HO-1/CO通过NF-κB-MLCK信号通路修复肝纤维化肠上皮屏障损伤的研究

The progress of liver fibrosis is often accompanied with intestinal flora imbalance, increased intestinal permeability and endotoxemia, which causes the second hit to the liver through the gut-liver axis, forms a vicious circle and aggravates liver injury. Therefore, the recovery of intestinal barrier is crucial to block the progression of liver fibrosis. It was confirmed by our previous experiments that the endotoxin level and the intestinal permeability increased in both the patient and the rat with liver fibrosis, as well as the expression of HO-1. Furthermore, to induce HO-1 and increase its product CO could inhibit the LPS/NF-κB p65, repair tight junction and improve the intestinal barrier function. On the basis of the above, in this project, Ad-HO-1 and HO-1 siRNA are respectively transfected into the isolated intestinal epithelial cells and the expression of NF-κB and MLCK is regulated for verifying that HO-1/CO regulates the MLC phosphorylation to repair tight junctions via the NF-κB and its downstream pathway. In vivo, the intestinal epithelial specific HO-1 overexpression transgenic mice is constructed, and the liver fibrosis model is prepared, then the wild type mice is given cobalt porphyrin or CO releasing molecules which respectively lead to the endogenous or exogenous increase of HO-1/CO. The repair effect of HO-1/CO on the intestinal barrier of liver fibrosis is determined through the three kinds of intervention. We look forward to providing an idea for the targeted therapy of intestinal barrier and a method to solve the problem of the shortage of these drugs in the treatment of liver diseases.

肠道菌群失调、肠黏膜通透性增加和高内毒素血症是肝纤维化进展中的病理生理改变，并可通过肠肝轴造成肝脏二次打击，形成恶性循环而加重肝损伤，故修复肠屏障对阻断肝纤维化进展至关重要。课题组前期研究证实肝纤维化患者和大鼠的内毒素水平、肠通透性均增加，且发现血红素氧合酶-1（HO-1）表达增多；诱导HO-1及其产物CO增多可抑制LPS/NF-κB p65、恢复紧密连接（TJ）蛋白表达及屏障功能。本课题进一步分离培养小鼠小肠上皮细胞，转染Ad-HO-1及HO-1 siRNA、调控NF-κB和MLCK，研究HO-1/CO通过NF-κB及其下游通路调节MLC磷酸化修复TJ的机制；构建肠上皮特异性HO-1过表达小鼠，制备肝纤维化模型，予野生鼠钴卟啉和CO释放分子分别从内/外源性增加HO-1/CO，验证三种干预方式对肝纤维化肠屏障的修复作用，期待为肠屏障的靶向治疗提供新思路，并解决肝病治疗中此类药物不足的问题。

肠道菌群失调、肠黏膜通透性增加和高内毒素血症是肝纤维化进展中的病理生理改变，并可通过肠-肝轴造成肝脏二次打击，形成恶性循环而加重肝损伤，故修复肠屏障对阻断肝纤维化进展至关重要。课题组前期研究证实肝纤维化患者和大鼠的内毒素水平、肠道通透性均增加，且发现血红素氧合酶-1（HO-1）表达增多；诱导HO-1及其产物CO增多可抑制LPS/NF-κB p65、恢复紧密连接（TJ）蛋白表达及屏障功能。基于前期系列研究，本课题进一步从体内、体外、分子机制、基因敲除、疾病验证等不同水平研究了HO-1/CO维持肝纤维化肠屏障完整性的作用。我们发现外源性上调HO-1（CoPP）和内源性补充CO（CORM-2）可显著减轻慢性CCl4注射所致肠道黏膜损伤、TNF-α分泌、TJ破坏和上皮NF-κB p65/MLCK/p-MLC-2信号通路激活；外源性下调HO-1（ZnPP）可完全逆转CoPP和CORM-2的作用。同样地，这些发现在体外TNF-α诱导的HO-1过表达或敲低细胞株中得到证实。采用JSH-23（NF-κB抑制剂）或ML-7（long MLCK抑制剂）预处理后，HO-1过表达阻止了TNF-α诱导的TJs破坏，而HO-1 shRNA促进了TNF-α所致TJs损伤即使在JSH-23或ML-7存在情况下。更重要的是，我们利用肠道特异性HO-1敲除小鼠进一步证实了HO-1在维持肠道屏障完整性中的重要作用及其分子机制。最后，我们通过减轻的肝脏纤维化程度和血清ALT水平验证了HO-1/CO修复肠屏障损伤的临床意义。总之，HO-1/CO通过NF-κB/MLCK信号通路维持肠屏障的完整性，从而延缓肝纤维化的进展。肠道HO-1/CO-NF-κB/MLCK系统是肠屏障功能障碍的潜在治疗靶点，本课题期待为靶向肠屏障的治疗肝纤维化提供新思路，并解决肝病治疗中此类药物不足的问题。