Effects of Shugan granule on hepatic stellate cells activation and its trans-membrane signal transduction stimulated by transforming growth factor beta 1 / 中国组织工程研究

ABSTRACT

BACKGROUND: Hepatic fibrosis is a reversible disease, interfering in course of disease promptly can decrease hepatic cirrhosis and fatal complication. Hepatic stellate cells (HSCs) is a key factor in pathogenesy of hepatic fibrosis.OBJECTIVE: To investigate the effects of Shugan granule on HSCs activation and trans-membrane signal transduction stimulated by transforming growth factor beta 1 (TGF-β1) in rats, and to explore the anti-fibrosis mechanism.DESIGN, TIME AND SETTING: Controlled observational trials based on cytology were performed in the Central Laboratory of Molecules, Luzhou Medical College between June 2008 and February 2009. MATERIALS: HSC-T6 cell line was purchased from Institute of Liver Diseases, Shanghai University of Traditional Chinese Medicine, its phenotype was the activated hepatic stellate cells. Shugan granule was offered by Drug Institute, Affiliated Hospital of Luzhou Medical College at a Batch No. 20071120.METHODS: The influence of different concentrations (0.01, 0.02, 0.04, 0.08 g/L) of Shugan granule on HSCs proliferation was determined by MTT. 0.01 g/L was defined as the dose of Shugan granule contributing no influence on cell proliferation. HSCs were cultured in a culture plate and then divided into 4 groups: control group without management, TGF-β1 group with 5 μg/L TGF-β1 solution in culture medium, Shugan granule group with 0.01 g/L Shugan granule in a culture medium, and TGF-β1 + Shugan granule group with 5 μg/L TGF-β1 solution and 0.01 g/L Shugan granule in culture medium. MAIN OUTCOME MEASURES: Morphological features of HSCs were detected by microscopic. Immunohistochemical method was used to detect the expression of Smad3 and Smad7 in HSCs. RT-PCR was applied to observe the HSCs activation and trans-membrane signal transduction. RESULTS: ①The cell morphology of TGF-β1 group was similar with that in the control group, and the extension was more obvious. In the TGF-β1 + Shugan granule group, the cell morphology was close to that in TGF-β1 group. There was no karyopyknosis or apoptosis observed in each group. ②Immunohistochemical method showed the expression of Smad3 and Smad7 in control groups were increased. TGF-β1 could slightly increase the expression of Smad3 and Smad7 (P < 0.05), while Shugan granule group and TGF-β1 + Shugan granule group increased the expression of Smad7 significantly, accounting for 1.99 times compared with control group (P < 0.01). ③RT-PCR result showed that Shugan granule could increase the expression of Smad7 (P < 0.05), but the expression of Smad3 was not regulated. 5 μg/L TGF-β1 could up-regulate the expression of Smad3 and Smad7 (P < 0.05). In the TGF-β1 + Shugan granule group, Smad7 expression was increased by 101% (P < 0.05), but Smad3 transcriptional level was not changed(P > 0.05). CONCLUSION: ①TGF-β can stimulate the gene expression of Smad3 and Smad7, it also obtain a balance of feedback regulation mechanism between R-Smads and I-Smads. ②Shugan granule may prevent and cure hepatic fibrosis through decreasing the proliferation of HSCs in a dose-dependent manner. ③Shugan granule can inhibit the TGF-β-Smad signaling pathway through increasing the expression of Smad7.