PPENK-MIDGE-NLS后处理对大鼠心肌缺血再灌注时线粒体的影响

Effects of PPENK-MIDGE-NLS post-conditioning on mitochondria during myocardial ischemia reperfusion in rats

目的观察PPENK-MIDGE-NLS基因载体对大鼠心肌缺血再灌注时线粒体的影响。方法成年雄性SD大鼠40只,随机分为假手术组(开胸不进行LAD血流阻断,sham组)、缺血再灌注组(I/R组)、PPENK-MLDGE-NLS载体组(PPENK组)及Control-MIDGE-NLS载体组(control组),后面3组均阻断LAD 30 min,分别于再灌注前给予生理盐水1.5 ml、PPENKMIDGE-NLS 200μg/1.5 ml及control-MIDGE-NLS 200μg/1.5 ml。再灌注24 h后,观察基因载体转染后在缺血区心肌组织表达情况;测定缺血区组织中亮氨酸脑啡肽(L-ENK)含量、血浆c Tn I浓度,心肌组织中丙二醛(MDA)、超氧化物歧化酶(SOD)及ATP含量,线粒体膜电位水平变化,电子显微镜下观察心肌细胞及线粒体超微结构变化。结果与sham组相比,I/R组、PPENK组及control组的L-ENK及、SOD活性、ATP含量降低,c Tn I浓度及MDA明显上升,线粒体膜电位水平下降(P<0.05);与I/R组及control组相比,PPENK组心肌L-ENK及SOD活性、ATP含量升高,c Tn I浓度及MDA明显降低,线粒体膜电位水平升高(P<0.05),心肌及线粒体超微结构也有明显改善。control组与I/R组间,L-ENK、SOD、ATP含量、c Tn I浓度及MDA、线粒体膜电位水平对比无统计学差异(P>0.05),两组超微结构改变也相似(P>0.05)。结论 PPENK-MIDGE-NLS基因载体处理缺血再灌注大鼠模型后,能够提高大鼠缺血区局部脑啡肽含量,减轻缺血区心肌氧化应激反应,改善心肌细胞及线粒体结构。

Objec t ive To observe the effects of preproenkephalin-minimalistic immunologically defined gene expression-nuclear localization signal （PPENK-MIDGE-NLS） vector on mitochondria during the myocardial ischemia reperfusion in rats. MeAods A total of 40 adult male Sprague Dawley （SD） rats were randomly divided into a sham-operated group 「with thoracotomy perfonned without occlusion of left anterior descending coronary artery （LAD）, the sham groupl, an ischemia reperfusion group （I/R group）, a PPENK-MLDGE-NLS vector group （the PPENK group） and a Control-MIDGE-NLS vector group （the control group）. The latter three groups were all treated with the LAD occlusion for 30 min and then respectively treated with 1.5 ml normal saline, 200 μ g/1.5 ml PPENK-MIDGE-NLS and 200 μ g/1.5 ml control-MIDGE-NLS before the reperfusion. After 24 h reperfusion, the expression of gene in the myocardial tissue of ischemic area after gene transfection was observed; the content of leucine enkephalin （L-ENK） and the concentration of serum cardiac troponin （cTnI） in the tissue of ischemic area, the content of malondialdehyde （MDA）, superoxide dismutase（SOD） and adenosine triphosphate（ATP） in myocardial tissue, and the change in the level of mitochondrial membrane potential （MMP） were measured; cardiomyocytes and mitochondria were observed under an electron microscope for changes in the ultrastructure. Results Compared with the sham group, the content of L-ENK, the activity of SOD and the content of ATP decreased, the concentration of cTnI and the content of MDA increased obviously, and the level of mitochondrial membrane potential decreased in the I/R group, the PPENK group and the control group （P 〈 0.05）; compared with the I/R group and the control group, the level of myocardial L-ENK, the activity of SOD and the content of ATP increased, the concentration of cTnI and the content of MDA decreased obviously, the level of mitochondrial membrane potential increased, and the ultrastructure of myocardium and mitochondria were markedly improved in the PPENK group （P〈 0.05）. There was no statistically significant difference in the content of L-ENK, the sensitivity of SOD, the content of ATP, the concentration of cTnI, the content of MDA and the level of mitochondrial membrane potential between the control group and the I/R group （P 〉 0.05） and the changes in ultrastructure in these two groups were similar to each other （P 〉 0.05）. Conclusion PPENK-MIDGE-NLS vector post-conditioning in the rat model of ischemia reperfusion can increase the local content of encephalin in the ischemic area of rats, reduce the myocardial oxidative stress, and improve the ultrastructure of cardiomyocytes and mitochondria.