ROCK介导的自噬抑制在冠脉微血管缺血/再灌注损伤中的作用及机制研究

Coronary microvascular dysfunction is the main cause of myocardial ischemia reperfusion (I/R) injury, and its mechanism has not been elucidated. Our previous studies showed that ROCK was activated in the coronary microvascular endothelial cells (CMVECs) after I/R. Inhibition of ROCK could ameliorate I/R-induced CMVEC injury. According to the literature, ROCK inhibitors can induce autophagy in many kinds of cells, such as cardiomyocytes, which play a protective role in cell stress and adaptation. Therefore, we hypothesized that inhibition of ROCK activity could induce protective autophagy and alleviate CMVEC damage induced by I/R. This project aims to establish I/R models both in vivo and in vitro. Molecular biological techniques, Western blot, iimmunofluorescence, cardiac ultrasonography are used to investigate the effect of fasudil on autophagy, coronary microvascular injury and cardiac function of the rat hearts after I/R; meanwhile, primary CMVECs are used for in vitro verification, and to further explore the underlying mechanism. This study aims to provide a new target for the treatment of myocardial infarction by elucidating the role and mechanisms of ROCK in I/R-associated coronary microvascular injury.

冠状动脉微血管完整性的破坏是造成心脏缺血后再灌注（I/R）损伤的重要原因，其机制尚未阐明。我们的前期研究发现，I/R后，冠脉微血管内皮细胞（CMVEC）中ROCK活性升高，抑制其活性可减轻I/R损伤。根据文献报道，ROCK抑制剂可诱导心肌细胞等多种细胞发生自噬，而自噬通常在细胞应激和适应过程中发挥保护作用。因此，我们推测抑制ROCK活性可通过诱导保护性自噬，缓解冠脉微血管I/R损伤。本项目拟从整体和细胞两个水平建立I/R模型，采用RNA干扰，Western blot、免疫荧光染色和心脏B超等手段，观察I/R条件下，ROCK抑制剂fasudil对大鼠心脏自噬体形成，自噬标志蛋白的表达以及冠脉微血管损伤、心脏功能的影响；同时在原代培养的CMVEC上进行验证，并进一步探讨ROCK调控自噬的机制。本课题通过阐明ROCK在I/R冠脉微血管损伤中的作用及机制，为临床心肌梗死的治疗提供新靶点。

冠脉微血管内皮损伤引起的供血功能障碍是导致缺血/再灌注后心肌坏死的重要因素，以往的研究多集中于心肌细胞，然而冠脉微血管内皮损伤的机制鲜有人研究。本项目通过综述Rho激酶（Rho-associated kinase，ROCK）在血管新生中的关键作用及机制，将ROCK作为研究对象，研究其在微血管损伤中的作用及机制。我们前期的研究表明，ROCK抑制缺氧/复氧引起的血管内皮细胞凋亡。通过本项目，我们还发现ROCK通过负调节VE-cadherin的蛋白表达参与血管内皮细胞通透性的调节。此外，我们还增加了对双氢青蒿素（dihydroartemisinin, DHA）的研究，结果表明，DHA显著地促进VECs发生自噬，同时产生大量活性氧，然而活性氧却不是导致自噬的原因。进一步的研究发现，DHA通过抑制Akt/mTOR信号通路参与诱导自噬。本研究详细阐明了ROCK在微血管功能损伤中的作用及其调控血管内皮通透性改变的分子调控机制，进一步完善了冠脉微血管损伤的分子生物学机制，并为疾病的防治提供新的作用靶点，具有重要的理论意义。