糖尿病因素取消大鼠缺血后处理心肌保护效应的线粒体机制:琥珀酸脱氢酶

Mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by ischemia postconditioning in rats:succinate dehydrogenase

目的评价糖尿病因素取消大鼠缺血后处理(IPO)心肌保护效应的线粒体机制与琥珀酸脱氢酶(SDH)的关系。方法SPF级雄性非糖尿SD大鼠36只,16~20周龄,体重约300 g,采用随机数字表法分为3组(n=12):假手术组(ND+Sham组)、缺血再灌注组(ND+I/R组)、ND+IPO组;取糖尿病模型制备成功的大鼠72只,采用随机数字表法分为6组(n=12):假手术组(DM+Sham组)、缺血再灌注组(DM+I/R组)、DM+IPO组、假手术+丙二酸二甲酯组(DM+Sham+Dme组)、缺血再灌注+丙二酸二甲酯组(DM+I/R+Dme组)、缺血后处理组+丙二酸二甲酯(DM+IPO+Dme组)。建立CBP模型,结扎升主动脉30 min恢复灌注60 min的方法制备全心缺血再灌注损伤模型。各IPO组于再灌注即刻给予3个循环的灌注30 s-缺血30 s处理。各Dme组于CPB开始时以4 mg·kg^(-1)·min^(-1)的速率经尾静脉输注丙二酸二甲酯40 min。于再灌注结束时取心肌组织,采用汉莎氧电极法测定线粒体呼吸控制率(RCR),JC-1法检测线粒体膜电位,吸光光度法检测线粒体通透性转换孔(mPTP)的开放程度,DHE荧光探针法检测ROS活性,分光光度法检测SDH活性、琥珀酸和延胡索酸含量。结果与ND+Sham组比较,ND+I/R组SDH和ROS活性、mPTP开放程度和延胡索酸含量升高,线粒体膜电位、RCR和琥珀酸含量降低(P<0.05);与ND+I/R组比较,ND+IPO组SDH和ROS活性、mPTP开放程度和延胡索酸含量降低,线粒体膜电位、RCR和琥珀酸含量升高(P<0.05)。与DM+Sham组比较,DM+I/R组SDH和ROS活性、mPTP开放程度和延胡索酸含量升高,线粒体膜电位、RCR和琥珀酸含量降低(P<0.05);与DM+I/R组比较,DM+IPO组上述指标差异无统计学意义(P>0.05)。与DM+IPO组比较,DM+IPO+Dme组SDH和ROS活性、mPTP开放程度和延胡索酸含量降低,线粒体膜电位、RCR和琥珀酸含量升高(P<0.05);与DM+I/R+Dme组比较,DM+IPO+Dme组SDH和ROS活性、mPTP开放程度和延胡索酸含量降低,线粒体膜电位、RCR和琥珀酸含量升高(P<0.05)。结论糖尿病因素取消大鼠IPO心肌保护作用的线粒体机制可能与增强SDH活性有关。

Objective To evaluate the relationship between the mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by ischemia postconditioning(IPO)and succinate dehydrogenase(SDH)in rats.Methods Thirty-six SPF male non-diabetic Sprague-Dawley rats,aged 16-20 weeks,weighing about 300 g,were divided into 3 groups(n=12 each)using a random number table method:sham operation group(ND+Sham group),ischemia-reperfusion(I/R)group(ND+I/R group)and IPO group(ND+IPO group).Seventy-two rats with diabetes mellitus were divided into 6 groups(n=12 each)using a random number table method:sham operation group(DM+Sham group),I/R group(DM+I/R group),DM+IPO group,sham operation+dimethyl malonate group(group DM+Sham+Dme),I/R+dimethyl malonate group(group DM+I/R+Dme)and IPO+dimethyl malonate group(group DM+IPO+Dme).The model of cardiopulmonary bypass(CPB)was established,and the model of total I/R injury was induced by ligating the ascending aorta for 30 min followed by 60 min of reperfusion.The animals underwent 3 cycles of 30-s reperfusion followed by 30-s ischemia starting from the onset of reperfusion in each IPO group.In each Dme group,dimethyl malonate was infused through the tail vein at a rate of 4 mg·kg^(-1)·min^(-1) for 40 min starting from the beginning of CPB.At the end of reperfusion,the myocardial tissues were taken for measurement of mitochondrial respiratory control ratio(RCR)(by the Lufthansa electrode method),mitochondrial membrane potential(MMP)(by the JC-1 method)and the opening of mitochondrial permeability transition pore(mPTP)(by absorptiometry)and for determination of the activity of reactive oxygen species(ROS)(with the fluorescent probe),succinate dehydrogenase(SDH)(using spectrophotometric method)and the contents of succinic acid and fumarate.Results Compared with ND+Sham group,the activities of SDH and ROS,opening of mPTP and content of fumarate were significantly increased,and MMP,RCR and succinic acid content were decreased in ND+I/R(P<0.05).Compared with group ND+I/R,the activities of SDH and ROS,opening of mPTP and content of fumarate were significantly decreased,and MMP,RCR and succinic acid content were increased in ND+IPO(P<0.05).Compared with group DM+Sham,the activities of SDH and ROS,opening of mPTP and content of fumarate were significantly increased,and MMP,RCR and succinic acid content were decreased in group DM+I/R(P<0.05).Compared with group DM+I/R,no significant change was found in the parameters mentioned above in group DM+IPO(P>0.05).Compared with group DM+IPO,the activities of SDH and ROS,opening of mPTP and content of fumarate were significantly decreased,and MMP,RCR and succinic acid content were increased in group DM+IPO+Dme(P<0.05).Compared with group DM+I/R+Dme,the activities of SDH and ROS,opening of mPTP and content of fumarate were significantly decreased,and MMP,RCR and succinic acid content were increased in group DM+IPO+Dme(P<0.05).Conclusion The mitochondrial mechanism of diabetic mellitus-caused abolition of cardioprotection induced by IPO may be related to the enhancement of SDH activity in rats.