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

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.

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。

急性心肌梗死是国民健康的头号杀手。心梗后，大量氧氮自由基的产生，花生四烯酸代谢的活化及炎性巨噬细胞的浸润，影响心肌的炎症损伤和修复。目前尚不清楚花生四烯酸代谢产物的作用靶点及对巨噬细胞分型和功能的影响。..本课题组前期利用含端基炔烃的花生四烯酸处理巨噬细胞，用点击化学引入生物素，经纯化及质谱鉴定明确了花生四烯酸代谢产物15-keto-PGF2α与PKM2共价结合。进一步做了以下三个方面的工作：1）阐明15-keto-PGF2α通过调控PKM2来促进M2型巨噬细胞的极化和吞噬能力的分子机制。在细胞水平上，观察到15-keto-PGF2α促进PKM2活性，但抑制PKM2参与的细胞信号传导、验证了15-keto-PGF2α与PKM2共价结合可改变巨噬细胞M1/M2极化，提高巨噬细胞吞噬能力。在动物水平上，利用免疫组化等技术等确认15-keto-PGF2α在小鼠心梗模型中对巨噬细胞极化和吞噬能力的影响。2）确定花生四烯酸代谢产物15-keto-PGF2α的共价结合靶点及相关的结构特异性。将15-keto-PGF2α与重组PKM2蛋白孵育后，质谱分析发现15-keto-PGF2α结合到含Cys48的多肽，用分子对接技术确认15-keto-PGF2α结合相应位点，通过定点基因突变技术确认15-keto-PGF2α对PKM2有关的细胞信号传导的影响。3）揭示15-keto-PGF2α调控巨噬细胞的极化和功能而减轻小鼠心肌缺血再灌注损伤的作用机制。发现15-keto-PGF2α促进巨噬细胞从促炎性M1型向抗炎性M2型转化，并且减少心脏梗死面积和炎性细胞浸润。..本课题验证了“花生四烯酸代谢产物15-keto-PGF2α可能通过共价结合丙酮酸激酶PKM2而调控巨噬细胞极化和功能，从而减少心肌缺血再灌注损伤”。有助以PKM2为靶标，以15-keto-PGF2α为先导化合物，开发治疗心肌梗塞疾病的新药。

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