AMPK参与程序性坏死及其作为心肌缺血再灌注损伤防护靶点的研究

Programmed necrosis (necroptosis) is one of the hallmarks of myocardial ischemia and reperfusion injury (IRI). Understanding the regulatory mechanism controlling the degree of necroptosis would provide insights into the protection of myocardial IRI as well as leads for identifying new therapeutic targets. Recent findings demonstrated that AMP-activated protein kinase (AMPK), an established cell metabolic sensor, plays a critical role in protection of apoptosis from injury in different cell and animal models. However, its function in necroptosis remains largely unclear. .The stimulus to propose this grant application is our preliminary findings that depletion of AMPK resulted in markedly increased necroptosis and cytotoxicity induced by pro-necrotic N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). More importantly, our previous results revealed that AMPK protects against necroptosis by regulating the expressions of PGAM5 and Keap1 proteins and the combination of those proteins. Finally, AMPK agonist metformin reduced myocardial IRI induced necroptosis through down-regulated the expression of PGAM5 in the Langendorff-perfused rat heart model. Thus, we hypothesize AMPK safeguards necroptosis through regulating Keap1/PGAM5 complex. As a corollary, activation of AMPK may possess a protective effect against necroptosis and serves as a target for the protection against myocardial IRI. To address our hypothesis, we designed 3 specific aims: (1) We will determine the role of AMPK in necroptosis and explore the activation of AMPK-mediated protection on the maintenance of cell survival (2) To explore the possible molecular mechanisms by which AMPK are activated under necroptosis induction and identify downstream targets of AMPK in necroptosis. (3) To validate the effect of metformin, an AMPK activator, on the protection against myocardial IRI..Results obtained from the proposed studies will yield novel findings that will suggest how AMPK integrates different signals in necroptosis in the maintenance of cell survival and provide valuable new information to further our current knowledge in myocardial IRI-induced necroptosis. These studies will also provide a rationale for pharmacologic activation of AMPK in the protection against myocardial IRI.

程序性坏死在缺血再灌注损伤（IRI）中发挥重要的作用。阐明其分子调控机制，有助于为心肌IRI提供新思路。研究表明AMPK不仅是调节细胞能量代谢的靶基因，而且是多种应激损伤的感受分子。但AMPK在程序性坏死中的作用至今不明。我们前期发现AMPK缺乏会加重DNA烷化剂诱导的细胞坏死。更重要的是，AMPK活化抵抗坏死是通过调节Keap1和PGAM5的蛋白表达，及蛋白间的相互结合。动物实验显示，AMPK激活剂二甲双胍预处理可有效减轻心肌IRI。据此，我们认为AMPK通过调节Keap1/PGAM5复合物在程序性坏死中发挥保护作用，应用二甲双胍可起到心肌坏死防护作用（假说）。本研究将明确AMPK表达、磷酸化及其上下游信号分子在程序性坏死中的变化和作用。探讨AMPK激活抵抗坏死的具体分子调控机制；评估AMPK作为心肌IRI防护靶点的可行性。研究结果对心肌IRI个体的防治具有高度的指导和应用价值。

心肌细胞程序性坏死及其带来的毁灭性损伤被认为是心肌缺血再灌注损伤（IRI）发生的重要原因。有研究表明AMP激活的蛋白激酶(AMPK)不仅是调节细胞能量代谢的靶基因，而且还是程序性坏死中的感受分子。但AMPK在心肌IRI诱导的程序性坏死中的作用一直不明。基于我们的前期研究结果，我们初始的研究假设是＂AMPK在IRI引起的心肌细胞程序性坏死中起重要作用，应用AMPK激活剂可起到防护作用。. 为明确AMPK在IRI引起的程序性坏死中的作用。首先我们通过药物诱导的细胞坏死中，激活AMPK在细胞活性、有毒物质释放、DNA 损伤修复等中的作用；观察在程序性坏死中，AMPK表达、磷酸化和上下游激酶水平改变，探讨AMPK与程序性坏死相关蛋白的相互作用，明确AMPK参与调控坏死相关蛋白的具体分子机制；最终全面评估AMPK激活剂二甲双胍在大鼠心肌IRI中的作用。. 我们的研究成果进一步拓展AMPK的功能研究，率先阐述该分子在细胞程序性坏死中的作用，为程序性坏死信号通路中的分子调控增加新的认识。此外，此项研究成果将为临床心肌IRI防护工作提供新的实验室依据和理论，为心肌IRI防护分子靶点的选择提供新的视角和思路。最后，二甲双胍治疗具有成本低廉、实施方便、安全系数高等的优点。研究证实二甲双胍通过激活AMPK促进Keap1介导的PGAM5降解而减少心肌IRI所致的心肌细胞程序性坏死，为将来临床应用二甲双胍辅助治疗冠心病等心肌缺血损伤等提供了崭新的前景和新思路，通过节省巨额医疗费用,获得巨大的社会效益。. 总之，本项目证实二甲双胍通过激活AMPK促进Keap1介导的PGAM5降解而减少心肌IRI所致的心肌细胞程序性坏死，项目发表多篇研究论文，已达到预定目标。此外，在本课题资助的计划外，本课题组联合院内兄弟学科和省外知名团队共同发表多篇论文，进一步探讨多种药物在代谢性疾病及心肌保护中的作用及分子机制。