sRAGE通过泛素蛋白酶体系统抑制心肌缺血再灌注损伤的机制研究

Severe myocardial dysfunction and tissue damage resulting from ischemia reperfusion is a common clinical scenario in patients with certain types of heart diseases and therapies such as thrombolysis, percutaneous coronary intervention,coronary artery bypass grafting, and cardiac transplantation. The underlining mechanism of endogenous cardiac protection after ischemia reperfusion injury has been a focus of current research.Growing evidences suggests that soluble receptor for advanced glycation end-products has a cardioprotective effect; however, its role in ischemia reperfusion injury remains unclear.Our previous study found that soluble receptor for advanced glycation end-products (sRAGE) could prevent from ischemia reperfusion injury through alleviating apoptosis of cardiomyocyte. The ubiquitin proteasome system is another important way of clearing away the damaged protein in the cell. Our previous work has found that ischemia reperfusion injury could inhibit activities of ubiquitin proteasome system (UPS) in the cardiomyocytes, while sRAGE can increase activities of the UPS. Our hypothesis is that sRAGE can protect the heart through activating UPS which could reduce apoptosis of cardiomyocyte. Our study included observing change of endogenous sRAGE, UPS, apoptosis in cardiomyocytes and effect of sRAGE on UPS, apoptosis when ischemia reperfusion injury happened and effect of sRAGE when activating or inhibiting UPS. The project will provide new thought for clarifying mechanism of myocardial ischemia reperfusion in patients with coronary heart disease and for prevention and treatment of the disease.

我们前期研究发现sRAGE通过抑制心肌细胞凋亡拮抗I/R损伤。UPS是清除细胞内受损蛋白的另一重要途径，我们前期工作发现I/R损伤可抑制心肌组织中UPS活性，而sRAGE可以增加UPS活性，因此我们推测sRAGE通过激活心肌细胞内UPS，增强细胞清除受损蛋白的能力，减少心肌细胞凋亡，起到保护心肌I/R损伤的作用。本项目拟通过观察I/R时sRAGE、UPS和心肌细胞凋亡的变化；sRAGE对I/R时UPS和心肌细胞凋亡的影响；干预UPS对sRAGE上述作用的影响；以及相关信号转导通路的变化，验证sRAGE通过激活UPS，减少心肌细胞凋亡发挥其心脏保护作用的假说。

心肌缺血再灌注损伤严重制约着冠心病的救治成功率，探讨sRAGE通过泛素蛋白酶体系统抑制心肌缺血再灌注心肌细胞凋亡的作用及机制，为临床防治心肌缺血再灌注损伤提供新的理论依据及靶点。本课题首先评价sRAGE对心肌缺血再灌注时泛素蛋白酶体系统活性和表达及心肌细胞凋亡的影响；其次明确sRAGE是否通过影响泛素蛋白酶体活性及表达抑制缺血再灌注诱导的心肌细胞凋亡；最后明确sRAGE通过激活泛素蛋白酶体减少缺血再灌注心肌细胞凋亡的细胞内信号通路。结果：①与对照组相比较，小鼠心肌缺血再灌注后心肌梗死面积增加、TUNEL阳性心肌细胞数目增高，caspase-3活性升高，p53蛋白表达和Bax/Bcl-2比值均增加(P < 0.05)。②与对照组相比，泛素蛋白酶体活性均明显降低，β亚基蛋白表达降低，即β1i蛋白水平和β5i蛋白水平均降低，总泛素蛋白表达增加（P<0.05）。③与单纯心肌缺血再灌注组相比较， sRAGE减少小鼠心肌缺血再灌注梗死面积、TUNEL阳性心肌细胞数目及caspase-3活性 (P<0.05)。④与单纯心肌缺血再灌注组相比较， sRAGE降低心肌缺血再灌注的p53表达和Bax/ Bcl-2比值。与sRAGE组比较，STAT3抑制剂（AG490、S3I-201）减少了sRAGE抑制体外心肌缺血再灌注心肌细胞p53表达及Bax/Bcl-2比值的作用(P <0.05)。⑤sRAGE抑制小鼠心肌缺血再灌注泛素蛋白酶体系统活性的降低；抑制β亚基蛋白表达的降低，即β1i蛋白水平和β5i蛋白水平；抑制总泛素蛋白表达的增加（P<0.05）。⑥泛素蛋白酶体系统抑制剂减弱sRAGE对体外心肌缺血再灌注TUNEL阳性心肌细胞数目和Caspase-3活性增加的抑制作用(p< 0.05)。⑦STAT3抑制剂AG490、PI3K/Akt抑制剂LY294002减弱sRAGE对心肌缺血再灌注TUNEL阳性心肌细胞数目和caspase-3活性的抑制作用(p < 0.05)，但ERK1/2抑制剂PD95089未能影响sRAGE对心肌缺血再灌注TUNEL阳性心肌细胞数目和caspase-3活性的抑制作用。结论：①sRAGE通过增加泛素蛋白酶体系统活性抑制p53/Bax通路的激活，继而减少心肌缺血再灌注诱导的心肌细胞凋亡。②sRAGE通过激活JAK2/STAT3通路抑制心肌缺血再灌注诱导的心肌细胞凋亡。