褪黑素通过调节SIRT3介导的氧化应激/线粒体自噬在心肌缺血再灌注损伤中的作用及其机制研究

Cardiac damage caused by myocardial ischemia is the main cause of cardiovascular disease. Restore myocardial perfusion as soon as possible is the key point for the clinic treatment. Recent studies found that myocardial ischemia with inadequate oxygen supply followed by successful reperfusion initiates a wide and complex array of inflammatory responses that may both aggravate myocardial injury as well as induce impairment of remote organ function, which is called ischemia-reperfusion injury (IRI). How to attenuate IRI has been becoming the key question in the treatment of myocardial ischemia...The mitochondria are the control center of energy supply for cardiocytes. Our previous studies found that IRI increases the expression of voltage-dependent anion channel 1 (VDAC1), the opening of mitochondrial permeability transition pore (mPTP) and the production of mitochondrial reactive oxygen species (ROS) and then aggravates mitochondrial injury. SIRT3 can attenuate mitochondrial injury by reducing mPTP opening. Melatonin (MT) plays an important role in the clearance of ROS in myocardial mitochondria. No matter the over-release or reduction of clearance for mitochondrial ROS will induce mitochondrial autophagy and finally aggravate mitochondrial injury. Thus, we imagine that MT may play a protective role through regulating SIRT3-mediated oxidative stress and mitochondrial autophagy on hypoxia / reoxygenation injured cardiomyocytes...In this study, we will first establish the hypoxia / reoxygenation injury model of isolated cardiomyocytes to verify whether MT may play a protective role through regulating SIRT3-mediated oxidative stress and mitochondrial autophagy on hypoxia / reoxygenation injured cardiomyocytes. siRNA, laser scanning confocal microscope and other technologies will be applied. Secondly, we will further study and confirm in vivo experiments using SIRT3-specific inhibitors in rat model of myocardial ischemia-reperfusion injury. The best dose and using time of MT will be investigated...Our study aims to find a new intervention and therapeutic target for the clinical treatment of myocardial ischemia-reperfusion injury.

心肌缺血导致心肌损伤是心血管疾病的主要病因，尽早恢复心肌灌注是关键。但当心肌组织重新获得血供时，却会发生缺血-再灌注损伤（IRI）。IRI是心肌缺血治疗中的难题。.线粒体是心脏能量产生的控制中心。我们前期的研究发现IRI时，线粒体电压依赖性阴离子通道表达升高，线粒体膜通透性转换孔（mPTP）开放增多，活性氧（ROS）产生增加，可诱导线粒体自噬，加重线粒体损伤。褪黑素（MT）在清除心肌线粒体过多ROS方面有重要作用，SIRT3可阻止mPTP开放。故我们提出MT通过调节SIRT3介导的氧化应激/线粒体自噬在心肌缺血再灌注损伤中发挥保护作用的设想。.我们将分别在心肌细胞缺氧/复氧损伤模型和大鼠心肌缺血再灌注损伤模型中应用细胞分子生物学等相关技术，用SIRT3特异siRNA处理细胞及SIRT3选择性抑制剂3-TYP在体进一步阐述这一设想。为临床心肌缺血再灌注损伤找到新的治疗靶点提供依据。

背景：缺血再灌注损伤（IRI）一直是心肌缺血治疗中难以解决的问题。IRI发生原因包括活性氧产生过多、线粒体动力学失调以及自噬紊乱等。沉默调节蛋白3（SIRT3）已被证实可通过改善线粒体自噬改善心肌IRI。褪黑素对于心肌IRI有保护作用，但褪黑素对线粒体途径的心肌保护作用尚不明确。本研究的研究目的是探究褪黑素能否通过SIRT3调控线粒体途径对IRI心肌细胞起保护作用.方法：建立离体心肌细胞缺氧/复氧损伤模型，进行褪黑素预处理及后处理，使用抑制剂或小干扰RNA对SIRT3的表达进行抑制，检测心肌细胞凋亡、氧化应激和线粒体功能、线粒体自噬等指标，探索褪黑素最佳剂量及使用时机，并观察SIRT3的作用。建立大鼠心肌IRI模型，进行在体实验，进一步研究和证实。.结果：褪黑素预处理/后处理减少心肌细胞凋亡，降低LDH和CK-MB水平，降低细胞内Ca2+浓度，降低ROS和MDA水平，升高SOD活性，同时稳定线粒体膜电势。褪黑素预处理及后处理可通过SIRT3显著降低LC3 II/LC3 I比值、增加p62蛋白表达水平。褪黑素预处理及后处理可通过SIRT3抑制线粒体通透性膜转换孔的开放，降低Bax、Cyt-C、caspase-3 和VDAC的表达。.结论：褪黑素预处理和后处理可通过SIRT3调节线粒体自噬通路发挥心肌保护的作用，这提示褪黑素可能成为减轻 MI/R 损伤的新型药物，为褪黑素在 I/R 损伤治疗中提供了新的理论基础。