花生四烯酸代谢产物15-keto-PGF2α通过结合共价丙酮酸激酶PKM2调控巨噬细胞M1/M2极化而降低心肌缺血再灌注损伤

花生四烯酸代谢产物多在机体免疫，炎症及心血管功能中起重要作用。鉴定其靶蛋白在生物学和药学上具重要价值。本课题组前期以端基炔烃衍生物为分子探针，巨噬细胞为模型，经生物素标记、亲和分离和蛋白组学鉴定，发现前列腺素衍生物15-keto-PGF2α主要与丙酮酸激酶PKM2共价结合。文献显示PKM2是治疗炎症性疾病的重要靶标。前期实验表明该衍生物不仅能显著促进巨噬细胞由促炎性向抗炎性转化和增强巨噬细胞的吞噬能力，而且能减轻心肌缺血再灌注损伤。因此本课题假设：前列腺素衍生物通过共价结合PKM2调控巨噬细胞极化而减轻缺血再灌注损伤。拟利用巨噬细胞和小鼠缺血再灌注模型，实现三个研究目标：研究该衍生物调控巨噬细胞极化及潜在分子机制；研究该衍生物与PKM2的结合位点及相关结构特征；研究该衍生物在体调控巨噬细胞与心肌损伤的关系。本课题为研究相关代谢产物的靶蛋白及为开发以调控巨噬细胞治疗心肌梗塞的药物提供新思路。

Arachidonic acid (AA) metabolites are implicated in various pathophysiological processes including immunity, inflammation and cardiovascular functions. It is of biological and pharmaceutical importance to identify the molecular targets and related signaling pathways for AA metabolites. In our pilot study, we employed ω-alkynyl arachidonic acid as molecular probe to identify the covalent AA metabolites-protein adducts in macrophage RAW264.7 cells as the cell model. Following Click chemistry biotinylation, affinity isolation and proteomic identification, we identified glycolytic enzyme pyruvate kinase M2 (PKM2) as the predominant protein target for 15-keto-PGF2α. We also demonstrated that 15-keto-PGF2α suppressed macrophage M1 polarization, promoted macrophage M2 polarization and markedly enhanced the phagocytosis. Importantly, we further discovered that 15-keto-PGF2α exhibited potent cardioprotective activities against acute myocardial ischemia-reperfusion injury. These results stimulate us to hypothesize that 15-keto-PGF2α may regulate the polarization and function of macrophages against myocardial ischemia-reperfusion injury via covalently binding to PKM2. We will pursue three specific aims: 1) to study the in vitro and in vivo effects of 15-keto-PGF2α on the polarization and function of macrophages; 2) to characterize the binding of 15-keto-PGF2α to PKM2 and related structural specificity; 3) to determine the in vivo cardioprotective activities of 15-keto-PGF2α against myocardial ischemia reperfusion injury via modulating macrophage polarization. The results of this project will provide the useful guidance to apply chemical biological approaches in the discovery of protein targets for related AA metabolites, and, importantly, pave the avenue for the development of novel macrophage-targeting drugs for the treatment of myocardial infarction and a broad spectrum of other inflammatory diseases.