基于肝脏卵圆细胞分化取向探讨绞股蓝总苷抗肝纤维化效应机制研究

Based on the results of the anti-fibrotic mechanism of Gypenosides (GPs) mainly through inhibiting differentiation into myofibroblast (MFB)from Hepatic Oval Cell (HOC) in progressive fibrosis, and the research progresses that Hedgehog signaling pathway play an important role in the process of repair and reconstruction of liver damage, a hypothesis “ GPs can inhibit differentiation into MFB from HOC may through inhibiting Hedgehog signaling pathway” is proposed. We propose a meaningful research project as follows: 2-Acetylaminofluorene/Carbon Tetrachloride-Induced fibrosis model is employed, by setting a Hedgehog signaling pathway inhibitor as control; TGF-β1 induced MFB from HOC is employed in vitro, and transfected Smo interfering or overexpressing lentiviral vector. To investigate the expression of the related proteins and mRNA of Hedgehog signaling pathway. Our results will clarify the mechanism of GPs on regulating the differentiation into MFB from HOC. To provide new ideas for the prevention and treatment of liver fibrosis. This project will provide experimental basis for analyzing the anti-fibrotic mechanism of effective compound of Chinese medicine and it’s clinical transformation and application.

基于绞股蓝总苷（GPs）抗肝纤维化的效应可能主要与抑制肝纤维化进展过程中肝脏卵圆细胞（HOC）向肌成纤维细胞（MFB）分化有关的前期研究结果，结合Hedgehog信号通路激活在肝损伤修复与重建过程中的重要作用及相关研究进展，提出并论证“抑制Hedgehog信号通路可能是GPs抑制HOC向MFB分化的主要效应机制之一”的科学假说。通过①建立二乙酰氨基芴/四氯化碳大鼠肝纤维化模型；②体外采用TGF-β1诱导原代HOC分化为MFB的细胞模型，以Hedgehog信号通路抑制剂为对照、体外细胞转染Smo干扰慢病毒质粒或Smo过表达慢病毒质粒；观测GPs抑制HOC向MFB分化过程中Hedgehog信号通路相关蛋白及其mRNA表达的变化，以期解析GPs抑制HOC向MFB分化的部分效应机制。为肝纤维化的防治提供新思路；为解析中药有效组分抗肝纤维化的效应机制及临床转化应用提供实验依据。

肝纤维化是严重危害人民健康和消耗社会资源的难治性疾病。迄今临床上仍缺乏直接针对纤维结缔组织增生的有效治疗肝纤维化的生物或化学药物。研究肝纤维化的发病机制和研发抗肝纤维化药物是慢性肝病的重要治疗环节。本项目通过体内采用2-AAF联合CCl4诱导的大鼠肝纤维化模型，分别予以绞股蓝总皂苷和扶正化瘀方干预，并以Gli1的小分子抑制剂GANT61为对照，观察不同处理因素对大鼠血液生化、肝组织病理、肝组织羟脯氨酸含量、肝脏卵圆细胞活化、增殖、分化以及Hedgehog信号通路的影响；并结合体外采用丁酸钠诱导肝脏卵圆细胞WB-F344向胆管上皮细胞分化模型，予以GANT61或干扰慢病毒抑制细胞Gli1的表达，或予以绞股蓝总皂苷和扶正化瘀方干预。结果显示在肝纤维化进展阶段，肝脏卵圆细胞的活化、增殖后主要向胆管上皮细胞分化发生胆管反应而促进肝纤维化的进展；而Gli1可介导肝脏卵圆细胞发生胆管反应促进肝纤维化的进展。绞股蓝总皂苷通过抑制肝脏卵圆细胞活化、增殖，并抑制其向胆管上皮细胞分化发生胆管反应而发挥抗肝纤维化作用，其作用机制可能与抑制Hh信号通路活化有关。此研究的阐明为临床治疗肝纤维化提供新靶标；为中药有效组分抗肝纤维化提供现代分子生物学的部分基础依据，推动中医药抗肝纤维化研究的发展,也为临床药物开发新用途奠定良好的科学基础。