膜联蛋白A1三肽在糖尿病心肌缺血-再灌注损伤中的保护作用及线粒体机制研究

The morbidity and mortality of myocardial ischemia-reperfusion injury (MIRI) are much higher in diabetic patients and the efficient prevention and cure are limited. We have reported that Annexin A1 short peptide (ANXA1sp) can efficiently attenuate MIRI via increasing the expression and the activity of Sirtuin 3 (Sirt3) which locates in mitochondria. Based on our previous findings, we hypothesize that ANXA1sp relieves MIRI by stabilizing the mitochondria homeostasis. The in vivo and ex vivo diabetic MIRI model and the in vitro adult diabetic cardiomyocyte ischemia-reperfusion injury model will be set, the ANXA1sp will be given at the onset of reperfusion. Then, the effects of ANXA1sp under the diabetic MIRI circumstance on 1) the short- and long-term cardiac function, biomarkers of heart ischemic injury, the infarct area and the ultrastructural changes of myocardium; 2) the cardiomyocyte viability, intracellular free calcium and ATP content; 3) the mitochondria structure, mitochondrial membrane potential (ΔΨm), the opening level of mitochondrial permeablity transition pore (mPTP), the respiratory function and the flowout of Cyt c; 4) the expression of Sirt3, the roles of mitochondrial ATP sensitive potassium channel (mitoKATP), the oxidative stress biomarkers and mitochondrial related apoptosis biomarkers will be evaluated. These studies will not only reveal the effects of ANXA1sp on MIRI in diabetic rats, but also help to develop drugs that targeting ANXA1sp, Sirt3 or mitoKATP.

糖尿病患者心肌缺血-再灌注损伤（MIRI）发病率及致死率高，且缺乏有效的防治手段。前期证实膜联蛋白（Annexin）A1短肽（膜联三肽）可通过线粒体内去乙酰化酶3（Sirt3）明显减轻MIRI，因此提出膜联三肽通过调控线粒体稳态减轻糖尿病MIRI这一假说。本研究拟建立糖尿病大鼠心脏在体、离体、成年鼠心肌细胞缺血-再灌注模型，并于再灌注即刻给予膜联三肽，对比观察糖尿病MIRI情况下，膜联三肽对①近远期心功能、心肌损伤标志物、心梗程度、心肌组织微结构；②心肌细胞活力、细胞內钙、ATP含量；③线粒体结构、膜电位、mPTP开放水平、呼吸功能、Cyt c溢出情况；④Sirt3表达量、线粒体ATP敏感性钾通道（mitoKATP）的作用及氧化应激、线粒体凋亡途径相关指标的影响。本项目将进一步阐明膜联三肽对糖尿病心肌的保护作用，也将有助于开发以膜联蛋白、Sirt3、mitoKATP为靶点的药物。

糖尿病患者心肌缺血-再灌注损伤（MIRI）发病率及致死率高，且缺乏有效的防治手段。前期证实膜联蛋白（Annexin）A1短肽（膜联三肽）可通过线粒体内去乙酰化酶3（Sirt3）明显减轻MIRI，因此提出膜联三肽通过调控线粒体稳态减轻糖尿病MIRI这一假说。本研究建立糖尿病大鼠心脏在体、离体、成年鼠心肌细胞缺血-再灌注模型，并于再灌注即刻给予膜联三肽，对比观察糖尿病MIRI情况下，膜联三肽对①近远期心功能、心肌损伤标志物、心梗程度、心肌组织微结构；②心肌细胞活力、细胞內钙、ATP含量；③线粒体结构、膜电位、mPTP开放水平、呼吸功能、Cyt c溢出情况；④Sirt3表达量、线粒体ATP敏感性钾通道（mitoKATP）的作用及氧化应激、线粒体凋亡途径相关指标的影响。本项目研究结果如下：.1、在糖尿病大鼠心肌细胞缺氧-再灌注模型中观察到，膜联三肽可降低糖尿病大鼠心肌细胞缺氧复氧后细胞内钙离子浓度，ROS水平；增加ATP含量，心肌细胞活力，心肌细胞存活率。膜联三肽对缺氧/复氧损伤的糖尿病大鼠心肌细胞具有一定保护效应。.2、在糖尿病大鼠心脏在体缺血-再灌注模型中观察到，再灌注3h及术后24h膜联三肽可减少心肌梗死面积及心肌损伤标志物CK-MB、LDH的含量，抑制氧化应激损伤，降低MDA、ROS水平，增加SOD水平，增加心肌组织中ATP含量。膜联三肽也可增加Sirt3活性，使mitoKATP亚基Kir6.1蛋白表达升高，改善糖尿病大鼠线粒体功能。以上结果说明膜联三肽能够抗糖尿病大鼠MIRI，其可能通过增加mitoKATP通道开放，激活线粒体内Sirt3产生抗糖尿病大鼠MIRI效应。.3、在糖尿病大鼠心脏离体Langendorff模型中观察到，再灌注1h后上述在体模型指标结果同样显示膜联三肽对MIRI糖尿病大鼠起到心肌保护作用，并且在分离得到的心肌线粒体中发现，膜联三肽增加线粒体内Cyt-c含量，提高线粒体复合物IV的活性，稳定线粒体呼吸链。表明膜联三肽促进线粒体内Sirt3表达，增加线粒体内Cyt-c含量，提高复合物Ⅳ活性，有助于改善缺血再灌注糖尿病大鼠心肌线粒体结构及功能，产生抗糖尿病大鼠MIRI效应；并且mitoKATP参与了膜联三肽对糖尿病大鼠的心肌保护作用。

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