RhoA/ROCK 信号通路介导的自噬障碍在2型糖尿病心肌损伤中的作用

High glucose and insulin resistance induced cardiac injury is the major cause of type 2 diabetic cardiomyopathy (DCM), the regulation mechanisms of which are not full clear. It has been known that in type 2 diabetes autophagy is increased, but the molecular mechanism and pathophysiological significance is unknown. ROCK1 kinase is a critical regulator of autophagy during metabolic stress in cancer cells and neurodegenerative disease. Elevated activity of the RhoA/ROCK pathway has been implicated in diabetic cardiomyopathy. Based on the previous study, we hypothesized that the RhoA/ROCK signaling pathway regulates autophagy in type 2 diabetic cardiac injury. We are going to make type 2 diabetic cardiac injury model in vitro and in vivo to address three questions: (1) if the alteration autophagy has an impact upon cardiac injury in type 2 diabetes mellitus and the RhoA/ROCK signaling pathway has an impact upon autophagy. (2) how altered autophagy was involved in the impact of altered RhoA/ROCK signaling pathway upon cardiac injury in type 2 diabetes mellitus. (3) what is the molecular mechanism underlying the observed autophagic activity change in response to altered RhoA/ROCK signaling pathway? in the present study, we will uncover the molecular mechanism for cardiac injury in type 2 diabetes mellitus from new point of view and provide a potential therapeutic target site for DCM prevention and treatment.

糖尿病性心肌病是独立存在的糖尿病并发症之一，目前对其机制和治疗的研究尚不充分。现有证据表明：2型糖尿病时心肌自噬增多，但自噬发生的分子机制与信号转导过程及其病理生理学意义未明。ROCK1对神经系统疾病、肿瘤自噬调控起重要作用，我们前期研究发现RhoA/ROCK信号通路参与2型糖尿病心肌病发生发展过程。但RhoA/ROCK信号通路是否通过调控心肌自噬参与2型糖尿病心肌损伤还不清楚。我们拟制备2型糖尿病心肌损伤在体和离体模型，从整体-细胞-分子层次阐明三个问题：(1)在2型糖尿病心肌损伤过程中自噬对心肌损伤的影响以及RhoA/ROCK信号通路对自噬的影响； (2) RhoA/ROCK信号通路是如何调控自噬参与2型糖尿病心肌损伤的；(3) 离体环境中RhoA/ROCK信号通路调控自噬的机制是什么；本课题将从新的视觉阐明2型糖尿病心肌损伤发生的分子机制，为糖尿病心肌病的防治提供新思路和新靶点。

糖尿病心肌病发病机制尚未阐明，是当前糖尿病和心脏病学界亟待解决的问题。近年来的研究发现，自噬在糖尿病心肌病发生和发展过程中也扮演着重要角色。本项目分别通过体外实验及2型糖尿病大鼠模型，利用细胞生物学、分子生物学等技术，对高糖高胰岛素环境下RhoA/ROCK信号通路调控心肌细胞自噬的分子机制作了涉入探讨。我们分别利用正常葡萄糖浓度（5.5mM）的培养液和含高浓度葡萄糖（30mM）高胰岛素（10-7M）的培养液体外培养原代乳大鼠心肌细胞，发现高糖高胰岛素促进心肌细胞凋亡及自噬，诱导心肌细胞损伤。高糖高胰岛素环境下，心肌细胞中ROCK1、ROCK2蛋白表达上调，抑制ROCK可以显著抑制减少细胞凋亡，减轻自噬，改善心肌细胞损伤。抑制ROCK可以显著抑制高糖高胰岛素对心肌细胞JNK、Beclin1蛋白表达的上调。通过JNK抑制剂阻断RhoA/ROCK信号通路下游调控分子JNK后，显著抑制信号通路中Beclin1蛋白的表达。利用STZ构建2型糖尿病大鼠，给予DM大鼠RhoA/ROCK信号通路抑制剂，可以通过调控心肌细胞自噬水平，抑制心肌细胞肥大，降低纤维化，抑制心脏重构，改善心脏收缩舒张功能。综上所述，RhoA/ROCK可能是通过ROCK／JNK／Beclin1通路调控凋亡和自噬参与高糖高胰岛素对心肌细胞的损伤。本研究从新的视觉阐明了高糖心肌损伤发生的分子机制，为糖尿病心肌病的防治提供新思路和新靶点。