线粒体调控NLRP3炎症小体活性在阿托伐他汀抗高血压血管内皮损伤的机制研究

Vascular endothelial injury is ubiquitous and appears to play an essential role in the pathophysiological progression of hypertension, but the mechanism of mitochondria-mediated NLRP3 inflammasome activation and associated inflammatory response in vascular endothelial injury is not clear. Previous studies have found that regulation of mitochondrial homeostasis exerts protective effects on ischemic myocardium and vascular endothelial cells. Therefore, this project will establish hypertensive rats/Ang II-induced vascular endothelial injury models combined with atorvastatin treatment. We will 1) examine the effect of atorvastatin treatment on the influence of hypertension-induced endothelial damage and mitochondria homeostasis by using vascular morphology and functional methods. 2) explore the signal transduction of NLRP3 inflammasome and related transport protein after mitochondrial homeostasis imbalance by molecular biology methods. 3) investigate the translocation of cardiolipin and interaction of cardiolipin and NLRP3 inflammasome by laser confocol microscopy and immunofluorescence and co-immunoprecipition technologies. 4) determine the mechanism of atorvastatin on inhibition of mitochondria-mediated NLRP3 inflammasome activation by applying siRNA/overexpression technologies. The project will explore the molecular mechanism of atorvastatin on inhibition of vascular endothelial injury induced by hypertension, offering the theoretical basis and new promising drug targets for therapy of hypertension.

血管内皮损伤是高血压的病理生理基础，线粒体介导NLRP3炎症小体活化及下游炎症反应在高血压内皮损伤中的作用尚未明确。项目组前期研究发现，线粒体稳态调控可修复缺血后的心肌和血管内皮细胞。本项目拟建立高血压大鼠/AngII诱导的内皮细胞损伤模型给予阿托伐他汀干预：采用血管形态学、功能学方法研究阿托伐他汀对高血压血管内皮损伤和线粒体稳态的影响；应用分子生物学方法阐明线粒体稳态失衡对NLRP3炎症小体功能蛋白信号转导的作用，运用免疫荧光，激光共聚焦和免疫共沉淀技术探讨线粒体特异性心磷脂转位对NLRP3炎症小体活化的影响，明确线粒体稳态调控与下游炎症反应及内皮损伤的内在联系；应用siRNA/高表达技术探究阿托伐他汀调控线粒体稳态影响NLRP3炎症小体的作用机制。本项目有助于阐明阿托伐他汀抑制高血压血管内皮损伤的分子机制，为高血压的防治提供理论依据和潜在药物靶点。

血管内皮损伤是高血压的病理生理基础，线粒体介导NLRP3炎症小体活化及下游炎症反应在高血压内皮损伤中的作用尚未明确。项目组前期研究发现，线粒体稳态调控可修复缺血后的心肌和血管内皮细胞。本项目拟建立高血压大鼠/AngII诱导的细胞损伤模型，结合阿托伐他汀（ATV）或胆碱干预，探讨NLRP3炎症小体活化及其介导的细胞凋亡在心肌和血管内皮损伤中的作用机制。研究表明：（1）ATV可改善自发性高血压大鼠的血管内皮损伤。ATV预处理逆转了高血压诱发的炎症因子含量的增加。高血压可活化NLRP3炎症小体以及血管内皮TUNEL阳性细胞率，而ATV干预可减轻这些变化。此外，ATV通过降低线粒体ROS和增加Bcl-2/Bax比值维持线粒体稳态，提示抑制线粒体功能障碍是ATV发挥血管内皮保护作用的机制。ATV可减轻AngII诱导的HUVECs损伤，包括炎症因子的释放，线粒体ROS以及细胞凋亡。ATV可抑制NLRP3炎症小体的活化，如下调IL-1β，caspase-1的蛋白表达。（2）胆碱促进心肌细胞HSF1的核转位以及NICD的蛋白表达。AngII可增加线粒体ROS的含量，促进线粒体促凋亡分子（包括细胞色素c，Omi/HtrA2和Smac/DIABLO）从线粒体释放到胞浆，这些作用在胆碱干预后降低。此外，胆碱调控M3AChR降低心肌细胞TUNEL阳性细胞率以及细胞表面积。沉默M3AChR和Notch1可废除胆碱的保护作用，提示激活M3AChR和Notch1/HSF1信号通路是胆碱心肌保护作用的分子机制。本项目有助于阐明阿托伐他汀/胆碱抑制血管内皮和心肌细胞损伤的分子机制，为高血压相关的心血管疾病的防治提供理论依据和潜在药物靶点。