电针、艾灸预处理对心肌缺血再灌注损伤大鼠心肌自噬相关蛋白LC 3、Beclin 1表达的影响

Effect of Electroacupuncture and Moxibustion Pretreatment on Expression of Autophagy Related Proteins LC 3 and Beclin 1 in Rats with Myocardial Ischemia-reperfusion Injury

目的:观察针灸预处理对心肌缺血再灌注损伤(MIRI)大鼠心肌自噬相关蛋白LC 3、Beclin 1表达的影响,探讨针灸预处理在MIRI过程中对自噬调控的作用机制。方法:SD大鼠随机分为假手术组、模型组、缺血预适应(IP)组、电针组和艾灸组,每组8只。冠状动脉结扎法建立大鼠MIRI模型。电针组及艾灸组造模前分别电针或艾灸"内关"穴,每次20min,每日1次,连续7d。HE染色法及透射电镜检测左心室心肌病理形态学的变化,TUNEL法检测心肌细胞凋亡情况及凋亡指数,Western blot法检测LC 3、Beclin 1蛋白在心肌细胞中的表达。结果:模型组心肌损伤较严重,心肌细胞排列紊乱、边界模糊,炎性细胞浸润,部分横纹坏死、溶解,线粒体结构紊乱、模糊,空泡化严重,并出现大量双层膜结构的自噬小体;IP组、电针组及艾灸组心肌损伤较模型组轻微,肌纤维大多正常,肌丝间局灶性水肿,线粒体丰富伴空泡增多,偶见自噬体。与假手术组比较,模型组凋亡指数、LC 3Ⅱ及Beclin 1蛋白表达水平、LC 3Ⅱ/LC 3Ⅰ均明显升高(P<0.01),LC 3Ⅰ蛋白表达显著下降(P<0.01);与模型组比较,IP组、电针组及艾灸组凋亡指数、LC 3Ⅱ及Beclin 1蛋白表达水平、LC 3Ⅱ/LC 3Ⅰ均明显降低(P<0.01),LC 3Ⅰ蛋白表达明显升高(P<0.05,P<0.01);电针组凋亡指数、LC 3Ⅱ及Beclin 1蛋白表达水平、LC 3Ⅱ/LC 3Ⅰ明显低于IP组及艾灸组(P<0.05),而LC 3Ⅰ蛋白表达水平则显著高于IP组及艾灸组(P<0.01)。结论:IP、电针或艾灸"内关"穴预处理对MIRI大鼠均具有保护作用,可对MIRI大鼠心肌的自噬产生调节作用,尤以电针最佳,而这种适度调节MIRI过程中的自噬水平可能与下调LC 3Ⅱ、Beclin 1蛋白的表达有关。

Objective To observe the effect of electroacupuncture （EA） and moxibustion （Moxi） pretreatment on myocardial pathological and structural changes and expression of autophagy related protein LC 3 Ⅰ / Ⅱ and Beclin 1 in rats with myo- cardial ischemia-reperfusion injury （MI/RI）, so as to explore their mechanisms underlying improving MI/RI. Methods Forty SD rats were randomly divided into sham operation, model, ischemic preconditioning （IP）, EA and Moxi groups （n = 8 in each group）. EA （10Hz/50 Hz, 1mA） or Moxi （ignited moxa stick） was respectively applied to bilateral ＂Neiguan＂（PC 6） for 20 min, once daily for 7 days. The MI/RI model was established by occlusion of the anterior descending branch of the left coronary artery for 40 min, followed by reperfusion for 60 min. The left ventricular （LV） tissue samples were collected and analyzed for pathological （H. E. staining） and ultrastructural changes, for myocardial apoptosis （apoptotic index= number of apoptotic cells/total num- ber of cardiomyocytes x 100 % ） with terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） method, and for the expression of LC 3 and Beclin 1 in myocardial cells with Western blot. Results Following MI/RI, H, E. staining revealed a disorder of arrangement of cardiomyocytes with vague border, inflammatory cell infiltration, intracellular swelling with bleeding, necrosis and dissolution of partial striated muscles of the left ventricle under light microscope, and dual staining of Uranyl acetate and leadnitrate showed atrophy, arrangement disorder, dissolution, necrosis, and interstitial edema of partial myocardial fibers, mitochondrial structural disorder, vacolation, and large body of autophagosomes with bilayers, etc. in ultrastructure, which was relatively lighter in both EA and Moxi groups. The apoptosis index, expression levels of myocardial LC 3Ⅱ and Beclin 1 and the ratio of LC 3 Ⅱ/LC 3Ⅰ were significantly higher in the model group than those in the sham operation group （P〈0.01）, but the expression level of LC 3Ⅰwas considerably down-regulated in the model group relevant to the sham operation group （P〈0.01）. Following the intervention and MI preconditioning, the increased apoptosis index and expression levels of LC 3Ⅱ and Beclin 1 proteins and the ratio of LC 3 Ⅱ/LO 3Ⅰ were obviously down-regulated in the IP, EA and Moxi groups relevant to the model group （P〈0.01）, and the decreased expression of LC 3Ⅰ protein was up-regulated obviously in the 3 treatment groups （P〈0.05, P〈0.01）. The effects of EA were significantly superior to those of IP and Moxi groups in down-regulating apoptosis index and expression of LO 3Ⅱ and Beclin 1 and the ratio of LO 3Ⅱ/LO 3Ⅰ and in up-regulating expression of LC 3 Ⅱ （ P〈0.05, P〈0.01 ）. Conclusion Both EA and Moxi preconditioning of PC 6 have a protective effect on ischemic myocardium in MI/RI rats, which is probably related to their effects in regulating expression of myocardial autophagy proteins as LC 3Ⅰ/ Ⅱ and Beclin 1.