过氧化氢酶通过AMPK/Raptor通路调控自噬的机制及其对糖尿病心肌保护作用的研究

Autophagy plays a critical role in the myocardial injury during diabetes mellitus. Catalase (CAT)，as a non-specific antioxidant，could reverse this injury via inhibition of excess autophagy, but the mechanism is still unclear. In previous work，we have demonstrated that cardiac-specific overexpression of CAT could down-regulate the phosphorylation levels of AMPK and Raptor in type I diabetic mice hearts. In addition, Raptor is a key regulatory factor of mTOR, which can negatively regulate autophagy. Therefore, we speculate that the protection effect of CAT on diabetic cardiomyopathy depends on its role in the inhibition of excess autophagy via down-regulating the phosphorylation levels of AMPK and Raptor. Based on our previous research, this project will further confirm that Raptor is the substrate of AMPK participating in modulation of excess autophagy. It would be performed by transfecting a dominant-negative AMPK or a constitutively active mutant form of AMPK in cardiomyocytes. Moreover, the targeted proteomics and site-directed mutation would be introduced to verify the influences of relative phosphorylation sites of Raptor on the formation of Raptor-mTOR complex, the autophagy flux and the cardiac function in both cardiomyocytes and mice hearts. This project will make a valuable contribution to clarify the mechanism of myocardium protection through inhibition of excess autophagy by CAT, and provide a novel target for the treatment of diabetic myocardium.

自噬在糖尿病心肌损伤中扮演重要角色，非特异性抗氧化分子过氧化氢酶（CAT）可抑制因过度自噬诱导的心肌损伤，但其调控机制尚不清楚。前期在糖尿病小鼠模型中研究发现，心脏特异性高表达CAT能下调AMPK及其底物Raptor的磷酸化水平，而Raptor是自噬负调控因子mTOR的上游关键蛋白。因此我们推测CAT可能通过AMPK介导Raptor磷酸化水平下调，抑制心肌过度自噬，从而发挥其对糖尿病心肌的保护作用。本项目将在前期研究基础上，通过细胞转染丧失激酶活性或具有持续激酶活性的AMPK，确认Raptor为AMPK参与调控糖尿病心肌自噬过度发生的底物。然后借助靶向蛋白质组学及定点突变技术分别在细胞及动物水平探究Raptor中不同靶位点的磷酸化修饰对Raptor-mTOR复合物形成、自噬及糖尿病心肌功能的影响，详细阐明CAT抑制糖尿病心肌中过度自噬产生的机理。本项目有望为糖尿病心肌病的防治提供新靶点。

本研究旨在探讨AMPK/Raptor通路在非特异性抗氧化分子过氧化氢酶（CAT）抑制糖尿病心肌中过度自噬产生心肌损伤的重要作用及潜在的分子机制。超声心动图，免疫组化和DHE染色结果表明CAT对糖尿病心肌具有保护作用。免疫印迹结果表明CAT通过抑制过度自噬实现保护功能，且伴随着AMPK及其底物Raptor磷酸化水平下调。转染丧失激酶活性的AMPK，可使Raptor磷酸化水平下调，引起mTOR激活，进而抑制过度自噬，减少细胞凋亡。而通过AMPKα1312持续激活AMPK，可使Raptor磷酸化水平上调，抑制mTOR活性，自噬激活，促进细胞凋亡。蛋白质组学结果显示，相较于对照组，CAT组中Raptor S722和S792位点磷酸化水平明显下调。此外，采用定点突变技术抑制Raptor S722和S792位点的磷酸化，可逆转过度自噬，减少细胞凋亡；而Raptor S722和S792位点的持续磷酸化激活，进一步增加自噬水平，促进凋亡。本研究表明，CAT通过介导AMPK/Raptor通路的激活抑制过度自噬，进而实现对糖尿病心肌的保护。