice respectively (Figures 1(e)

and 1(f)) For investigating the inflammation and collagenaccumulation in the liver tissue HampE and Sirius Red stainingwere observed respectively (Figures 1(g) and 1(h)) InNormalcontrol and CMCS control groups the hepatocytes wereradially arranged around a central vein with no collagenfibers present respectively In Model control hepatic plateswere diffusely disruptedwith pronounced balloonednecrotichepatocytes containing acidophilic hyaline inclusions whichwere degenerated with widely mononuclear cell infiltrationpresent in the portal area and fibrous septa (Figure 1(g))Hepatic parenchyma was expanded by collagen fibers andlobules were separated by fibrous septa that surroundedthe hepatocytes (Figure 1(h)) Hepatic inflammation andfibrosis were significantly alleviated by CMCS treatmentas evidenced by reduced inflammatory cell infiltration andthinner fibrous septa (Figures 1(h) and 1(j)) Similarly hepatichydroxyproline content was dropped by CMCS treatment inmodel mice (Figure 1(l))

32 CMCS Reduced Expression of 120572-SMA In Vivo SinceCMCS could attenuate CCl

4-induced liver fibrosis we next

examined the effect of CMCS on the expression of hepatic 120572-SMA the gold marker of liver fibrosis and HSCs activationInNormal control andCMCS control group positive stainingfor 120572-SMA was mainly observed in vascular walls of centralveins and portal regions while the stronger 120572-SMA stainingwas seen in Model control especially in the vicinity of thebile ductules and the fibrous septa (Figures 1(i) and 1(l))The expression of 120572-SMA was significantly decreased inCMCS treatment group comparedwith that inModel control(Figures 1(i) and 1(l))These results revealed that CMCS couldsignificantly inhibit the activation of HSCs

33 Ergosterol Inhibited Activities of HSCs In Vitro Wepreviously detected the active compounds of CMCS byHPLC finding thatCMCSmainly contained four ingredientsadenosine ergosterol vernine and uridine In an effort todefine the potential active substances of CMCS that mightplay the critical roles in resisting liver fibrosis we observedthe effects of these four monomers on human hepatocytesand HSC cell lines in vitro respectively Firstly HL-7702


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Figure 1 Continued


120572-S

MA

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Figure 1 Antifibrotic effects of CMCS on CCl4-induced liver fibrosis in mice Mice of ten-week old were treated as described in the legend

Serum ALT (a) AST (b) Alb (c) and TBil (d) were assessed by liver function tests kits respectively Levels of serum liver functions weredecreased and liverBW (e) and spleenBW (f) were alleviated by CMCS treatment in CCl

4-induced liver fibrosis mice Histologic evaluation

of liver tissues were stained with HampE ((g) times200) Collagen deposition was revealed with Sirius Red staining ((h) times100) and was shown as theproportion of Sirius red-positive area (j) Expressions of 120572-SMA in liver tissues were analyzed by immunofluorescent staining Representativebright-field and fluorescent micrographs were shown ((i) times100) Semiquantification of 120572-SMA expression was evaluated and shown aspercentage of 120572-SMA-positive areas (k) Hydroxyproline content was quantified from 100mg liver samples and was measured by Jamallrsquosmethod (l) lowast119875 lt 005 lowastlowast119875 lt 0001 versus Normal control 119875 lt 005 119875 lt 0001 versus Model control

Table 1 IC50 values (120583M) of active compounds in CMCSa

Cell line Active compoundsAdenosine Ergosterol Vernine Uridine

HL-7702 nc 3710 (plusmn207) nc ncLX-2 nc 5551 (plusmn713) nc ncaValues are means of at least three experiments standard deviation is givenin parentheses (nc = no toxic)

and LX-2 cells were preincubated with 25ndash80 120583M of adeno-sine ergosterol vernine and uridine for 24 h respectively

After 24 h the survival ratio of HL-7702 and LX-2 cellsin incubations with 25ndash20120583M of adenosine ergosterolvernine and uridine for 24 h exceeded 80 respectively(Figures 2(a) and 2(b)) which showed no obvious toxic tothe cells (Table 1) Secondly HL-7702 cells were exposedto 05mM H

2O2for 30min to induce hepatocellular injury

in vitro Subsequently cells were incubated with 25ndash20120583Madenosine ergosterol vernine and uridine for 24 h 24 hourslater the four active ingredients of CMCS were not able toprotect against hepatocellular injury induced byH

2O2in vitro

(Figure 2(c)) Finally LX-2 cells were incubated with 5 ngmL


25 5 10 20 40 800

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120

140

VernineUridine

AdenosineErgosterol

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wth

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)

Concentration (120583M)

(a)

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406080

100120140160180200

Adenosine Ergosterol Vernine Uridine

Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

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25120583

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(d)

Normal control Ergosterol 25 120583M Ergosterol 5120583M Ergosterol 10120583M

Ergosterol 20120583M Ergosterol 40120583M Ergosterol 80120583M

(e)

Figure 2 Continued


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lowast

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(f)

Figure 2 Cytotoxicity and proliferation of adenosine ergosterol vernine and uridine in HL-7702 and LX-2 cell lines ((a) and (b)) Cells werecultured in a 96-well plate at a density of 4000 cellswell and incubated with 25ndash80120583M of adenosine ergosterol vernine and uridine for24 h respectively ForHL-7702 cell cytotoxicity was detected byCCK8 For LX-2 cell cytotoxicity was analyzed byMTT Representative dose-response curves inHL-7702 cell (a) and LX-2 cell (b) culture with adenosine ergosterol vernine and uridine for 24 h were represented by linechart respectively ((c) and (d)) Cells were cultured in a 96-well plate at a density of 4000 cellswell HL-7702 cells were exposed with 05mMH2O2for 30min and LX-2 cells were incubated with 05 ngmL TGF-1205731 for 24 hThe following cells were cultured with 25ndash20 120583Madenosine

ergosterol vernine and uridine for 24 h respectively For HL-7702 cell proliferation was detected by CCK8 For LX-2 cell proliferationwas analyzed by MTT Representative dose-response curves in HL-7702 cell (c) and LX-2 cell (d) were represented in comparison with theModel control (100) and were shown as growth of the cells respectively (e) LX-2 cells were cultured in a 96-well plate at a density of 4000cellswell Cells were incubated with 25ndash80120583M of ergosterol for 24 h Images of permeability of the lysosomal membrane were investigatedby Cellomics ArrayScan VTI HCS ReaderThe nuclear and lesioned lysosomal membranes were stained with blue and green respectively (f)The image analysis of immunofluorescence used by Cellomics Cell Health Profiling BioApplication Software lowast119875 lt 005 lowastlowast119875 lt 001 versusNormal control 119875 lt 005 119875 lt 001 versus Model control

TGF-1205731 for 24 h to induce HSC activation Then cells werecultured with 25ndash20120583M adenosine ergosterol vernine anduridine for 24 h respectively 24 hours later HSCs activationcould not be restrained by the other monomers of CMCSexcept ergosterol (Figure 2(d)) Surprisingly ergosterol had adose-dependent inhibition onHSCs activation in vitro whichrevealed that ergosterol might be able to act a vital role inresisting liver fibrosis in vitro To confirm the antifibroticeffect of ergosterol in vitro we proceeded to explore the effectof ergosterol on permeability of the lysosomal membrane inLX-2 cells As expected the permeability of the lysosomalmembrane in LX-2 cells was altered by treatment with ergos-terol for 24 h (Figures 2(e) and 2(f)) Incubating with 10 120583Mergosterol decreased the level of permeability of the lysosomalmembrane but increased in the concentration of 20120583M(Figures 2(e) and 2(f)) Besides proliferation of activated LX-2 cells stimulated by 5 ngmL TGF-1205731 for 24 h was inhib-ited after treatment with 10 120583M ergosterol for another 24 hunsurprisingly (Figure 3) In addition cytoskeleton proteinexpression of LX-2 cells was significantly downregulated andlevels of 120572-SMA showed a decreasing trend by cultured withergosterol in vitro respectively which further proved thatergosterol as a highly efficacious substance in CMCS couldinhibit HSCs activation in vitro (Figure 3)

34 Ergosterol Protected Liver against CCl4-Induced Hepatic

Fibrosis In Vivo Treatment of intragastric administrationwith 50mgkg ergosterol was given to investigate the pre-vention of liver fibrosis induced by 10 CCl

4in vivo Model

control group had much higher serum levels of ALT ASTTBil liverBW and spleenBW and lower Alb than thoseof the Normal control group (Figures 4(a) 4(b) 4(c) 4(d)4(e) and 4(f)) Furthermoremore severe liver inflammationcollagen deposition and expression of 120572-SMA were seen inthe Model control group respectively (Figures 4(g) 4(h)4(i) and 4(j)) compared to those in the Normal controlgroup Ergosterol treatment ameliorated serum liver func-tion decreased liverBW or spleenBW attenuated hepaticinflammation decreased collagen deposition and improvedexpression of 120572-SMA in Model group which obviouslyindicated that ergosterol could protect against hepatic fibrosisin vivo

4 Discussion

Liver fibrosis represents chronic wound repair following liverinjury [13] Upon liver injury quiescent HSCs the most rele-vant cell type for hepatic fibrogenesis become active convertinto myofibroblast-like cells and overproduce extracellularmatrix (ECM) [14] which actually worsen the liver damage


Normal control Model controlEd

UF-

actin

Ergosterol 25 120583M Ergosterol 5120583M Ergosterol 10120583M Ergosterol 20120583M120572

-SM

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Figure 3 Effects of ergosterol on expressions of EdU F-actin and 120572-SMA induced by TGF-1205731 in LX-2 cell (a) LX-2 cells were culturedin a 96-well plate at a density of 4000 cellswell Cells were stimulated by 5 ngmL TGF-1205731 for 24 h and then incubated with 25ndash20 120583M ofergosterol for 24 h Images of EdU F-actin and 120572-SMAwere investigated by Cellomics ArrayScan VTI HCS Reader respectivelyThe nuclearand proliferating cells were stained with blue and pink respectively (b) The image analysis of immunofluorescence used by Cellomics CellHealth Profiling Bio Application Software lowast119875 lt 005 lowastlowast119875 lt 001 versus Normal control 119875 lt 005 119875 lt 005 versus Model control

The terminal outcome of liver fibrosis is the formation ofnodules encapsulated by fibrillar scar matrix [15] Thereforeit is imperative that inhibition of HSCs activation is urgentlyneeded

Natural Cordyceps sinensis (Berk) Sacc has been widelyused in China for potentially vulnerable people with chronicdiseases in all ages and its use is strongly supported byclinical application Nowadays CMCS is widely used as thealternative of natural Cordyceps sinensis (Berk) Sacc [7]which has better curative effect and lightens the economicburden of patients in low income group CMCS containsan array of active substances which reveal the advantage ofpotential synergy between the various componentsMore and

more research information has convinced its significant phar-macological properties Some CMCS related nutraceuticalsavailable in the market are considered to relieve the ldquostressfor humans of living in technologically developed societiesrdquoby stimulating basic and secondary responses of the immunesystem [16]

To explore the antifibrotic effect on CMCS we haveinvestigated the role of CMCS and its active compounds inhepatic fibrosis in vivo and in vitro In our study we firstlyconfirmed earlier findings that the extractum of CMCS couldalleviate levels of serum liver function in CCl

4-induced liver

fibrosis mice in vivoThen we further analyzed the pathologyof liver tissue to evaluate the efficacy of CMCS on treatment


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Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)

Normal control Model control Ergosterol treatment

Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued

10 Evidence-Based Complementary and Alternative Medicine120572

-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)

Figure 4 Antifibrotic effects of ergosterol on CCl4-induced liver fibrosis in mice Mice of ten-week old were treated as described in the

legend Serum ALT (a) AST (b) Alb (c) and TBil (d) were assessed by liver function tests kits respectively Levels of serum liver functionswere decreased and ratios of liverBW (e) and spleenBW (f) were alleviated by Ergosterol treatment in CCl

4-induced liver fibrosis mice

Histologic results of liver tissues were stained with HampE ((g) times200) Quantification of the same three groups in (g) with respect to Sirius Redstaining ((h) times100) Collagen deposition was shown as percentage of Sirius red-positive area (j) Liver tissues were analyzed by staining for120572-SMA immunofluorescence Representative bright-field and fluorescent micrographs were shown ((i) times100) Semiquantification data for120572-SMA expressions in the liver tissue were evaluated and were shown as percentage of 120572-SMA-positive areas (k) Hydroxyproline contentwas quantified from 100mg liver samples and measured by Jamallrsquos method (l) lowastlowast119875 lt 001 versus Normal control 119875 lt 001 versus Modelcontrol

of liver fibrosis It was obvious that CMCS could relieve theinfiltration of inflammatory cells and collagen depositionBesides expression of 120572-SMA the goldenmarker of activatedHSC was decreased by CMCS treatment To observe thetoxicity of CMCS normal mice were administrated withthe same dosage of CMCS in the meantime The resultsof serum liver function and histopathological assessmentindicated that treatment with CMCS for four weeks remainedno hepatotoxicity for normal mice These results suggestedthat CMCS had potential antifibrotic effect on CCl

4-induced

liver fibrosisWe previously made a quantitative analysis on the freeze

drying powder of CMCS extractum by HPLC to detect themain active compounds Subsequently our TCM fingerprintrevealed that CMCS mainly contained four active ingredi-ents adenosine ergosterol vernine and uridine Herein weobserved the toxicological and pharmacological studies onthese four monomers on HSCs (LX-2 cell lines) in vitro inorder to discover the main potential active substances in

CMCS that played critical roles in resisting liver fibrosisMoreover the active antifibrotic substance should be withno hepatotoxicity Hence toxicological study of the fourmonomers was performed on hepatocytes (HL-7702 celllines) in vitro simultaneously The results of cytotoxicity andproliferation in HL-7702 and LX-2 implied that ergosterolhad no hepatotoxicity on hepatocytes In addition it tookeffects in manner of dose-dependent inhibition on activatedHSCs in vitro

Lysosomes are involved in the cellular degradation ofmaterial either initially present in the cell or brought intothe cell by phagocytosis or endocytosis [17] Lysosomaldestabilization is critical for the organelle and living cells [18]In our study we found ergosterol could decrease the level ofpermeability of the lysosomal membrane for HSCs at a lowconcentration which revealed that ergosterol might be liablefor damage on cellmembrane ofHSCs in vitro Evidently per-meation of EdUand expression of120572-SMAofHSCs stimulatedby TGF-120573 were repressed by ergosterol at a concentration


of 20120583M in vitro respectively In nonmuscular cells F-actinforms a system of fibers that provide mechanical support forseveral structures and in association with myosin assemblescontractile units responsible for cellular movements In ourstudy the expression of F-actin was increased in HSCsstimulated by TGF-120573 while it was decreased after ergosterolintervention which indicated that ergosterol might disruptthe cytoskeleton of HSCs

In the following studies in vivo ergosterol like CMCScould ameliorate serum liver function attenuate hepaticinflammation decrease collagen deposition and improvethe expression of 120572-SMA in mouse model of liver fibrosisrespectively which obviously confirmed that ergosterol couldprotect liver against hepatic fibrosis in vivo This finding wasconformed to that of experiments in vivo

Our results demonstrated that CMCS and ergosterolattenuate hepatic fibrosis induced by CCl

4in mice We con-

firmed the pharmacological effects ofCMCSon inhibiting theactive HSCs We further defined that the antifibrotic effect ofergosterol was associated with the restriction of proliferationwreck of cytoskeleton and activation of HSCs Our studyis the first to report that ergosterol is the active compoundof CMCS on antiliver fibrosis However as a therapeuticagent the accurate effect of CMCS and ergosterolrsquos effecton restraining liver fibrosis still need to be investigatedFurther studies and other animal models of liver fibrosisare necessary to elucidate the precise mechanisms of theirantifibrosis effects

5 Conclusions

CMCS is effective in alleviating liver fibrosis induced by CCl4

in vivo and ergosterol might be the efficacious antifibroticsubstance of CMCS in vivo and in vitro which might providealternative treatment options for hepatic fibrosis

Abbreviations

CMCS Cultured mycelium Cordyceps sinensisCCl4 Carbon tetrachloride

ALT Alanine aminotransferaseAST Aspartate aminotransferaseAlb AlbuminTBil Total bilirubinHyp HydroxyprolineHampE Hematoxylin and eosin120572-SMA Alpha smooth muscle actinHPLC High performance liquid chromatographyCCK8 Cell Counting Kit-8MTT Thiazolyl Blue Tetrazolium BlueHSCs Hepatic stellate cellsDMEM Dulbeccorsquos modified Eaglersquos mediumFBS Fetal bovine serumDMSO Dimethyl sulfoxideBSA Bovine plasma albuminPBS Phosphate-buffer salineDAB 331015840-Diamino BenzedrineDAPI 410158406-Diamidino-2-phenylindole

TGF-120573 Transforming growth factor-betaBW Body weight

Conflict of Interests

The authors declare no conflict of interests

Authorsrsquo Contribution

Chenghai Liu and Qinglan Wang designed the project YuanPeng Li Shen and Tao Yang performed experiments YuanPeng and Li Shen analyzed the data Yuan Peng YanyanTao and Zulong Liu prepared the paper Chenghai Liu madecritical revision for paper Yuan Peng and Yanyan Tao equallycontributed as the first author

Acknowledgments

This work was supported by the following Grants NationalNatural Science Foundation of China (nos 8117340581102701 and 81270053) National Science and TechnologyMajor Project (2014ZX1005001) National Key New DrugsCreation Project Innovative Drug Research and Devel-opment Technology Platform (no 2012ZX09303009-001)ZYSNXD-CC-YJXYY E-Institute (E03008) and InnovativeResearch Team in Universities of Shanghai MunicipalEducation Commission

References

[1] S L Friedman ldquoMechanisms of hepatic fibrogenesisrdquoGastroen-terology vol 134 no 6 pp 1655ndash1669 2008

[2] M BallinD EGomez CC Sinha andU PThorgeirsson ldquoRasoncogene mediated induction of a 92kDa metalloproteinasestrong correlation with the malignant phenotyperdquo Biochemicaland Biophysical Research Communications vol 154 no 3 pp832ndash838 1988

[3] R Bataller and D A Brenner ldquoLiver fibrosisrdquo The Journal ofClinical Investigation vol 115 no 2 pp 209ndash218 2005

[4] S L Friedman F J Roll J Boyles and D M Bissell ldquoHepaticlipocytes the principal collagen-producing cells of normal ratliverrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 82 no 24 pp 8681ndash8685 1985

[5] T Kisseleva M Cong Y Paik et al ldquoMyofibroblasts revertto an inactive phenotype during regression of liver fibrosisrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 109 no 24 pp 9448ndash9453 2012

[6] R Bataller and D A Brenner ldquoHepatic stellate cells as a targetfor the treatment of liver fibrosisrdquo Seminars in Liver Disease vol21 no 3 pp 437ndash451 2001

[7] L Z Shen Xiaoyun and T Jun ldquoChemical component com-parison between Cordyceps sinensis and Mycelia of Cordycepssinensisrdquo Journal of Shanxi University vol 21 pp 80ndash85 1998

[8] J X Nan E J Park B K Yang C H Song G Ko and DH Sohn ldquoAntifibrotic effect of extracellular biopolymer fromsubmerged mycelial cultures of Cordyceps militaris on liverfibrosis induced by bile duct ligation and scission in ratsrdquoArchives of Pharmacal Research vol 24 no 4 pp 327ndash332 2001

[9] S-Y Won and E-H Park ldquoAnti-inflammatory and relatedpharmacological activities of cultured mycelia and fruiting


bodies of Cordyceps militarisrdquo Journal of Ethnopharmacologyvol 96 no 3 pp 555ndash561 2005

[10] Y Peng Q Chen T Yang Y Tao X Lu and C Liu ldquoCulturedmycelium Cordyceps sinensis protects liver sinusoidal endothe-lial cells in acute liver injured micerdquoMolecular Biology Reportsvol 41 no 3 pp 1815ndash1827 2014

[11] S Radaeva R Sun B Jaruga V T Nguyen Z Tian and BGao ldquoNatural killer cells ameliorate liver fibrosis by killing acti-vated stellate cells in NKG2D-dependent and tumor necrosisfactor-related apoptosis-inducing ligand-dependent mannersrdquoGastroenterology vol 130 no 2 pp 435ndash452 2006

[12] I S Jamall V N Finelli and S S Que Hee ldquoA simple methodto determine nanogram levels of 4-hydroxyproline in biologicaltissuesrdquo Analytical Biochemistry vol 112 no 1 pp 70ndash75 1981

[13] S L Friedman ldquoCytokines and fibrogenesisrdquo Seminars in LiverDisease vol 19 no 2 pp 129ndash140 1999

[14] S L Friedman ldquoMolecular regulation of hepatic fibrosis anintegrated cellular response to tissue injuryrdquo The Journal ofBiological Chemistry vol 275 no 4 pp 2247ndash2250 2000

[15] H Tsukamoto ldquoCytokine regulation of hepatic stellate cells inliver fibrosisrdquo Alcoholism Clinical and Experimental Researchvol 23 no 5 pp 911ndash916 1999

[16] T N Lakhanpal and M Rana ldquoMedicinal and nutraceuticalgenetic resources of mushroomsrdquo Plant Genetic ResourcesCharacterisation andUtilisation vol 3 no 2 pp 288ndash303 2005

[17] K Docherty G A Maguire and C N Hales ldquoPermeabilityproperties of lysosomal membranesrdquo Bioscience Reports vol 3no 3 pp 207ndash216 1983

[18] S-J Hao J-F Hou N Jiang and G-J Zhang ldquoLoss of mem-brane cholesterol affects lysosomal osmotic stabilityrdquo GeneralPhysiology and Biophysics vol 27 no 4 pp 278ndash283 2008

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


Whether the active ingredients of CMCS could inhibit HSCsactivation and liver fibrosis remained unknown

In this study we aimed to elucidate the effects of CMCSon CCl

4-induced liver fibrosis in vivoThen we evaluated the

effects of the active compounds of CMCS onHSCs activationand hepatic fibrosis in vitro and in vivo To clarify our aimseveral questions should be formulated Specifically theywere as follows (1) Could CMCS inhibit hepatic fibrosis (2)What were the ingredients of CMCS (3)Whichmight be theactive antifibrotic compound of CMCS that inhibited HSCsactivation and hepatic fibrosisThe results demonstrated thatboth CMCS and ergosterol an active ingredient of CMCSreduced liver injury andhepatic fibrosisThese findingsmightsuggest its potent effects on antifibrosis and exceed its role fordesigning better antifibrotic drugs

2 Material and Methods

21 Reagents CCl4of analytical reagent grade was obtained

from Sinopharm Group Co Ltd All the other chemicals andsolvents used were of analytical grade

22 Drug Preparation The powder of cultured myceliumCordyceps sinensis was purchased from Shanghai SundiseChinese Medicine Technology Development Co Ltd Theextraction process of CMCS and determination and quan-titation of active compounds in CMCS analyzed by HPLCwere all as previously described [10] Adenosine ergosterolvernine and uridine were all purchased from ShanghaiWinherb Medical Science Co Ltd The purity of thesestandards determined by HPLC was more than 98 as perthe manufacturerrsquos specification

23 Mice Male C57BL6 mice (10 weeks of age) wereobtained from Shanghai SLAC Laboratory Animal Co Ltd(Shanghai China) Mice were housed in a room undercontrolled temperature (22ndash25∘C) humidity (46ndash52) andlighting (12-hour artificial light and dark cycle) with freeaccess to tap water andmouse chowThe standard diet pelletscontained not less than 20 protein 5 fibers 35 fatsand 65 ash and vitamins mixture Our experiment wasconducted according to the local ethical guidelines (ShanghaiUniversity of TCM Shanghai China)

24 Induction of Hepatic Fibrosis For liver fibrosismicewereintraperitoneally injected with 10 CCl

4olive oil 2mLkg

body weight three times a week for 4 weeks [11] Followingpentobarbital sodium anesthesia all mice were sacrificed24 hours after the last injection Serum and liver sampleswere harvested For histology tissue specimens were fixed inbuffered formalin and embedded in paraffinwax For fluores-cence liver specimens were embedded in OCT compoundSerum and liver samples were kept frozen at minus70∘C untilassayed

25 Animal Groups and Experimental Design To evaluate theeffect of CMCS against liver fibrosis mice were randomlydivided into 4 groups (10 mice per group) normal controlgroup (Normal control) CMCS-treated group (CMCS con-trol) CCl

4-treated group (Model control) and CCl

4plus

CMCS-treated group (CMCS treatment) Mice in CMCScontrol and CMCS treatment were treated orally with CMCSat a daily dose of 120mgkg of body weight respectivelywhich was equivalent to the dosage of a 60 kg adult Mice inNormal control and Model control were treated with ddH

2O

by gavage To investigate the effect of ergosterol against liverfibrosis mice were randomly divided into 3 groups (10 miceper group) normal control group (Normal control) CCl

4-

treated group (Model control) and CCl4plus ergosterol-

treated group (Ergosterol treatment) Mice in Ergosteroltreatment were administered orally by gavage with ergosterolat a daily dose of 50mgkg of body weight

26 Measurements of Serum Liver Function and Hydroxypro-line Content The activities of serum alanine aminotrans-ferase (ALT) and aspartate aminotransferase (AST) and thelevels of serum albumin (Alb) and total bilirubin (TBil) werequantitated by following the instructions provided by themanufacturer (Nanjing JianCheng Bioengineering InstituteNanjing China) including use of standardization Levels ofhydroxyproline (Hyp) in tissue were measured by Jamallrsquosmethod [12]

27 Histopathological Assessment of Liver Injury Liver spec-imens were fixed in 10 formaldehyde solution and dehy-drated in a graded alcohol series embedded in paraffinblocks and cut into 4 120583m thick slices For histopathologicalexamination slices were stained by using standard procedureof hematoxylin and eosin (HampE) For investigation of thehepatic collagen deposition Sirius red-polarization stainingwas performed and a red color staining was consideredwherethe collagen deposition Images were analyzed with lightmicroscope (Olympus BX40 Japan)

28 Cell Culture The LX-2 cell line a spontaneously immor-talized human hepatic stellate cell was a gift from Dr ScottL Friedman (Mount Sinai School of Medicine New YorkNY) LX-2 cells were cultured in Dulbeccorsquos modified Eaglersquosmedium (DMEM) supplemented with 10 fetal bovineserum (FBS) 100 unitsmL of penicillin and 100 unitsmLof streptomycin The human hepatic cell line HL-7702an immortal cell line derived from human adult hepatictissue was purchased from Institute of Biochemistry andCell Biology SIBS CAS (Shanghai China) HL-7702 cellswere incubated in RPMI 1640 medium containing 10 heat-inactivated FBS 100UmL penicillin 100 120583gmL strepto-mycin and 2mM glutamine All the cells were grown in ahumidified incubator at 37∘C in a 5 CO

2atmosphere

29 Drug Treatments Cells were treated with 6 concentra-tions (25 5 10 20 40 and 80120583M) of each compoundin duplicate for a period of 24 h The compounds wereinitially dissolved as concentrated stock solutions in dimethylsulfoxide ([DMSO] Sigma-Aldrich USA)

210 Cell Viability and Proliferation Assay ForHL-7702 cellscell viability and proliferation assays were performed by usingthe Cell Counting Kit-8 (CCK8 Beyotime BiotechnologyJiangsu China) according to the manufacturerrsquos protocol







3 Results










0

50

100

150

200

Seru

m A

LT le

vels

(IU

L)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(a)

0

50

100

150

200

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(b)

0

5

10

15

lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(c)

0

10

20

30

40

Seru

m A

lb le

vels

(gL

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(d)

0

2

4

6

8

Live

rBW

()

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(e)

0

2

4

6

8

10

Sple

enB

W (permil

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 1 Continued


120572-S

MA

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(j)

0

5

10

15

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(k)

0

200

400

600

800

1000

lowastlowast

Hyd

roxy

prol

ine (120583

gg)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(l)












2O2in vitro



25 5 10 20 40 800

20

40

60

80

100

120

140

VernineUridine

AdenosineErgosterol

Gro

wth

of H

L-77

02 ce

lls (

)


(a)

0

20

40

60

80

100

120

140

Gro

wth

of L

X-2

cells

()

25 5 10 20 40 80

VernineUridine

AdenosineErgosterol


(b)

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

406080

100120140160180200


Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(c)

40

60

80

100

120

Gro

wth

of L

X-2

cells

()

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l


25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(d)



(e)

Figure 2 Continued


Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






















3 Results










0

50

100

150

200

Seru

m A

LT le

vels

(IU

L)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(a)

0

50

100

150

200

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(b)

0

5

10

15

lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(c)

0

10

20

30

40

Seru

m A

lb le

vels

(gL

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(d)

0

2

4

6

8

Live

rBW

()

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(e)

0

2

4

6

8

10

Sple

enB

W (permil

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 1 Continued


120572-S

MA

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(j)

0

5

10

15

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(k)

0

200

400

600

800

1000

lowastlowast

Hyd

roxy

prol

ine (120583

gg)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(l)












2O2in vitro



25 5 10 20 40 800

20

40

60

80

100

120

140

VernineUridine

AdenosineErgosterol

Gro

wth

of H

L-77

02 ce

lls (

)


(a)

0

20

40

60

80

100

120

140

Gro

wth

of L

X-2

cells

()

25 5 10 20 40 80

VernineUridine

AdenosineErgosterol


(b)

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

406080

100120140160180200


Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(c)

40

60

80

100

120

Gro

wth

of L

X-2

cells

()

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l


25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(d)



(e)

Figure 2 Continued


Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

50

100

150

200

Seru

m A

LT le

vels

(IU

L)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(a)

0

50

100

150

200

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(b)

0

5

10

15

lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(c)

0

10

20

30

40

Seru

m A

lb le

vels

(gL

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(d)

0

2

4

6

8

Live

rBW

()

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(e)

0

2

4

6

8

10

Sple

enB

W (permil

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 1 Continued


120572-S

MA

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(j)

0

5

10

15

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(k)

0

200

400

600

800

1000

lowastlowast

Hyd

roxy

prol

ine (120583

gg)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(l)












2O2in vitro



25 5 10 20 40 800

20

40

60

80

100

120

140

VernineUridine

AdenosineErgosterol

Gro

wth

of H

L-77

02 ce

lls (

)


(a)

0

20

40

60

80

100

120

140

Gro

wth

of L

X-2

cells

()

25 5 10 20 40 80

VernineUridine

AdenosineErgosterol


(b)

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

406080

100120140160180200


Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(c)

40

60

80

100

120

Gro

wth

of L

X-2

cells

()

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l


25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(d)



(e)

Figure 2 Continued


Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















120572-S

MA

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(j)

0

5

10

15

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(k)

0

200

400

600

800

1000

lowastlowast

Hyd

roxy

prol

ine (120583

gg)

Normalcontrol

CMCScontrol

Modelcontrol

CMCStreatment

(l)












2O2in vitro



25 5 10 20 40 800

20

40

60

80

100

120

140

VernineUridine

AdenosineErgosterol

Gro

wth

of H

L-77

02 ce

lls (

)


(a)

0

20

40

60

80

100

120

140

Gro

wth

of L

X-2

cells

()

25 5 10 20 40 80

VernineUridine

AdenosineErgosterol


(b)

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

406080

100120140160180200


Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(c)

40

60

80

100

120

Gro

wth

of L

X-2

cells

()

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l


25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(d)



(e)

Figure 2 Continued


Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















25 5 10 20 40 800

20

40

60

80

100

120

140

VernineUridine

AdenosineErgosterol

Gro

wth

of H

L-77

02 ce

lls (

)


(a)

0

20

40

60

80

100

120

140

Gro

wth

of L

X-2

cells

()

25 5 10 20 40 80

VernineUridine

AdenosineErgosterol


(b)

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

406080

100120140160180200


Gro

wth

of H

L-77

02 ce

lls (

)

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(c)

40

60

80

100

120

Gro

wth

of L

X-2

cells

()

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l

Nor

mal

cont

rol

Mod

el co

ntro

l


25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

25120583

M5120583

M10120583

M20120583

M

(d)



(e)

Figure 2 Continued


Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Nor

mal

cont

rol

0

500

1000

1500

2000

Fluo

resc

ent i

nten

sity

of m

embr

ane

perm

eabi

lity

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Ergo

stero

l40120583

M

Ergo

stero

l80120583

M

lowast

lowast

lowast

lowast

(f)






4in vivo Model


4 Discussion




UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















UF-

actin


-SM

A

(a)

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of E

du

0

50

100

150

200

Fluo

resc

ent i

nten

sity

of F

-act

in

0

500

1000

1500

2000

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

lowast

lowastlowast

lowastlowast

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

Fluo

resc

ent i

nten

sity

of120572

-SM

A

Nor

mal

cont

rol

Mod

el co

ntro

l

Ergo

stero

l25120583

M

Ergo

stero

l5120583

M

Ergo

stero

l10120583

M

Ergo

stero

l20120583

M

(b)






4-induced liver



0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















0

20

40

60

80

100

Seru

m A

LT le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(a)

0

5

10

15

20

25

Seru

m A

ST le

vels

(IU

L) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(b)

0

5

10

15

Seru

m A

lb le

vels

(gL

)

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(c)

0

1

2

3

4

5 lowastlowast

Seru

m T

Bil

leve

ls (120583

mol

L)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(d)

0

2

4

6

8

Live

rBW

()

lowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(e)

0

2

4

6

Sple

enB

W (permil

)

Normalcontrol

Modelcontrol

Ergosteroltreatment

(f)


Hamp

E

(g)

Siriu

s red

(h)

Figure 4 Continued


-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















-SM

A

(i)

0

2

4

6

8

10

Posit

ive a

rea (

)

lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(j)

0

5

10

15

Posit

ive a

rea (

) lowastlowast

Normalcontrol

Modelcontrol

Ergosteroltreatment

(k)

0

200

400

600

800

Normalcontrol

Modelcontrol

Ergosteroltreatment

lowastlowast

Hyd

roxy

prol

ine (

120583g

g)

(l)






4-induced









4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















4in mice We con-


5 Conclusions



Abbreviations








Acknowledgments


References


























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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