Caveolin-1调控PKCβ2/p66shc通路在肠缺血再灌注损伤中的作用及分子机制

Oxidative stress and apoptosis after intestinal ischemia reperfusion （I/R）are critical to intestinal mucosa barrier damage. Caveolin-1 (Cav-1),an important structural protein of Caveolae, is associated with tissue I/R injury, while the mechanism is remain not clear. Our research shows that selective blocking PKCβ2/p66shc pathways significantly reduce intestinal mucosal epithelial cells apoptosis after intestinal I/R. Additionally, some studies show there are some structure region to activate PKCs or p66shc in the Caveolae. Our preliminary experiments show that, the express of Cav-1 on cell membrane is down-regulating after intestinal I/R, with negative correlation to the up-regulating the express of PKCβ2; Meanwhile, PKCβ2 shifting to cell membranes combines with Cav-1 to form immune co-precipitation. Accordingly, we put forward the hypothesis that Cav-1 negatively regulates PKCβ2/p66shc pathway, inhibiting the intestinal mucosa oxidative stress and apoptosis mediated by intestinal I/R injury. Combining with animals and isolated cell model, using molecular biological techniques, this project aims to reveal the role of Cav-1 in intestinal I/R injury and Cav-1 regulates PKCβ2/p66shc pathways after intestinal I/R, providing evidence for clinical prevention and treatment to intestinal I/R.

氧化应激和细胞凋亡是肠缺血再灌注（I/R）后肠黏膜屏障损伤的关键环节。小窝蛋白-1（Cav-1）是小窝的结构蛋白，与组织、器官I/R损伤密切相关，但具体机制不清。我们研究表明，特异性阻断PKCβ2/p66shc通路显著减轻肠I/R后肠黏膜上皮细胞凋亡。文献报道PKCs家族、p66shc的激活位于小窝内。我们预实验显示，肠I/R后细胞膜Cav-1表达下调，与PKCβ2高表达负相关；同时Cav-1与细胞膜转位的PKCβ2结合形成免疫共沉淀。据此提出假说：Cav-1负性调控PKCβ2/p66shc通路，抑制氧化应激和细胞凋亡介导的肠I/R后肠黏膜屏障损伤。本项目拟结合动物、离体细胞模型，运用分子生物技术，研究Cav-1在肠I/R损伤中的作用，并揭示Cav-1是调控PKCβ2/p66shc通路的重要靶点，为临床防治肠I/R损伤提供科学依据。

氧化应激和细胞凋亡是肠缺血再灌注（I/R）后肠黏膜屏障损伤的关键环节。小窝蛋白-1（Cav-1）是小窝的结构蛋白，与组织器官I/R损伤密切相关。Cav-1在I/R中的保护作用与其抑制PKCs家族及p66shc介导的氧化应激和细胞凋亡相关。本项目结合动物、离体细胞模型，运用分子生物技术，研究Cav-1在肠I/R损伤中的作用，并揭示Cav-1是调控PKCβ2通路的重要靶点，为临床防治肠I/R损伤提供科学依据，阐明Cav-1抑制PKCβ2通路，抑制氧化应激和细胞凋亡介导的肠I/R后肠黏膜屏障损伤。.主要研究成果：Cav-1在肠I/R损伤中的保护作用：I/R后诱导肠组织中Cav-1高表达，而Cav-1的基因敲出鼠表现出对肠I/R的明显不耐受。此外，我们首次发现Cav-1与PKCβ2形成免疫共沉淀结合，且肠I/R后两者结合明显增多。通过体内基因敲出及体外RNA干扰技术，我们发现Cav-1敲除后，肠I/R中PKCβ2的大量激活并介导下游的氧化应激和细胞凋亡。此外，我们还发现，PKCβ2特异性阻断剂LY333531可减少I/R后诱导肠组织中Cav-1表达。. 本项目采用 Cav-1 基因敲除小鼠制备肠 I/R 模型及肠黏膜上皮细胞缺氧复氧（H/R）模型，从分子-组织-动物整体水平研究研究 Cav-1 在肠 I/R 损伤中的作用，并揭示 Cav-1 是肠 I/R 后调控 PKCβ2/p66shc 通路的重要靶点，为临床防治肠 I/R 损伤提供科学依据。