髓样分化蛋白1对心肌缺血再灌注损伤的作用及其机制的研究

Myocardial ischemia–reperfusion (I/R) injury severely impacts the postoperative survival rate of coronary atherosclerotic heart disease. The pathogenesis of myocardial I/R injury involves various mechanisms including inflammatory responses, accumulated free radical, and cell apoptosis. Toll-like receptor 4 (TLR4) signaling pathway plays an important role in inflammatory responses. Previous studies showed that excessive activation of Toll-like receptor 4 (TLR4) was close related to myocardial I/R injury. Studies have tested the efficacy of inhibiting TLR4- mediated inflammatory responses in treatment of myocardial I/R injury. Myeloid Differentiation 1 (MD-1), also known as Lymphocyte Antigen 86 (Ly86), a secreted protein interacting with radioprotective 105 (RP105), plays an important role in Toll-like receptor 4 (TLR4) signalling pathway. Recent studies have shown that the RP105–MD-1 complex is a physiologically negative regulator of TLR4 signalling. However, we then ask whether MD-1, as an inhibitor of the TLR4 signaling pathway, plays a critical role in myocardial I/R injury. Our recent study showed that MD-1 inhibits cardiac hypertrophy and postpones cardiac dysfunction during the remodeling process, which is dependent on its modulation of the TLR4 signaling axis. Therefore, in the present study, we will investigate the effects and mechanisms of MD-1 on myocardial ischemia–reperfusion (I/R) injury using in vivo and in vitro techniques, and molecular biology methods in transgenic mice with cardiac-specific overexpression of human MD-1 gene and MD-1 knockout mice. We aim to clarify the effects and mechanisms of MD-1 on myocardial ischemia–reperfusion (I/R) injury and provide experimental evidence and new molecular target for treatment and prevention of myocardial ischemia–reperfusion (I/R) injury.

心肌缺血再灌注损伤严重影响着冠心病术后的生存率。其病理机制包括炎症反应、自由基的增加和细胞凋亡。TLR4信号通路是炎症反应的重要通路。既往研究显示在心肌缺血再灌注损伤中TLR4出现了过度的激活，阻止TLR4介导的炎症反应能有效的阻止缺血再灌注损伤。髓样分化蛋白1(MD-1)，它与放射性保护蛋白105结合是TLR4信号通路的天然负性调节因子。然而，MD-1能否通过抑制TLR4信号通路发挥抗心肌缺血再灌注损伤的作用？目前国内尚无报道。最近我们的研究显示MD-1能通过抑制TLR4信号通路发挥改善心肌重构的作用。有鉴于此，本项目拟运用MD-1基因敲除小鼠和心脏特异性MD-1过表达小鼠，通过在体、离体和细胞水平的研究，结合分子生物学研究，对MD-1在心肌缺血再灌注损伤中的作用及其机制展开系统研究，力图明确MD-1 对心肌缺血再灌注损伤的影响及其机制，为有效防治心肌缺血再灌注损伤提供理论依据和新靶点。

急性心肌梗死是一种最常见而且很严重的疾病，具有较高的死亡率。既往研究暗示心肌缺血再灌注损伤可能与多种机制相关，如氧化应激及炎症反应等。因此，更好的理解和探索心肌梗死的机制显的尤其重要。.我们应用野生型和MD-1敲除小鼠建立了缺血再灌注模型。我们EB/TTB染色评价心肌梗死、超声及血流动力学监测评价心脏功能。应用病理及分子生物学技术检测心肌细胞凋亡。同时我们检测心肌损伤标志物、炎症因子、氧化应激标志物的表达， WB方法检测了p-P38、p-JNK、p-MEK、p-ERK、p-NF-κB/P65、PI3K、p-AKT蛋白的表达。.我们将构建的过表达MD-1（Ad MD-1）或MD-1 RNA干扰腺病毒（Adsh MD-1），及相应的对照组AdGFP、AdshRNA腺病毒感染原代乳鼠心肌细胞。应用TUNEL、CCK8及Caspase3 活性检测评价了心肌细胞凋亡，应用WB方法检测Bax、Bcl-2凋亡相关蛋白的表达。同时检测了氧化应标志物SOD及MDA的水平。WB方法检测了p-P38、p-JNK、p-NF-κB/P65蛋白的表达。.结果：1）敲除MD-1基因加重了缺血再灌注心肌细胞凋亡及氧化应激，同时心功能恶化更明显。机制研究缺血再灌注组p-P38、p-JNK、p-MEK、p-ERK、p-NF-κB/P65、PI3K、p-AKT表达上调，MD-1 敲除后p-P38、p-JNK、p-MEK、p-ERK、p-NF-κB/P65、PI3K、p-AKT的表达更明显。2）过表达MD-1减轻缺血再灌注心肌细胞凋亡及氧化应激。在缺血再灌注组MD-1的缺失上调了p-P38、p-JNK and p-NF-κB/P65蛋白的表达。过表达MD-1则发挥相反的作用。结论：1）MD-1基因缺失进一步加重心肌缺血再灌注后的氧化应激、炎症反应、心肌细胞凋亡、心肌组织的损伤以及心肌梗死面积，从而恶化心脏功能及降低小鼠的远期生存率。2）MD1基因缺失进一步激活 MAPK 、NF-κB 及PI3K/AKT信号通路。3）过表达MD-1减轻心肌缺血再灌注损伤，MD-1缺失则加重了心肌缺血再灌注损伤。4）过表达MD-1通过抑制p38、JNK1/2 and NF-κB通路保护心肌缺失再灌注损伤，而MD-1缺失起相反作用。.MD-1在心肌缺血再灌注损伤中起着重要的作用，MD-1可能成为治疗心肌缺血再灌注损伤的新靶点。

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