基于肠源性内毒素血症探讨参苓白术散对NAFLD大鼠TLR4/NLRP3信号通路作用的分子机制

It is reported that: ① Intestinal endotoxemia (IETM) plays an important role in the development of NAFLD. ② Inflammation is a key factor in the development of hepatocellular steatosis to NASH and cirrhosis. ③ There intersection between the TLR4 and NLRP3 pathway is closely related to the development of NAFLD. We found that Shenling Baizhu Powder had a definite effect in the treatment of NAFLD, and its mechanism may be through inhibition of hepatic inflammatory factors, but the detailed mechanism is unknown. We plan to use high-fat diet induced rat model of NAFLD in experimental models, and use various methods including 16S rDNA sequencing, Western Blot, and Quantitative Real-time PCR. We will take further experiments to observe intestinal microenvironment, endotoxemia, TLR4/NLRP3 pathways in hepatocytes and Kupffer cell and its role of related inflammatory cytokines on NAFLD treated with Shenling Baizhu Powder in vivo. Thereby the new molecular mechanisms of Shenling Baizhu Powder in treatment of NAFLD may be well illuminated.

研究表明：①肠源性内毒素血症（IETM）在NAFLD的发生发展中具有重要作用。②炎症是单纯性脂肪肝向NASH甚至肝硬化、肝癌发生发展的关键因素。③有交汇协同作用的TLR4和NLRP3通路与NAFLD发生发展密切相关。我们研究发现，参苓白术散对NAFLD的防治效果肯定，其对有关炎症因子的调控作用可能是该方取效的重要机制之一，但详细作用机制未明。本项目采用高脂饮食诱导大鼠NAFLD模型，运用16S rDNA测序、Western Blot、PCR等技术，观察参苓白术散对NAFLD大鼠肠道微环境、IETM的作用及肝细胞、Kupffer细胞TLR4/NLRP3通路和炎症因子的影响。预期：揭示参苓白术散抗NAFLD作用的分子机制。

①项目背景：肠源性内毒素血症在非酒精性脂肪性肝病（NAFLD）的发生发展中具有重要作用，与肝脏TLR4/NLRP3通路及炎症反应密切相关。本项目在前期基础上，以肠源性内毒素血症为切入点，进一步深入探讨参苓白术散抗NAFLD的作用机制。②主要研究内容：采用高脂饮食建立NAFLD大鼠模型，通过检测常规生化指标，观察肝和结肠组织病理变化，检测血清有关炎症因子水平和肝组织TLR4/NLRP3通路有关蛋白的表达，16S rDNA测序分析肠道菌群变化，研究参苓白术散对NAFLD大鼠肝脏TLR4/NLRP3通路、血清炎症因子和肠道微环境的影响。③主要结果：肝组织病理显示模型组大鼠肝细胞呈脂肪变性，细胞肿胀、脂滴积聚严重，线粒体淡化肿胀，参苓白术散组肝细胞脂肪变性、细胞结构和线粒体形态等情况明显改善；结肠病理显示模型组部分粘膜上皮细胞脱落，occludin表达减少，参苓白术散组粘膜较完整，occludin表达升高；模型组大鼠血清ALT、AST、TC、TG和肝组织TC、TG与正常组比明显升高，参苓白术散组大鼠血清ALT、AST、TC及肝脏TC、TG含量水平与模型组比明显下降；模型组大鼠门静脉血清LPS和腹主动脉血清TNF-α、IL-1β、IL-18与正常组比明显升高，参苓白术散组LPS、TNF-α和IL-1β与模型组比显著降低；模型组大鼠肝组织TLR4/NLRP3通路相关蛋白表达与正常组比明显升高，参苓白术散组TLR4、MyD88、TRAF6、TRAM、TRIF、p-IRF3、NLRP3、ASC蛋白表达水平与模型组比明显下降；肠道菌群测序结果显示，参苓白术散组肠道菌群丰度高于模型组，在属水平上Anaerostipes属及Bifidobacterium属菌群丰度显著升高。④主要结论及科学意义：16周高脂饮食能够导致大鼠血清炎症因子含量和门静脉LPS含量明显升高，肝脏TLR4/NLRP3通路相关蛋白表达明显上调，同时导致肠道菌群结构改变和肠黏膜屏障受损；参苓白术散能够明显降低NAFLD大鼠血清炎症因子含量和肝脏TLR4/NLRP3通路相关蛋白表达水平，其机制可能与参苓白术散改善NAFLD大鼠肠道微环境，从而降低门静脉LPS含量有关。肠道微环境紊乱可能是NAFLD脾虚病机的重要表现，参苓白术散作为健脾法的代表方，其改善肠道微环境的作用可能是健脾法抗NAFLD的部分现代生物学机制。