Effects Of PPAR-γ Ligand Rosiglitazone On Expression Of MMPs, TIMPs And Collagens In Hepatic Stellate Cells

Objective:Hepatic fibrosis (HF) is a pathologic change after many forms of chronic hepatic injury, it can proceed to hepatic cirrhosis and even hepatocellular carcinoma , and it's a severe disease with a strong impact on human health. As we know, compared with hepatic cirrhosis, HF is reversible. Activated hepatic stellate cell (HSC) is crucial in the process of HF. Activated HSC could produce excessive extracellular matrixes (ECM) and numerous tissue inhibitors of metalloprot -einases (TIMPs),which can inhibit the activity of matrix metalloproteinases (MMPs). The decrease of ECM degradation could lead to the disequilibrium of ECM production and degradation, eventually induce the formation of hepatic fibrosis. Peroxisome proliferators activated receptor gamma (PPAR-γ) is a transcription factor of nuclear receptor, which is activatd by ligands.The relation between PPAR-γand activation of HSC is the focal question in researches on PPAR-γand HF. Some researches indicated that: quiescent HSC expressed PPAR-γ, but its expression in activated HSC is obviously diminished. The expression of PPAR-γmight play an important role on the maintenance of quiescent HSC, the reduction of PPAR-γexpre -ssion is closely correlated with the activation of HSC. The functions of PPAR-γand its ligand are hotspot on hepatic fibrogenesis , but the mechanism isn't clearly understood. It is now considered that: as excitomotor, PPAR-γligand inhibits HSC proliferation , reduces collagen deposition and increases collagenase activity in liver. The goal of this study was to evaluate the effects of different levels of PPAR-γligand rosiglitazone on expression of MMPs, TIMPs and collagens in HSC , and to investingate the regulation of PPAR-γon HSC biologic characterristics.Methods :①Cell culture: HSC-T6 was cultivated by Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum(FBS).After passaged 4-5 generations,HSCs were cultivated in plastic culture flask by DMEM without FBS. After adherencing , cells were randomly divided into 5 groups: A: normal control group; B: 5μmol/L rosiglitazone group; C: 10μmol/L rosiglitazone group; D:15μmol/L rosiglitazone group; E: 20μmol/L rosiglitazone group. After cultivated 24h, cells were collected in order. Each group has six flasks respectively.②Detecting the levels of MMPs,TIMPs and collagens in each group : Total RNA was isolated using TRI-Reagent , following the protocol provided by the manufacturer .The expression levels of TIMP-1, TIMP-2, MMP-2, MMP9, COLⅠ and COLⅢwere measured by semi-quantitative RT-PCR.The images of agarose electrophoresis were analyzed by Quantity One system.③Statistical analysis: The results were analyzed by variance analysis with SPSS10.0, p<0.01 suggested that the difference was statistically significant.Results:The results demonstrated that TIMP-1: A > B > C > D > E; TIMP-2: A > B > C > D > E; COLⅠ: A > B > C > D > E; COLⅢ: A > B > C > D > E; MMP-2: A < B < C < D < E; MMP-9: A < B < C < D < E. Rosiglitazone, a ligand of PPAR-γ, increased the activity of MMP-2 and MMP-9 in HSC, decreased the expression levels of TIMP-1, TIPM-2, COLⅠand COLⅢ(P<0.01), and the effects were dose-dependent in certain extent(P<0.01).Conclusion:By activating PPAR-γ, rosiglitazone could inhibit HSC proliferation, activation and induce HSC apoptosis. The decrease of TIMPs expression could lead to the increase of MMPs expression and ECM degradation, thus HF may be reversed. The regulation of PPAR-γfor activated HSC is responsible for the process of hepatic fibrogenesis, and PPAR-γligand may be as a potential candidate for the treatment of HF.