柔肝方经FBRS/PI3K/Hedgehog信号通路调控肝星状细胞骨桥蛋白表达抗肝纤维化作用机制研究

Activation of resident hepatic stellate cells(HSC) and then more extracellular matrix production is the central part of hepatic fibrosis. Osteopontin(OPN)，synthesed by HSC, maybe regulated by the PI3K/AKT/Hedgehog signal pathway，can induce the activation of HSC. Our previous studies showed that Fibrosin(FBRS)upregulates the expression of OPN,RouGan formula played a role in anti-hepatic fibrosis by downregulating the synthesis of OPN, its mechanism maybe based on the inhibition of PI3K/AKT signal. To this end, we propose the hypothesis: The PI3K/AKT/Hedgehog signal may upregulate the expression of OPN, leading the development of liver fibrosis; while RouGan formula can prevent its process by inhibiting the PI3K/AKT/Hedgehog signal, downregulating the expression of OPN and inhibiting the activation of HSC. In This study, we apply hepatic fibrosis mouse model induced by ConA in vivo and normal mice primary HSC and HSC cell line in vitro. Beginning with some key molecules on FBRS/PI3K/Hedgehog signal pathway, recombinant Shh, Shh ShRNA, Hhip and RouGan formula were used. We also use RT-PCR, Westernblot, ELISA, histopathology, immunofluorescence and some more experimental methods, To confirm whether the mechanism of Rougan formula on anti-hepatic fibrosis is through inhibiting the activation of the FBRS/PI3K/Hedgehog signal pathway and then downregulating the OPN expression and HSC activation. This study will in further elucidate the modern biological mechanism of Chinese herbs on anti-hepatic fibrosis.

肝星状细胞（HSC）活化并合成大量细胞外基质是肝纤维化的核心，骨桥蛋白（OPN）可由HSC合成并促进其活化；PI3K/AKT/Hedgehog(Hh)信号通路可能参与了该过程。前期研究证实FBRS可上调OPN表达，促进肝纤维化；柔肝方可抑制PI3K/AKT信号而下调OPN表达。由此提出假说：柔肝方通过干预FBRS/PI3K/Hh信号通路，下调OPN表达、抑制HSC活化而抗肝纤维化。本研究拟采用ConA诱导的肝纤维化小鼠模型、正常小鼠原代HSC及HSC细胞株，从影响FBRS/PI3K/Hh信号通路若干关键分子入手，用重组Wortmannin、Shh、Shh ShRNA、Hhip及柔肝方进行干预，通过RT-PCR、Westernblot、ELISA、组织病理、免疫荧光染色等方法，分析FBRS/PI3K/Hh信号通路对OPN的调控作用及柔肝方的干预机制，为阐释柔肝方的分子药理作用提供科学依据。

肝星状细胞（HSC）活化并合成大量细胞外基质是肝纤维化的核心，前期研究证实FBRS、OPN蛋白是肝纤维化形成过程中的关键蛋白，PI3K/AKT/Hedgehog信号通路可能参与了肝纤维化的形成。本研究通过尾静脉注射刀豆蛋白A构建肝纤维化小鼠模型、FBRS刺激星状细胞株LX-2，从影响FBRS/PI3K/Hh信号通路若干关键分子入手并予柔肝方进行干预，通过RT-PCR、Westernblot、组织病理、免疫荧光染色等方法，分析FBRS/PI3K/Hh信号通路对OPN的调控作用及柔肝方的干预机制。研究结果显示：与模型组相比，柔肝组小鼠脾脏重量、脾脏指数、ALT、AST及Hyp水平明显下降（P<0.01）；病理染色可见柔肝组小鼠肝组织炎症、胶原沉积显著改善；柔肝组小鼠FBRS、α-SMA、TGFβ1、Cols、MMPs、TIMPs表达以明显下调（P<0.05或P<0.01）；FBRS促进LX-2细胞活化，具有时间和剂量依赖性；FBRS可诱导LX-2细胞p38、ERK及PI3K/AKT/Hedgehog信号通路活化；柔肝方抗肝纤维化作用与抑制FBRS/PI3K/Hedgehog信号通路相关。本项目资助发表核心论文2篇，待发表SCI论文1篇。培养博士生1名，已毕业；培养硕士生3名，其中1已经取得硕士学位，2名在读。项目投入经费20万元，支出13.8166万元，各项支出基本与预算相符。剩余经费6.1834万元，剩余经费计划用于本项目研究后续支出。