缺血后处理中miR-499调控的热休克蛋白90信号通路对缺血再灌注损伤免疫炎症反应的影响

Reperfusion injury may cause myocardial cell death and limit the benefit achieved by restoration of coronary artery patency in patients with acute myocardial infarction. Activation of the innate immune system and inflammation are some of the prominent features of myocardial ischemia-reperfusion injury. microRNA-499 (miR-499) is highly expressed in hearts and has been implicated in suppression of apoptosis in myocardial infarction and after ischemia-reperfusion. Ischemic postconditioning, which is a series of brief (a few seconds) reperfusion/ischemia cycles at reperfusion onset, attenuates also ischemia-reperfusion injury. The mechanism whereby postconditioning relieves ischemia-reperfusion injury is not well known. Our previous studies have shown that protein kinase C (PKC) - signaling pathway mediated by heat shock protein (HSP) 90 played a important role in postconditioning cardioprotection. Recently, in a study conducted on human and murine it has been reported that HSP90 phosphorylation was directly affected by miR-499 in cardiomyopathy. Therefore, in the present study, we tested the hypothesis that miR-499 is involved in postconditioning cardioprotection by targeting HSP90, which are linked to PKC, nuclear transcription factor (NF-ΚB), c-jun N-terminal kinase (JNK) signaling pathways, ultimately inhibiting innate immune system including Toll-like receptors (TLRs), complements (Cs), and inflammatory cytokines such as interleukin (IL), tumor necrosis factor (TNF)-α, intercellular adhesion molecules (ICAM-1). Specifically, the model of cardiomyocytes with hypoxia-reoxygenation and rats with ischemia-reperfusion were established. The systemic and local immune and inflammatory factors such as TLR2, TLR4, C3, C5, IL-1, IL-6, TNF-α, ICAM-1 were detected after intervention with overexpression and underpression of microRNA-499 viral vectors and HSP90 inhibitors. PKC, NF-ΚB, and JNK signaling pathways was also investigated.

天然免疫系统激活和炎症反应是心肌缺血再灌注损伤(IRI）的重要特征，miR-499水平调控与IRI密切相关。缺血后处理可有效减轻IRI，然而其机制不明。我们前期研究显示热休克蛋白（HSP）90介导PKC信号通路参与缺血后处理心脏保护。已有研究证实miR-499对HSP90具有调控作用。因此我们假设缺血后处理中miR-499通过调控HSP90，进而调节天然免疫系统Toll样受体（TLR）、补体（C），减少炎症因子释放，减轻IRI。本研究通过建立大鼠离体和在体IRI模型，采用miR-499过表达、抑制表达病毒载体及HSP90抑制剂干预，检测全身及心脏免疫炎症因子TLR2，TLR4，C3，C5，IL-1，IL-6，TNF-α，ICAM-1的表达，同时检测PKC,核转录因子, c-jun氨基末端激酶信号通路，探讨后处理中miR-499调控的HSP90信号与免疫炎症间内在联系及信号转导机制

本研究通过建立离体心肌细胞缺氧复氧（H/R）和大鼠在体缺血再灌注（I/R）模型，实施缺氧（血）后处理，予miR-499过、抑制表达病毒载体及HSP90抑制剂干预，检测全身及局部免疫炎症因子TLR2，TLR4，C3，C5a, IL-1β，IL-6，TNF-α，ICAM-1的表达，探讨后处理中miR-499调控的HSP90信号与免疫炎症间内在联系。同时检测PKC, 核转录因子, c-jun氨基末端激酶信号通路，从细胞和动物水平探讨后处理中miR-499调控的HSP90信号转导机制。我们发现：.（1）后处理在上调HSP90表达的同时显著减少H/R及I/R心肌细胞及组织C3、C5a、 TLR-2、TLR-4及炎症因子TNF-α、IL-1β、IL-6、ICAM表达，减少心肌细胞凋亡及心梗面积，而应用HSP90特异性抑制剂GA显著抑制后处理上述效应，提示后处理通过上调HSP90表达抑制H/R及I/R心脏补体及TLRs介导的免疫炎性损伤。首次揭示了HSP90介导的后处理对天然免疫系统的调控机制。.（2）过表达miR-499可显著上调后处理组心肌细胞和组织HSP90表达，同时显著增强后处理对TLR2，TLR4，C3，C5a，IL-1β，TNF-α表达的抑制，显著减轻心肌细胞凋亡、心梗面积；抑制miR-499表达，上述作用显著减弱。提示后处理通过miR-499上调HSP90减轻TLRs及补体介导心脏炎性损伤和细胞凋亡。首次揭示了缺血后处理中miR-499通过HSP90调控心脏天然免疫系统发挥心脏保护作用。.（3）miR-499/HSP90介导的后处理通过抑制补体/JNK通路和TLR/NF-κB通路活化，减轻心脏免疫炎症损伤和细胞凋亡。此发现是对缺血后处理心肌保护作用机制的新补充。