基于RhoA/ROCK信号通路研究祛湿化瘀方调节非酒精性脂肪肝中肠紧密连接功能的机制

In the previous study on the mechanisms of Quhi Huayu (QSHY) decoction anti-nonalcoholic fatty liver disease (NAFLD) based on the “gut-liver axis”, we found that: 1) the pathological progression from nonalcoholic fatty liver (NAFL) to steatohepatitis (NASH) is relative to the increase in the abundance of the bacterium containing lipopolysaccharides (LPS) and the decrease of which contributes to protecting intestine barrier function; 2) in NAFLD, liver injury and the gut-derived LPS is linked by LPS gut-leakage induced by intestinal mucosal barrier injury; 3) QSHY ameliorating liver injury of NAFLD was significantly relative to its inhibition on LPS gut-leakage; 4) After QSHY treatment, the abundance of Gram-negative bacterium containing LPS decreased and that of contributing to protecting intestinal mucosal barrier increased obviously. Additionally, intestinal ultrastructure injury and the down-regulated mRNA of tight junction (TJ) were all recovered with QSHY treatment, which indicates that protection on intestinal mucosal barrier function by QSHY probably involved in the mechanisms of inhibition LPS gut-leakage by QSHY besides its regulation on gut microbiota. .LPS was found to activate Rho family of guanosine triphosphatase A (Rho A) / Rho-associated kinase (ROCK) signaling by up-regulation inducible nitric oxide synthase (iNOS) in the intestinal epithelium and eventually phosphorylate TJ. Based on the results found previously and the mechanism of LPS opening TJ, the hypothesis was proposed that the events of gut-derived LPS activating Rho A/ROCK pathway and opening TJ were involved in the mechanisms of intestinal mucosal barrier function injury in NAFLD and the effects of QSHY on the TJ and the Rho A/ROCK signaling were probably relative to its inhibition LPS gut-leakage. .To confirm the effect of QSHY on TJ in NAFLD, the model of NAFLD in C57/6J mouse induced by high-fat diet intake for 16 weeks and the NAFLD compound with colitis model in C57/6J mouse induced by high-fat diet and dextran sulphate sodium (DSS) were employed. To investigate the targets of QSHY on the Rho A/ROCK pathway, the intestinal mucosal barrier injury model in C57/6J mouse induced by LSP intraperitoneal injection and the Rho A/ROCK signaling activation model in Caco-2 cell were employed.

前期发现：1非酒精性脂肪肝(NAFLD)的病理进展与肠道含内毒素(LPS)细菌增加显著相关；2肠黏膜屏障损伤-LPS肠渗漏是联系NAFLD肠源性LPS与肝损伤的关键因素；3祛湿化瘀方(QSHY)防治NAFLD与抑制LPS肠渗漏密切相关；4QSHY调节NAFLD肠道菌群的同时，改善小肠超微结构上调肠紧密连接(TJ)的表达。结合LPS损伤TJ的机制进展提出：肠源性LPS激活RhoA/ROCK信号打开肠TJ，是NAFLD肠黏膜屏障损伤的重要因素，QSHY对肠TJ及RhoA/ROCK信号通路的调节可能是其保护NAFLD肠粘膜屏障的作用机理之一。拟采用：1NAFLD模型及NAFLD合病肠炎模型，观察QSHY对NAFLD肠TJ及RhoA/ROCK信号通路的影响；2剔除QSHY对肠道菌群的调节因素，采用LPS诱导肠黏膜损伤及体外RhoA/ROCK活化模型，阐明QSHY对RhoA/ROCK通路的作用环节。

项目背景：在前期发现祛湿化瘀方可通过调整肠道菌群减少内毒素肠渗漏从而防治非酒精性脂肪性肝病（nonalcoholic fatty liver disease, NAFLD）的基础上，针对“肠源性内毒素（Lipopolysaccharide, LPS）激活RhoA / Rho相关蛋白激酶（Rho-associated protein kinase, ROCK）信号打开肠紧密连接，是NAFLD肠黏膜屏障损伤的重要因素”这一核心机制，提出“祛湿化瘀方可能通过调节RhoA / ROCK信号通路而保护肠粘膜屏障，进而防治NAFLD”的假说。主要研究内容：通过1）NAFLD模型及NAFLD合病肠炎模型，观察祛湿化瘀方对NAFLD肠紧密连接及RhoA/ROCK信号通路的影响；2）剔除肠道菌群因素，采用LPS诱导肠黏膜损伤及体外RhoA/ROCK活化模型，阐明祛湿化瘀方对RhoA/ROCK信号通路的作用环节。重要结果及关键数据：1）祛湿化瘀方改善NAFLD小鼠以及NAFLD合病肠炎小鼠的肠粘膜损伤，并且降低肠组织ROCK活性及其下游肌球蛋白磷酸酶目标亚单位1（myosin phosphatase target subunit 1, MYPT1）的磷酸化，该作用与其治疗NAFLD作用密切相关；2）祛湿化瘀方保护LPS腹腔注射诱导的小鼠肠粘膜损伤，抑制结肠组织ROCK活性及其下游信号通路的活化；祛湿化瘀方药物血清抑制体外RhoA / ROCK通路的活化。科学意义：上述结果提示，祛湿化瘀方除调整肠道菌群以外，还可以通过保护肠粘膜屏障，抑制肠道细菌产物的肠渗漏，从而抑制NAFLD动物的肝损伤，该药理作用是通过抑制ROCK活性继而调节Rho A / ROCK信号通路实现的。