Toxicology and Applied Pharmacology 265 (2012) 51–60

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Toxicology and Applied Pharmacology

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Ligustrazine attenuates oxidative stress-induced activation of hepatic stellate cells byinterrupting platelet-derived growth factor-β receptor-mediated ERK andp38 pathways

Feng Zhang a, Chunyan Ni a,d, Desong Kong a, Xiaoping Zhang a, Xiaojing Zhu a, Li Chen a,Yin Lu a,b,c, Shizhong Zheng a,b,c,⁎a Department of Clinical Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029, Chinab Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing 210046, Chinac National First-Class Key Discipline for Traditional Chinese Medicine of Nanjing University of Chinese Medicine, Nanjing 210046, Chinad The First People's Hospital of Changzhou, Changzhou 213003, China

Abbreviations: α-SMA, α-smooth muscle actin; CTGF,DMEM,Dulbecco'smodified eaglemedium;ECM, extracelluulated protein kinase; FBS, fetal bovine serum; GAPDH, Glycgenase; H2O2, hydrogen peroxide; HSC, hepatic stellate ceMAPK, mitogen-activated protein kinase; MMP, matrix mdimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4NF-κB, nuclear factor-κB; PDGF-βR, platelet-derivedpropidium iodide; PPARγ, peroxisomeproliferator-activatygen species; RTK, receptor tyrosine kinases; TIMP, tissue⁎ Corresponding author at: Department of Clinical Ph

Nanjing University of Chinese Medicine, 282 HanzhongChina. Fax: +86 25 86798188.

E-mail address: nytws@163.com (S. Zheng).

0041-008X/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.taap.2012.09.016

a b s t r a c t

a r t i c l e i n f o

Article history:Received 31 July 2012Revised 14 September 2012Accepted 18 September 2012Available online 27 September 2012

Keywords:LigustrazineHepatic stellate cellExtracellular regulated protein kinasep38Oxidative stress

Hepatic fibrosis represents a frequent event following chronic insult to trigger wound healing reactions with ac-cumulation of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is the pivotalevent during liver fibrogenesis. Compelling evidence indicates that oxidative stress is concomitant with liver fi-brosis irrespective of the underlying etiology. Natural antioxidant ligustrazine exhibits potent antifibrotic activ-ities, but the mechanisms are poorly understood. Our studies were to investigate the ligustrazine effects on HSCactivation stimulated by hydrogen peroxide (H2O2), an in vitro model mimicking the oxidative stress in liverfibrogenesis, and to elucidate the possiblemechanisms. Our results demonstrated that H2O2 at 5 μMsignificantlystimulated HSC proliferation and expression of marker genes of HSC activation; whereas ligustrazinedose-dependently suppressed proliferation and induced apoptosis in H2O2-activated HSCs, and attenuated ex-pression of fibrotic marker genes. Mechanistic investigations revealed that ligustrazine reduced platelet-derived growth factor-β receptor (PDGF-βR) expression and blocked the phosphorylation of extracellular regu-lated protein kinase (ERK) and p38 kinase, two downstream effectors of PDGF-βR. Further molecular evidencesuggested that ligustrazine interruption of ERK and p38 pathways was dependent on the blockade of PDGF-βRand might be involved in ligustrazine reduction of fibrotic marker gene expression under H2O2 stimulation.Furthermore, ligustrazine modulated some proteins critical for HSC activation and ECM homeostasis in H2O2-stimulated HSCs. These data collectively indicated that ligustrazine could attenuate HSC activation caused by ox-idative stress, providing novel insights into ligustrazine as a therapeutic option for hepatic fibrosis.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Hepatic fibrosis is an excessive wound-healing response occurringin most forms of chronic liver injury and leads tomassive accumulationof extracellular matrix (ECM) in the liver. Central to the fibrotic cas-cades is the activation of hepatic stellate cells (HSCs) (Hernandez-Gea

connective tissue growth factor;larmatrix; ERK, extracellular reg-eraldehyde phosphate dehydro-ll; JNK, c-Jun N-terminal kinase;etalloproteinase; MTS, 3-(4,5--sulfo-phenyl)-2H-tetrazolium;growth factor-β receptor; PI,ed receptor-γ; ROS, reactive ox-inhibitor of metalloproteinase.armacy, College of Pharmacy,Road, Nanjing 210029, Jiangsu,

rights reserved.

and Friedman, 2011). Upon injury, HSCs undergo a remarkable transfor-mation to an activated α-smooth muscle actin (α-SMA) positive phe-notype. These highly proliferative cells are the major source of ECMcomponents like type I collagen and structural glycoprotein fibronectin(Friedman, 2008). Activated HSCs also overexpress connective tissuegrowth factor (CTGF), a master molecule contributing to ECM accumu-lation (Gressner andGressner, 2008), and the expression pattern ofma-trix metalloproteinases (MMPs) and their inhibitors (TIMPs) by HSCs isaltered shifting the balance of ECM components towards fibrogenesis(Hemmann et al., 2007).

Although the underlying mechanisms remain incompletely under-stood, compelling evidence indicates that oxidative stress plays a crit-ical role in HSC activation and ECM synthesis irrespective of theunderlying etiology (Novo and Parola, 2008). Oxidative stress is a del-eterious imbalance between the production and removal of reactiveoxygen species (ROS) (Finkel, 2011). Studies have demonstratedthat ROS such as superoxide anion and hydrogen peroxide (H2O2) isa direct or indirect relevant pro-fibrogenic stimulus for HSC activation

52 F. Zhang et al. / Toxicology and Applied Pharmacology 265 (2012) 51–60

(Parola and Robino, 2001). Furthermore, oxidative stress represents anovel class of “the third messenger,” leading to activation of severalsignal pathways in the molecular networks of hepatic fibrosis(Zheng et al., 2007a), for example, receptor tyrosine kinases (RTKs)are able to respond to oxidative stress by enhancing their activity(Chen et al., 2003; Filosto et al., 2011; Takeyama et al., 2000). Abun-dant data also demonstrate that oxidative stress influences themitogen-activated protein kinase (MAPK) cascades that consist of ex-tracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase(JNK) and p38 kinase (McCubrey et al., 2006). The ERK signaling hasbeen found to be critical for HSC survival and proliferation (Zhenget al., 2007b). The JNK and p38 kinase pathways are sometimesgrouped together and referred to as the stress-activated protein ki-nases (Matsuzawa and Ichijo, 2005). Activation of MAPK cascadesby oxidative stress may be pivotally involved in the determinationof cell fate in response to reactive oxygen intermediates.

Currently, there are few breakthroughs in the therapeuticintervention of hepatic fibrosis. Therefore, research identifyingantifibrogenic agents that are innocuous is urgently needed. Naturalproduct ligustrazine has shown a therapeutic promise for hepaticfibrosis. Ligustrazine has been identified to be a potent antioxidantimplicated in multiple pathophysiological conditions covering kid-ney, cardiovascular system and immunity (Guo et al., 2012; Jiang etal., 2011; Liu et al., 2008). Recent studies revealed that ligustrazineimproved liver histology and reduced fibrosis (Chen et al., 2007,2010; Lu et al., 2010) and protected mice against acute hepatotoxic-ity induced by thioacetamide associated with oxidative stress (So etal., 2002). These discoveries strongly suggest ligustrazine as ahopeful antifibrotic candidate. However, the underlying mecha-nisms remain to be defined. The present study was designed toinvestigate the ligustrazine effects on HSC activation under H2O2

stimulation in vitro, which could mimic the oxidative stress in liverfibrogenesis since H2O2 is the most stable ROS and diffuses readilyin and out of cells (Oya-Ohta et al., 1995) and to elucidate the molec-ular mechanisms. The results demonstrated that ligustrazine couldsuppress proliferation and induce apoptosis in H2O2-stimulatedHSCs, and that disruption of platelet-derived growth factor-β recep-tor (PDGF-βR)-mediated ERK and p38 pathways was required forligustrazine reduction of HSC activation under oxidative stress.

Materials and methods

Reagents and antibodies. Ligustrazine was purchased from Sigma (StLouis, MO, USA) and was dissolved in dimethylsulfoxide (SinopharmChemical Reagent Co., Ltd, Shanghai, China) for all experiments.Analytical grade 30% H2O2 was obtained from Sinopharm ChemicalReagent Co., Ltd. (Shanghai, China), and was diluted with deionizedwater to the indicated concentrations for experiments. Recombinantrat PDGF was from Cell Sciences (Canton, MA, USA). PDGF-βR blockerimatinib was from Nanjing Norris-Pharm Technology Co., Ltd.(Nanjing, China). MEK inhibitor U0126 and p38 kinase inhibitorSB203580 were from Cell Signaling Technology (Danvers, MA, USA).The primers used in real-time PCR were from GenScript Co., Ltd.(Nanjing, China). The primary antibodies used in western blot analy-ses against α-SMA, fibronectin, α1(I) procollagen, MMP-2, TIMP-1,CTGF, NF-κB p65, and p-NF-κB p65 were from Santa Cruz Biotechnol-ogy (Santa Cruz, CA, USA). The primary antibodies against p-ERK,ERK, p-JNK, JNK, p-p38, and p38 were from Cell Signaling Technology(Danvers, MA, USA). The primary antibodies against PDGF-βR andPPARγ were from Epitomics (San Francisco, CA, USA). The primaryantibody against β-actin was from Sigma (St Louis, MO, USA), andthe horseradish peroxidase-conjugated secondary antibodies werefrom Cell Signaling Technology (Danvers, MA, USA).

Cell isolation and culture conditions. Primary HSCs were isolated frommale Sprague–Dawley rats (200–250 g; Nanjing Medical University,

Nanjing, China) as previously described (Chen, 2002). Freshly isolat-ed HSCs were cultured in Dulbecco's modified eagle medium(DMEM; Invitrogen, Grand Island, NY, USA) supplemented with 10%fetal bovine serum (FBS; Sijiqing Biological Engineering MaterialsCo., Ltd., HangZhou, China), 100 U/mL penicillin and 100 mg/mLstreptomycin, and grown in a 95% air and 5% CO2 humidified atmo-sphere at 37 °C. HSCs aged at passages 4–8 were used for the experi-ments (Zheng et al., 2007a). Cell morphology was assessed using aninverted microscope with a Leica Qwin System (Leica, Germany).

MTS assay. Passaged HSCs in logarithmic growth were seeded in96-well plates and cultured in DMEM supplemented with 10% FBSfor 24 h, and then in low-serum medium (0.5%, v/v) for an additional12 h. HSCs were treated with H2O2 (or PDGF in certain experiment)and/or ligustrazine at the indicated concentrations for 24 h.Treatment with DMSO (0.02%, w/v) was set up as the negativecontrol, and this was performed throughout the study. After treat-ment, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS; Sigma, St Louis, MO, USA)and phenazine methosulfate (Promega Corporation, Madison, WI,USA) were added, and the cells were further incubated for 3 h at37 °C. The spectrophotometric absorbance at 490 nm was measuredby a SPECTRAmax™ microplate spectrophotometer (Molecular De-vices, Sunnyvale, CA, USA). Six duplicate wells were set up for eachgroup.

Flow cytometric analyses of apoptosis. Apoptosis was determined byFITC labeled Annexin-V/PI double staining and flow cytometry analy-sis. Passaged HSCs were seeded in 6-well plates and cultured inDMEM supplemented with 10% FBS for 24 h, and then in low-serummedium (0.5%, v/v) for an additional 12 h. HSCs were treated withH2O2 (or PDGF in certain experiment) and/or ligustrazine at the indi-cated concentrations for 24 h. An Annexin V-FITC apoptosis assay kit(Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) was usedaccording to the protocol. Only fluorescein-positive cells without PIstaining were regarded as apoptotic cells and the percentages weredetermined by flow cytometry (FACSCalibur; Becton, Dickinson andCompany, Franklin Lakes, NJ, USA). The data were analyzed usingthe software CELLQuest. The experiments were performed intriplicate.

RNA isolation and real-time PCR. Total RNA was isolated from treatedHSCs using Trizol reagent (Sigma, St Louis, MO, USA) following theprotocol provided by themanufacturer. Real-time PCRwas performedas we described previously (Fu et al., 2006). Glyceraldehyde phos-phate dehydrogenase (GAPDH) was used as the invariant control.Fold changes in the mRNA levels of target genes related to the invari-ant control GAPDH were calculated as suggested by Schmittgen et al.(2000). The following primers were used in Real-time PCR: α-SMA:(forward) 5′-CCGACCGAATGCAGAAGGA-3′, (reverse) 5′-ACAGAGTATTTGCGCTCCGGA-3′; α1(I) procollagen: (forward) 5′-CCTCAAGGGCTCCAACGAG-3′, (reverse) 5′-TCAATCACTGTCTTGCCCCA-3′; fibronectin:(forward) 5′-TGTCACCCACCACCTTGA-3′, (reverse) 5′-CTGATTGTTCTTCAGTGCGA-3′; PDGF-βR: (forward) 5′-CTGCCACAGCATGATGAGGATTGAT-3′, (reverse) 5′-GCCAGGATGGCTGAGATCACCAC-3′; and GAPDH:(forward) 5′-GGCCCCTCTGGAAAGCTGTG-3′, (reverse) 5′-CCGCCTGCTTCACCACCTTCT-3′.

Western blot analyses. Whole cell protein extracts were preparedfrom treated HSCs. The protein levels were determined using a BCAassay kit (Pierce, USA). Proteins (50 μg/well) were separated bySDS-polyacrylamide gel, transferred to a PVDF membrane (Millipore,Burlington, MA, USA), and blocked with 5% skim milk in Tris-bufferedsaline containing 0.1% Tween 20. Target proteins were detected bycorresponding primary antibodies, and subsequently by horseradishperoxidase-conjugated secondary antibodies. Protein bands were

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visualized using chemiluminescence reagent (Millipore, Burlington,MA, USA). Equivalent loading was confirmed using an antibody againstβ-actin. The levels of target protein bandswere densitometrically deter-mined using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA,USA). The variation in the density of bands was expressed as foldchanges compared to the control in the blot after normalized toβ-actin or the total proteins in some experiments.

Statistical analysis. Data were presented as mean±SD, and resultswere analyzed using SPSS16.0 software. The significance of differencewas determined by one-way ANOVA with the post-hoc Dunnett's test.Values of pb0.05 were considered to be statistically significant.

Results

H2O2 significantly stimulates HSC activation

Activation of HSCs can be mimicked in vitro by culturing freshlyisolated HSCs that are quiescent on plastic, a well-established modelfor investigating the mechanisms of HSC activation and therapeuticintervention (Friedman, 2004). We first assessed the stimulatory ef-fects of H2O2 on HSC activation and attempted to establish an invitro model for evaluating ligustrazine intervention under oxidativestress. Microscopic evaluation showed that stimulation with H2O2 at10 μM resulted in remarkable changes in HSC morphology. Comparedwith the control HSCs that were relatively quiescent, H2O2-treatedHSCs were more spindle-shaped with well-developed stress fibersof actin cytoskeleton and long cytoplasmic processes, indicating thatthey were more “activated” or “transdifferentiated” under oxidativestress (Fig. 1A). Since proliferation is a striking feature of HSC activa-tion (Friedman, 2004), we thus examined the pro-proliferative effectsof H2O2 on HSCs. The results demonstrated that H2O2 increased HSCviability in a dose-dependent manner and H2O2 at 5 μM led to asignificant effect (Fig. 1B). Western blot assays showed that the ex-pression of α-SMA, a marker of HSC activation (Sato et al., 2003),and ECM components α1(I) procollagen and fibronectin wasupregulated in H2O2-stimulated HSCs, providing further evidence

Fig. 1. H2O2 stimulates HSC activation. Low-serum cultured HSCs were treated with phosphawith an inverted light microscope (×100). (B) MTS assay for evaluation of cell viability. Datablot analysis for protein levels of fibrotic marker genes. β-Actin was used as an invariant conData are expressed as mean±SD and *pb0.05 vs control.

that HSCs were markedly activated by H2O2 in culture (Fig. 1C).Collectively, these data indicated that H2O2 was a potent stimulatorfor HSC activation and that H2O2-treated HSCs represented a cell cul-ture model of oxidative stress-induced HSC activation.

Ligustrazine inhibits proliferation and induces apoptosis in H2O2-activat-ed HSCs

We subsequently investigated the ligustrazine effects on oxidativestress-induced HSC activation using 5 μMH2O2-stimulated HSCs in cul-ture system. MTS assay showed that H2O2-enhanced HSC viability wasabrogated by ligustrazine dose-dependently. Ligustrazine at dosesover 10 μM significantly suppressed the pro-proliferative effects ofH2O2, suggesting that ligustrazine could inhibit HSC growth under oxi-dative stress (Fig. 2A). We next assessed whether ligustrazine could in-duce apoptosis in H2O2-treatedHSCs. Results from FITC labeledAnnexinV/PI staining using flow cytometry demonstrated that the numberof apoptotic HSCs (i.e. apoptotic rate) was increased in a dose-dependent manner, although H2O2 conferred the resistance to apopto-sis, and that ligustrazine at 25 μM led to significant apoptosis, indicatingthe pro-apoptotic effects of ligustrazine on HSCs under oxidative stress(Fig. 2B). Altogether, these data suggested that ligustrazine could inhibitproliferation and facilitate apoptosis in HSCs activated by oxidativestress. We also performed additional experiments where growth factorPDGF, one of the most potent pro-fibrogenic cytokines (Pinzani, 2002),was used to induce HSC activation complementary to H2O2-induced ox-idative stress. Ligustrazine dose-dependently abrogated PDGF-enhancedHSC viability (Fig. 2C) and stimulatedHSC apoptosis despite the apoptosisresistance caused by PDGF (Fig. 2D). The resultswere consistentwith thatunder oxidative stress and could confirm the anti-proliferative effects ofligustrazine on HSC growth.

Ligustrazine inhibits expression of fibrotic marker genes in H2O2-activatedHSCs

HSC activation is characterized by overexpression of α-SMA andresults in excessive production of ECM components such as α1(I)

te buffered saline as negative control or H2O2 for 24 h. (A) Cell morphology assessmentare expressed as mean±SD, *pb0.05 vs control, and **pb0.01 vs control. (C) Westerntrol for equal loading. Representative blots were from three independent experiments.

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procollagen and glycoprotein fibronectin (Friedman, 2008). We nextassessed the ligustrazine effects on the expression of these markers ofHSC activation under H2O2 stimulation at both gene and protein levels.Real-time PCR analysis showed that the mRNA levels of α-SMA, α1(I)procollagen and fibronectin were all significantly elevated by H2O2 stim-ulation, but ligustrazine downregulated their mRNA expressiondose-dependently, suggesting that the transcription of these genes wasinhibited (Fig. 3A). Western blot assays demonstrated that the elevatedprotein abundance of the three fibrotic markers by H2O2 stimulationwas also reduced by ligustrazine in a dose-dependent manner(Fig. 3B). Thesefindings suggested that ligustrazinewas able to eliminatethe de novo synthesis of some fibrotic marker genes during HSCactivation induced by oxidative stress. Further, we recaptured similar re-sults in the PDGF-activated HSCs. The elevated mRNA and protein levelsof the three marker genes by PDGF were also reduced by ligustrazinedose-dependently (Figs. 3C, D). These results could expand theantifibrotic potential of ligustrazine targeting HSC activation.

Disruption of PDGF-βR-mediated ERK and p38 pathways is involved inligustrazine attenuation of fibrotic marker genes in H2O2-activated HSCs

Numerous studies have described the potent fibrogenic role of PDGFsignaling transmitted by PDGF-βR, a RTK that can activate MAPKcascades, in HSC activation (Pinzani, 2002). In addition, activation ofMAPK pathways via RTKs has also been linked to oxidative stress(McCubrey et al., 2006; Torres and Forman, 2003). Therefore, wehypothesized that the PDGF-βR/MAPK cascades might be involved inH2O2-treated HSCs associatedwith HSC activation and ECM production,and that ligustrazine could disrupt these pathways exerting itsantifibrotic effects. To test this hypothesis, we first detected the mRNAlevel and protein abundance of PDGF-βR. The results showed thatH2O2 treatment indeed increased PDGF-βR expression at both geneand protein levels, but ligustrazine dose-dependently reduced the ex-pression of PDGF-βR mRNA and protein in H2O2-activated HSCs(Figs. 4A, B). These data led us to postulate that the downstreamMAPK cascades might be affected by H2O2 and ligustrazine. Our subse-quent western blot assays showed that the phosphorylation of ERK andp38was significantly enhanced in the presence of H2O2, but ligustrazineled to a significant reduction in the phosphorylated ERK and p38 in adose-dependent manner (Fig. 4C). These data suggested that the ERKand p38 pathways could be selectively disrupted by ligustrazine inHSCs activated by oxidative stress. We next used PDGF-βR blockerimatinib and activator PDGF to examine whether the disruption ofERK and p38 pathways was secondary to the blockade of PDGF-βR.The results showed that imatinib led to reduced phosphorylation ofERK and p38 and its combinationwith ligustrazine produced synergisticeffects under H2O2 stimulation (Fig. 4D). However, PDGF rescuedligustrazine inhibition of ERK and p38 pathways and significantlyenhanced the phosphorylation of ERK and p38 in the absence ofligustrazine (Fig. 4E). These data clearly indicated that ligustrazine in-terruption of ERK and p38 pathways was mediated by the blockade ofPDGF-βR inH2O2-activatedHSCs. To further testify the association of in-hibition of PDGF-βR-mediated ERK and p38 pathways with ligustrazineattenuation of H2O2-induced HSC activation, the specific MEK inhibitorU0126 for blocking ERK signaling and p38 kinase inhibitor SB203580were used respectively. The data demonstrated that these two agentsdownregulated the expression of α-SMA, α1(I) procollagen and fibro-nectin dose-dependently and their combination with ligustrazineresulted in more significant effects (Figs. 4F, G). Both U0126 andSB203580 could mimic the ligustrazine effects on H2O2-activatedHSCs due to the blockade of ERK and p38 cascades, respectively.Taken together, these findings suggested that PDGF-βR-mediatedERK and p38 pathways modulated the marker gene expression andwere involved in ligustrazine attenuation of HSC activation underoxidative stress.

Ligustrazine modulates some key proteins related to HSC activation andECM homeostasis under H2O2 stimulation

Wenext investigated ligustrazine regulation of some key effectors im-plicated in HSC activation. HSC activation is accompanied by a dramaticreduction in transcription factor peroxisome proliferator-activatedreceptor-γ (PPARγ). Activation of PPARγ can inhibit HSC collagen pro-duction and modulate HSC adipogenic phenotype at transcriptional andepigenetic levels (Zhang et al., 2012). Our data showed that ligustrazinedose-dependently increased PPARγ expression in H2O2-activated HSCs(Fig. 5A). Nuclear factor-κB (NF-κB) is critically involved in the “inflam-mation–fibrosis–cancer axis” in the diseased liver and its activity is re-quired for persistent inflammatory and mitogenic phenotype of HSCsduring liver fibrogenesis (Elsharkawy andMann, 2007). Our results dem-onstrated that the phosphorylation of NF-κBwas inhibited by ligustrazinedose-dependently inH2O2-stimulatedHSCs, suggesting the eliminationofNF-κB transcriptional activity (Fig. 5A). Furthermore, CTGF expression inHSCs is significantly enhanced during the process of activation in vitroand in vivo and plays a key role in ECM overproduction in activatedHSCs (Gressner and Gressner, 2008). Our results demonstrated thatH2O2 stimulated CTGF expression, but this elevation was eliminated byligustrazine in a dose-dependent manner (Fig. 5A).

Compelling evidence has documented the association of MMP/TIMPsystemwith liver fibrosis in controlling ECM synthesis and degradation.MMP-2 is a gelatinasewith proteolytic activity and can degrade gelatinsand ECM molecules including type I collagen; whereas TIMP-1 of highlevel deteriorated fibrogenesis due to its broad inhibitory effects onMMPs (Han, 2006; Hemmann et al., 2007). Our present data showedthat ligustrazine could dose-dependently reduce TIMP-1 protein abun-dance, but had no obvious effects onMMP-2 expression inH2O2-treatedHSCs (Fig. 5B). These effects could upregulate the ratio of MMP-2/TIMP-1 and compromise the inhibitorymachinery for ECMdegradationin the fibrotic liver, although the fibrinolytic processmight not be accel-erated directly by ligustrazine. Taken together, these data indicated thatligustrazine modulation of some critical effectors related to HSC activa-tion and ECM homeostasis probably contributed to its inhibition of HSCactivation and ECM expression under oxidative stress.

Discussion

Increasing studies indicate that oxidative stress is associated withhepatic fibrosis of different etiologies. Oxidative stress may representa direct or indirect pro-fibrogenic stimulus for HSC activation(Hernandez-Gea and Friedman, 2011; Novo and Parola, 2008). Cur-rently, most evolving antifibrogenic therapies are aimed at inhibitingHSC activation (Popov and Schuppan, 2009). In search for potentialantifibrotic agents, the antioxidant ligustrazine has shown a promisein many in vivo studies (Chen et al., 2007, 2010; Lu et al., 2010; So etal., 2002), but few reports are seen to address its effects on HSCactivation under oxidative stress and to elucidate the molecularmechanisms. Thus, our present investigations were designed to ex-plore the antifibrotic properties of ligustrazine and the underlyingmechanisms in a culture model of oxidative stress-induced HSCactivation.

Signs of oxidative stress are concomitant or precede HSC activa-tion and collagen deposition. There was evidence that exposure ofcultured human or rat HSCs to pro-oxidant systems or to mediumcontaining products released from hepatocytes undergoing oxidativestress was followed by increased type I procollagen gene synthesis(Parola et al., 1993; Svegliati-Baroni et al., 1999). H2O2 is a well-known oxidative stress-related molecule generated in the extracellu-lar environment or within the cell (Parola and Robino, 2001). Ourpresent data showed that the primary rat HSCs exposed to H2O2 at5 or 10 μM exhibited a dramatic myofibroblast phonotype with en-hanced proliferation and upregulated expression of fibrotic markergenes (Fig. 1). These results were consistent with the previous reports

Fig. 2. Ligustrazine inhibits proliferation and induces apoptosis in H2O2-activated HSCs. Low-serum cultured HSCs were treated with stimuli (H2O2 or PDGF) and/or ligustrazine for 24 h. Treatment with DMSO (0.02%, w/v) was negativecontrol. (A) MTS assay for evaluation of cell viability. Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (B) Flow cytometric analyses of apoptosis. HSCswere double stained with FITC labeled Annexin-V/PI. Only fluorescein-positive cells (in the right-bottom panel of each plot) without PI staining were regarded as apoptotic cells and the apoptosis rates were determined by flow cytometry.Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (C) MTS assay for evaluation of cell viability. Data are expressed as mean±SD, #pb0.05 vs DMSO withoutPDGF, *pb0.05 vs DMSO plus PDGF, and **pb0.01 vs DMSO plus PDGF. (D) Flow cytometric analyses of apoptosis. HSCs were double stained with FITC labeled Annexin-V/PI. Only fluorescein-positive cells (in the right-bottom panel of eachplot) without PI staining were regarded as apoptotic cells and the apoptosis rates were determined by flow cytometry. Data are expressed as mean±SD, #pb0.05 vs DMSO without PDGF, and *pb0.05 vs DMSO plus PDGF.

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Fig. 3. Ligustrazine reduces the expression of fibrotic marker genes in H2O2-activated HSCs. Low-serum cultured HSCs were treated with stimuli (H2O2 or PDGF) and/or ligustrazine for 24 h. Treatment with DMSO (0.02%, w/v) was negativecontrol. (A) Real-time PCR analysis for transcript levels of fibrotic marker genes. GAPDHwas used as the invariant control for calculating fold changes in mRNA levels (n=3). Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2,*pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (B) Western blot analysis for protein levels of fibrotic marker genes. β-Actin was used as an invariant control for equal loading. Representative blots were from three inde-pendent experiments. Data are expressed as mean±SD #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (C) Real-time PCR analysis for transcript levels of fibrotic marker genes. GAPDHwas used as the invariant control for calculating fold changes in mRNA levels (n=3). Data are expressed as mean±SD, #pb0.05 vs DMSO without PDGF, ##pb0.01 vs DMSO without PDGF, *pb0.05 vs DMSO plus PDGF, and **pb0.01 vsDMSO plus PDGF. (D) Western blot analysis for protein levels of fibrotic marker genes. β-Actin was used as an invariant control for equal loading. Representative blots were from three independent experiments. Data are expressed asmean±SD, #pb0.05 vs DMSO without PDGF, ##pb0.01 vs DMSO without PDGF, *pb0.05 vs DMSO plus PDGF, and **pb0.01 vs DMSO plus PDGF.

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Fig. 4. Disruption of PDGF-βR-mediated ERK and p38 pathways is involved in ligustrazine attenuation of HSC activation under H2O2 stimulation. Treatment with DMSO (0.02%, w/v)was negative control. (A) Real-time PCR analysis for transcript levels of PDGF-βR. Low-serum cultured HSCs were treated with H2O2 and/or ligustrazine for 24 h. GAPDH was usedas the invariant control for calculating fold changes in mRNA levels (n=3). Data are expressed as mean±SD, ##pb0.01 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and**pb0.01 vs DMSO plus H2O2. (B) Western blot analysis for protein levels of PDGF-βR. Low-serum cultured HSCs were treated with H2O2 and/or ligustrazine for 24 h. Data areexpressed as mean±SD, ##pb0.01 vs DMSO plus H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (C) Western blot analysis for protein levels of ERK, JNKand p38 pathways. Low-serum cultured HSCs were pretreated with ligustrazine for 3 h and then treated with H2O2 for an additional 1 h. Data are expressed as mean±SD,#pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (D) Western blot analysis for protein levels of ERK and p38 pathways.Low-serum cultured HSCs were pretreated with ligustrazine plus imatinib for 3 h and then treated with H2O2 for an additional 1 h. Data are expressed as mean±SD, #pb0.05vs DMSO without H2O2, ##pb0.01 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (E) Western blot analysis for protein levels of ERKand p38 pathways. Low-serum cultured HSCs were pretreated with ligustrazine plus PDGF for 3 h and then treated with H2O2 for an additional 1 h. Data are expressed asmean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and §pb0.05 vs ligustrazine plus H2O2. (F) Western blot analysis for protein levels of fibrotic markergenes. Low-serum cultured HSCs were treated with H2O2 and/or ligustrazine plus U0126 for 24 h. Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vsDMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2. (G) Western blot analysis for protein levels of fibrotic marker genes. Low-serum cultured HSCs were treated with H2O2

and/or ligustrazine plus SB203580 for 24 h. Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2, and **pb0.01 vs DMSO plus H2O2.For all western blot assays in this figure, β-actin was used as an invariant control for equal loading and representative blots were from three independent experiments.

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that H2O2 could act as an important mediator of the pro-fibrogenicactions of transforming growth factor-β (De Bleser et al., 1999;Maher et al., 1994) or acetaldehyde (Greenwel et al., 2000) leading

to type I collagen gene expression in rodent HSCs. Importantly,these results suggested a culture model of oxidative stress-inducedHSC activation. Of note, some studies demonstrated that H2O2 at

Fig. 5. Ligustrazine modulates some key proteins related to HSC activation and ECM homeostasis in H2O2-activated HSCs. Low-serum cultured HSCs were treated with H2O2 and/orligustrazine for 24 h. Treatment with DMSO (0.02%, w/v) was negative control. (A, B) Western blot analysis with the indicated antibodies. β-Actin was used as an invariant controlfor equal loading. Representative blots were from three independent experiments. Data are expressed as mean±SD, #pb0.05 vs DMSO without H2O2, *pb0.05 vs DMSO plus H2O2,and **pb0.01 vs DMSO plus H2O2.

58 F. Zhang et al. / Toxicology and Applied Pharmacology 265 (2012) 51–60

concentrations higher than 200 μM could result in cell death in HSCs(Dunning et al., 2009), presumably because all the major cellularstructures, particularly mitochondria and cytoskeletal proteins, mac-romolecules and metabolic pathways can be directly or indirectly ox-idized, damaged and then blocked or inactivated, leading eventuallyto necrotic cell death (Kaplowitz, 2000). However, this was not ob-served in our experiments. Therefore, treatment of HSCs with H2O2

at 5 μM in our studies could well reflect the pro-fibrogenic effects ofoxidative stress without cytotoxicity on HSCs.

Recent studies have described the antifibrotic activity of ligustrazinein vivo, suggesting it as a hopeful option for liver fibrosis. Ligustrazinecould downregulate a series of serum markers of fibrogenesis, such ashyaluronic acid, procollagen III, collagen IV and laminin, and improveliver functions (Chen et al., 2007, 2010; Lu et al., 2010). These beneficialeffects might be associated with inhibited HSC activation and reducedproduction of ECM components in the fibrotic liver. This could besupported by our present studies. We found that ligustrazine hadanti-proliferative and apoptosis-inducing effects on HSCs treated withH2O2 (Fig. 2) and decreased the expression of fibrotic marker genes(Fig. 3). These data demonstrated that ligustrazine was able to attenu-ate HSC activation and inhibit ECMproduction possibly leading tofibro-sis resolution under oxidative stress. Our additional experiments withPDGF stimulation could strengthen the antifibrotic potential ofligustrazine. Furthermore, our studies suggested the antioxidant thera-py for hepatic fibrosis. Indeed, a growing number of studies in vivo haveshown that administration of antioxidants, most of them were fromnatural resources, prevented oxidative stress and fibrogenesis in vari-ous experimental liver fibrosis due to suppression of HSC activation aswell as protection of hepatocytes (Calleja et al., 2012; Hong et al.,2009; Mandal et al., 2007; Phung et al., 2009). Our data indicated thatligustrazine could be a novel candidate falling into this category.

Few data are available on the mechanisms responsible for oxidativestress-mediated upregulation ofmarker genes during liverfibrogenesis.Some studies have outlined a close relationship between oxidativestress and RTK-mediated activation of MAPK signaling cascades(Guyton et al., 1996; Wang et al., 1998). MAPK pathways also play anessential role in the initiation of cellular processes such as proliferation,differentiation and apoptosis (Boutros et al., 2008). In the present study,we found that exogenous H2O2-driven oxidative stress increased theexpression of PDGF-βR (Figs. 4A, B), a RTK well-known for itspro-fibrogenic role in HSCs (Pinzani, 2002), and significantly activatedthe downstream ERK and p38 cascades manifested by the enhancedphosphorylation of the two kinases (Fig. 4C). Subsequent evidencethat pharmacological blockade of ERK or p38 pathways downregulatedthe protein abundance of fibrotic marker genes confirmed the associa-tion of ERK and p38 with HSC activation due to oxidative stress. Itcould also be extrapolated that H2O2-stimulated HSC proliferation andresistance to apoptosis were probably attributed to the activation ofERK signaling, which could also be supported by the emerging datathat PDGF-βR-mediated ERK signaling is a powerful pro-fibrogenic cas-cade that promotes HSC activation (Pinzani, 2002). Furthermore, ourobservation that p38 phosphorylation was increased under H2O2 stim-ulation was also in line with previous reports that the p38 MAPK path-way can be activated in a number of different cell types in response toreactive oxygen intermediates (McCubrey et al., 2006). In the presentstudy, activation of p38 was shown to be required for overexpressionof fibrotic marker genes in HSCs under H2O2 stimulation, which wasalso consistent with the recent published data that p38 MAPK was apro-fibrogenic cascade responsible for HSC activation and collagen pro-duction during hepatic fibrogenesis (Che et al., 2007; Thirunavukkarasuet al., 2006; Varela-Rey et al., 2002; Yan et al., 2012). However, there aresome investigations suggesting an apoptotic role for p38 in response to

59F. Zhang et al. / Toxicology and Applied Pharmacology 265 (2012) 51–60

oxidative stress in different cell types (Tobiume et al., 2001). Our pres-ent data probably could not describe a definite role for p38 in control-ling the fate of H2O2-treated HSCs, given the finding that p38activation might not be sufficient in itself for apoptosis to occur in thepresence of high enough levels of ERK activation in certain experimen-tal conditions (Birkenkamp et al., 1999). This merits additional investi-gation. Furthermore, of interest in this study was that the JNK pathwayappeared to be not involved in H2O2-stimulated expression of fibroticmarker genes in HSCs, although it is established that many of the initialproteins and activation events in the p38 pathway are also involved inthe activation of the JNK pathway (McCubrey et al., 2006; Reeves etal., 2000). This could be explained by the recognition that many cyto-skeletal proteins, protein kinases and transcription factors are sub-strates for the ERK or p38 MAPK while only a few transcription factorsare targets for the JNK (Torres and Forman, 2003).

Our molecular evidence showed that ligustrazine could inhibitPDGF-βR expression at both mRNA and protein levels in H2O2-activatedHSCs and also disrupt the downstream ERK and p38 cascades (Figs. 4A,B, C). Inhibition or activation of PDGF-βR provided clear evidence thatblockade of PDGF-βR was required for ligustrazine interruption of ERKand p38 pathways in HSCs under oxidative stress (Figs. 4D, E). BothU0126 and SB203580 could independently mimic the ligustrazineeffects (Figs. 4F, G), indicating that ligustrazine reduction offibroticmark-er genes was associated with disruption of PDGF-βR-mediated ERK andp38 cascades. These findings were also consistent with some previousstudies, for example, ERK inhibition by curcumin (Chen and Zheng,2008) and p38 inhibition by butein (Szuster-Ciesielska et al., 2012)were reported to reduce HSC activation and collagen production. In addi-tion, it was possible that ligustrazine blockade of ERK and p38 pathwayscould play a role in the induction of apoptosis in H2O2-treated HSCs ob-served in this study. Our further findings suggested that ligustrazinecould be a more potent inhibitor for HSC activation, because some keyfibrosis-related effectors such as PPARγ, NF-κB and CTGF in H2O2-activated HSCs were affected by ligustrazine (Fig. 5A). Of note, NF-κB isa ubiquitous transcription factor that can be activated by a large numberof extracellular stimuli including oxidative stress (Elsharkawy andMann, 2007). Recent evidence showed that NF-κB transcriptional activitywas required for CTGF expression in activated HSCs (Chen and Zheng,2008). It thus could be presumed that ligustrazine blockade of NF-κBphosphorylation probably contributed to the inhibited CTGF expressionin H2O2-treated HSCs. Furthermore, the MMP/TIMP system is altered inactivated HSCs favoring ECM deposition in the fibrotic liver (Han, 2006;Hemmann et al., 2007). In the present study, ligustrazine downregulationof TIMP-1 leading to elevated ratio ofMMP-2/TIMP-1might contribute in-directly to the decomposition of ECM components (Fig. 5B). Whetherligustrazine interruption of PDGF-βR-mediated ERK and p38 cascadeswas involved in the modulation of ECM homeostasis under oxidativestress needs further investigation.

In summary, our results demonstrated that H2O2 potently promot-ed the activation of HSCs in vitro, whereas ligustrazine suppressedproliferation and stimulated apoptosis in H2O2-treated HSCs.Ligustrazine reduced the expression of marker genes of HSC activa-tion under H2O2 stimulation, which was possibly associated with in-terruption of PDGF-βR-mediated ERK and p38 pathways. These dataprovided novel insights into the underlying mechanisms by whichligustrazine inhibits HSC activation and thereby exerts its antifibroticeffects.

Conflict of interest

The authors have no conflicts of interest to disclose.

Acknowledgments

The financial support was from the National Natural Science Founda-tion of China (81270514, 30873424), the Doctoral Discipline Foundation

of theMinistry of Education of China (20103237110010), Jiangsu NaturalScience Foundation (BK2008456), Project for Supporting JiangsuProvincial Talents in Six Fields (2009-B-010), theOpenProgramof JiangsuKey Laboratory of Integrated Acupuncture and Drugs (KJA200801), theOpen Project Program of the National First-Class Key Discipline for Tradi-tional Chinese Medicine of Nanjing University of Chinese Medicine(2011ZYX4-008), and the “Eleven-Five”National Science and TechnologySupporting Program (2008BAI51B02).

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