脑缺血再灌注后线粒体氧化应激损伤的动态变化

Dynamic Characteristics of Mitochondria Damage Induced by Oxidative Stress after Cerebral Ischemia Reperfusion

目的探讨脑缺血再灌注后线粒体氧化应激损伤的动态变化特点。方法利用线栓法制备大鼠大脑中动脉缺血模型,通过线粒体分离技术测定线粒体丙二醛、三磷酸腺苷、线粒体膜电位水平,应用Realtime PCR仪测定线粒体基因(线粒体DNA)拷贝数,分析脑缺血再灌注后不同时间点缺血周围皮质线粒体的氧化应激水平及线粒体失功能的动态变化特点。结果①缺血再灌注后线粒体丙二醛水平呈渐进性增高,6 h为(4.61±0.83)nmol·mg-1 prot,72 h达(6.94±0.96)nmol·mg-1 prot,均较对照组显著升高(P<0.05);②缺血再灌注6 h后mtDNA拷贝数开始下降,24 h轻度回升,72 h显著下降水平为对照组的62%(P<0.01);③脑缺血再灌注后6 h线粒体三磷酸腺苷水平即显著下降,24 h出现短暂升高,再灌后72 h下降水平为对照组的42%(P<0.01);④荧光探针检测显示脑缺血再灌注后线粒体膜电位持续下降,再灌注后72h线粒体膜电位水平显著下降至对照组的43%(P<0.01)。结论线粒体损伤随缺血再灌注时间延长而呈渐进性加重,氧化应激损伤是构成其损伤的主要因素。缺血后脑组织线粒体存在短暂的内源性代偿修复机制,但不足以逆转氧化应激对线粒体的持续损伤。

Aim To explore the dynamic characteristics ofmitochondria damage induced by oxidative stress after cerebral ischemia reperfusion.Methods Transient middle cerebral artery occlusion （MCAO） model was conducted by the intraluminal filament technics.Mitochondrial MDA,ATP,mitochondrial membrane potential （△Ψm） level were determined by using isolated mitochondria from brain tissue in perifocal ischemic area.The mtDNA copy number was measured with realtime PCR.And dynamic characteristic of mitochondria damage and mitochondria dysfunction induced by oxidative stress after cerebral ischemia reperfusion were explored by utilizing the above measurement.Results Mitochondrial MDA level increased gradually with the time extending after cerebral ischemia-reperfusion.It was 4.61 ± 0.83 at 6 hours post reperfusion,and was 6.94 ± 0.96 at 72 hours,which both were dramatically higher compared with the sham-operated control （P〈0.01）.Realtime PCR results showed the mtDNA copy number decreased at 6 hours,partly recovered at 24 hours,and declined markedly to 62% of the control group at 72 hours after ischemia （P〈0.01）.Mitochondrial ATP level decreased significantly at 6 hours post repersuion,obtained transient increasement at 24 hours,and declined markedly to 42% of the control group at 72 hours after ischemia （P〈0.01）.Fluorescence probe JC-1 results indicated the sustained reduction of △Ψm with the time extending after ischemia reperfusion,it reduced significantly to 43% of the control group at 72 hours after ischemia （P〈0.01）.Conclusion Mitochondria damage is gradually exacerbated with the time extending after ischemia reperfusion,which was induced by oxidative stress injury.Mitochondria of brain tissue have transient endogenous repair mechanism after cerebral ischemia-reperfusion,but it can＇ t resist the durable damage of oxidative stress.