靶向脑啡肽减轻心肌缺血再灌注损伤及其线粒体自噬机制

It has been well documented that enkephlin can initiate potent endogenous protective mechanism to ameliorate myocardial ischemic injury by specific opioid receptor. However, the clinical application of opioids still have many limitations at ischemic circumstances. Although it has been demonstrated that leukocyte can produce and secrete endogenous opioid peptide, but without specificity and the amount was also not abundant. The effects of enkephlin on mitophagy during myocardial ischemia have not been illustrated till now. We have constructed PPENK-MIDGE-NLS gene vector of enkephalin by minimalistic immunologically defined gene expression (MIDGE) and verified that gene can successfully transfect leukocyte. The purpose of this study is to intravenously inject PPENK-MIDGE-NLS during myocardial ischemic reperfusion. Combining the advantages of leukocytic aggregation and infiltration to the myocardial injury site, PPENK-MIDGE-NLS induces leukocyte continues pruduce and targeted release endogenous enkephalin in myocardial injury area. The effects of PPENK-MIDGE-NLS on gene expression and content of myocardial enkephalin precursor protein proenkephalin(PENK), ENK and metabolites are detected by RT-PCR, Western Blot, immunohistochemistry and radioimmunity. The feasibility and efficacy of PPENK-MIDGE-NLS gene vector inducing leukocyte to increase content of endougenous enkephlin for myocardial protection are also evaluated by the changes of haemodynamics, infarction area, cTnI and NF-κB. In addition, the other purpose is to investigate the relation of enkephlin and PI3k/Akt/mTOR intracellular signal pathway as well as mitophagy PINK1/Parkin pathway during myocardial ischemic reperfuion. Based above experimental results, we try to prove that PPENK-MIDGE-NLS is an innovative method for preventing and treating of myocardial ischemia-reperfusion injury and the regulation of mitophagy is an important mechanisms for enkephalin afforded cardioprotection.

脑啡肽启动细胞内强效保护机制、减轻器官缺血再灌注损伤的特性日益突显，但阿片类制剂的临床应用仍受到诸多限制，而且脑啡肽对与缺血细胞存活密切相关的线粒体自噬的影响尚无研究。课题组前期构建了PPENK-MIDGE-NLS脑啡肽基因载体并初步证实了其转染效果。本课题在大鼠心肌缺血再灌注模型，将该载体中心静脉注射转染粒细胞，利用粒细胞向缺血区"靶向"聚集、浸润的特性，在缺血局部持续合成分泌内源性脑啡肽。通过评价心肌功能及损伤程度；分子生物、免疫组化、透射电镜等方法测定心肌梗死区转基因脑啡肽的表达及心肌细胞线粒体功能和自噬水平；药理学实验设计分析脑啡肽与心肌损伤、脑啡肽与PI3k/Akt/mTOR自噬信号转导通路和线粒体自噬Parkin/Pink1蛋白表达的内在联系，旨在证实PPENK-MIDGE-NLS基因载体是治疗心肌缺血再灌注损伤的有效措施，创新性地阐明调节线粒体自噬是脑啡肽心肌保护的重要机制。

脑啡肽启动细胞内强效保护机制、减轻器官缺血再灌注损伤的特性日益突显，但阿片类制剂的临床应用仍受到诸多限制。课题组前期构建了PPENK-MIDGE-NLS脑啡肽基因载体并初步证实其对白细胞、心肌细胞转染能力，利用载体转染后粒细胞向缺血区“靶向”聚集、浸润的特性，证实其在缺血局部持续合成分泌内源性脑啡肽的能力。线粒体自噬是线粒体质量控制体系中重要的调节机制，直接参与调节心肌缺血再灌注损伤。然而，脑啡肽在心肌缺血再灌注过程中对线粒体自噬的影响尚未研究。为明确心肌缺血局部过表达脑啡肽含量产生心肌保护作用的线粒体自噬机制，本研究通过评价心肌功能及损伤程度；分子生物、免疫组化、透射电镜等方法测定心肌转基因脑啡肽的表达及心肌细胞线粒体功能和自噬水平；分析脑啡肽和线粒体自噬Parkin/Pink1蛋白表达的内在联系，证实PPENK-MIDGE-NLS基因载体是治疗心肌缺血再灌注损伤的有效措施，线粒体自噬是脑啡肽心肌保护的重要机制。结论：PENK-MIDGE-NLS基因载体过表达脑啡肽可减轻大鼠心肌缺血再灌注损伤，其机制与增强线粒体自噬，保护线粒体功能与结构完整有关。