异位表达于肝细胞的Tim-4负调控NLRP3炎性体参与NAFLD的作用研究

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease, and molecular mechanism underlying the pathogenesis of NAFLD remains unclear so far. NLRP3 inflammasome and autophagy activations are the critical steps for NALFD progression. Recently, proteomic analysises reveal that multiple Tim-4 SNPs are associated with lowered LDL and triglycerides. Enhanced Tim-4 mRNA is found in rat liver tissues with fatty liver disease induced by high fat diet (HFD). However, no report is found whether Tim-4 participates in NAFLD pathogenesis. In the previous study, we found that Tim-4 gene knockout mice showed impaired glucose tolerance and insulin sensitivity. Tim-4 expression was increased in hepatocytes with adipose degeneration, which had beed verified in HFD induced fatty liver disease of mice as well as HepG2 cell model induced by oleic acid (OA). In addition, Tim-4 overexpression pretected HepG2 cells from adipose degeneration induced by OA stimulation. Tim-4 suppressed NLRP3 inflammasome activation, but promoted AMPKα activation which is a main way triggering autophagy. Based on above foundings, we put forward the hypothesis of this proposal: Tim-4 expressed in hepatocytes might involve in lipid metabolism and fatty liver disease by negatively regulating NLRP3 inflammasome activation. This study aims to identify the key role of Tim-4 in the development of NAFLD and related mechanisms, and further explore the regulation mechanism of Tim-4 expression in hepatocytes induced by high fat microenviroments. Hepatocyte-specific Tim-4 knockout mice, which have been developed successfully, and more models would be used to address the hypothesis. This study would unveil the novel function of Tim-4 in regulating lipid metabolism on the one hand. On the other hand, this study might provide new theory for NALFD pathogenesis, provide new target for NAFLD intervention.

非酒精性脂肪肝（NAFLD）是最常见慢性肝病，发病机制尚未阐明。NLRP3炎性体活化及自噬通路激活是新近被揭示的NAFLD进展关键环节。近期组学分析显示，Tim-4多个SNP位点与脂代谢相关，大鼠脂肪肝组织Tim-4表达上调，但Tim-4是否参与NAFLD未见报道。申请者前期发现，Tim-4敲除小鼠出现糖耐量及胰岛素敏感性异常，高脂诱导肝细胞表达Tim-4，Tim-4过表达抑制肝细胞脂肪变及NLRP3炎性体活化，并激活自噬通路激酶AMPKα，进而提出Tim-4可能通过AMPKα通路激活自噬抑制NLRP3炎性体调节脂代谢及脂肪肝进程的假说。本项目拟利用肝脏特异性Tim-4基因敲除小鼠及多种模型，明确肝细胞表达Tim-4在NAFLD中的作用及分子机制，阐明高脂微环境对Tim-4表达的调控机制。本研究将揭示Tim-4调节脂代谢的新型生物学功能，为NAFLD致病机制提供新学说，为其干预提供新靶点。

NAFLD是世界上最常见的慢性肝病，将成为肝癌增加的最主要原因，是21世纪肝病领域面临的新挑战。NAFLD的发病机制复杂，明确调节自噬及NLRP3炎性体活化的关键靶点有望为早期干预NAFLD提供新策略。通过本项目研究，发现Tim-4敲除可加重MCD诱导的脂肪肝进程，HFD和MCD诱导的NAFLD小鼠模型肝组织中Tim-4与LKB1、AMPKα的共定位明显增强。利用人、小鼠脂肪肝组织，证实Tim-4在脂肪肝组织中表达水平显著高于正常肝组织，HFD或MCD诱导的脂肪肝组织匀浆上清均可刺激Tim-4的表达。Tim-4过表达可显著抑制肝细胞脂肪变性，而干扰Tim-4则可加重肝细胞脂肪变性。利用细胞模型证实Tim-4可抑制NLRP3炎性体的活化，IgV结构域在Tim-4抑制NLRP3炎性体活化中发挥关键作用。另外发现，Tim-4可抑制炎性体各组分的表达及IL-1β的活化，Tim-4过表达可促进AMPK磷酸化、激活自噬，证实Tim-4通过LKB1途径促进AMPK磷酸化，该作用依赖于PS结合位点。通过该研究，揭示了Tim-4调节NLRP3炎性体活化参与脂代谢的新型生物学功能，初步阐明了其在脂肪肝发生中的作用机制，为早期干预NAFLD提供新靶点，具有重要的理论研究价值及潜在的临床应用前景。.通过拓展研究，探索了Tim-4调控巨噬细胞线粒体代谢在肥胖中的作用并获国家自然科学基金面上项目1项、探索sTim-4在肺癌转移中的作用并获山东省自然科学基金联合基金重点项目，通过该研究不但拓展了免疫分子Tim-4的生物学作用，而且对于肥胖、病毒感染、肺癌的发病机制、诊断、治疗提供了新的线索。. 在本项目的资助下，培养博士研究生3名、硕士研究生5名，项目负责人及课题组成员多次参加国际及全国性会议，多次就本项目相关研究结果进行交流，得到本领域专家的认可与高度评价，部分研究结果发表SCI收录论文3篇。.