lncRNA-GAS5/NLRP3/Caspase-1途径介导的肝细胞焦亡在肝纤维化中的作用及其机制研究

Liver injury, as a key event in liver fibrosis, has been reported to induce hepatocyte pyroptosis. However, whether long non-coding RNA (lncRNA) can regulate hepatocyte pyroptosis to control the progression of liver fibrosis during liver fibrosis is still not clear. Previously, the screening results of Chip showed that lncRNA-GAS5 was obviously down-regulated in hepatocytes in mouse liver fibrosis model. In vitro, our results showed that GAS5 inhibited cell apoptosis in hepatocytes and was associated with Caspase-1. Bioinformatic analysis suggested an interaction between GAS5 and NLRP3 inflammasome. In addition, there was a relation between GAS5 expression and methylation. These data suggest a key role of GAS5 in regulating hepatocyte pyroptosis. Currently, there is no report about GAS5-mediated hepatocyte pyroptosis. Based on these, we raised up a hypothesis that epigenetically-regulated GAS5 plays a role in hepatocyte pyroptosis via NLRP3/Caspase-1 pathway, contributing to the regulation in the activation of HSCs and the progression of liver fibrosis. Our study will confirm the hypothesis through many factors such as molecules, cells and animals. In this study, we will confirm the role of GAS5 in liver fibrosis, the significance of GAS5/NLRP3/Caspase-1 signalling in hepatocyte pyroptosis as well as the association between GAS5-mediated hepatocyte pyroptosis and activation of HSCs. This study explores the mechanisms of liver fibrosis from the new perspective of GAS5-mediated hepatocyte pyroptosis, and provides new ideas for the prevention and treatment of liver fibrosis.

肝损伤是肝纤维化中的重大事件，其会引起肝细胞焦亡。但肝纤维化时lncRNA能否调控肝细胞焦亡影响纤维化尚不清楚。前期芯片结果表明小鼠肝纤维化模型中lncRNA-GAS5在肝细胞内明显下调；体外实验表明GAS5抑制肝细胞凋亡且与Caspase-1有关；生物信息学预测GAS5与NLRP3炎症小体相互作用及GAS5表达与甲基化有关。结果提示GAS5在肝细胞焦亡中起重要作用。目前尚未见其相关研究报道，就此我们提出假说：甲基化修饰的GAS5经NLRP3/Caspase-1影响肝细胞焦亡，进而调控肝星状细胞活化及肝纤维化进程。本课题拟从分子、细胞及动物水平等方面明确GAS5在肝纤维化中作用；揭示GAS5/NLRP3/Caspase-1通路在肝细胞焦亡中的意义；明确GAS5介导的肝细胞焦亡对肝星状细胞活性的影响。本研究从GAS5介导肝细胞焦亡这一新角度探讨肝纤维化的发生机制，为肝纤维化的防治提供新思路。

肝损伤是肝纤维化中的重大事件，其会引起肝细胞焦亡。本研究通过检测肝细胞焦亡指标、肝星状细胞活化指标如α-SMA及COL1A1的表达、细胞增殖检测、细胞周期检测、GAS5下游靶基因预测和确证、以及GAS5启动子甲基化检测等方式评估GAS5在肝细胞焦亡中的生物学功能以及对肝星状细胞活化的抑制作用。本研究首次明确了GAS5在肝细胞焦亡中的作用及意义，证实了GAS5能通过抑制肝细胞焦亡进而改善肝纤维化进程的新机制，阐明了GAS5不仅是肝细胞焦亡负性调控因子，也是肝纤维化的抑制因子。在CCl4诱导形成的小鼠肝纤维化模型中，GAS5有效抑制肝纤维化进程且下调了肝细胞焦亡水平。在体外原代肝细胞中，GAS5抑制肝细胞焦亡水平；同时将GAS5处理后的肝细胞与肝星状细胞共培养，可有效地抑制肝星状细胞EMT进程及其活性。GAS5显著下调了肝细胞焦亡，这一抑制机制与NLRP3蛋白的下调存在密切关系。肝细胞焦亡水平的下降可通过抑制EMT进程调节肝星状细胞活性。GAS5在肝纤维化期间的表达水平下降被证实与其启动子存在高甲基化有关。此外，本研究还发现血清GAS5是一个不错的肝纤维化标记物，可有效区分纤维化患者和正常人群。本项目还发现了Liquiritigenin等中草药的抗纤维化新机制以及包含CircMTO1在内的关键纤维化分子。. 在国自然基金的资助下，以第一作者或通讯作者身份发表SCI 14篇，累计IF>100分（平均IF>7.2分，单篇最高IF: 20.693），以第一发明人获授国内专利11项（其中转化1项发明专利）。