miR-378靶向TGF-β1信号通路抑制肝星状细胞活化的抗肝纤维化作用研究及黄芪汤的干预

TGF-β1 and hepatic stellate cells (HSC) activation play important role in the progress of liver fibrosis. Our previous results showed: miR-378 was down-regulated in fibrotic liver and the serum of cirrhosis patients, indicating it may has relationship with liver fibrosis; the Chinese formula- HuangQi decoction has effect on liver fibrosis through regulating TGF-β1 signaling. This study will focus on the main line: “miR-378/target gene: CREBBP, FZR1/TGF-β1 signaling/HSC activation/liver fibrosis”, (1) Rat liver fibrosis model and HSC activation model will be established in vivo and in vitro respectively, and intervened by miR-378 agomir or antagomir, the dynamic changes of related parameters in the main line will be observed; (2) The above in vivo and in vitro model will be established and intervened by HuangQi decoction, miR-378 agomir will be used as positive control, the expression of miR-378, TGF-β1 signaling, HSC activation and liver fibrosis will be analyzed. This study will verify the hyposis: (1) miR-378 plays an important role in the development of liver fibrosis through targeting TGF-β1 signaling and inhibiting HSC activation; (2) Up-regulating miR-378 expression, regulating TGF-β1 signaling, thus inhibiting HSC activation may be the mechanism of action of HuangQi decoction against liver fibrosis. This study will illuminate the effects of miRNA during liver fibrogenesis, provide basis for finding diagnosis marker and treatment target of liver fibrosis.

基于TGF-β1/肝星状细胞（HSC）活化在肝纤维化中的关键作用，结合miR-378及黄芪汤的前期结果，本课题拟围绕“miR-378/靶基因/TGF-β1通路/HSC活化/肝纤维化”这一主线，①制备肝纤维化动物模型及HSC活化细胞模型，以miR-378模拟物/抑制剂使其过表达/低表达，动态观察该主线相关指标变化，并对miR-378的靶基因进行验证；②采用上述体内体外模型，黄芪汤干预，miR-378模拟物为对照，观察药物对上述主线相关指标的影响。验证以下假说：①miR-378通过下调靶基因表达影响TGF-β1信号通路抑制HSC活化参与了肝纤维化的发生发展；②上调miR-378影响TGF-β1通路从而抑制HSC活化是黄芪汤抗肝纤维化的重要作用机制。该工作将部分阐明miRNA在肝纤维化中的调控作用及中药复方的干预，为探索肝纤维化发病机制、寻找肝纤维化分子诊断靶标及治疗靶点提供基础。

MicroRNA(miRNA)在肝纤维化的发生发展中发挥重要作用，前期miRNA芯片分析发现miR-378在CCl4诱导的小鼠纤维化肝组织及肝硬化患者血清中均下调，提示其对肝纤维化的保护作用。本研究旨在进一步研究miR-378在肝纤维化形成中的作用并探讨其作用机制；同时探讨抗肝纤维化中药复方黄芪汤调节miR-378及其相关通路的抗肝纤维化作用机制。首先体内复制四氯化碳(CCl4)染毒小鼠肝纤维化模型，以miR-378激动剂干预，体外采用人肝星状细胞LX-2，以miR-378模拟物或抑制剂转染细胞，分别使其过表达或低表达，观察miR-378对肝纤维化及肝星状细胞活化的影响，结果发现miR-378对CCl4诱导的小鼠肝纤维化具有抑制作用，其作用机制为抑制其靶基因生长因子受体结合蛋白2（Grb-2）表达，调节Grb-2/PI3K/AKT信号通路，下调TGF-β1通路，从而抑制肝星状细胞活化。继之，体内复制CCl4染毒小鼠肝纤维化模型，体外采用LX-2细胞，黄芪汤干预，探讨黄芪汤抗肝纤维化的作用及其作用机制，结果发现黄芪汤对CCl4诱导的小鼠肝纤维化具有抑制作用，其作用机制可能与上调抗纤维化miRNA-miR-378表达，抑制Grb-2/PI3K/Akt通路，从而抑制HSC活化相关。该工作的完成部分阐明miRNAs在肝纤维化中的调控作用，为探索肝纤维化发病机制、寻找肝纤维化分子诊断靶标及治疗靶点提供基础；同时本课题研究发现调节miRNA表达是中药复方防治肝纤维化的重要作用环节，为肝纤维化的治疗研究提供新的依据。本课题已发表论文6篇，其中SCI期刊收录2篇，待发表论文2篇；参加国际国内学术会议交流5次；协助培养博士1名、硕士2名。