Parkin/DJ-1调控的线粒体自噬在恢复糖尿病心肌对缺血后处理敏感性中的作用及机制

Diabetic patients are more vulnerable to myocardial ischemia reperfusion injury (IRI),but less or not responsive to therapeutic interventions such as ischemic postconditioning (IPO) as compared with non-diabetics. Our previous study showed this was associated with mitophagy dysfunction, but its underlying mechenisms are still unknown.Recent studies indicated that Parkin and DJ-1 were the key factor regulating mitophagy.In our preliminary study, the protein complex of Parkin and DJ-1 was found in diabetic myocardium,though their expression was significantly downregulated, and autophagy inducers could inhibit myocardial IRI and restore the sensitivity to IPO in the diabetic myocardium. Therefore, we hypothesize that Parkin/DJ-1 regulated mitophagy is the key way to reduce IRI and restore the sensitivity of IPO in diabetic myocardium.In the current proposal, we will use the selective gene knockout mice and primary cultured cardiomyocytes to make diabetic model or high glucose with hypoxia/reoxygenation model, to elucidate the roles and mechanisms of Parkin/DJ-1 regulated mitophagy restoring IPO senstivity to diabetic myocardium, and invetigate its effects on the mitophagy sigaling pathway, such as AMPK/Akt/TSC/mTOR. This proposed studies will improve our understanding of diabetic myocardial IRI and may help facilitate the development of novel and optimal therapies in diabetic and ischemic heart disease.

糖尿病患者心脏较非糖尿病患者更不易耐受缺血/再灌注损伤(IRI),且对缺血后处理（IPO）的敏感性降低,我们前期研究发现其与线粒体自噬功能紊乱有关，但其具体调控机制仍然未明。新近研究表明Parkin与DJ-1是调控线粒体自噬的关键因子。我们预实验表明, 糖尿病心肌Parkin与DJ-1虽可形成蛋白复合体但两者的表达水平显著降低,应用自噬诱导剂可抑制糖尿病心肌IRI并可恢复糖尿病心肌对IPO的敏感性。据此，我们推测Parkin/DJ-1调控的线粒体自噬是糖尿病心肌抗IRI并恢复对IPO敏感性的关键途径。本项目拟用特异性基因敲除小鼠、原代心肌细胞为研究对象，探讨Parkin/DJ-1调控的线粒体自噬在恢复IPO对糖尿病心肌保护中的作用及分子机制, 并观察其对线粒体自噬信号通路AMPK/Akt/TSC/mTOR的影响，以期为临床开发糖尿病心肌IRI保护药物或措施提供实验依据。

糖尿病患者心脏对缺血/再灌注损伤的耐受性降低且缺血后处理对其保护作用丧失，可能与线粒体自噬功能紊乱以及内源性保护通路的失代偿密切相关，但详细机制未明。我们的研究阐明了Parkin通过促进DJ-1线粒体转移减少糖尿病心肌缺血再灌注损伤的机制，同时发现了在氧化应激状态下，DJ-1通过其半胱氨酸C106位点的抗氧化能力恢复IPO的新机制。我们的研究表明：（1）糖尿病大鼠心肌DJ-1和Parkin的蛋白表达均下调，同时IPO失活；（2）Parkin过表达能够促进DJ-1的线粒体转移减少心肌缺血性损伤，同时DJ-1的线粒体转移依赖于Parkin S56位点的磷酸化；（3）DJ-1过表达通过激活自噬通路AMPK/mTOR恢复IPO的保护作用；（4）DJ-1能够转移到线粒体和细胞核，促进Nrf2/HO-1活化以及减轻线粒体氧化应激恢复IPO的保护作用，这一作用与DJ-1的C106位点密切相关；（5）DJ-1能够直接抑制PTEN的活性，同时介导Akt的磷酸化，从而恢复IPO的保护作用，而合并给予PTEN抑制剂能抑制这一过程。基于我们的上述研究，以期待为糖尿病心脏缺血/再灌注损伤的防治提供有效的治疗靶点。