调控Gasdermin D的去泛素化酶参与非酒精性脂肪性肝炎病程的机制研究

Pyroptosis mediated by Gasdermin D (Gsdmd) involved in the pathogenesis of non-alcoholic steatohepatitis (NASH). In our previous study, Gsdmd protein level was found up-regulated in NASH, while Gsdmd mRNA level was down-regulated. Furthermore, we found that the K48 polyubiquitination of Gsdmd which could mediate the degradation of protein through proteasomes was decreased in NASH. In addition, we found that specific knock-down of hepatocellular Gsdmd could ameliorate the severity of NASH. However, roles of deubiquitinating enzyme of Gsdmd in NASH remain unknown. According to our previous study, we hypothesize that the deubiquitinating enzyme of Gsdmd involves in the pathogenesis of NASH. In this study, we aim to identify the deubiquitinating enzyme of Gsdmd, illustrate the exact mechanisms of Caspase1-dependent deubiquitinating of Gsdmd, and investigate roles of deubiquitinating enzyme of Gsdmd in NASH. This study is projected to illustrate the exact mechanisms of NASH and find novel NASH treatment targets.

Gasdermin D（Gsdmd）介导的焦亡在非酒精性脂肪性肝炎（NASH）的发病中起到重要作用。我们前期的研究发现NASH小鼠体内Gsdmd蛋白上调而Gsdmd mRNA下调，进一步研究发现NASH小鼠体内Gsdmd的K48位泛素化水平下降，提示NASH状态下泛素-蛋白酶体途径介导的Gsdmd蛋白的降解减少；还发现Gsdmd的去泛素化过程依赖于Caspase1；靶向敲低肝细胞的Gsdmd，可以显著改善NASH。但调控Gsdmd的去泛素化酶在NASH中的作用尚不清楚。我们推测调控Gsdmd的去泛素化酶参与NASH的发病。本项目中，我们将明确调控Gsdmd的去泛素化酶，阐明调控Gsdmd的去泛素化酶依赖Caspase1的机制，研究调控Gsdmd的去泛素化酶参与NASH病程的机制，为深入理解NASH的发病机制和寻找治疗NASH新靶点提供实验础。

非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）波及全球近四分之一的成年人，在我国，NAFLD也已经成为引起慢性肝病的首要病因之一。相较于单纯性脂肪变，非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）更容易进展为纤维化、肝硬化甚至肝细胞肝癌等终末期肝病。肝细胞的损伤与死亡是NASH的重要病理特征。我们的前期研究结果表明Gasdermin D（GSDMD）介导的焦亡参与NASH的病程进展，且在NASH小鼠肝脏中，GSDMD存在泛素化调控并受caspase1的影响，但具体机制未明。本研究在MCD喂养的NASH动物模型及PA和LPS诱导的AML12细胞脂肪变模型中明确了GSDMD及其剪切体GSDMD-N的蛋白表达水平增加。通过Co-IP结合多维质谱鉴定出多个与GSDMD共作用的泛素化酶/去泛素化酶。通过进一步免疫共沉淀确定了E3泛素连接酶NEDD4与GSDMD具有相互作用。体外研究发现，AML12细胞中NEDD4的敲减可上调GSDMD的蛋白表达水平，NEDD4的过表达可降低GSDMD的蛋白表达水平，明确了NEDD4对GSDMD的蛋白表达具有调节作用。AML12细胞过表达质粒转染结合免疫共沉淀明确了NEDD4能够泛素化GSDMD，且经PA和LPS处理后GSDMD的泛素化显著降低，与动物体内实验表型一致。在MCD饮食喂养小鼠中，研究发现NEDD4随着NASH进展蛋白水平不断升高，体外AML12细胞脂肪变模型中与对照组相比，PA和LPS的刺激可诱导NEDD4的蛋白水平升高，与动物实验趋势一致。通过AML12细胞的慢病毒敲减和过表达瞬转NEDD4，结果显示经PA和LPS处理后，NEDD4的敲低可减少肝细胞脂质沉积，NEDD4的过表达可增加肝细胞脂质沉积，表明NEDD4与NASH的进展密切相关。