电针预处理对心肌缺血再灌注损伤大鼠线粒体介导的细胞凋亡的影响

关键词：心肌缺血再灌注损伤  电针预处理  线粒体膜电位  细胞凋亡

Keywords:myocardial ischemia reperfusion injury  electroacupuncture pretreatment  mitochondrial membrane potential  cell apoptosis

摘要：

      摘要 目的：探讨电针预处理对心肌缺血再灌注损伤大鼠的线粒体膜电位（?Ψm）及其介导的细胞凋亡的影响。 方法：将60只SPF级雄性Wistar大鼠随机分为假手术组（Sham）、缺血再灌注组（MIRI）和电针预处理组（EA），每组20只。Sham组、MIRI组均采用自制鼠衣捆绑，固定于自制鼠台7天，1次/天，20min/次，第8天，Sham组开胸暴露心脏50min后、MIRI组开胸缺血20min再灌注30min后取材。EA组，电针预处理“内关”（双侧）、“足三里”（双侧）、“关元”7天，1次/天，20min/次，第8天开胸缺血20min，再灌注30min后取材。采用TTC染色法测定缺血再灌注损伤后心肌梗死的面积和重量，采用JC-1染色法检测?Ψm的水平，采用实时荧光定量PCR法检测第二个线粒体衍生的半胱氨酸蛋白酶激活剂（Smac/Diablo）、天冬氨酸特异性半胱氨酸蛋白酶-7（Caspase-7）、天冬氨酸特异性半胱氨酸蛋白酶-9（Caspase-9）基因表达水平。 结果：①心肌梗死面积和重量：与Sham组相比，MIRI组和EA组的梗死面积和重量均增加（均P＜0.05），且EA组的梗死面积和重量低于MIRI组（均P＜0.01）。②线粒体膜电位：与Sham组相比，MIRI组和EA组的红/绿荧光比值均明显下降（均P<0.01）；与MIRI组比较，EA组红/绿荧光比值明显升高（P<0.01）。③Smac/Diablo基因表达水平：与Sham组相比，MIRI组和EA组的Smac/Diablo、Caspase-7、Caspase-9的基因表达水平显著升高（均P＜0.01）；与MIRI组相比，EA组的Smac/Diablo、Caspase-7、Caspase-9基因表达明显下降（均P＜0.01）。 结论：电针预处理可有效缩小心肌梗死范围，提高线粒体膜电位水平，下调促凋亡基因Smac/Diablo、Caspase-7、Caspase-9的表达，电针预处理的保护作用可能是基于改善线粒体膜电位水平、抑制Smac/Diablo介导的线粒体Caspase凋亡通路产生的。

Abstract:

      Abstract Objective：To investigate the effect of electroacupuncture preconditioning on mitochondrial membrane potential(?Ψm) during myocardial ischemia reperfusion injury in rats. Method：Sixty SPF male Wistar rats were randomly divided into sham operation group(Sham Group)， myocardial ischemia reperfusion injury group (MIRI Group) and electroacupuncture pretreatment group (EA Group)， 20 in each group.The Sham group and MIRI group were both bound with self-made rat clothes and fixed on a self-made rat platform for 7 days. On the 8th day， the materials were collected after opened the chest and exposed the heart for 50min in the Sham group，and after ischemia for 20min and reperfusion for 30min in the MIRI group. In the EA group， electroacupuncture was used to pretreat "Neiguan" (PC 6，bilateral)， "Zusanli"（ST36，bilateral） and "Guanyuan" （RN 4） once a day， 20min/time， for 7 consecutive days. The myocardia infarction area and weight were measured by TTC staining，?Ψm level was detected by using JC-1 staining method， Smac/Diablo、Caspase-7、Caspase-9 gene expression level was detected by using real-time fluorescent quantitative PCR. Result：①Myocardial infarct area and weight：Compared with the sham group，the myocardial infarct area and weight of rats in the MIRI group and EA group were larger(P<0.01)，and the myocardial equality area and weight of rats in the EA group was obviously smaller than that in the MIRI group (P<0.01). ②Mitochondrial membrane potential(?Ψm)： Compared with the Sham group， red/green fluorescence ratio of the MIRI group and EA group significantly decreased (P<0.01).Compared with the MIRI group， the red/green fluorescence ratio in the EA group was significantly increased (P<0.01). ③The Smac/Diablo，Caspase-7，Caspase-9 gene expression level：Compared with the Sham group，Smac/Diablo，Caspase-7 and Caspase-9 gene expression in the MIRI group and the EA group were observably increased (P<0.01).The genes expression level in the EA group was lower than that in the MIRI group (P<0.01). Conclusion：Electroacupuncture preconditioning can effectively narrow infarction scope， improve the level of mitochondrial membrane potential，down-regulate the expression of Smac/Diablo， caspase-7 and caspase-9. The protective effect may be based on the improvement of mitochondrial membrane potential level and the inhibition of Smac/Diablo-mediated mitochondrial Caspase apoptosis pathway.