肠肝轴：从临床患者分离的肠道致病菌株Escherichia coli NF73-1对非酒精性脂肪性肝病的作用及机制研究

Gut microbiota is involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). However, current researches are limited to phylum and species level. Few research focus on the clinically isolated pathogenic bacterial strains. It is vital to find specific pathogenic strains which is involved in NAFLD. So we isolated Escherichia coli strains from a patient with nonalcoholic steatohepatitis and then identified the most pathogenic strain with the most abundancy Escherichia coli NF73-1. Whole genome sequence indicated components of Type VI Secretion System (T6SS) in genetic annotations. Our research will focus on how NF73-1 disrupts intestinal barrier function, facilitates hepatic inflammatory responses, and aggravates NAFLD. Besides, we will also study how NF73-1 directly cause inflammatory responses to hepatocytes. Furthermore, we will explore how NF73-1 contributes to NAFLD through T6SS function. Our research will provide important clues to identify specific pathogenic strain that will assist in diagnosis and prediction for disease activity score of NAFLD. Besides, evidence is also provided for therapies targeting pathogenic bacterial strain upon our research.

研究表明肠道微生态（尤其肠道菌群）改变参与了非酒精性脂肪肝（NAFLD）的发生和发展，但目前研究大多局限于门及属水平。对于肠道特定、致病菌株的研究仍然匮乏。寻找与NAFLD相关的关键致病菌株是亟待解决的重要问题。我们从一名非酒精性脂肪性肝炎（NASH）患者肠黏膜中分离出大肠杆菌菌株，通过前期研究筛选出致病性最强且丰度最高的致病菌株Escherichia coli NF73-1，并进行基因组测序发现其存在重要的致病因素——VI型分泌系统（T6SS）。本研究着重探讨NF73-1如何通过破坏肠黏膜屏障功能、引起肝脏炎症反应从而加重NAFLD进展；是否可以直接对肝细胞造成损伤；深入研究NF73-1是否通过T6SS功能参与NAFLD的发生发展。该研究为将来明确特定致病菌株用于临床协助诊断及预测NAFLD的疾病活动程度提供了重要的线索，并为通过靶向干预致病菌株治疗NAFLD提供了坚实的依据。

肠道微生态紊乱是非酒精性脂肪性肝病（NAFLD）的重要致病因素之一，既往研究多从“门”“属”的水平进行分析，而对 “种”“株”水平的研究不足，故无法揭示真正关键的致病菌。我们课题组前期发现了NAFLD相关的重要致病菌株E. coli NF73-1，本研究将深入探讨该菌促进NAFLD进展的机制。结果发现：1）NF73-1可引起NAFLD小鼠肠黏膜屏障功能受损，从受损的肠黏膜移位至肝脏，导致肝脏炎症及脂质沉积，加重NAFLD。2）NF73-1移位至肝脏后可激活TLR2-NF-κB/NLRP3-Caspase-1诱导产生促炎性M1型巨噬细胞，并与M1型巨噬细胞协同调控肝细胞mTOR-S6K1-SREBP-1/PPAR-α信号通路，促进肝细胞的甘油三酯合成，抑制其氧化分解，从而导致肝细胞内脂质沉积。3）T6SS基因簇是NF73-1的毒力基因，敲除T6SS基因簇的NF73-1变异菌株导致NAFLD发展的能力减弱。4）在完成既定研究内容的前提下，课题组还发现肠血管屏障受损是NAFLD发生的早期事件；NF73-1可能通过加重肠血管屏障损伤，介导NAFLD的发生发展。综上所述，E. coli NF73-1可破坏肠黏膜屏障，移位至肝脏促进肝脏炎症及脂质沉积；其毒力与T6SS有关，敲除该基因后E. coli NF73-1的致病力减弱。本研究证实了E. coli NF73-1是导致NAFLD进展的重要致病菌株。研究结果为靶向NF73-1菌株的NAFLD临床诊断和治疗提供了坚实的理论依据。在本项目资助下，我们发表了7篇期刊论文、6篇会议论文（发言3次，壁报交流3次），获国家发明专利1项（2020年，ZL201610591293.4），培养博士研究生3名。同时，我们应用获得授权的专利菌——E. coli NF73-1，在NAFLD患者无创诊断和NAFLD不同疾病进程鉴别过程中已经取得了重要突破，已逐步开展临床无创诊断。