Caveolae和Rho激酶信号传导通路在PPARs调节血管内皮细胞中缝隙连接蛋白的作用

Caveolae, Rho kinase signaling and connexins in endothelium play key roles in the pathogenesis of atherosclerosis, which is known as a chronic in?ammatory disease. Our previous data show that peroxisome proliferator-activated receptor (PPAR)γ agonist protected the endothelium by mitogen-activated protein kinase(MAPK) signaling pathway. To further study the mechanism of Caveolae、Rho kinase signaling and connexins in the endothelial in?ammation and the effect of PPARs on connexins，this propasal will block the Rho kinase signaling pathway, Cav-1(Cav-1) and Connexin (Cx) 37/43 with specific siRNA or shRNA or inhibitor，then explore the interrelation of Cav-1, Cx37/Cx43, proteins of Rho kinase signaling as well as vascular in?ammatory mediators in endothelial cells with PPARγ overexpression via Western Blotting, Immunofluorescent stainning, ELISA、Electrophoretic Mobility Shift Assay (EMSA)、Zymography and Luciferase assay. Finally, Cav-1 knockout mice and its control C57BL/6 mice are fed with high lipid food with addition of PPARγ agonist or antagonist, Rho kinase inhibitor to confirm PPARs on attenuating connexins disruption and vascular inflammation by inhibiting Cav-1 or Rho Rho kinase signaling. With techniques of molecular biology, cytology and histology，this study will indicate that PPARs regulate connexins expression in endothelium by modulating caveolae and Rho kinase signaling pathway, therefor affect the gap between the endothelial cells, eventually prevent the atherogenesis. Taken together, this data will provide information for prevention and potential therapeutic target for atherosclerosis.

caveolae、Rho激酶信号通路和血管内皮的连接蛋白对动脉粥样硬化的形成产生重要的作用，本项目将siRNA或shRNA或抑制剂阻断Rho信号通路、Cav-1及Cx37/43后，蛋白质印迹法、免疫荧光技术、ELISA、凝胶迁移迁移实验、明胶酶谱法和荧光素酶等方法检测超表达PPARγ的血管内皮细胞中Cav-1、Cx37/Cx43、Rho信号传导蛋白的相互关系及血管炎性介质反应。高脂饲养的Cav-1基因敲除小鼠和正常对照小鼠，分别给予PPARγ激动剂/拮抗剂、Rho激酶抑制剂干预，以证实抑制Cav-1或Rho激酶信号通路对PPARs调节血管内皮细胞连接蛋白及抑制血管炎症反应的作用。本项目从分子生物学、细胞学、组织学等方面阐明PPARs通过caveolae和Rho激酶信号通路调节血管内皮连接蛋白的表达，影响血管内皮细胞的间隙，减少动脉粥样硬化的发生，结果对防治动脉粥样硬化有理论意义。

Caveolae、Rho激酶信号通路和血管内皮的connexins对动脉粥样硬化的形成产生重要的作用。我们研究发现peroxisome proliferator-activated receptor (PPAR)γ激动剂通过MAPK信号传导通路而发挥血管内皮的保护功能，为进一步探明Caveolae、Rho激酶信号通路和connexins在血管内皮细胞炎症反应中的作用机制和PPARs对血管内皮细胞connexins的影响，本项目将siRNA或shRNA或抑制剂阻断Rho信号通路、Cav-1（Cav-1）及Connexin(Cx)37/43后，检测超表达PPARγ的血管内皮细胞中Cav-1、Cx37/Cx43、Rhoqwa2w信号传导蛋白的相互关系及血管炎性介质反应。本项目发现：①FTS通过阻断Ras信号通路可以增加CX43总蛋白表达， PPAR激动剂可协同调节，Ras抑制剂对LPS诱导的Cx43总蛋白表达的抑制作用可被PPAR拮抗剂消除，Ras-Rho信号传导通路对Cx43总蛋白的表达作用可能由PPARs介导。②LPS可明显增加冠脉 内皮细胞VCAM-1、MCP一1的表达。法舒地尔联合罗格列酮可协同抑制 u)S诱导的HCAEC中VCAM-1、MCP-1的表达，可能与 两者共同抑制p38h咄K、NF—KB信号通路有关。③LPS可诱导HCAEC中Cx43蛋白表达量增高，促进血管内膜损伤，增加白细胞向内膜迁移和聚集，增加平滑肌细胞的迁移和增殖及斑块的不稳定性；HF可使 HCAEC Cx43蛋白表达量下调，起到保护血管作用。HF与LPS共同作用可使HCAEC中Cx43蛋白表达量下调，提示炎症刺激可能增强HF保护血管的作用。④在LPS作用SD大鼠6 h后，心肌组织中ROCKl的表达水平显著上升，心肌组织中Cx43的表达显著降低，而HF 预处理0．5 h可以干预上述结果。可见，HF通过Rho信号通路调节损伤的心肌组织中Cx43 表达，从而改善心肌功能障碍。⑤脂多糖组主动脉组织中R0CKl的mRNA及蛋白表达水平均高于对照组，而脂多糖+HF组的ROCKl mRNA及蛋白表达水平均低于脂多糖组。研究表明，HF能抑制 RhoA／ROCK信号通路，减少ROCKl的生成，从而减轻ROCKl对血管内皮功能的损伤。本项目完成首次阐明：Caveolae通过Rho激酶信号通路调节血管内皮细胞Cx4