sRAGE通过整合素调控自噬抑制心肌缺血再灌注损伤作用及机制研究

Myocardial ischemia/reperfusion (I/R) injuriy induced by percutaneous coronary intervention (PCI) result in irreversible tissue damage during the clinical treatment of coronary heart disease. It’s extremely urgent to explore effective strategies for inhibiting I/R injuries. Our previous studies funded by NSFC(30801217，81370313) have concluded that sRAGE is an endogenous protective substance that is antagonistic to myocardial ischemia reperfusion injury. sRAGE reduced I/R injury through activating JAK2/STAT3 pathways and up-regulating ubiquitin proteasome system. Our preliminary experiment showed sRAGE inhibited excessive autophagy triggered by I/R, which was proved by inhibition of autophagy related protein expression, reduction of autophagosomes formation and improvement of myocardial ultrastructure with the treatment of sRAGE during I/R. Meanwhile, our preliminary experiment also showed that sRAGE increased both mRNA and protein levels of integrin β3 subunit after I/R. Integrin is transmembrane receptors that mediates bidirectional signal transmission between the cell and its environment and can be involved in mediating autophagy in cardiomyocyte. Thus it is speculated that sRAGE regulates autophagy during I/R through integrin β3/STAT3 pathway. Integrin β3 subunit gene knockout mice as well as its cardiomyocytes will be used in this project to explore the RAGE-independent pathway of sRAGE involved in reducing autophagy during I/R. Markers will be detected in both wild type mice and integrin β3 subunit gene knockout mice including the gene and protein levels of integrin β3 subunit; cardiac function, myocardial infarct size, cardiomyocytes apoptosis; the levels of autophagy and STAT3 activation. Clinical observation, Biochemistry, molecular biology techniques and immuno-electron microscope will be used. It will be clarified that sRAGE can reduce damage during I/R through regulating autophagy and explored that sRAGE regulates autophagy during I/R through integrin pathway to antagonize myocardial I/R injury. The results of this project will provide a new approach to reveal the mechanism of sRAGE on myocardial protective effect and provide an effective intervention for clinical prevention of I/R injury.

我们已结题国自然(30801217，81370313)证实sRAGE是拮抗心肌缺血再灌注（I/R）损伤的内源性保护物质，通过JAK2/STAT3激活泛素蛋白酶体系统参与保护作用。预实验发现，sRAGE减少自噬相关蛋白表达、自噬小体形成和心肌细胞自噬流水平，改善组织超微结构的紊乱，提示能抑制心肌I/R自噬过度激活；且sRAGE增加I/R心肌细胞整合素β3表达。据此认为sRAGE可能通过整合素β3/FAK-STAT3调控自噬水平拮抗心肌I/R损伤。本研究拟在前期工作基础上采用临床观察，利用整合素β3基因敲除小鼠和心肌I/R模型，应用生物化学、分子生物学及免疫电镜等方法，明确sRAGE通过调控细胞自噬拮抗心肌I/R损伤，阐明整合素在sRAGE调节自噬水平拮抗I/R损伤中的分子调控机制。研究成果将丰富人们对sRAGE心肌保护机制的认识，并为临床防治心肌I/R损伤寻找生物标记物或治疗靶点提供依据。

冠心病是全球重大慢病之一，严重影响人民生命健康。最新公布数据急性心肌梗死死亡率快速上升，严重威胁国民健康。其救治需尽早开通血管，但应用冠脉介入治疗等开通血管同时亦会导致心肌缺血再灌注损伤（MIRI），甚至危及生命。因此如何减轻MIRI是降低急性心肌梗死死亡率和改善预后亟待解决的重要临床难题。深入阐明MIRI机制并明确有效干预靶点尤为重要且极为迫切。内源性心肌保护机制有望攻克此难题。本课题组前期研究结果证实sRAGE是拮抗心肌缺血再灌注（I/R）损伤的内源性保护物质，通过JAK2/STAT3激活泛素蛋白酶体系统发挥心脏保护作用。本研究在此基础上采用临床观察，利用心肌细胞特异性过表达sRAGE小鼠和心肌细胞复制I/R模型，应用生物化学、分子生物学及免疫电镜等方法。探讨sRAGE通过调控细胞自噬拮抗心肌I/R损伤的作用，阐明整合素在sRAGE调节自噬水平拮抗I/R损伤中的分子调控机制，明确sRAGE水平与冠状动脉病变严重程度的关系。研究结果发现（1）sRAGE通过抑制心肌I/R时自噬过度激活，拮抗心肌I/R损伤；（2）sRAGE通过心肌细胞表面整合素β3亚基抑制自噬过度激活，拮抗I/R损伤；（3）sRAGE通过整合素β3亚基激活STAT3信号通路抑制心肌I/R时自噬过度激活，减少I/R损伤；（4）sRAGE通过抑制Bnip3向线粒体易位，减少心肌细胞自噬过度激活导致的线粒体损伤，拮抗心肌I/R损伤。（5）急性心肌梗死患者血浆sRAGE和IFN-γ水平升高，并与cTnI等心肌损伤标志物及冠状动脉病变严重程度呈正相关。本研究阐明sRAGE通过与心肌细胞膜表面整合素β3亚基结合激活下游STAT3信号通路，活化的STAT3调节Beclin-1和Bnip3等蛋白的转录，抑制心肌I/R过程中自噬的过度激活，减少细胞内线粒体与自噬小体融合，降低线粒体损伤促进细胞存活而拮抗心肌I/R损伤。sRAGE水平与冠状动脉病变程度呈正相关。研究成果将丰富人们对sRAGE心肌保护机制的认识，并为临床防治心肌I/R损伤寻找生物标记物或治疗靶点提供依据，为急性心肌梗死早期诊断提供新型标志物，为急性心肌梗死提供新型干预策略，改善预后，降低死亡率。