非酒精性脂肪肝小鼠TLR4 的MyD88和TRIF信号通路与胰岛素抵抗的相关研究

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is unclear, and there is no specific treatment for NAFLD now. The imbalance of inflammatory factors and insulin resistance (IR) play important role in NAFLD. TLR4 / MyD88 and TRIF / TRAF6 / NF -κB signal pathway regulate the release of inflammatory factors such as TNF-α, IL-18 and so on, which could cause IR and lead to the hepatic inflammation, necrosis. However, the different effects between MyD88 and TRIF signal pathways on inflammatory, IR and NAFLD formation remain unclear. Artesunate has the effects on lipid-lowering and downregulation of the inflammatory factors. TLR4 knockout, antibodies against TLR4, MyD88, TRIF and different doses of artesunate are used to observe the effects of blockade of the above three signals and artesunate on the expression of signals of TLR4 inflammatory pathway in NAFLD KCs and IR in hepatic cells. In addition, the hypothesis is tested in vivo.The aim of our study is to identify the effect of TLR4 inflammatory pathway in NAFLD, and the further pathogenesis of NAFLD, as well as the mechanism and effect of artesunate intervention on it. These results would provide a theory basis for clinical practice.

非酒精性脂肪性肝病（NAFLD）的发病机制尚未阐明，也无特殊治疗方法。炎症因子失衡和胰岛素抵抗（IR）是导致NAFLD的重要因素。TLR4/MyD88和TRIF/TRAF6/NF-κB信号通路调控TNF-α、IL-18等炎症因子的释放，推测可能引起IR，导致肝细胞炎症、坏死。而且，MyD88 、TRIF两条信号通路对炎症、IR和NAFLD形成有何不同尚不清楚。青蒿琥酯具有降脂、下调炎症因子的作用。本研究拟采用TLR4基因敲除、抗TLR4、 MyD88、TRIF的抗体和不同剂量的青蒿琥酯，从体内外两个层面，首次观察此三个信号阻断和药物干预是否影响NAFLD的库普弗细胞（KCs）TLR4炎症通路信号的表达和肝细胞IR，以及对肝脏炎症和脂肪病变的作用。以阐明TLR4炎症通路在NAFLD的作用，探索 NAFLD的发生机制。同时，探讨青蒿琥酯干预NAFLD的作用机制和效果，为其临床应用提供理论依据。

非酒精性脂肪肝病（NAFLD）的发病机制尚未阐明，也无特殊治疗方法。炎症因子失衡是导致NAFLD的重要因素。本研究用高脂饮食法构建TLR4基因野生型NAFLD小鼠模型，用油酸在人正常肝细胞LO2细胞制备NAFLD的细胞模型，从体内外两个层面，观察不同剂量青蒿琥酯对NAFLD的TLR4炎症通路的影响。应用光学显微镜、Western blot、ELISA、免疫荧光和二氯荧光素二乙酸酯测定等多种方法检测，结果显示青蒿琥酯可通过TLR4 / PI3K / Akt通路调节下游炎症细胞因子（TNF-α，IL-6，IL-8和IL-18）和氧化应激改善非酒精性脂肪肝疾病。首次系统观察了青蒿琥酯对肝细胞脂滴形成、肝酶水平、TLR4 / PI3K / Akt通路、炎性细胞因子产生和氧化应激在NAFLD细胞中的作用。在NAFLD小鼠模型中，显示类似的结果：青蒿琥酯可以通过降低TLR4、 TRAF6的表达，调节TLR4信号通路，减少炎症因子的释放，影响炎症因子的表达，进而减少细胞膜的损伤，显著降低血清ALT、AST水平，对肝脏炎症病变有着显著的抗炎作用，对脂肪变有一定的改善作用，并呈一定的剂量-效应关系。这些结果表明青蒿琥酯具有预防非酒精性脂肪性肝病进展和治疗疾病的潜在价值。研究为进一步明确非酒精性脂肪性肝病发病机制奠定了的理论基础，为青蒿素的在非酒精性脂肪肝疾病临床应用提供了一定的理论依据。