组织激肽释放酶1改善大鼠心脏缺血/再灌注损伤后线粒体功能障碍

Tissue kallikrein 1 mitigates mitochondrial dysfunction after cardiac ischemia/reperfusion injury in rats

目的 探讨组织激肽释放酶1(KLK1)对心脏缺血/再灌注损伤大鼠线粒体功能的影响及其机制。方法 通过KLK1重组腺病毒感染实现大鼠心脏KLK1过表达,然后采用冠状动脉左前降支结扎和再灌注的方法建立大鼠心脏缺血/再灌注损伤模型,检测梗死区面积和心脏缺血危险区细胞凋亡情况;分离大鼠心脏缺血危险区心肌组织线粒体,检测线粒体功能(线粒体过氧化物生成量、线粒体膜电位、线粒体ATP生成量)。分离新生大鼠心肌细胞,通过KLK1重组腺病毒感染实现KLK1过表达,然后建立心肌细胞缺氧/复氧损伤模型,对心肌细胞缺氧/复氧损伤模型应用缓激肽1型受体(B1R)阻断剂R715或缓激肽2型受体(B2R)阻断剂HOE140处理,利用MTT法检测细胞活力,并观察线粒体功能的变化。结果 在大鼠心脏缺血/再灌注损伤模型中,KLK1过表达能减轻心肌缺血/再灌注损伤,使心肌梗死区面积减小、缺血危险区细胞凋亡减少(P均<0.01);改善心脏缺血/再灌注损伤后线粒体功能障碍,降低过氧化物生成量、增加线粒体膜电位和线粒体ATP生成量(P均<0.01)。在离体大鼠心肌细胞缺氧/复氧模型中,KLK1过表达能减轻心肌细胞损伤(P<0.05)、改善线粒体功能障碍(P<0.05,P<0.01),并且其作用可被B2R阻断剂HOE140抑制。结论 KLK1能改善心脏缺血/再灌注损伤后线粒体功能障碍,这可能是其具有心脏保护作用的重要机制。

Objective To explore the effect of tissue kallikrein 1(KLK1)on mitochondrial function after cardiac ischemia/reperfusion(I/R)injury and its mechanism.Methods After KLK1 overexpression by KLK1 recombinant adenovirus infection,the cardiac I/R rat model was established by ligation of left anterior descending coronary artery and reperfusion.The cardiac infarction area and the apoptosis of cardiomyocytes were detected.The mitochondria were isolated from injured myocardial tissues,and mitochondrial functions(mitochondrial superoxide production,membrane potential and ATP production)determined.The neonatal rat cardiomyocytes were isolated and infected with KLK1 recombinant adenovirus to achieve KLK1 overexpression,and then hypoxia/reoxygenation(H/R)cell model was established.The H/R cells were treated with the media containing bradykinin receptor type 1(B1R)antagonist R715 or bradykinin receptor type 2(B2R)antagonist HOE140.The cell viability was determined with MTT assay,and the mitochondrial functions were observed.Results In I/R rats,KLK1 overexpression could alleviate the cardiac injury,and decrease infarction area and cell apoptosis(all P<0.01)in cardiac ischemic risk area;moreover,KLK1 overexpression could improve mitochondrial dysfunction,decrease mitochondrial peroxide production,and increase mitochondrial membrane potential and ATP production(all P<0.01).In vitro cardiomyocyte model,KLK1 overexpression could also attenuate cardiomyocyte injury(P<0.01)and mitochondrial dysfunction(P<0.05,P<0.01),which could be inhibited by B2R antagonist HOE140.Conclusion KLK1 mitigates mitochondrial dysfunction after cardiac I/R injury,which may be an important mechanism of its cardioprotective effect.