心肌线粒体复合体在高脂模型小鼠心脏缺氧-复氧中的作用

Effect of mitochondrial complex activity on heart hypoxia-reoxygenation in high-fat-diet mice model

目的:研究高脂模型小鼠心肌线粒体复合体功能变化,并探讨其在心肌缺氧/复氧(模拟缺血再灌注)中的作用。方法:将12只雄性2周龄C57B6小鼠随机分为高脂饮食组和对照组,进行20周高脂饮食和正常饮食喂养,并记录体重。实验结束后,提取心脏线粒体,将其在密闭空间内耗尽氧气后,维持缺氧状态30 min再通入氧气以模拟心脏缺血再灌注过程。通过氧气浓度检测探针测量并记录线粒体的耗氧率(oxygen consumption rate,OCR),荧光比色法检测线粒体复合体活性,荧光分光光度计测量活性氧(reactive oxygen species,ROS)的产生速率。结果:高脂饮食组心肌线粒体的OCR明显高于对照组(P <0.05),同时线粒体复合体Ⅱ的活性在高脂饮食组中明显升高(P <0.05)。OCR在两组中均升高,尤其是在高脂饮食组,且这种升高可以被复合体Ⅱ抑制剂Malonate抑制。比较线粒体ROS的产生速率也发现相同倾向。结论:线粒体复合体Ⅱ的活性在高脂模型小鼠中有较大变化,且这种变化可能是心肌缺氧/复氧(模拟缺血再灌注)损伤加剧的机制。

Objective:This study aims to detect the effect of mitochondria from high fat diet mice heart,and the effects of hypoxiareoxygenation.Methods:A total of 12 2-week-old mice(male,C57 B6)were randomly divided into high-fat-diet(HFD,n=6)group and control(CTR,n=6)group,and they were fed with high fat diet and normal diet respectively for 20 weeks.Hearts were used to isolate pure mitochondria.After the oxygen used up in the chamber,the mitochondria were subjected to 30 minutes of hypoxia-reoxygenation to simulate ischemia-reperfusion.Mitochondrial oxygen consumption rate(OCR)was measured using oxygen monitor system.Mitochondrial complex enzyme activity was assessed using microplate colorimetric assay kit.Reactive oxygen species(ROS)was measured by fluorimeter.Results:The mitochondrial OCR was greater in HFD group compared to that of CTR group(P<0.05).Similarly,mito-complexⅡactivity was significantly increased in HFD group compared to that of CTR group(P<0.05).Furthermore,reoxygenation of purified mitochondria following 30 min hypoxia transiently increased OCR,with significantly higher increase in HFD group.Pre-treatment of mito-complexⅡinhibitor,malonate diminished reoxygenation-induced OCR increase in both groups.The similar tendency was also detected in ROS.Conclusion:Mito-complexⅡactivity was totally enhanced in the HFD model,which could be involved in the injury of hypoxia-reoxygenation(which simulated ischemia-reperfusion)in heart.