Rap1介导NFkB信号通路及NLRP3炎症小体的活化加剧心肌缺血再灌注损伤

Myocardial ischemic disease is a leading cause of death all of the world. In clinical, the best effective way is restoration of blood flow, but reperfusion itself may cause additional tissue injury called myocardial “ischemia/reperfusion (I/R) injury”. Our preliminary work demonstrated that in H9C2 cardiomyocytes, hypoxia/reoxygenation (H/R, 6 hours hypoxia followed by 12 hours reoxygenation) significantly increased both mRNA and protein levels of the telomeric protein repressor activator protein 1 (Rap1). Rap1 knockdown significantly suppressed H/R-induced cell injury and cell apoptosis when compared to mock-transfected cardiomyocytes. Rap1 also enhance NFkB signaling pathway and facilitate the activation of NLRP3 inflammasome in THP-1 macrophages. Of note, plenty of studies demonstrated that, during myocardial I/R injury, there is strong enhancement of NFkB-mediated pro-inflammatory cytokines and chemokines expression and activation of NLRP3 inflammasome as well. Thus, it is feasible to hypothesize that Rap1 plays an active role in modulating myocardial I/R injury, and that elevated Rap1 expression contributes to the activation of inflammatory responses during myocardial I/R injury. The present proposal is designed to test the above hypothesis using both loss- and gain-of-function approaches. In particular, the specific questions will be addressed as follows: (1) Does Rap1 exacerbate myocardial I/R injury in mice? (2) Does Rap1 contribute to inflammatory responses through NFkB signaling or NLRP3 inflammasome formation during myocardial I/R injury? The proposed studies will improve our understanding of the pathophysiology of myocardial I/R injury and may help facilitate the development of novel and better therapies for the management of ischemic heart disease.

临床上心脏再灌注是降低心肌缺血损伤最有效的办法，但其本身却可诱导额外的心脏损害和综合症，称为“心肌缺血再灌注（I/R）损伤”。我们前期工作发现在大鼠H9C2心肌细胞中，伴随着缺氧复氧（H/R）诱导的细胞损伤及凋亡，端粒结合蛋白Rap1表达显著升高。Rap1基因敲减后，H/R诱导的细胞损伤及凋亡显著降低，表明Rap1可能介导I/R损伤。同时Rap1也能在巨噬细胞中增强NFkB炎症信号通路以及促进NLRP3炎症小体的活化。鉴于NFkB, NLRP3信号通路的增强同样能加剧I/R损伤，由此我们假设，Rap1可能介导NFkB信号通路及NLRP3炎症小体的活化加剧I/R损伤。本项目拟用Rap1敲除小鼠及在小鼠上建立Rap1过表达模型，通过离体在体I/R模型，干扰NFkB及NLRP3活性实验，及成年小鼠原代心肌细胞，心脏成纤维细胞H/R实验，为I/R损伤的治疗提供新的实验及理论依据。

端粒结合蛋白Rap1不仅仅在细胞核内发挥着维护端粒长度及功能的作用，同时也具有非端粒的功能。研究发现Rap1参与缺氧诱导的细胞凋亡以及NFkB介导的炎症反应，而心肌细胞凋亡及炎症反应是心肌缺血/再灌注损伤(I/RI)的重要分子机制。因此本研究利用体外、离体和体内实验探讨 了Rap1在心肌I/RI中的作用，并确定其潜在调控分子机制。 研究发现在心肌I/RI的小鼠模型中(左冠状动脉降支结扎30分钟，然后再灌注2小时)，Rap1缺乏显著降低了心肌梗死面积并改善了心脏收缩/舒张功能，且伴随着缺血后心肌细胞凋亡和促炎反应的降低。外源性补充Rap1水平取消了Rap1缺乏对心肌I/RI的保护作用。在心肌细胞中，Rap1敲低通过增加STAT3在Ser727位点的磷酸化/活化，显著抑制缺氧/复氧(H/R)诱导的细胞损伤和细胞凋亡。而 Stattic(选择性 STAT3 抑制剂)预处理取消了上述保护作用。此外，免疫共沉淀实验揭示了Rap1和STAT3直接结合。虽然Rap1敲低不影响H/R或脂多糖的H9C2心肌细胞的促炎反应，或心脏成纤维细胞中NLRP3炎症小体的形成，但Rap1的敲低显著抑制了THP-1巨噬细胞中NLRP3炎症小体的形成。此外，当缺氧后H9C2心肌细胞与Rap1-siRNA 转染的THP-1巨噬细胞共培养时，巨噬细胞中Rap1的敲低显著减弱了巨噬细胞介导的心肌细胞损伤。由此我们推断Rap1缺乏可以通过至少两种不同的分子机制保护心脏免受缺血性损伤，包括 通过激活STAT3信号通路减少心肌细胞凋亡和抑制巨噬细胞对缺血心肌的浸润导致的炎症反应。综上所述，本项目的研究发现有助于设计靶向 端粒结合蛋白Rap1的药物，从而治疗缺血性心脏病。