Hif-1调控自噬影响肝星状细胞脂滴降解和活化的机制研究

Liver fibrosis is a very important pathological process of different chronic liver diseases, and hepatic stellate cells (HSCs) are the main sources of extracellular matrix (ECM). In quiescent state, HSCs are the main lipid droplet storing cells. Once activated, the lipid droplet contracts, and even disappears. In addition, the skeleton protein of HSCs undergo rearrangement, with the upregulation of ability of proliferation, mobility and contraction of HSCs, finally resulting in enhanced secretion of cytokines, chemokines and ECM. We have previously discovered that hif-1 plays a key role in the activation of HSC during hepatic injuries and hypoxia microenvironment and hif-1 regulated mechanism might be related with autophagy. Thus, we raise a hypothesis that as a transcriptional regulator, hif-1 might target on some autophagy-related genes and start HSC autophagy activity which induces the decrease and disappearance of lipid droplet, therefore, promoting the progression of liver fibrosis. Based on these observations, we plan to target HSC, the main executor of hepatic fibrosis, as studying object, to investigate the mechanism of hif-1 regulated HSC autophagy which affects the metabolism of lipid droplet in HSC and its activation, in order to understand the mechanism of HSCs activation, development of liver fibrosis in depth and search for new target and new strategy for treatment of hepatic fibrosis.

肝纤维化是各种慢性肝脏疾病的重要病理特征，HSC是肝纤维化进展中生成细胞外基质的主要执行细胞。静息状态的HSC富含脂滴，脂滴缩小、甚至丧失，是HSC特征性的活化标志，活化后细胞内骨架蛋白重排，细胞增殖、迁移、收缩等功能增强，分泌大量细胞因子、趋化因子和ECM等。我们前期研究发现缺氧诱导因子-1在肝脏受损及继发性缺氧条件下HSC的活化中发挥重要的调控作用，其机制可能与自噬相关。我们推测，处于肝脏局部微环境中的HSC在肝脏受损、继发性缺氧或其他应激因素刺激时，可能活化Hif-1调节机制，Hif-1作为转录调控因子，靶向某些自噬相关基因，启动自噬，造成脂滴丢失、细胞活化，推动肝纤维化的发生发展。本课题以参与肝纤维化的主要执行细胞肝星状细胞为研究对象，探索Hif-1调控自噬影响HSC脂滴降解和细胞活化的机制，以深入理解肝星状细胞的活化机制、肝纤维化的发生发展机制、寻找治疗肝纤维化的新靶点新策略。

本课题基于缺氧诱导因子Hif-1是肝损伤时调节肝星状细胞活化导致肝纤维化发生发展的关键调控因子这一发现，在细胞模型和动物模型上研究肝星状细胞（HSC）中Hif-1信号途径调控HSC活化的机制。本研究初步筛选了肝星状细胞内受Hif-1调控的分子，如与自噬有关的Bnip3蛋白、与表观遗传-组蛋白甲基化修饰有关的OGT蛋白、与细胞代谢有关的GLUT1蛋白等；阐明了肝星状细胞活化过程中Hif-1转录复合体发生组蛋白甲基化修饰，该表观遗传学机制在Hif-1入核、肝星状细胞自噬及活化等细胞活动中具有重要作用；建立原代肝星状细胞分离和体外培养自然活化的细胞模型，在该模型上明确了肝星状细胞活化过程中Hif-1及其调控的靶分子-自噬相关蛋白Bnip3（Bcl-2/E1B-19kDa 相互作用蛋白3）表达升高，并伴随细胞自噬发生和活化，Hif-1通过靶分子Bnip3调节肝星状细胞自噬发生和活化；发现肝星状细胞活化过程中外泌体分泌增多，且外泌体分泌与肝星状细胞活化相关，活化的原代肝星状细胞分泌的外泌体中包含Hif-1靶分子-GLUT1（葡萄糖转运蛋白-1）和PKM2（M2型丙酮酸激酶），Hif-1、GLUT1及PKM2是参与葡萄糖有氧糖酵解，即Warburg effect（瓦尔堡效应）的关键调控分子，提示了外泌体这一细胞通讯方式在肝星状细胞代谢及活化中的作用，以及肝星状细胞外泌体在肝脏内传递信息的可能作用。进一步在小鼠肝损伤模型上通过干预外泌体分泌、糖酵解发生等Hif-1调控的细胞生物学活动，观察肝脏病理学改变和肝星状细胞活化指标。该课题研究了Hif-1对肝星状细胞自噬、外泌体分泌和活化等多个细胞生物学活动的影响和机制，可帮助深入理解肝星状细胞活化、肝纤维化发生发展机制、为寻找治疗肝纤维化的新靶点提供新策略。