sRAGE通过IFN-γ/STAT影响泛素蛋白酶体系统抑制心肌缺血再灌注细胞凋亡的机制研究

The treatment and prognosis of coronary heart disease is restricted by ischemia/reperfusion(I/R) injury. It’s important to explore the treatment strategies for I/R. The endogenous protective substances are involved in reducing I/R injury. From our previous projects, we have found a positive correlation between increasing of sRAGE after PCI for acute myocardial infarction and improving of heart function in clinical research, and sRAGE could alleviate I/R injury through inhibiting cardiomyocytes apoptosis in the animal study. The effect of sRAGE inhibition on I/R-induced apoptosis through increasing the activity of the ubiquitin-proteasome system(UPS) in vivo. However, the mechanism of sRAGE inhibiting I/R-induced apoptosis via UPS is unclear. Other study has showed that sRAGE can promote the secretion of IFN-γ, and another research have demonstrated that IFN-γ increases the subunit expression of UPS by activating STAT1. The previous pilot study has shown that sRAGE could increase expression of STAT. This project will use RAGE gene knockout mice to simulate model, left anterior descending artery occlusion induced I/R in vitro as well as ischemic buffer in isloated myocytes induced I/R in vivo. Markers will be detected including heart fuction, myocardial infarct size, cardiomyocytes apoptosis; the activity and subunit expression of UPS; the gene and protein experssion of IFN-γ, STAT1 and STAT3. The signaling pathways for sRAGE cardioprotective, and the molecular biological mechanism of sRAGE induced reduction on apoptosis through increasing the activity and expression of UPS will be systematically explored by using the techniques of biochemistry and molecular biology. Achievements of this research will enrich our understanding of sRAGE cardioprotective involved in I/R injury, and provide effective intervention for prevention and treatment of myocardium I/R injury.

我们前期临床研究发现急性心肌梗死患者PCI术后sRAGE增加与心功能改善呈正相关，实验发现sRAGE通过抑制心肌细胞凋亡拮抗I/R损伤，并证实sRAGE可通过增加UPS活性发挥作用。但sRAGE通过UPS抑制I/R损伤机制尚不明确。传统认为sRAGE阻断RAGE起作用,而其非RAGE途径还.不清楚。有研究表明sRAGE促进IFN-γ分泌，另有报道IFN-γ激活STAT1，而STAT1和STAT3增加UPS亚基表达。我们预实验发现sRAGE增加STAT3表达。据此，本研究拟在前期基础上利用RAGE基因敲除小鼠和心肌细胞I/R模型，应用生物化学和分子生物学等方法，明确sRAGE心脏保护作.用的具体信号途径，阐明sRAGE通过IFN-γ/STAT激活UPS抑制I/R心肌细胞凋亡的分子生物学机制。所获成果将丰富人们对sRAGE心脏保护作用机制的认识，并将为临床防治心肌I/R损伤提供有效的干预措施。

缺血性心脏病是当今严重威胁人类健康的疾病之一,现已成为我国发病率、致残率和死亡率最高的心脏重大疾病。尽管早期开通血管是治疗本病的有效手段，但是缺血心肌的再灌注损伤仍是影响预后的重要因素。本课题组既往研究表明急性心肌梗死患者血清可溶性高级糖基化终产物受体（soluble receptor for advanced glycarion end-products, sRAGE）水平显著增高，动物实验表明sRAGE可抑制小鼠缺血再灌注（Ischemia/reperfusion, I/R）后心肌凋亡水平，改善小鼠心功能，然而其内在机制并不明确。本项目利用药物及基因手段干预小鼠sRAGE表达水平，结合体内体外实验，探究sRAGE通过调节IFN-γ/STAT抑制I/R损伤的机制。研究结果显示sRAGE显著激活STAT3信号通路并上调心肌细胞I/R后免疫型蛋白酶体β1i、β2i、β5i的表达水平，利用ChIP技术验证STAT3可与β1i、β2i、β5i的启动子结合。此外，sRAGE上调免疫型蛋白酶体亚基的功能与其增加巨噬细胞向缺血区域招募，增加IFN-γ表达水平相关。使用IFN-γ中和抗体干扰其活性后，sRAGE抑制I/R损伤及其上调免疫蛋白酶体亚基的作用显著减弱。体外实验利用小干扰RNA干扰免疫蛋白酶体亚基表达水平，结果显示干扰β5i亚基表达水平显著减弱sRAGE抑制I/R诱导的心肌细胞凋亡的作用，显著减弱sRAGE抑制p53减少的作用。进一步，体外培养巨噬细胞并给予sRAGE过表达处理，显著激活巨噬细胞NF-κB信号通路并增加IFN-γ表达水平。本项目研究结果说明存在非RAGE依赖的作用途径：sRAGE可通过增加巨噬细胞向缺血区域募集激活NF-κB信号通路，上调IFN-γ表达水平；继而增加细胞内蛋白酶体亚基的表达水平及活性，促进p53降解，发挥抑制心肌I/R损伤的作用。本项目的研究结果再次验证了sRAGE抑制心肌I/R损伤的作用，丰富了sRAGE调控心肌凋亡机制的研究，开创了sRAGE非RAGE依赖性作用机制的空白，为sRAGE在临床医学中的应用提供新的理论依据。