RhoA/ROCK信号通过自噬促进血管平滑肌细胞凋亡在缺氧性肺动脉高压中的作用及机制研究

The treatment and rehabilitation of pulmonary artery hypertension is a tough problem in the clinic. The expression of RhoA/ROCK signaling increases under hypoxia induction in pulmonary arteriolar（shang, et al, JCP, 2015）and suppress this signal can improve the symptom of pulmonary arterial hypertension,however, its mechanism is unclear. In our preliminary research, autophagy occurred in pulmonary arteriolar smooth muscle cells and increased after hypoxia induction, which was associated with RhoA/ROCK signaling.This project is a in-depth study of the regulating mechanism of RhoA/ROCK signaling on autophagy promoting apoptosis of smooth muscle cells in the formation process of pulmonary arterial hypertension. In-vivo and in-vitro mice models of hypoxia-induced pulmonary artery hypertension are used. RhoA/ROCK signaling is regulated by antagonist to change the condition of autophagy and autophagy itself is also activated. We evaluate 1) the regulating mechanism of RhoA/ROCK signaling on autophagy suppressing proliferation of smooth muscle cell; 2)the regulating mechanism and effect of both RhoA/ROCK signaling and autophagy on pulmonary arterial hypertension. Findings of this study will be beneficial to explore new mechanism of pulmonary arterial hypertension formation, and the two targeted intervention also provide new thinking for the clinical treatment of pulmonary artery hypertension.

肺动脉高压的治疗和康复一直是临床上的一个难题。肺小动脉在低氧诱导下会出现RhoA/ROCK信号表达增高（shang, et al, JCP, 2015），且抑制该信号能缓解肺动脉高压，但其机制尚不明确。我们的前期工作发现，肺小动脉平滑肌细胞在低氧诱导后出现自噬水平增高，且和RhoA/ROCK信号相关。本项目拟进一步研究RhoA/ROCK介导的自噬促进平滑肌细胞凋亡在肺动脉高压形成中的作用机制。采用小鼠体内外低氧诱导模型，通过拮抗剂调控RhoA/ROCK信号通路以改变自噬状态，并激活自噬本身进行二次调控，评估1）RhoA/ROCK信号调控自噬，抑制平滑肌细胞增殖的机制；2）RhoA/ROCK和自噬信号联合调控干预肺动脉高压的机制和疗效。本研究的发现有利于寻找肺动脉高压形成的新机制，联合双靶点的干预可为肺动脉高压的临床治疗提供新的思路。

研究背景:本研究旨在探讨Rho激酶信号转导自噬和凋亡在缺氧诱导的肺动脉平滑肌细胞(PASMCs)中的作用机制，探索一种新的联合治疗方法以改善低氧性肺动脉高压。.主要研究内容:将雄性Sprague-Dawley大鼠和PASMCs随机分为对照组、缺氧组、法舒地尔组和法舒地尔联合氯喹组。检测右心室/左心室重量+中隔(RV/LV+S)比值及肺动脉形态特征。α-平滑肌肌动蛋白(α-SMA)免疫荧光染色法进行评估。Western blotting分析Rhoa、Rock、P-MYPT1、LC3-II、beclin-1、P62、c-caspase3、bax、bcl-2。.重要结果:我们发现抑制Rho激酶信号可以通过自噬激活PASMCs来改善缺氧诱导的凋亡抵抗，从而抑制肺动脉高压的发展。法舒地尔和氯喹联合治疗对肺动脉高压有良好的效果。.关键数据及科学意义:我们的结果表明，fasudil显著增加了LC3B-II/-I比值和beclin-1的表达，也显著降低了P62的表达，与缺氧组相比，自噬增加(P<0.01)。法舒地尔可显著增加c-caspase3和bax蛋白的积累，降低bcl-2蛋白的积累，与缺氧组相比，凋亡增加(P均<0.01)。此外，联合自噬拮抗剂氯喹和法舒地尔治疗可导致缺氧条件下PASMCs自噬下调和凋亡上调。缺氧大鼠肺小动脉壁增厚，RV/LV+S比值升高，经法舒地尔治疗后降低。而联合使用法舒地尔和氯喹治疗后，肺小动脉壁增厚和RV/LV+S比值进一步明显降低。本研究为肺动脉高压的临床靶向治疗提供了实验依据。