抗糖尿病心肌缺血/再灌注损伤新机制：褪黑素膜受体-PKGIα-SIRT1介导褪黑素心脏保护作用的关键信号通路研究

Mitochondrial dysfunction in diabetic state aggravates myocardial ischemia/reperfusion injury (MI/RI). However, there is still a lack of effective therapeutic strategy. We previously found that melatonin could preserve myocardial mitochondrial function and reduce MI/RI in diabetic state, while melatonin membrane receptors and its downstream cGMP-PKGIα signaling pathway might mediate this effect. However, the specific type of melatonin receptor involved in this process and the detailed mechanisms have not been reported previously, which severely limit the further clinical application of melatonin. Silent information regulator 1 (SIRT1) plays an important role in the regulation of myocardial mitochondrial function and the nuclear translocation of SIRT1 is a critical process for its cytoprotective actions. The result of our pre-experiment showed that melatonin could promote SIRT1 nuclear translocation in diabetic ischemic myocardium, while the melatonin receptor antagonist inhibited this effect, suggesting that activation of melatonin membrane receptor and its downstream PKGIα pathway is likely to activate the SIRT1 signaling, thus preserving the myocardial mitochondrial function. The aim of this study was to elucidate the mechanisms of melatonin against diabetic MI/RI in the in vitro and in vivo settings by employing cardiac-specific SIRT1 knockout mice and melatonin membrane receptor knockout mice. This study will not only provide a theory for further clinical trials, but also provide new strategies and new targets for the prevention and treatment of diabetic myocardial ischemic injury.

线粒体功能障碍加重糖尿病心肌缺血/再灌注损伤（MI/RI），临床缺乏有效干预策略。我们发现褪黑素可维护心肌线粒体功能、减轻糖尿病MI/RI，且褪黑素膜受体及下游cGMP-PKGIα通路可能介导此作用，但参与此过程的褪黑素受体具体亚型及确切机制尚无报道，限制褪黑素临床应用。沉默信息调控因子1（SIRT1）对心肌线粒体功能发挥重要调节作用且SIRT1细胞核转位是其发挥细胞保护作用的关键过程。我们预实验发现褪黑素可促进糖尿病缺血心肌SIRT1核转位，而褪黑素膜受体拮抗剂抑制此作用，提示活化褪黑素膜受体及下游PKGIα通路很可能是激动SIRT1信号进而维护糖尿病心肌线粒体功能的关键机制。本项目拟利用心肌特异性SIRT1基因敲除小鼠和褪黑素膜受体基因敲除小鼠，在细胞与在体水平阐明褪黑素膜受体介导褪黑素抗糖尿病MI/RI的详细机制，为其临床试验提供理论依据，为防治糖尿病心肌缺血损伤提供新策略和新靶点。

心肌缺血/再灌注损伤（MI/RI）是糖尿病缺血性心脏病预后不良的主要原因，而线粒体功能障碍是MI/RI发生的关键机制，近年来外源性线粒体靶向药物越来越受到重视但仍无法取得理想的效果，加之外源性药物存在安全性、伦理学等潜在问题，寻找人体内源性分子维护糖尿病心肌线粒体功能、减轻缺血损伤对糖尿病心肌缺血损伤的防治具有重要的意义。为阐明褪黑素通过其细胞膜受体调控糖尿病心肌线粒体功能进而抗糖尿病心肌缺血损伤的作用和详细机制，本项目进行了下列工作：构建在体动物糖尿病模型及高糖体外培养的心肌细胞模型研究心脏cGMP-PKG1α、Nrf-2-HO-1及SIRT1信号活水平在糖尿病状态下的改变，确定褪黑素对糖尿病动物心室SIRT1细胞定位及线粒体功能的调控作用，在体及细胞水平明确了MT2是参与褪黑素糖尿病心肌保护作用的受体亚型，揭示了cGMP-PKGIα促进SIRT1核转位的关键作用，阐明了褪黑素通过其细胞膜受体激动SIRT1信号进而调控糖尿病心肌线粒体功能的详细分子机制。该项目为褪黑素应用于糖尿病缺血心肌损伤的防治提供关键的理论及实验证据，为内源性小分子抗糖尿病心肌线粒体损伤治疗提供了新思路，同时，本项目的顺利完成也进一步揭示了糖尿病MI/RI加重的分子机理及褪黑素的生物作用的多样性。