nih public access 1 reiichi higashiyama kinji asahina ...rosmarinic acid and baicalin epigenetically...

Rosmarinic acid and baicalin epigenetically de-repress Pparγ inhepatic stellate cells for their anti-fibrotic effect

Melissa D. Yang1, Yi-Ming Chiang2, Reiichi Higashiyama1, Kinji Asahina1, Derek A. Mann3,Jelena Mann3, Clay Wang2, and Hidekazu Tsukamoto1,4,*

1Southern California Research Center for ALPD and Cirrhosis and Department of Pathology,Keck School of Medicine, University of Southern California, Los Angeles, California, USA2Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University ofSouthern California, Los Angeles, California, USA3Institute of Cellular Medicine, Faculty of Medical Sciences, Newcastle University, UK4Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles,California, USA

AbstractHepatic stellate cells (HSCs) undergo myofibroblastic trans-differentiation (activation) toparticipate in liver fibrosis, and identification of molecular targets for this cell fate regulation isessential for development of efficacious therapeutic modalities for the disease. Peroxisomalproliferator-activated receptor γ (PPARγ) is required for differentiation of HSCs and its epigeneticrepression underlies HSC activation. The herbal prescription Yang-Gan-Wan (YGW) preventsliver fibrosis, but its active ingredients and molecular mechanisms are unknown. Here wedemonstrate YGW prevents and reverses HSC activation via epigenetic de-repression of Pparγinvolving reductions in MeCP2 expression and its recruitment to Pparγ promoter, suppressedexpression of PRC2 methyltrasferase EZH2 and consequent reduction of H2K27di-methylation atthe 3’ exon. HPLC/MS and NMR analyses identify polyphenolic rosmarinic acid (RA) andbaicalin (BC) as active phytocompounds. RA and BC suppress the expression and signaling bycanonical Wnts, which are implicated in the aforementioned Pparγ epigenetic repression. RAtreatment in mice with existing cholestatic liver fibrosis inhibits HSC activation and progressionof liver fibrosis. In conclusion, these results demonstrate a therapeutic potential of YGW and itsactive component RA and BC for liver fibrosis via Pparγ de-repression mediated by suppressionof canonical Wnt signaling in HSCs.

Keywordsliver fibrosis; MeCP2; EZH2; Wnt; H3K27-methylation

INTRODUCTIONExcessive scarring of the liver results in cirrhosis, the end-stage liver disease of highmortality for which efficacious medical treatments are not currently available except forliver transplantation. Central to the pathogenesis of the disease is trans-differentiation oractivation of hepatic stellate cells (HSCs), vitamin-A storing liver pericytes, intomyofibroblastic cells with increased capacity for extracellular matrix (ECM) production and

*Correspondence via [email protected].

NIH Public AccessAuthor ManuscriptHepatology. Author manuscript; available in PMC 2013 April 1.

Published in final edited form as:Hepatology. 2012 April ; 55(4): 1271–1281. doi:10.1002/hep.24792.

NIH

-PA Author Manuscript

NIH


NIH


contractility. For better understanding of HSC trans-differentiation, primary efforts havebeen made on gene regulation and intracellular signaling for expression of activation-associated molecules such as collagens, cytokines (TGF-β, PDGF), chemokines (MCP-1),ECM degradation enzymes and inhibitors (MMPs, TIMPs), NADPH oxidase, renin-angiotensin system, and TLR4 ((1),(2) for review). Yet, fundamental questions concerningcell fate regulation of HSCs, remain largely underexplored. HSCs express many neuronal orglial cell markers, and their neuroectoderm origin was proposed with a subsequent failure tovalidate this notion using the Wnt1-Cre and ROSA26 reporter mice (3). This findinglogically favored a hypothesis of mesoderm-derived multipotent mesenchymal progenitorcells (MMPC) as the origin of HSCs since MMPC also give rise to neural cells besides othermesenchymal lineages for smooth muscle cells, chondrocytes, osteoblasts, and adipocyteswhose markers are also expressed by HSCs (4). In consistent with this notion, a recent studyby Asahina, et al demonstrates the mesoderm origin of mouse fetal HSCs (5).

A fat-storing phenotype is a unique and distinct feature of quiescent HSCs, and ourlaboratory proposed a decade ago that there is a regulatory commonality between adipocytesand quiescent HSCs (6). Germane to this proposal is the expression and regulation by themaster adipogenic transcription factor PPARγ, which is essential for both adipocytedifferentiation (7) and HSC quiescence (8, 9). PPARγ promotes storage of intracellular fatincluding retinyl esters in HSCs (7) while suppressing α1(I) collagen promoter via inhibitionof p300-facilitated NF-I binding (10). As shown for inhibition of adipogenesis, canonicalWnt signaling suppresses the expression and promoter activation of Pparγ in HSC trans-differentiation (11). Necdin, a member of the melanoma antigen family (MAGE) of proteins,inhibits differentiation of adipocytes (12) but promotes that of neurons (13), skeletal andsmooth muscle cells (14, 15). Our recent study demonstrates Wnt10b, one of canonical Wntsexpressed by activated HSCs, is a direct target of necdin and the necdin-Wnt pathway causesHSC trans-differentiation via epigenetic repression of Pparγ (16). This epigenetic regulationinvolves induction and recruitment of the methyl-CpG binding protein MeCP2 to the Pparγpromoter and concomitant H3K27 di- and tri-methylation in the 3’ exons of Pparγ, resultingin formation of a repressive chromatin structure as recently demonstrated by Mann, et al(17). Intriguingly, this study also demonstrates MeCP2-mediated induction of EZH2, aH3K27 methyltransferase of the polycomb repressive complex 2 (PRC2), responsible forH3K27 di- and tri-methylation (17). Most recently, this paradigm of the MeCP2-EZH2regulatory relay has elegantly been characterized in neuronal differentiation where MeCP2-mediated epigenetic repression of miR137 is shown to result in EZH2 induction (18).

This epigenetic mechanism of Pparγ repression involving the MeCP2-EZH2 relay, identifiespotential new therapeutic targets for liver fibrosis. To this end, the present study discoversthat the herbal prescription Yang-Gan-Wan (YGW) which has been known for its protectiveeffects on the liver (19), targets and abrogates the MeCP2–EZH2 relay of epigenetic Pparγrepression to reverse activated HSCs to their quiescent phenotype in culture. Our HPLC-MSand NMR analyses coupled with bioassays with primary HSCs, identify rosmarinic acid(RA) and baicalin (BC), the active component of Sho-Saiko-To, as the main activephytocompounds of YGW. RA and BC achieve the anti-fibrotic effect by supression ofcanonical Wnt signaling and epigenetic Pparγ de-repression.

MATERIALS AND METHODSAnimal Experiment

Male C57Bl/6 and collagen α1(I) promoter-GFP (Coll-GFP; kindly provided by Prof. DavidBrenner of UC San Diego) mice were subjected to ligation and scission of the common bileduct (BDL) to induce cholestatic liver fibrosis for HSC isolation or testing the therapeuticefficacy of RA. For the latter, after one week following BDL, RA was intraperitoneally

Yang et al.

Hepatology. Author manuscript; available in PMC 2013 April 1.

NIH


NIH


NIH


injected daily at the dose of 0.1mg/25g body weight until the animals were sacrificed oneweek later for Sirius-red staining morphometry, immunohistochemistry, and qPCR analysisof the livers as described below.

Hepatic Stellate Cell Isolation and CultureHSCs were isolated from normal male Wistar rats, C57Bl/6 and Coll-GFP mice by in situdigestion of the liver and arabinogalactan gradient ultracentrifugation by the Non-Parenchymal Liver Cell Core of the Southern California Research Center for ALPD andCirrhosis as described previously (11, 16). The purity of the cells as determined by phasecontrast microscopy and ultraviolet-excited fluorescence microscopy, exceeded 96%, andthe viability as determined by trypan blue exclusion exceeded 94%. In vitro activation ofHSC was achieved by culturing rat HSCs in Dulbecco’s modified Eagle’s medium (DMEM)with 1.0 g/liter glucose, 10% fetal bovine serum and 1% antibiotics on plastic dish for 3, 5or 7 days. Culture-activated rat primary HSCs were treated with the YGW or starch (control)aqueous extract at 25% (v/v). To obtain the extract, the YGW or starch powder (provided byS.P. Pharmaceutics Inc.) was suspended in DMEM at the concentration of 35mg/ml, mixedthoroughly with a vortex for 5 min, and centrifuged at ×150g for 30 min to collect thesupernatant. This supernatant was designated as 100% extract and used after filter-sterilization. RA and BC (Sigma Chemical Co) were dissolved in DMSO and tested at theconcentration of 67.5~270 µM.

Fluorescence-Activated Cell Sorting (FACS)Two weeks after BDL or sham operation, nonparenchymal cells (NPCs) were isolated fromthe Coll-GFP mice and subjected to FACS using FACS AriaII sorter (BD Bioscience) at theUSC-CSCRM/NCCC Flow Cytometry Core. GFP expression was analyzed by an argonlaser at 488 nm and a 530 nm filter. Vitamin A autofluorescence was analyzed by a solid-state laser at 350 nm and a 450 nm filter. As a negative control for vitamin Aautofluorescence, we used the spontaneously immortalized rat HSC line (BSC) establishedfrom cholestatic liver fibrosis in rats (20).

Immunohistochemistry, TUNEL and Lipid StainingAfter 3 days of the extract treatment, the cells were washed with cold phosphate-bufferedsaline (PBS) and fixed in 4% paraformaldehyde (PF). To stain α-smooth muscle actin(SMA), a fluorescein isothiocyanate (FITC) conjugated antibody (1:200, Sigma, SaintLouis, MO) was added as a primary antibody at 4°C for overnight. After washing andblocking with 5% nonfat milk, fluorescence images were viewed by a Nikon microscope asdescribed above. For intracellular lipid staining, HSCs treated with the extract for 3 days,were cultured with retinol (5µM) and palmitic acid (100µM) (Sigma, Saint Louis, MO) for48 hr, and fixed with 10% formalin in PBS. Oil Red O (0.5%w/v in isopropanol) was dilutedwith 67% volume of water, filtered, and added to the fixed HSCs. Apoptosis was detected incultured HSCs and liver sections from BDL mice using a Cell Death Detection kit fromRoche. For liver section immunostaining, liver tissues were fixed with 4% PF and embeddedin freezing medium. Cryosections (7 µm) were washed with PBS, digested with 20 µg/mlproteinase K (Invitrogen, Carlsbad, CA), and blocked with 5% goat serum and 0.2% bovineserum albumin. The sections were then incubated with mouse anti-SMA antibodyconjugated with FITC (Sigma, 1:400) and rabbit anti-desmin antibody (Thermo Scientific,Rockford, IL, 1:400). After washing, the sections were incubated with goat anti-rabbitantibody conjugated with AlexaFluor 568 (Invitrogen, 1:400) and mouse anti-FITC antibodyconjugated with DyLight 488 (Jackson ImmunoResearch, West Grove, PA, 1:400). Thesections were mounted with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen) andfluorescence images were visualized under a microscope. To quantify the percentage anddensity of HSCs in the liver after BDL with or without treatment of RA, 6 images were

Yang et al.


NIH


NIH


NIH


randomly captured using a 10× objective lens in 3 different sections and SMA+ and desmin+ HSCs in the parenchyma were counted.

Real Time Quantitative PCRTotal RNA was extracted from the cells using TRIzol reagent (Invitrogen) or RNeasy Minikit (Qiagen). One microgram of RNA was reverse transcribed to cDNA by usingSuperScript III First-Strand Synthesis System (Invitrogen) and amplified by 40 cycles usingprimers listed below and the SYBR Green PCR Master mix reagent (AB AppliedBiosystem). Each threshold cycle (Ct) value was first normalized to the 36B4 Ct value of asample and subsequently compared between the treatment and control samples. Primersequences used are shown in Supplemental Information : Pparγ, CCT GAA GCT CCAAGA ATA CCA AA; and 5’-AGA GTT GGG TTT TTT CAG AAT AAT AAGG;α1(I)Coll, 5’-TCG ATT CAC CTA CAG CAC GC and 5’- GAC TGT CTT GCC CCAAGT TCC; 36B4, 5’- TTC CCA CTG GCT GAA AAG GT and 5’- CGC AGC CGC AAATGC; Ezh2, 5’-AGT GGA GTG GTG CTG AAG and 5’-GCC GTC CTT TTT CAG TTG;Tgfβ1, 5’-AGA AGT CAC CCG CGT GCTA and 5’-TGT GTG ATG TCT TTG GTT TTGTCA; Suz12, 5’-GTG AAG AAG CCG AAA ATG and 5’-AAT GTT TTC CTT TTG ATG;Eed, 5’-ATC CTA TAA CAA TGC AGT and 5’-TTC ATC TCT GTG CCC TTC; α-Sma,5’-TGT GCT GGA CTC TGG AGA TG and 5’-GAT CAC CTG CCC ATC AGG; Wnt10b,5’-CGA GAA TGC GGA TCC ACAA and 5’-CCG CTT CAG GTT TTC CGTTA; Wnt3a,5’-CAT CGC CAG TCA CAT GCA CCT and 5’-CGT CTA TGC CAT GCG AGC TCA;Desmin, 5’-CAG GAC CTG CTC AAT GTG and 5’-GTA GCC TCG CTG CTG ACAACC TC; Gapdh, 5’-CTG CCC GTA GAC AAA ATG GT and 5’-GAA TTT GCC GTGAGT GGA GT; Sma, 5’-CTG AGC GTG GCT ATT CCT TC and 5’-CCT CTG CAT CCTGTC AGC AA; Timp1,5’-CAG TAA GGC CTG TAG CTG TGC and 5’-CTC GTT GATTTC GG GGA AC.

Transfection and reporter assay and IKK assayTCF promoter-luciferase construct TOPFLASH (a gift from Dr. Randall Moon, Univ. ofWashington, Seattle, WA) or a κB-luciferase construct was used for transient transfection inthe rat primary HSCs by electroporation using the Neon™ Transfection System (Invitrogen).The Renilla pRL-TK construct was used for standardization for transfection efficiency. Celllysates were analyzed by the dual luciferase assay (Promega) on a luminometer. To assessthe activity of IKK, IKK was immunoprecipitated by IKKα antibody and protein G-Sepharose, and the assay was performed at 30°C for 1hr in buffer containing 20 mM TrisHCl, pH 7.5, 20 mM MgCl2, 2mM dithiothreitol, 20 µM ATP, 2 µg GST-IκBα, and[γ-32P]ATP. The reaction was stopped by addition of Laemmi buffer and was resolved by10% SDS-PAGE followed by a transfer onto a membrane for imaging.

Immunoblot AnalysisWhole cell extracts were prepared as previously described (8). Equal amount of the extract(20µg) was separated by 8–15% SDS-PAGE and the proteins were transferred tonitrocellulose membranes (Bio-Rad, Hercules, CA). MeCP2, type I collagen, and β-actinwere detected by incubating with rabbit polyclonal anti-MeCP2 (1:1000) (Abcam), anti-typeI collagen (1:4000), and anti-β-actin (1:5000) primary antibodies (Santa CruzBiotechnology) in TBS (100 mM Tris-HCl, 1.5 M NaCl, pH 7.4) with 5% non fat milkovernight at 4°C followed by incubation with horseradish peroxidase conjugated goat anti-rabbit secondary antibodies (1:4000) (Sigma) at room temperature for 2 hr. The antigen-antibody complexes’ chemiluminescence was detected by using the ECL detection kit(Pierce).

Yang et al.


NIH


NIH


NIH


Chromatin immunoprecipitation (ChIP)For assessing Pparγ epigenetic regulation, carrier ChIP was performed using Raji cells asthe source of carrier chromatin. For native ChIP, 20µg of HSC chromatin was mixed with80µg of Raji cell chromatin. For cross-link ChIP, Raji cells (1.4 × 107 cells) were mixed toHSCs (0.2 × 106 cells) and fixed with 1% formaldehyde on the rotating platform for 5–10minutes at room temperature followed by addition of glycine to a final concentration of0.125M. After lysis of the cells with SDS buffer (1% SDS, 10mM EDTA, 50mM Tris- HClpH8.1) with protease inhibitors, the lysates were sonicated and snap frozen in aliquots. Forchromatin IP, diluted samples were first pre-cleared using protein G-agarose beads and thenincubated with antibody against Ser2P RNApolyII, MeCP2, H3K27me2, H3K4me2 andH3Kacetylated (Abcam) at 1 µg/µl at 4°C for overnight followed by precipitation withprotein G-agarose beads. After elution of immunoprecipitated complex, crosslinking wasreversed with 5N NaCl and proteins digested with protease K. Extracted chromatin wassubjected to real-time PCR using the primers flanking a segment within Pparγ promoter orexon as previously described (17). Ct values of the samples with non-immune IgG weresubtracted and compared to their respective input Ct values.

Active compounds isolation and identificationThe aqueous YGW extract (350mg/ml in PBS) was applied to size exclusionchromatography using Super Prep Grade gel in XK 16/70 column (Amersham PharmaciaBiotech, Piscataway, NJ) and PBS as a mobile phase solvent. The fractions were tested fortheir bioactivity toward activated HSCs as determined by the morphological reversal of thecells to their quiescence under microscope, and the fractions with the molecular size rangeof 200–750 Da, were shown to contain the most of the activity. To improve the extractionefficiency of both water soluble and lipophilic phytocompounds and to allow their structuralelucidation, n-butanol (BuOH) was added to the YGW water suspension. Briefly, 10 gramsof YGW powder suspended in 200 ml of ddH2O were partitioned with 200 ml of BuOH.After centrifugation and phase separation, BuOH and ddH2O were evaporated in vacuo andlyophilized to yield water (3.59 g) and BuOH (0.54 g) soluble part. Based on the bioactivity-guided fractionation, BuOH soluble phytocompounds (500 mg) were fractionated by columnchromatography on RP-18 gel (COSMOSIL 75C18-OPN, 20 by 70 mm, Nacalai, USA)eluting with MeCN-H2O mixtures of decreasing polarity. Fraction A (250 ml of 10%MeCN-H2O, 192.3 mg), B (250 ml of 40% MeCN-H2O, 196.6 mg), and C (250 ml of 100%MeCN, 64.2 mg) were then subjected to bioassay and high performance liquidchromatography-photodiode array detection-mass spectrometry (HPLC-DAD-MS) analysisafter removing the solvent by using the rotavapor and lyophilizer. HPLC-DAD-MS analysiswas carried out on a ThermoFinnigan LCQ Advantage ion trap mass spectrometer with a RPC18 column (Alltech Prevail C18 3 µm 2.1 × 100 mm) at a flow rate of 125 µl/min with a 10µl injection. The solvent gradient system and the conditions for MS analysis were asdescribed (21). For quantification of RA and BC in each fraction, linear curves of eachcompound were generated by using extract ion chromatograms (EIC) in negative mode atthe molecular weight of each corresponding parent ion. For identification of the majorphytocompounds in fraction A, fraction A (135.0 mg) was purified by reverse phase HPLC[Phenomenex Luna 5µm C18 (2), 250 × 10 mm] with a flow rate of 5.0 ml/min andmeasured by a UV detector at 254 nm. The gradient system was MeCN (solvent B) in 5%MeCN/H2O (solvent A) both containing 0.05% TFA: 10% B from 0 to 5 min, 10 to 30% Bfrom 5 to 25 min, 30 to 100% B from 25 to 27 min, 100% B from 27 to 30 min, 100 to 10%B from 30 to 32 min, and re-equilibration with 20% B from 32 to 35 min. RA (4.3 mg) andBC (8.7 mg) were eluted at 22.1 and 23.6 min, respectively. NMR spectral data werecollected on a Varian Mercury Plus-400 spectrometer. The structures were elucidated bytheir mass, 1H-, 13C-, and 2D-NMR data and also confirmed by comparing their

Yang et al.


NIH


NIH


NIH


spectroscopic data with those of literatures (22, 23) and commercial authentic samples fromSigma.

Data AnalysisData were presented as the means - S.E. Student’s t test was performed to assess thestatistical significance between the two sets of data, and p values less than 0.05 wereconsidered significant.

RESULTSYGW reverses activated HSCs to quiescent cells

We previously demonstrated attenuation of liver fibrosis in two etiologically distinct animalmodels (porcine serum-induced liver fibrosis in rats and CCl4-induced liver fibrosis in mice)by administration of the YGW aqueous extract (19). In order to understand the mechanismsof the demonstrated anti-fibrotic effect of YGW at the cellular level, primary cultures of ratHSCs were treated with the YGW extract or the solvent as a control. Rat HSCs cultured onplastic dish spontaneously undergo myofibroblastic transdifferentiation (“activation”) fromday 2~3 and become fully activated by day 5~7. Upon treatment of day 3 activating or day 7fully activated HSCs with the YGW extract for 2 days, activation of HSC is morphologicallyattenuated as compared to the cells treated with the solvent control or no treatment (Fig.1A). The YGW decreases the expression of SMA, the bona fide marker for the HSCactivation as detected by immunohistochemistry (Fig. 1B) and increases oil red O stainingupon addition of retinol and palmitic acid, the parameter for vitamin A storage and theunique feature of quiescent HSCs (Fig. 1C). In addition, the YGW treatment markedlysuppresses mRNA expression of markers for HSC activation such as α1(I) procollagen,SMA, and TGF-β1 while upregulating the HSC quiescence marker PPARγ (Fig. 1D). Asrestored expression of PPARγ reverses activated HSCs to quiescent cells (8, 9), the observedYGW’s effect to prevent or reverse culture-activation of HSCs, is most likely mediated viaPPARγ induction.

YGW epigenetically de-represses PparγOur recent study revealed the epigenetic mechanisms of Pparγ repression in HSC activationinvolving upregulation and recruitment of the DNA methyl-CpG binding protein MeCP2 tothe Pparγ promoter resulting in the recruitment of the HP-1α corepressor (17). This studyalso demonstrated MeCP2-dependent upregulation of EZH2, the histone H3 lysine 27(H3K27) methyltransferase of polychrome repressor complex 2 (PRC2), increasing H3K27di- and tri-methylation in the Pparγ exons with consequent formation of a repressivechromatic structure (17). Thus, we tested whether YGW’s inductive effect on Pparγ isassociated with epigenetic effects on this gene. First, we examined the recruitment ofelongating RNA polymerase II (Ser2-p RNAPoly II) to the Pparγ gene. As previouslyshown, culture-activated HSCs at day 7 have a markedly reduced recruitment of the Ser2-pRNAPoly II as compared to day 1 quiescent HSCs, and this suppression is attenuated by theYGW treatment (Fig. 2A). MeCP2 enrichment to the Pparγ promoter is increased in Day 7culture-activated HSCs but reduced by the YGW treatment to the level seen in Day 1 HSCs(Fig. 2B). This reduction is associated with abrogation of MeCP2 protein induction seen inday 5 HSCs subsequently incubated with the YGW extract for 24 or 48 hr (Fig. 2C).Increased H3K27 di-methylation (H3K27me2) noted at the exon 2 of Pparγ in culture-activated HSCs (17) with or without the solvent, is also normalized by the YGW extract(Fig. 2D), most likely attributable to suppressed expression of PRC2 components, EZH2,Suz12, and EED (Fig. 2E). H3K4 di-methylation (H3K4me2) and H3 acetylation (H3Ac),the histone modifications for active transcription, are both increased at the Pparγ promoterlocus by the YGW treatment (Fig. 2F and G). These data collectively demonstrate that

Yang et al.


NIH


NIH


NIH


epigenetic repression of the Pparγ gene in culture-activated HSCs is lifted by the YGWextract treatment, and this effect must be responsible for restored PPARγ expression andHSC quiescence.

YGW suppresses IKK and NF-κB activity in HSCAnother important biochemical feature of activated HSCs is increased activity of NF-κB(24). We tested how the YGW extract affects this parameter. The treatment with the YGWextract markedly inhibits the activity of IκB kinase (IKK) as assessed by phosphorylation ofIκBα-GST fusion protein (Fig. 3A), the expression of IκBα and β, both targets of NF-κB(Fig. 3B) in day-5 HSCs, and NF-κB promoter activity in the rat HSC line (BSC) (Fig. 3C).The demonstrated suppressive effects of YGW on IKK and NF-κB suggest that it maypromote apoptotic death of HSCs. Only after a prolonged extract treatment exceeding 4–5days with replenishment of the medium containing the extract every 2 days, apoptosis ofcultured HSCs begins to appear and becomes apparent after 8 days as assessed by TUNELstaining (Suppl Fig. 1A).

Identification of YGW’s active ingredientsAs the first step in identifying active ingredients of YGW rendering the above reversaleffects on activated HSCs, we first tested different fractions of gel filtration of the YGWwater extract in culture-activated HSCs. This analysis reveals a fraction with a molecularmass range of 200~750Da, reproduces the YGW effects including the morphologicalreversal (Fig. 4A), down-regulation of α1(I)procollagen mRNA (Fig. 4B), and decreasedMeCP2 enrichment at the Pparγ promoter (Fig. 4C). This gel filtration fraction was nextapplied to LC/MS for identification of active ingredients. This analysis identifies smallpeaks with the retention time of 14~15 min (boxed in the UV254 tracing of Fig. 4D). Due tolow amounts of these molecules detected in the water extract to allow their purification andidentification, we next analyzed YGW ingredients extracted with butanol (BuOH). Thismethod ensures that most hydrophilic and lipophilic organic compounds are extracted intothe butanol layer while most of the sugar and ionic inorganic components remain in thewater layer. After lyophilization, the water-soluble portion of YGW shows reduced activityof the HSC morphologic reversal when compared with the YGW water extract before thebutanol partitioning. In contrast, the butanol-soluble portion of YGW shows clear bioactivitytoward HSCs (data not shown), suggesting that the bioactive phytocompounds are enrichedin the butanol soluble portion. We further fractionated the butanol soluble portion by reversephase chromatography eluted with 10% (A fraction), 40% (B fraction), and 100% (Cfraction) acetonitrile-water mixtures (Fig. 4D). The butanol A fraction shows a reproducibleeffect on HSC morphologic reversal (Fig. 4E) while the C fraction causes immediatecytotoxicity evident by detachment of the cells (data not shown). The B fraction shows amoderate reversal effect (Fig. 4E). The HPLC profiles clearly show the metabolitesdistribution of each fraction and suggest that the bioactive compound(s) may be eluted from15 min to 20 min in fraction A (Fig. 4F). In order to identify the bioactive phytocompoundsin the A fraction, total of eight subfractions were further purified by semi-preparative HPLC(data not shown). Two major compounds were then isolated and identified to be thebioactive principles. They are rosmarinic acid (RA) and baicalin (BC) (Fig. 4G) byanalyzing their mass, 1H-, 13C-, and 2D-NMR data as well as by comparing their 1H-, 13C-NMR data with those of commercial authentic samples (data not shown).

In vitro effects of RA and BC on HSCsWe tested next whether authentic RA and BC reproduce the effects observed with the YGWextract by testing a wide range of concentrations for HSC morphologic reversal. Indeed,both RA and BC morphologically reverse activated HSCs to quiescent cells with increasedUV-excited autofluorescence at the concentration of 135 and 270 µM (Fig. 5A). Using the

Yang et al.


NIH


NIH


NIH


concentration of 270 µM, RA and BC are shown to downregulate α1(I) procollagen 2~3 foldand to induce PPARγ 3~4 fold (Fig. 5B). Both RA and BC reduce MeCP2 protein level (Fig.5C) and its enrichment in the Pparγ promoter (Fig. 5D). RA and BC also reduce EZH2expression and H3K27me2 at the Pparγ exon (Fig. 5E and F). Collectively, these resultssupport that RA and BC are indeed active phytocompounds that render the YGW’s effect toinhibit or reverse HSC activation via epigenetic de-repression of Pparγ.

We have previously shown that activation of canonical Wnt signaling underlies HSCactivation (11) via epigenetic repression of Pparγ involving MeCP2 and H3K27me2 (16).Thus we thought epigenetic de-repression of Pparγ achieved by RA and BC is due to theirability to inhibit canonical Wnt signaling. Indeed, both RA and BC suppress the expressionof Wnt10b and Wnt3a (Fig. 5G), the canonical Wnts upregulated in HSC activation (11) andTOPFLASH activity (Fig. 5H). Expression of Necdin which transcriptionally upregulatesWnt10b (16), is also reduced by RA and BC (Fig. 5G), suggesting that thesephytocompounds target the Necdin-Wnt-MeCP2 pathway for reversal of HSC activation.

RA inhibits HSC activation and progression of biliary liver fibrosis in miceBC is the active ingredient of Sho-Saiko-To, a Japanese herbal medicine which has beentested for its anti-fibrotic effects in experimental models (25) and patients (26). In contrast,studies on the effects of RA on liver fibrosis are limited to a few recent reports (27, 28). Inone of these studies, RA was shown to prevent the development of CCl4-induced liverfibrosis in rats (27). As RA is an anti-oxidant, this effect on CCl4-induced oxidative liverdamage and consequent liver fibrosis are rather expected. To extend this observation in adifferent etiological model, we considered testing the efficacy of RA for inhibitingprogression of pre-existing cholestatic liver fibrosis induced by BDL in mice. As portalmyofibroblasts (MFs) rather than HSCs are thought to be the primary source of the fibroticresponse in the BDL model (29), we first examined whether HSCs are activated in themodel by analyzing HSCs isolated by FACS from α1(I) collagen promoter-GFP (Coll-GFP)mice subjected to 2-wk BDL. As shown in Suppl. Fig. 2A, the percentage of GFPlow cells(minimal collagen promoter activity) in UV+ (vitamin A containing) HSCs is reduced from9.5% to 2.1% while the percentage of GFPhigh/UV+ HSCs increases in BDL mice ascompared to sham-operated animals, indicating activation of HSCs in the model. Further,qPCR analysis of all UV+ HSCs from BDL vs. sham mice, reveals induction of HSCactivation markers such as α1(I)procollagen (Col1a1), Sma, and Timp1 in BDL HSCs butnot Desmin (Suppl Fig. 2B). Having confirmed that HSCs are indeed activated in the model,we tested the effects of daily intraperitoneal administration of RA vs. vehicle during thesecond week of BDL. The liver to body weight percentage is not different between RA orvehicle-treated mice (6.8+0.7 vs. 6.3+0.3), nor are the plasma ALT levels (157+71 vs.283+95, p=0.29). However, the digital morphometric analysis of Sirius red-stained collagenfibers shows a significant attenuation of liver fibrosis by RA treatment (Fig. 5I). To examinewhether this anti-fibrotic effect of RA is associated with suppressed activation of HSCs invivo, immunohistochemistry for SMA and Desmin were performed (Suppl. Fig. 2C). In thesham-operated liver, expression of SMA is primarily seen in the hepatic artery and a veryfew cells around the bile duct, but not in HSCs in the sinusoid (Suppl. Fig. 2C, upper andlower left panel). In the vehicle-treated BDL liver, expression of SMA increases in Desmin+portal MFs and HSCs (Suppl. Fig. 2C, upper and lower middle panel). RA treatment reducesthe percentage of SMA+ MFs by 40% and that of SMA+ HSCs by 75% (Suppl. Fig. 2C and2D). The density of Desmin+ HSCs increases by BDL, but RA treatment has no effect onthis change (Suppl. Fig. 2D). No TUNEL+ HSCs or hepatocytes are detected in the liverparenchyma of either RA- or vehicle-treated BDL livers. These data indicate that RAsuppresses activation of both portal MFs and HSCs in BDL-induced liver injury. HepaticmRNA levels of α1(I)procollagen and SMA are also significantly reduced by RA treatment

Yang et al.


NIH


NIH


NIH


(Fig. 5J), further supporting anti-fibrotic effects of RA in this model. Taken together, thesedata indicate that RA suppresses activation of HSCs and liver fibrosis in BDL-induced liverinjury.

DISCUSSIONThe present study demonstrates that the MeCP2 -EZH2 relay of Pparγ epigenetic repressionis an important target for the anti-fibrotic effect of the herbal prescription YGW.Polyphenolic RA and flavonoid BC are identified as active phytochemicals in YGW thatreverse epigenetic Pparγ repression and activated phenotype of HSCs. Both RA and BCinhibit MeCP2 induction and its recruitment to the Pparγ promoter while suppressing theexpression of PRC2 components including the H3K27 methyltrasferase EZH2 resulting inreduced H3K27me2 at the Pparγ exon locus. These epigenetic effects which result in theformation of eurochromatin at the Pparγ locus, increase a recruitment of RNA polymeraseto Pparγ and its transcription, and restore expression of the gene which is essential for HSCdifferentiation (8, 9). In essence, these results provide the molecular basis of the anti-fibroticeffects of YGW and its ingredients, RA and BC at the epigenetic level.

Due likely to the ability to suppress NF-κB, the prolonged treatment of cultured HSCs withthe YGW extract for 8 days, causes apoptosis in cultured HSCs (Suppl Fig. 1). However, noapoptosis is evident during the first 2 days of the treatment when the epigenetic Pparγ de-repression and phenotypic reversal of HSCs are achieved. RA treatment of BDL miceattenuates liver fibrosis, and this effect is accompanied by suppressed activation of HSCs asdemonstrated by a marked reduction in SMA+ HSCs. In these livers, apoptosis of HSCs isnot evident and the number of HSCs is not reduced (Suppl Fig. 2C and 2D). Thus, theseresults suggest that suppressed activation rather than apoptosis of HSCs is responsible atleast in part for RA’s anti-fibrotic effect in the BDL model. Portal MFs which areconsidered as a major source of a fibrogenic response in the BDL model (29), are indeedincreased in number after BDL (Suppl Fig. 2D), and this change is attenuated by RAtreatment. At present, we do not know the molecular basis of this suppression of MFs byYGW and its active ingredients RA and BC, and a future study will need to address thisquestion.

BC is an active ingredient of Sho-saiko-to, a Japanese herbal medicine known for its anti-fibrotic effects, and its mechanism of action has primarily been ascribed to its antioxidantproperty and its ability to reduce lipid peroxidation (25). RA is also a polyphenolicantioxidant which may also render the similar protective effects against oxidant liverdamage and fibrosis. Suppression of IKK and NF-κB activities by YGW shown in HSCs isalso consistent with its ability to suppress oxidant stress which is a well known signal foractivation of IKK. Oxidant stress generated by NADPH oxidase is recognized as a keysignaling event in activation of HSC induced by a wide array of agonists such as angiotensinII (30), PDGF (31), and leptin (32). Accordingly, antioxidants which scavenge NADPHoxidase-derived ROS are expected to suppress activation of HSCs. However, the presentstudy demonstrates that BC and RA inhibit the canonical Wnt signaling which we haverecently shown to mediate epigenetic repression of Pparγ involving MeCP2 and EZH2 (16).Further, Necdin which transcriptionally activates Wnt10b via its binding to a GN box in itsproximal promoter (16), is also reduced by both RA and BC. Taken together, these resultssuggest that both phytocompounds target the Necdin-Wnt-MeCP2-EZH2 pathway for theirepigenetic effects. Whether and how this novel effect is related to antioxidant activity of thephytocompounds, are yet to be determined.

Yang et al.


NIH


NIH


NIH


Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsThe present study was supported by NIH grants: U01AA018663 (HT and DM), P50AA11199 (HT), R24AA12885(HT), and R01AA020753 (KA) and by Medical Research Service of Department of Veterans Affairs (HT); MedicalResearch Council, Welcome Trust, and British Liver Trust; and the Newcastle Health Care Charity and Newcastleupon Tyne Hospitals NHS Charity (JM and DM); and a grant from S.P. Pharmaceutics, Inc. (HT).

REFERENCES1. Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.

Physiol Rev. 2008 Jan; 88(1):125–172. [PubMed: 18195085]2. Brenner DA. Molecular pathogenesis of liver fibrosis. Trans Am Clin Climatol Assoc. 2009;

120:361–368. [PubMed: 19768189]3. Cassiman D, Barlow A, Vander BS, Libbrecht L, Pachnis V. Hepatic stellate cells do not derive

from the neural crest. J Hepatol. 2006 Jun; 44(6):1098–1104. [PubMed: 16458991]4. Geerts A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate

cells. Semin Liver Dis. 2001 Aug; 21(3):311–335. [PubMed: 11586463]5. Asahina K, Tsai SY, Li P, Ishii M, Maxson RE Jr. Sucov HM, et al. Mesenchymal origin of hepatic

stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liverdevelopment. Hepatology. 2008 Nov 5; 49(3):998–1011. [PubMed: 19085956]

6. Miyahara T, Schrum L, Rippe R, Xiong S, Yee HF Jr. Motomura K, et al. Peroxisome proliferator-activated receptors and hepatic stellate cell activation. J Biol Chem. 2000 Nov 17; 275(46):35715–35722. [PubMed: 10969082]

7. Spiegelman BM, Flier JS. Adipogenesis and obesity: rounding out the big picture. Cell. 1996 Nov 1;87(3):377–389. [PubMed: 8898192]

8. Hazra S, Xiong S, Wang J, Rippe RA, Krishna V, Chatterjee K, et al. Peroxisome proliferator-activated receptor gamma induces a phenotypic switch from activated to quiescent hepatic stellatecells. J Biol Chem. 2004 Mar 19; 279(12):11392–11401. [PubMed: 14702344]

9. She H, Xiong S, Hazra S, Tsukamoto H. Adipogenic transcriptional regulation of hepatic stellatecells. J Biol Chem. 2005 Feb 11; 280(6):4959–4967. [PubMed: 15537655]

10. Yavrom S, Chen L, Xiong S, Wang J, Rippe RA, Tsukamoto H. Peroxisome proliferator-activatedreceptor gamma suppresses proximal alpha1(I) collagen promoter via inhibition of p300-facilitatedNF-I binding to DNA in hepatic stellate cells. J Biol Chem. 2005 Dec 9; 280(49):40650–40659.[PubMed: 16216869]

11. Cheng JH, She H, Han YP, Wang J, Xiong S, Asahina K, et al. Wnt antagonism inhibits hepaticstellate cell activation and liver fibrosis. Am J Physiol Gastrointest Liver Physiol. 2007 Nov 15.

12. Tseng YH, Butte AJ, Kokkotou E, Yechoor VK, Taniguchi CM, Kriauciunas KM, et al. Predictionof preadipocyte differentiation by gene expression reveals role of insulin receptor substrates andnecdin. Nat Cell Biol. 2005 Jun; 7(6):601–611. [PubMed: 15895078]

13. Kuwajima T, Nishimura I, Yoshikawa K. Necdin promotes GABAergic neuron differentiation incooperation with Dlx homeodomain proteins. J Neurosci. 2006 May 17; 26(20):5383–5392.[PubMed: 16707790]

14. Kuwajima T, Taniura H, Nishimura I, Yoshikawa K. Necdin interacts with the Msx2homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells. J BiolChem. 2004 Sep 24; 279(39):40484–40493. [PubMed: 15272023]

15. Brunelli S, Tagliafico E, De Angelis FG, Tonlorenzi R, Baesso S, Ferrari S, et al. Msx2 and necdincombined activities are required for smooth muscle differentiation in mesoangioblast stem cells.Circ Res. 2004 Jun 25; 94(12):1571–1578. [PubMed: 15155529]

16. Zhu NL, Wang J, Tsukamoto H. The Necdin-Wnt pathway causes epigenetic peroxisomeproliferator-activated receptor gamma repression in hepatic stellate cells. J Biol Chem. 2010 Oct 1;285(40):30463–30471. [PubMed: 20663865]

Yang et al.


NIH


NIH


NIH


17. Mann J, Chu DC, Maxwell A, Oakley F, Zhu NL, Tsukamoto H, et al. MeCP2 controls anepigenetic pathway that promotes myofibroblast transdifferentiation and fibrosis.Gastroenterology. 2010 Feb; 138(2):705–714. 714. [PubMed: 19843474]

18. Szulwach KE, Li X, Smrt RD, Li Y, Luo Y, Lin L, et al. Cross talk between microRNA andepigenetic regulation in adult neurogenesis. J Cell Biol. 2010 Apr 5; 189(1):127–141. [PubMed:20368621]

19. Yang MD, Deng QG, Chen S, Xiong S, Koop D, Tsukamoto H. Hepatoprotective mechanisms ofYan-gan-wan. Hepatol Res. 2005 Aug; 32(4):202–212. [PubMed: 16107322]

20. Sung CK, She H, Xiong S, Tsukamoto H. Tumor necrosis factor-alpha inhibits peroxisomeproliferator-activated receptor gamma activity at a posttranslational level in hepatic stellate cells.Am J Physiol Gastrointest Liver Physiol. 2004 May; 286(5):G722–G729. [PubMed: 14656714]

21. Bok JW, Chiang YM, Szewczyk E, Reyes-Dominguez Y, Davidson AD, Sanchez JF, et al.Chromatin-level regulation of biosynthetic gene clusters. Nat Chem Biol. 2009 Jul; 5(7):462–464.[PubMed: 19448638]

22. Lin YL, Wang WY, Kuo YH, Chen CF. Nonsteroidal constituents from Solanum incanum L.[Abstract]. Journal of the Chinese Chemical Society. 2000 Feb 1.(47):247–251.

23. Tezuka Y, Kasimu R, Li JX, Basnet P, Tanaka K, Namba T, et al. Constituents of roots of Salviadeserta Schang. (Xinjiang-Danshen). [Abstract]. Chemical and Pharmaceutical Bulletin. 1998;(46):107–112.

24. Oakley F, Mann J, Ruddell RG, Pickford J, Weinmaster G, Mann DA. Basal expression ofIkappaBalpha is controlled by the mammalian transcriptional repressor RBP-J (CBF1) and itsactivator Notch1. J Biol Chem. 2003 Jul 4; 278(27):24359–24370. [PubMed: 12700242]

25. Shimizu I, Ma YR, Mizobuchi Y, Liu F, Miura T, Nakai Y, et al. Effects of Sho-saiko-to, aJapanese herbal medicine, on hepatic fibrosis in rats. Hepatology. 1999 Jan; 29(1):149–160.[PubMed: 9862861]

26. Stickel F, Brinkhaus B, Krahmer N, Seitz HK, Hahn EG, Schuppan D. Antifibrotic properties ofbotanicals in chronic liver disease. Hepatogastroenterology. 2002 Jul; 49(46):1102–1108.[PubMed: 12143213]

27. Li GS, Jiang WL, Tian JW, Qu GW, Zhu HB, Fu FH. In vitro and in vivo antifibrotic effects ofrosmarinic acid on experimental liver fibrosis. Phytomedicine. 2010 Mar; 17(3–4):282–288.[PubMed: 19524418]

28. Zhang JJ, Wang YL, Feng XB, Song XD, Liu WB. Rosmarinic acid inhibits proliferation andinduces apoptosis of hepatic stellate cells. Biol Pharm Bull. 2011 Mar; 34(3):343–348. [PubMed:21372382]

29. Wang B, Dolinski BM, Kikuchi N, Leone DR, Peters MG, Weinreb PH, et al. Role of alphavbeta6integrin in acute biliary fibrosis. Hepatology. 2007 Nov; 46(5):1404–1412. [PubMed: 17924447]

30. Bataller R, Schwabe RF, Choi YH, Yang L, Paik YH, Lindquist J, et al. NADPH oxidase signaltransduces angiotensin II in hepatic stellate cells and is critical in hepatic fibrosis. J Clin Invest.2003 Nov; 112(9):1383–1394. [PubMed: 14597764]

31. Adachi T, Togashi H, Suzuki A, Kasai S, Ito J, Sugahara K, et al. NAD(P)H oxidase plays acrucial role in PDGF-induced proliferation of hepatic stellate cells. Hepatology. 2005 Jun; 41(6):1272–1281. [PubMed: 15915457]

32. De MS, Seki E, Oesterreicher C, Schnabl B, Schwabe RF, Brenner DA. Reduced nicotinamideadenine dinucleotide phosphate oxidase mediates fibrotic and inflammatory effects of leptin onhepatic stellate cells. Hepatology. 2008 Dec; 48(6):2016–2026. [PubMed: 19025999]

Yang et al.


NIH


NIH


NIH


Fig. 1.YGW prevents and reverses hepatic stellate cell (HSC) activation in culture. A. Phasecontrast microscopy of activating day 3 or fully-activated day 7 rat HSCs cultured for thelast 48 hr with YGW extract, vehicle control, or no addition. Note a morphologic reversal ofactivated HSCs to quiescent cells. B. Immunostaining for SMA. Note a marked reduction inSMA with YGW extract. C. Oil red O staining after retinol and palmitate addition isincreased in YGW-treated 7 day HSCs. D. The mRNA levels for activation marker genes,α1(I)collagen, αSMA, TGF-β1 are conspicuously suppressed in day 7 HSC by the 48-hrtreatment with YGW extract while PPARγ mRNA is induced. *p<0.05, **p<0.01 comparedto the vehicle control treatment.

Yang et al.


NIH


NIH


NIH


Fig. 2.PPARγ epigenetic repression is lifted with YGW extract. A. Recruitment of Ser2-p RNApolymerase II to the Pparγ gene is significantly reduced in day 7 culture-activated HSCswith no addition or with the vehicle control treatment, and this reduction is attenuated by theYGW extract treatment. B. Increased MeCP2 recruitment to Pparγ promoter in day 7culture-activated HSCs is normalized with the YGW extract. C. MeCP2 protein detected byimmunoblotting in day 5 HSCs cultured for 24 and 48 hr with the vehicle control becomesundetectable by the YGW treatment. D. Increased H3K27me2 at the Pparγ exon 2 locus inday 7 HSCs is reduced with the YGW extract. E. Increased mRNA levels of the PRC2component EZH2, Suz12, and EED in day 7 HSCs are reduced by the YGW treatment. F.H3K4me2 at the Pparγ promoter locus is increased by the YGW extract treatment in day 7HSCs compared to HSCs treated with the vehicle. G. Reduced H3 acetylation (H3Ac) in 7day HSCs is attenuated with the YGW extract. *p<0.05 compared to day 1 HSCs, †p<0.05compared to the vehicle control.

Yang et al.


NIH


NIH


NIH


Fig. 3.Suppression of IKK and NF-κB with YGW. A. Day 5 HSCs cultured with the YGW extractvs. the vehicle control for 6 or 24 hr in serum-free medium, show reduced IKK activity asassessed by phosphorylation of IκBα-GST fusion protein. A positive control for IKKactivation is shown with LPS-stimulated RAW macrophage cell line (last lane). B. Day 5HSCs cultured with the YGW extract for 24 or 48 hr, show marked reductions in the levelsof IκBα and IκBβ proteins, as well as in type I collagen protein. C. The activity of κBpromoter is significantly reduced by the YGW extract in the rat HSC line (BSC) as assessedby a transient transfection-reporter analysis. *p<0.05.

Yang et al.


NIH


NIH


NIH


Yang et al.


NIH


NIH


NIH


Fig. 4.Identification of active components. A. Treatment of day 7 HSCs with a gel filtrationfraction with a molecular mass range of 200~750Da, causes a morphologic reversal of HSCsas compared to the cells treated with the elution buffer control (phase contract microscopy).B and C. Addition of the fraction to 7 day HSC culture reduces increased α1(I)collagenmRNA and MeCP2 enrichment to the Pparγ promoter as shown with the YGW extract. D.A summary of chromatographic methods for separation of YGW’s active ingredients. E.Butanol (BuOH) fraction A and B eluted with 10% acetonitrile-90% water and 40%acetonitrile-60% water, respectively, produce reproducible effects of HSC morphologicreversal as shown by phase contrast microscopy and oil red O staining. F. LC/MS tracing of

Yang et al.


NIH


NIH


NIH


butanol fractions identifies 5 peaks of which two are identified to be RA and BC. G.Molecular structures of rosmarinic acid and baicalin.

Yang et al.


NIH


NIH


NIH


Fig. 5.Rosmarinic acid (RA) and baicalin (BC) are the YGW’s active components to renderepigenetic de-repression of Pparγ. A. Note both rosmarinic acid (RA) and baicalin (BC)reverse activated HSC to quiescent cells as shown by phase contrast and UV-excitedautofluorescence microscopy. B. RA and BC (270 µM) reduce mRNA expression forα1(I)collagen and increase that for PPARγ. C. RA and BC reduce MeCP2 protein level inday 7 HSCs. D. MeCP2 enrichment to the Pparγ promoter is reduced with RA and BC. E.EZH2 mRNA level is reduced equally by RA and BC. F. H3K27me2 at the Pparγ exon 2 isreduced by RA and BC. * p<0.05 compared to the solvent control. G. RA and BC (270 µM)reduce the expression of Wnt10b, Wnt3a, and Necdin in day 7 HSCs compared to thevehicle control as determined by qPCR analysis. H. RA (shaded bar) and BC (black bar)reduces the TOPFLASH promoter activity in day 7 primary HSCs ad determined by atransient transfection using an electroporation method. I. RA treatment (ip injection daily at0.1mg/25g body weight) given during the last one week of the 2-week cholestasis caused bythe bile duct ligation, attenuates liver fibrosis in mice as assessed by digital morphometricanalysis of Sirius red staining. *p<0.05 compared to the vehicle control. J. Hepaticexpression of α1(I)procollagen and SMA are also significantly reduced by the RA treatment.*p<0.05 compared to Sham. +p<0.05 compared to vehicle-treated (Cont) mice.

Yang et al.


NIH


NIH


NIH


nih public access 1 reiichi higashiyama kinji asahina ...rosmarinic acid and baicalin epigenetically...

Documents