肠道菌群代谢产物琥珀酸通过NLRP3/GSDMD介导的肝细胞焦亡促进非酒精性脂肪性肝炎的作用及机制研究

肠道菌群失调是促进非酒精性脂肪性肝炎（NASH）进展的重要原因，菌群代谢产物是菌群发挥作用的重要机制。课题组发现：粪菌移植后，使高脂喂养的无菌小鼠（HF-GF）NASH表型较严重组所移植的粪菌样本代谢产物中，琥珀酸含量明显高于对照组；进而，发现琥珀酸促进HF-GF小鼠肝脏甘油三酯沉积、NLRP3炎症小体激活及细胞焦亡指标（GSDMD，casp-1 p20）表达。据此，我们推测琥珀酸通过激活NLRP3/GSDMD介导肝细胞焦亡促进NASH。项目拟分析临床粪便及血液样本中琥珀酸含量与NASH的相关性；在NASH动物模型中证明琥珀酸促进NASH；在动物及肝细胞系中下调NLRP3及细胞焦亡执行分子GSDMD观察琥珀酸对NASH表型及细胞焦亡的影响，同时分离小鼠原代肝细胞验证琥珀酸对肝细胞焦亡的作用，阐明琥珀酸通过NLRP3/GSDMD介导肝细胞焦亡促进NASH的机制，为NASH治疗提供新的策略。

Microbiota dysbiosis is an important etiology in non-alcoholic steatohepatitis (NASH) progression, metabolite is a major mechanism that microbiota exerts its role in NASH. In our previous studies, we found the succinate, a metabolite produced by gut microbiota, was significantly increased in fecal samples of high fat-diet (HF) mice after sitagliptin (Sit) treatment. Transplantation of fecal samples from Sit treated mice promoted NASH progression in HF-germ free (GF) mice. Then, we found hepatic triglyceride (TG) content, NLRP3 inflammasome and the biomarkers of pyroptosis, gasdermin D (GSDMD) and caspase-1 p20, were upregulated in the liver of HF-GF mice after succinate treatment. Previous studies have shown that pyroptosis is a type of programmed cell death with inflammatory response. Pyroptosis has been reported to be regulated by NLRP3 inflammasome activation and play an important role in NASH progression. Therefore, we speculate that succinate promotes NASH by regulating NLRP3/GSDMD-mediated hepatocyte pyroptosis. .In this study, we will detect succinate content in human fecal and serum specimens, and analysis the relevance of succinate content and NASH progression. Then, we will investigate the promoting effect of succinate in NASH using experimentally-induced steatohepatitis mouse model (methionine and choline deficient diet-induced NASH mouse model, MCD mice). Moreover, we will explore the potential mechanism of this effect by combining with succinate treatment and downregulation of NLRP3 or GSDMD in HepG2 liver cells and MCD mice, meanwhile, detect the pyroptosis process. Finally, we will also observe the alteration of pyroptosis process in mouse primary hepatocytes separated from mice mentioned above to further explicit the role of succinate in primary hepatocyte pyroptosis. These results could provide a new insight into the potential treatment of NASH.