eIF2α乙酰化修饰在尿毒症小鼠心肌缺血再灌注损伤中的作用机理研究

Uremia increases the susceptibility to myocardial ischemia reperfusion (I/R) injury, and acetylation of eukaryotic initiation factor 2α (eIF2α) is a potential key target for myocardial I / R injury. However, its role and mechanism in uremic myocardium injury are not clear. Previous studies showed that eIF2αdeacetylation significantly reduced myocardial I / R injury; eIF2α acetylation in uremic myocardium was significantly increased, and Sirt1 expression was significantly reduced. We presumed that targeting eIF2α acetylation modification might significantly reduce uremic myocardial I / R injury. The uremia model was constructed in mice by 5/6 subtotal nephrectomy. By myocardial specific eIF2α acetylation site mutation and co-immunoprecipitation techniques, we will observe the ATF4/CHOP signal pathway mediated myocardial apoptosis after I/R injury, explore the upstream regulatory target of eIF2α acetylation modification, and clarify the mechanism of eIF2α acetylation modification in uremic myocardial I / R injury. This project will reveal the mechanism of increased susceptibility to myocardial I / R injury in uremic myocardium from the new perspective of eIF2α acetylation modification, and provide an effective intervention target for uremic myocardial protection.

尿毒症增加心肌缺血再灌注（I/R）损伤易感性，真核细胞翻译启始因子2α（eIF2α）乙酰化修饰是影响心肌I/R损伤的潜在关键靶点，然而其在尿毒症心肌损伤中的作用及机制尚不明确。前期研究显示，eIF2α去乙酰化明显减轻心肌I/R损伤；尿毒症心肌eIF2α乙酰化水平明显上调，而去乙酰化酶Sirt1表达显著下调，基于此，本项目提出“eIF2α乙酰化靶向修饰显著减轻尿毒症心肌I/R损伤”的科学设想。拟借助5/6肾切除构建小鼠尿毒症模型，从细胞和整体水平，采用心肌特异性eIF2α乙酰化位点突变及免疫共沉淀等技术，观察I/R损伤后ATF4/CHOP信号通路介导的心肌细胞凋亡，明确eIF2α乙酰化修饰的上游调控靶点，阐明eIF2α乙酰化修饰在尿毒症心肌I/R损伤中的作用机理。本项目将从eIF2α乙酰化修饰这一全新角度，揭示尿毒症增加心肌I/R损伤易感性的机制，为尿毒症心肌保护提供有效干预靶点。

胸痛中心建设明显提高急性心肌梗死患者预后，然而再灌注损伤作为一个重要的临床问题至今未有效解决，尤其是临床患者常合并慢性肾脏疾病甚至尿毒症等心血管疾病危险因素，明显增加了心肌缺血再灌注损伤易感性。真核细胞翻译启始因子2α（eIF2α）乙酰化修饰是影响心肌I/R损伤的潜在关键靶点，因此研究eIF2α乙酰化修饰对尿毒症个体心肌缺血再灌注损伤的作用及机制具有重要价值。我们发现与正常个体对照，尿毒症明显增加心肌缺血再灌注损伤eIF2a（K143）乙酰化水平，激活下游ATF4/CHOP信号通路，增加内质网应激介导的心肌细胞凋亡，而其上游调控蛋白Sirt1被明显抑制。.通过腺相关病毒定向突变心肌细胞eIF2a（K143）乙酰化位点，我们发现，在正常与尿毒症小鼠，eIF2a（K143R）乙酰化程度均明显抑制，显著降低心肌梗死面积，减少心肌细胞凋亡，改善心功能，同时抑制ATF4/CHOP信号通路，而eIF2a（K143Q）乙酰化程度均明显激活，加重心肌缺血再灌注损伤，同时激活ATF4/CHOP信号通路。.Sirt1基因敲除显著加重正常小鼠心肌缺血再灌注损伤程度，明显增高eIF2 α乙酰化水平，激活ATF4/CHOP信号通路。腺相关病毒定向突变心肌细胞eIF2a（K143R）去乙酰化，我们发现，SIRT1敲除加重心肌缺血再灌注损伤的作用被取消，ATF4/CHOP信号通路被明显抑制。尿毒症小鼠在基础状态及缺血再灌注后Sirt1表达均明显低于正常小鼠，给予尿毒症小鼠Sirt1激动剂SRT1720预处理后行缺血再灌注损伤，Sirt1蛋白表达显著增加，心肌梗死面积明显降低，心肌细胞凋亡显著减少，心功能明显改善，同时eIF2 α乙酰化水平及ATF4/CHOP信号通路相关蛋白表达明显降低。暗示Sirt1调控的eIF2α乙酰化修饰可以作为一个潜在的尿毒症心肌缺血再灌注损伤的治疗靶点。