血清和糖皮质激素诱导的蛋白激酶1（SGK1)通过激活NLRP3炎症小体介导急性心肌梗死后心脏损伤的机制研究

Recently, increasing evidence demonstrates that the inflammasome as central mediators of inflammatory immune response, could play an important role in development of cardiac remodeling. However, which intracellular signals activate the NLRP3 inflammasome contributing to postinfarction inflammatory response and cardiac adverse remodeling remains unclear. Our preliminary studies suggested that SGK1 expression was elevated after myocardial infarction (MI); SGK1-/- mice showed reduced infarct size and improved cardiac function compared with WT mice post-MI. Importantly, RNA-Seq data indicated that deficiency of SGK1 inhibited NLRP3 or IL-1β expression in the infarct myocardium after MI. Thus, we hypothesized that SGK1 might be fundamentally involved in activation of the NLRP3 inflammasome, trigger cytokines production contributing to adverse cardiac remodeling after infarction. The goals of this project are: 1) to determine the critical roles of bone marrow-derived SGK1 in postinfarct inflammatory response leading to cardiac injury; 2) to explore the mechanism by which SGK1 regulates the NLRP3 inflammasome; 3) to elucidate how SGK1 promotes postinfarct injury via the NLRP3 inflammasome. We will use WT and SGK1-/- mice undergoing permanent ligation of the left anterior coronary artery in combining with bone marrow transplant, Bio-plex assay and NLRP3shRNA injection in vivo. Moreover, to evaluate the effect of SGK1 on NLRP3 gene expression on the transcriptional level, the DNA binding activity of transcription factors will be investigated by dual-luciferase reporter assay, CHIP assay and EMSA assy. Furthermore, we will examine the expression levels of SGK1 and NLRP3 in periphery blood monocytes which from AMI patients or healthy people, and will further evaluate the relationship between SGK1 or NLRP3 and myocardial injury biomarkers. The proposed studies will provide new knowledge regarding the molecular mechanisms of how inflammatory response participates the pathogenesis of postinfarct injury, and provide the theoretical and experimental basis for intervention of adverse remodeling after MI.

近期发现炎症免疫反应的核心炎症小体是促进心血管重塑的重要机制，但是急性心梗后通过激活哪些关键分子活化NLRP3炎症小体进而介导心脏损伤仍不清楚。我们前期研究发现，心梗可激活心脏组织SGK1，敲除SGK1可减少梗死面积及NLRP3基因表达。据此我们假设：SGK1通过激活NLRP3炎症小体，释放大量炎症因子导致心梗后心脏损伤。本申请通过骨髓移植、Bio-plex悬液芯片、体内注射NLRP3干扰载体、流式细胞术结合病理等技术研究骨髓来源的巨噬细胞中SGK1 活化对NLRP3炎症小体及心梗的影响；采用分子克隆、荧光素酶活性检测、ChIP及EMSA等技术探索SGK1调控NLRP3表达的分子机制；进一步检测急性心梗病人SGK1及NLRP3炎症小体表达，并分析与心梗后心肌酶水平的相关性。本研究将阐明心梗激活SGK1调节NLRP3炎症小体介导心脏损伤的分子机制，为寻找和筛选新的药物靶点提供重要的科学依据。

炎症反应在心肌梗死后心室重塑和心力衰竭的发生发展过程中起重要作用，对急性心肌梗死患者的预后有重要的影响。因此，如何控制心肌梗死后心脏的炎症反应过度激活，则可能成为未来临床治疗的潜在亮点。大量的证据显示了以中性粒细胞、巨噬细胞浸润为主的炎症反应是造成心梗后心脏组织损伤的重要机制。但是，炎症细胞如何在心肌损伤中发挥关键作用，以及重要的信号调控途径目前仍不清楚。.SGK1是应激条件下快速诱导产生的蛋白激酶，我们的前期研究发现SGK1在心脏纤维化及血管重塑的形成、发展中发挥重要作用。此外，新近的研究发现：炎症小体是天然免疫系统的重要组成部分，炎症小体可能参与多种心血管疾病的发生及发展过程。本项目中我们以SGK1基因敲除鼠为基础，并采用SGK1特异性抑制剂处理，深入研究SGK1在心梗导致心脏损伤中的作用以及调控机制，发现SGK1通过激活、放大炎症反应，在心梗后心肌损伤中发挥关键作用；SGK1抑制剂可对心肌损伤发挥明显的保护作用。通过分析SGK1对NLRP3炎症小体的调控作用，揭示了SGK1活化通过激活NLRP3炎症小体，导致心脏损伤的生物学过程；通过抑制SGK1可通过阻断炎症小体的激活，减轻心脏纤维化。进一步研究发现，SGK1活化可诱导心脏成纤维细胞合成大量的趋化因子，通过招募大量巨噬细胞的迁移，扩大炎症反应。此外，本研究在不同的微环境刺激下，进一步探索介导巨噬细胞活化的关键靶点，研究发现粘附分子ICAM-1通过阻滞巨噬细胞的吞噬功能，抑制巨噬细胞向M2型分化。.本研究阐明了SGK1在心梗导致心脏损伤中的作用及分子机制，明确SGK1对巨噬细胞炎症小体活化、心脏成纤维细胞放大炎症反应的调控机制，将为心脏损伤的预防、检测、治疗及新的药物靶点的研发提供重要的科学依据。通过本课题的研究，SGK1可能成为今后临床防治心力衰竭的一个新的治疗靶点。此外，本研究在不同的微环境刺激下，进一步阐明介导巨噬细胞活化的关键靶点，为炎性微环境作为疾病治疗目标提供更多的依据，并且可能提供新的潜在治疗靶点。