过表达前脑啡肽原(PPENK)增强线粒体自噬减轻大鼠心肌缺血再灌注损伤

Overexpression of PPENK reduces myocardial ischemia reperfusion injury by promoting mitophagy in rats

目的探讨前脑啡肽原-最低限度免疫定义基因表达-核定位信号(PPENK-MIDGE-NLS)基因载体后处理对大鼠心肌缺血再灌注线粒体自噬的影响。方法成年雄性SD大鼠随机分为假手术对照组(sham)组:开胸不进行冠状动脉左前降支(LAD)血流阻断及再灌注;缺血再灌注(I/R)组、PPENK-MLDGE(MIDGE)-NLS载体(PPENK)组及Control-MIDGE-NLS载体(对照)组均阻断LAD 30 min,分别于再灌注前给予生理盐水、PPENK-MIDGE-NLS 200μg及Control-MIDGE-NLS 200μg,股静脉注射各1.5 m L。再灌注24 h后取材。ELISA测定血浆心肌肌钙蛋白I(c Tn I)含量;2,3,5-氯化三苯基四氮唑(TTC)法测定心肌梗死面积;透射电镜下观察心肌细胞超微结构及线粒体自噬并分析线粒体损伤评分;Western blot法检测线粒体自噬相关蛋白微管相关蛋白1轻链3B(LC3B)、p62、线粒体外膜转位酶20(TOM20)、PTEN诱导推定激酶1(PINK1)、帕金森病蛋白(parkin)水平。结果 PPENK组血浆c Tn I含量降低,心肌梗死面积减少,线粒体损伤评分改善,心肌组织及线粒体超微结构明显改善,线粒体自噬体增多。线粒体PINK1、parkin、p62、LC3B表达增强。对照组与I/R组间无显著性差异。结论 PPENK-MIDGENLS基因载体处理后可减轻大鼠心肌I/R损伤,可能与增强线粒体自噬,保护线粒体结构完整有关。

Objective To investigate the effect of preproenkephalin-minimalistic immunologically defined gene expression-nuclear localization signal（ PPENK-MIDGE-NLS） vector postconditioning on mitophagy during myocardial ischemia reperfusion in rats. Methods Forty male SD rats were randomly divided into 4 groups： sham operation group,ischemia reperfusion group,PPENK-MIDGE-NLS group and Control-MIDGE-NLS group. Myocardial ischemia reperfusion injury model was induced by ligating left anterior descending branch of coronary artery（ LAD）. Sham operation group was treated identically with the ischemia reperfusion group except that LAD was not tied and occluded. Ischemia reperfusion group,PPENK-MIDGE-NLS group,and Control-MIDGE-NLS group were treated with ligation and occlusion of LAD for 30 minutes followed by 24 hour-reperfusion,with 1. 5 m L saline,or 1. 5 m L PPENK-MIDGE-NLS vectors（200 μg）,or 1. 5 m L ControlMIDGE-NLS vectors（200 μg） administered respectively right before reperfusion started. Serum c Tn I was assayed by ELISA and myocardial infarct size was measured by TTC. Ultrastructural changes of mitochondria and mitophagy were observed using electron microscope,and mitochondrial damage scores were analyzed. Expressions of the mitophagy-related proteins such as PINK1,parkin,p62,TOM20 and LC3 B were measured by Western blotting. Results Compared with the ischemia reperfusion group,both serum c Tn I content and myocardial infarct size in the PPENK group decreased; the expression of PINK1,parkin,p62 and LC3 B were all up-regulated. Mitophagy was enhanced and mitochondrial damages were alleviated,with conspicuous improvement in mitochondria ultrastructure. Conclusion PPENK-MIDGE-NLS vector postconditioning can mitigate myocardial ischemia reperfusion injury by promoting mitophagy in rats.