RIP1/RIP3-PARP1信号通路调控Necroptosis发生在肠缺血再灌注肠损伤中的作用及机制

Intestinal ischemia/reperfusion (I/R) is one of the main causes contributing to multiple organ failure during perioperative period. However, the exact mechanism remains an enigma. We had already revealed that intestinal injury after intestinal I/R is related to necrosis and apoptosis of intestinal epithelial cells, and apoptosis inhibitation can reduce intestinal injury caused by intestinal I/R. In addition to necrosis and apoptosis, a novel form of caspase-independent cell death has been described recently, termed necroptosis. Evidences demonstrated that the morphological features of necroptosis are different from necrosis and receptor-interacting protein kinases (RIP1/RIP3) are specifically involved in regulating necroptosis. Previous studies had documented that Nec-1, the specific necroptosis inhibitor, significantly attenuated I/R insults of heart, brain, and so on. Our recent study also found that intestinal I/R-indcued intestinal injury was markedly ameliorated after Nec-1 administration, suggesting that necroptosis might be an important mode of cell death in intestinal I/R model. Hence, we hypothesize that RIP1/RIP3-PARP1 signaling pathway induced necroptosis plays a pivotal role in the progression of intestinal I/R-caused pathophysiology and the molecular mechanisms that are responsible for intestinal I/R injury in vivo and in vitro. Finally, our study attempts to provide new targets and ideas for the prevention and treatment of intestinal injury after intestinal I/R.

肠缺血再灌注（I/R）是围术期患者发生多脏器功能衰竭的主要原因之一，其机制尚未明确。申请者过去发现肠I/R后肠黏膜细胞发生坏死与凋亡，且抑制凋亡有一定的肠保护作用。近年来研究证实细胞缺血损伤后不仅产生坏死和凋亡，还存在另一种新的死亡形式，其不依赖于调节凋亡的因素且与传统的坏死细胞形态学不同，被称为程序性坏死（Necroptosis），而且其发生受上游的受体相互作用蛋白1和3（RIP1/RIP3）激酶调控。已证实Necroptosis特异抑制剂Nec-1能显著减轻心、脑I/R损伤。我们新近实验亦发现Nec-1能减轻肠I/R后肠损伤，提示Necroptosis可能是肠I/R后肠损伤的机制之一。据此，本项目提出RIP1/RIP3-PARP1信号调控Necroptosis发生是肠I/R肠损伤的一个重要分子机制的假设，并采用一系列的体内外实验来验证该假设，为肠I/R后肠损伤的防治提供新靶点和思路。

本项目从机体氧化应激平衡失调、肠道免疫屏障和机械屏障损伤角度探讨了肠缺血再灌注（I/R）后肠损伤的新机制，为临床寻找防治肠I/R损伤可供干预的靶点提供了理论基础。取得的学术成果如下：.（1）证实大鼠I/R损伤过程中细胞膜脂质花生四烯酸过氧化所产生的15-F2t-Isoprostane不仅与机体的氧化应激损伤程度相关，而且其自身的强生物活性进一步介导了I/R后的肠道损伤。.（2）证实肠I/R损伤能激活肠道巨噬细胞并促进M2向M1型巨噬细胞转变；重组旋毛虫组织蛋白酶B样蛋白（rTsCPB）免疫接种能促进小鼠体内Th2型免疫反应，逆转肠I/R损伤所致M2向M1型巨噬细胞的转变，并通过依赖STAT6的方式促进M1向M2型巨噬细胞转变，从而减轻肠I/R所致的肠损伤并促进肠道功能恢复。.（3）证实RIP1/RIP3介导的细胞程序性坏死（Necroptosis）参与了肠I/R后的肠损伤过程，明确使用特异性抑制剂Necrostatin-1能阻断Necroptosis的发生而产生抗肠I/R肠损伤的作用；同时联合使用凋亡抑制剂Z-VAD-fmk治疗能对肠I/R后的黏膜组织损伤产生更好的保护作用，而且单用其中一种抑制剂时能使黏膜上皮细胞的死亡方式发生互相转换。高迁移率族蛋白1（HMGB1）作为重要的效应分子介导的下游TLR4/RAGE信号通路的激活及其核内转位在肠黏膜上皮细胞的Necroptosis发生过程中起了重要的作用。.（4）证实肝脏细胞发生程序性坏死（Necroptosis）后参与了大鼠肠I/R致远程肝损伤的过程。肝脏汇管区周围肝细胞内HMGB1蛋白的核外转位可能是其分子机制之一。