Hye Kyung Kim, Department of Food and Biotechnology, Hanseo University, Seosan, Chungnam 356-706, South Korea
Taik-Hoon Yang, Hong-Yon Cho, Department of Food and Biotechnology, College of Science and Technology, Korea University, Jochiwon, Chungnam 339-700, South Korea
Author contributions: Kim HK and Cho HY designed the study, data analysis, interpretation and drafting of the manuscript; Yang TH performed measurements and data analysis.
Supported by A Korea University Grant
Correspondence to: Hong-Yon Cho, Professor, Department of Food and Biotechnology, College of Science and Technology, Korea University, Jochiwon, Chungnam 339-700, South Korea. email@example.com
Telephone: +82-41-8601433 Fax: +82-41-8650220
Received: July 9, 2009 Revised: August 4, 2009
Accepted: August 11, 2009
Published online: November 7, 2009
AIM: To examine the protective effect of green tea extract (GT) on hepatic fibrosis in vitro and in vivo in dimethylnitrosamine (DMN)-induced rats.
METHODS: HSC-T6, a rat hepatic stellate cell line, was used as an in vitro assay system. Cell proliferation, collagen content, and type 1 collagen expression were examined in activated HSC-T6 cells. Collagen was determined by estimating the hydroxyproline content. In rats with DMN-induced hepatic fibrosis, serum aspartate aminotransferase and alanine aminotransferase concentrations, liver hydroxyproline and lipid peroxides were determined. Pathologic changes were examined by hematoxylin & eosin staining.
RESULTS: GT administration prevented the development of hepatic fibrosis in the rat model of DMN-induced liver fibrosis. These results were confirmed both by liver histology and by quantitative measurement of hepatic hydroxyproline content, a marker of liver collagen deposition. Accordingly, inhibition of proliferation, reduced collagen deposition, and type 1 collagen expression were observed in activated HSC-T6 cells following GT treatment. These results imply that GT reduced the proliferation of activated HSC and down regulated the collagen content and expression of collagen type 1, thereby ameliorating hepatic fibrosis.
CONCLUSION: This study demonstrates that green tea administration can effectively improve liver fibrosis caused by DMN, and may be used as a therapeutic option and preventive measure against hepatic fibrosis.
© 2009 The WJG Press and Baishideng. All rights reserved.
Key words: Dimethylnitrosamine; Green tea extract; HSC-T6 cell; Liver fibrosis; Rat model; Type 1 collagen
Peer reviewer: Fabio Grizzi, PhD, Laboratories of Quantitative Medicine, Istituto Clinico Humanitas IRCCS, Via Manzoni 56, 20089 Rozzano, Milan, Italy
Kim HK, Yang TH, Cho HY. Antifibrotic effects of green tea on in vitro and in vivo models of liver fibrosis. World J Gastroenterol 2009; 15(41): 5200-5205 Available from: URL: http://www.wjgnet.com/1007-9327/15/5200.asp DOI: http://dx.doi.org/10.3748/wjg.15.5200
Hepatic fibrosis is a consequence of severe liver damage and occurs in many forms of chronic liver damage, including virus infection, autoimmune liver diseases and sustained alcohol abuse. Hepatic stellate cells (HSC) are recognized as the primary cellular source of matrix components in chronic liver diseases, and therefore play a critical role in the development and maintenance of liver fibrosis. The key cellular and molecular events involved in the pathogenesis of liver fibrosis include activation of HSC to a myofibroblast-like phenotype, production of excess matrix proteins, and increased cell proliferation. Overproduction of extracellular matrix (ECM) components, particularly collagen, is a characteristic of activated HSC, and activation and proliferation of HSC have been implicated in the pathogenesis of liver fibrosis. Therefore, suppression of HSC activation has been proposed as a therapeutic target against hepatic fibrosis.
Acetaldehyde, a highly reactive compound produced by alcohol metabolism, stimulates the deposition of ECM proteins. Acetaldehyde also stimulates type 1 collagen synthesis and gene transcription in cultured rat and human HSC and in human liver fibroblasts.
Several studies have shown that lipid peroxidation stimulates collagen production in fibroblasts and HSC, and plays an important role in the development of liver fibrosis. Lipid peroxidation has been shown to stimulate the expression of collagen gene transcripts. It has recently been shown that stellate cells are activated by free radicals as well as by malondialdehyde (MDA), a product of lipid peroxidation. In addition, stellate cell activation by type 1 collagen has been shown to be blocked by antioxidants, suggesting that lipid peroxidation may play a role in hepatofibrogenesis.
Green tea, which is a widely consumed drink, has received much attention due to its beneficial biological effects. Polyphenols, often collectively referred to as catechins, account for up to 30% of the dry weight and serve as a major effective component of green tea. The effects of green tea have been widely studied and antioxidative, antiallergic, antimutagenic/anticarcinogenic, and antibacterial effects have been documented[10-12]. It has been shown that an aqueous extract of polyphenols from green tea (Camellia sinensis) reduces liver fibrosis in rats induced by bile duct ligation, and epigallocatechin gallate (EGCG), the major component in green tea, was implicated as the main active ingredient. EGCG has been reported to suppress cell proliferation and collagen production in HSC. In addition, the hepatoprotective effects of green tea against carbon tetrachloride, cholestasis and alcohol induced liver fibrosis were reported in many studies[13,15,16]. However, the hepatoprotective effect of green tea in dimethylnitrosamine (DMN)-induced models has not been studied. The DMN-induced liver fibrosis model can reproduce most of the features observed during human liver fibrosis. Furthermore, this model has other advantages such as progressive and remarkable pathological alterations, a high fibrosis reproduction rate, and a low mortality rate in experimental animals. This model is also stable even after termination of DMN administration and is a reliable tool for screening antifibrotic agents. Therefore, the aim of the present study was to examine the protective effect of green tea extract (GT) on hepatic fibrosis in a rat HSC line and in a rat model of DMN-induced hepatic fibrosis.
MATERIALS AND METHODS
Preparation of GT
Green tea, cultivated from Cheju island, Korea, was extracted with 80% methanol and freeze-dried.
In vitro experiment
Cell culture: HSC-T6 cells, an immortalized rat HSC line, were cultured in Dulbecco’s minimal essential medium (DMEM, Gibco, Grand Island, NY, USA) supplemented with 10% FBS (Gibco) and 0.5% antibiotics. Cultures were placed in a humidified atmosphere of 5% CO2 at 37℃, and the medium was changed twice a week. Acetaldehyde (175 mmol/L) was added to induce collagen type 1 and morphological features of activated stellate cell.
Cell viability: HSC-T6 cells were seeded into 96-well plates at a density of 1.5 × 104 cells/well until 50% confluence. Cells treated with GT (10, 50, 100 m