Melatonin prevents H2O2-induced activation of rat hepatic stellatecells

Introduction

Liver cirrhosis is a critical stage of chronic liver diseasescaused by a variety of insults including chronic viralhepatitis, alcohol and nonalcohol steatohepatitis [1]. Hep-atic stellate cells (HSCs) play key roles in hepatic fibrosis

and liver cirrhosis, as activated HSCs are the major sourcesof collagens and other extracellular matrices (ECM) infibrotic livers. Thus, inhibiting HSCs activation and ECM

production are regarded as useful antifibrotic strategies tohamper fibrogenesis [2].

Evidence shows that melatonin prevents thioacetamide-

or tetrachloride-induced hepatic fibrosis in rats via inhibi-tion of oxidative stress [3, 4]. Transforming growth factorbeta 1 (TGF-b1) is the most important cytokine contribu-

ting to the activation of HSCs and liver fibrogenesis [1, 2]. Itis involved in H2O2-stimulated synthesis of ECM in HSCsand modulates gene expression of CCAAT/enhancer-bind-ing proteins including C/EBP-a [5–7]. The latter may reverse

the phenotype of activated HSCs in vitro and amelioratescarbon tetrachloride-induced rat liver fibrosis in vivo [8].

The present studies were designed to investigate the

protective effect of melatonin on H2O2-stimulated activa-tion of HSCs in vitro and the involvement of TGF-b1 andC/EBP-a proteins.

Material and methods

Reagents

Collagenase type IV, pronase E and Nycodenz, glycerol,H2O2 and melatonin were purchased from Sigma-Aldrich,

Inc. (St Louis, MO, USA). Deoxyribonucleate 5¢-oli-gonucleotidohydrolase (DNase, from beef pancreas) wasfrom Faizyme Laboratories (Philippi, Cape Town, South

Africa). Dubocco’s minimum essential medium (DMEM),newborn bovine serum and fetal calf serum were purchasedfrom Invitrogen Corporation (Carlsbad, CA, USA). Trizol,RNasin, Taq polymerase, dNTP, Oligo(dT)16 were from

Shanghai Sangon Biological Engineering & TechnologyServices Co., Ltd (Shanghai, China). Mu-MLV ReverseTranscriptase was purchased from Promega Corporation

(Madison, WI, USA). SYBR Green PCR Master Mix wasfrom Applied Biosystems (Foster City, CA, USA). Anti-bodies to collagen type I, TGF-b, and C/EBP-a were

purchased from Santa Cruz Biotechnology, Inc. (SantaCrutz, CA, USA). FITC- and Texas Red-labeled secondantibodies were purchased from Proteintech Group, Inc.(Chicago, IL, USA).

Isolation and culture of rat HSCs

Normal male Sprague–Dawley rats (body weight about450 g) were cared for according to the principles of Guidefor the Care and Use of Laboratory Animals prepared by

Shanghai Medical College, Fudan University. HSCs wereisolated from the livers through a two-step of digestionaccording to Weiner et al. [9] with minor modification as

stated previously [8]. Cells were suspended in DMEMcontaining 10% newborn bovine serum, 100,000 units/Lpenicillin, 100 mg/L streptomycin, and 2 mm glutamine,seeded into 25-cm2 plastic tissue culture flasks (Corning

Incorporated, Corning, NY, USA), or 12-well tissue culturemultiplates (Corning Incorporated) with 18-mm circle

Abstract: Considerable evidence shows therapeutic effects of melatonin on

liver injury and the involvement of hepatic stellate cells (HSCs) in vivo. In the

present studies, we investigate the protective effect of melatonin on H2O2-

induced activation of HSCs in vitro. Compared with that in control HSCs,

synthesis of collagen type I was increased in H2O2-treated cells. Melatonin

pretreatment significantly inhibited the above effects of H2O2 in HSCs.

CCAAT/enhancer-binding protein alpha (C/EBP-a), which could partially

reverse the phenotype of activated HSCs, augmented in HSCs pretreated

with melatonin. Moreover, secretion of the most important fibrotic cytokine

transforming growth factor beta 1 (TGF-b1) diminished in melatonin-

pretreated HSCs. These results suggest that melatonin prevents H2O2-

induced activation of HSCs and that the mechanism involves, at least in part,

differential regulation of TGF-b1 and C/EBP-a gene expression.

Jing Gu1*, Li Zhuang2* andGuang-Cun Huang2

1National Key Laboratory of Medical

Neurobiology, and 2Department of Pathology,

Shanghai Medical College, Fudan University,

Shanghai, China

Key words: CCAAT/enhancer-binding protein,

extracellular matrix, H2O2, hepatic stellate

cell, melatonin, transforming growth factor

Address reprint requests to Guang-Cun

Huang, Department of Pathology, Shanghai

Medical College, Fudan University, 138

Yixueyuan Road, Shanghai 200032, China.

E-mail: gc_huang@fudan.edu.cn

*These authors contributed equally to this

work.

Received April 13, 2006;

accepted June 14, 2006.

J. Pineal Res. 2006; 41:275–278Doi:10.1111/j.1600-079X.2006.00364.x

� 2006 The AuthorsJournal compilation � 2006 Blackwell Munksgaard

Journal of Pineal Research

275

coverslips predisposed in, and incubated in 5% CO2/95%air atmosphere at 37�C. The medium was changed at 24 hrafter seeding, and thereafter at every 48 hr.

After they reached confluence (10 days after planting),activated HSC were subcultured onto plastic tissue culturemulti-plates with or without coverslips. Experiments wereperformed on cells between serial passage 1 and 3 using

three independent cell lines.

Isolation of cellular total RNA and real-time RT-PCR

Total cellular RNA was isolated by Trizol following themanufacturer’s instructions. The templates and primer sets

(Table 1) were mixed with SYBR Green PCR Master Mix,and real-time PCR were performed using a real-time PCRmachine Rotor-Gene 3000 (Corbett Research, Sydney,Australia) in quantitation mode, and the cycling parameters

were: initial denaturing at 94�C for 4 min, followed by 40cycles of 94�Cdenaturing for 45 s, primer annealing for 30 s,primer extension at 72�C for 30 s, and final extension at 72�Cfor 7 min (see Table 1 for the details of annealing temper-atures, and the cycle numbers). The fluorescence intensity ofthe double-strand specific SYBR Green reflecting the

amount of formed amplicon was read after each elongationstep at additional acquisition temperature (Table 1) using theRotor-Gene software (release version 6.0 Build 21) (Corbett

Research, Sydney, Australia). A negative control was inclu-ded in each set of experiments. To enhance specificity of theamplification reaction,melting curve analysis was performedand the PCR products were separated by electrophoresis

through 1.5% agarose gels containing 0.1 lg/mL ethidiumbromide to ensure production of correct sized product.

Immunofluorescence staining

Cultured HSCs grown on coverslips were fixed with 4%

paraformaldehyde for 15 min at room temperature, fol-lowed by incubation with first antibodies respectively at37�C for 1 hr and FITC- or Texas Red-labeled secondantibody at 37�C for 45 min, and finally mounted with

glycerol followed by sealed with transparent nail polish.Cells were observed and images taken under a fluorescencemicroscopy (Nikon Corporation, Tokyo, Japan).

ELISA assay

Transforming growth factor beta 1 was assayed by ELISAfrom the culture supernatants according to directions of itsmanufacturer.

Statistical analyses

The data were expressed as mean ± standard deviation

(S.D.), and significant differences were evaluated by one-way ANOVA with SPSS 14.0 for Windows (SPSS Inc.,Chicago, IL, USA). Differences were considered significantif P < 0.05. All the experiments were repeated three times

with independent isolated cells.

Results

Incubation with 100 lm H2O2 for 24 hr did not inducenecrotic cell death or apoptosis in HSCs (data not shown).

This is consistent with the results from Novo et al. [10].The protein level of collagen type I in HSCs exposed to

H2O2 for 24 hr decreased when compared with controlHSCs (Fig. 1). However, pretreatment of 1 lm melatonin

inhibited H2O2-stimulated upregulation of collagen type I(Fig. 1).Secretion of TGF-b1, the most important fibrotic cyto-

kine in liver fibrogenesis, declined in melatonin pretreatedHSCs. In addition, C/EBP-a gene expression, which hasbeen shown to reverse the activated phenotype of HSCs in

our previous studies [8], was induced by pretreatment ofmelatonin (Figs 2 and 3).

Discussion

Hepatic stellate cells activation is a critical event during thedevelopment of liver fibrogenesis [1, 2]. While HSCs are

activated by stimuli such as damaged hepatocytes, Kupffercells, soluble cytokines or growth factors, e.g. TGF-b1, theytransform to ECM- and cytokines-producing myofibro-

blast-like cells characterized by expression of a-SMA.Protective roles of melatonin in liver injury and associatedliver cirrhosis have been shown and evidence supports the

involvement of melatonin in inhibiting oxidative stress,partially by indirectly restraining secretion of tumornecrosis factor alpha and interleukin-1 beta [3, 4]. However,the direct effects of melatonin on ECM production in HSCs

remain unclear.Our present studies show that melatonin inhibits H2O2-

stimulated synthesis of collagen type I in activated cultured

rat HSCs (Fig. 1), which indicates that melatonin preventsH2O2-induced activation of HSCs.Pretreatment of melatonin reduced secretion of TGF-b1.

The latter is the most important fibrotic cytokine in liverfibrogenesis, so melatonin suppresses ECM synthesis, atleast partially, via inhibition of autocrine TGF-b in HSCs.

Moreover, the current results show that C/EBP-a protein

Table 1. Primers for PCR used in thecurrent studies

Targetgene Primer sequence (5¢-3¢)

Annealingtemperature

(�C)

Acquisitiontemperature

(�C)

C/EBP-a For: TCA CTT GCA GTT CCA GAT CGRev: TTG ACC AAG GAG CTC TCA GG

62 79

b-actin For: AGG ATG CAG AAG GAG ATT ACT GCRev: AAA ACG CAG CTC AGT AAC AGT CC

55 76

For, forward primer; Rev, reverse primer.

Gu et al.

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and mRNA levels are augmented in HSCs pretreated with

melatonin (Figs 2 and 3), which implies that up-regulationof C/EBP-a gene expression is also involved in the protect-ive effects of melatonin in H2O2-induced activation of

HSCs, as C/EBP-a gene hampers the activation of HSCs [8].

Fig. 1. Melatonin prevents H2O2-stimulated synthesis of collagentype I in hepatic stellate cells (HSCs). Control HSCs (A), andH2O2-stimulated HSCs without (B) or without (C) pretreatment ofmelatonin were fixed and immunofluorescence staining was per-formed to detect collagen type I. The photographs shown arerepresentative of three independent experiments. Original magni-fications were 400·.

Fig. 2. Melatonin induces the mRNA level of CCAAT/enhancer-binding protein alpha (C/EBP-a) in hepatic stellate cells (HSCs).Total cellular RNA was isolated from H2O2-stimulated HSCswithout (a) or with (b) pretreatment of melatonin, and quantitationRT-PCR was performed to determine C/EBP-a mRNA levels byusing Rotor-Gene Application Software (Release Version 6.0 Build21). The columns represent mean values and the error bars indicatestandard deviations of three experiments performed in triplicate.P < 0.05.

Fig. 3. CCAAT/enhancer-binding protein alpha (C/EBP-a) pro-tein augments in HSCs pretreated with melatonin. H2O2-stimulatedHSCs without (A) or with (B) pretreatment of melatonin were fixedand immunofluorescence staining was performed to detect C/EBP-a. The photographs shown are representative of three independentexperiments. Original magnifications were 400·.

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In summary, therapeutic effects of melatonin in liverinjury and associated liver cirrhosis is, at least in part,related to modulation of autocrine of TGF-b1 and gene

expression of C/EBP-a.

Acknowledgments

The authors thank Mr Teng-Fan Zhu and Ms Wen-yan Lifor technician help in using fluorescence microscope.

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