Smurf2参与肠道稳态失衡所致肝脏“二次打击”的机制研究

The “first-hit” to liver is caused by a varity of pathogenesis, while in-testinal endotosemia (IETM) induced by enteric microbiota imbalance give the fatal “two-hits” injury to liver. Lipopolysaccharide(LPS) activate Toll like receptor 4 (TLR4)in kupffer cells(KCs), and through Myeloid Differentiation Factor 88(MyD88) depending signaling pathway, contribute to transcription of downstream cytokines and liver injury.LPS/TLR4 signal patheway plays an important role in “two-hits” injury. Smad ubiquitination regulatory factor 2(Smurf2) is an E3 ubiquitin ligase of ubiquitin-proteasome degradation pathway.It is newly reported that Smurf2 could inhibit TLR4 signaling pathway through ubiquitin depending prodeasome degradation of MyD88 in colon cance cell line CMT93. Our preliminary work found homeostasis of enteric microbiota could change Smurf2 levels and extent of liver injury. We also found alterations in KCs activation and MyD88 protein levels. This study is to use the CCL4 plus DSS induced liver “two-hits” injury rat model, undergo Smurf2 over-expression and RNAi, and utilize RT-PCR,WB,IHC etc. to clarify the interaction of enteric microbiota with Smurf2; to analyze the action of Smurf2 to “two-hit” liver injury induced by IETM. This study presents one new idea that enteric microbiota can influence liver “two-hit” injury through regulation of LPS/TLR4 pathway by Smurf2, and provides a new target for diagnosis and treatment to liver injury.

继各种因素的炎症损伤造成对肝脏的“首次打击”后，肠道稳态失衡引起的肠源性内毒素血症(IETM)，给肝脏致命的“二次打击”。IETM导致的LPS/TLR4信号紊乱在“二次打击”中起重要作用。Smad泛素化调节因子2(Smurf2)在结肠癌细胞株中，可降解髓样分化因子88(MyD88)，抑制TLR4信号传导。前期实验中，我们发现肠道稳态变化可改变大鼠肝脏Smurf2表达，KCs活性、MyD88蛋白水平和肝损伤程度随之变化。本研究拟利用CCl4加DSS诱导“二次打击”肝损伤大鼠模型，在Smurf2过表达/干扰表达后，用RT-PCR、WB、IHC等方法，从体内和体外研究Smurf2参与LPS/TLR4信号调节、改变IETM所致大鼠肝脏“二次打击”后果的分子机制。本研究提出了肠道稳态水平可通过调控Smurf2表达，作用于LPS/TLR4信号通路，影响肝脏“二次打击”的新机制，为肝病诊治提供了新靶点。

肝硬化所致肠道粘膜通透性增加和肠动力不足，可诱发肠道稳态失衡，肠道细菌入血，引起肠源性内毒素血症(IETM)，给肝脏致命的“二次打击”。 LPS/TLR4信号紊乱在IETM导致的肝脏“二次打击”中起重要作用，髓样分化因子88(MyD88)是该信号通路的关键因子。Smad泛素化调节因子2(Smurf2)是一种泛素E3连接酶，可降解MyD88，抑制TLR4信号传导。本项目首先构建了CCl4诱导肝硬化小鼠模型，发现清洁肠道抗生素的使用有助于维持肠道稳态、减轻肠源性内毒素血症、改善肝功能和延缓肝纤维化的进展；同时证明肠道稳态平衡可上调Smurf2在肝脏中的表达，进而通过降解MyD88来抑制LPS-TLR4信号传导，减轻肝脏炎症和纤维化的进展。本研究进一步采用Cre-LoxP重组酶系统构建了Smurf2肝脏特异性转基因小鼠，采用CCl4和胆管结扎(BDL)诱导肝纤维化小鼠模型，在动物体内和细胞实验确立了Smurf2抑制结缔组织生长因子(CTGF)、抗肝纤维化的作用；并采用miRNA array和生物信息学分析，筛选出miR-132是该抑制效应的中介子。通过进一步荧光素酶报告分析、免疫共沉淀等方法，发现Smurf2抑制CTGF、阻断肝纤维化的具体机制为：Smurf2通过泛素蛋白水解酶途径促进肝细胞内PDE4B的泛素化和降解，提高胞内cAMP的水平；增加的cAMP促进蛋白激酶A(PKA)的活化，随后诱导cAMP反应元件结合(CREB)蛋白转录因子的磷酸化；磷酸化CREB的与cAMP反应元素(CRE) 在miR-132启动子位点结合，激活miR-132的转录；最后miR-132诱导CTGF mRNA的降解，启动抗肝纤维化的进程。本研究提出了肠道稳态水平的改变可调控Smurf2表达；进一步提出了Smurf2不仅作用于LPS/TLR4-MyD88信号通路，还通过对细胞内PDE4B的泛素化降解、诱导cAMP在肝细胞内的富集、通过cAMP-PKA-CREB信号途径来激活miR-132、诱导CTGF mRNA的降解，影响肝脏“二次打击”所致纤维化进展的新机制，为肝纤维化的治疗提供了新靶点。