自噬在缺血预处理减轻大鼠肢体缺血再灌注心肌损伤中的作用

Effects of autophagy on myocardial injury induced by limb ischemia reperfusion in ischemic preconditioning in rats

目的探讨自噬在缺血预处理减轻大鼠肢体缺血再灌注心肌损伤中的作用。方法雄性SD大鼠28只,8周龄,体重200~250 g,采用随机数字表法,将其分为4组(ni=7):假手术组(S组)、肢体缺血再灌注组(IR组)、肢体缺血预处理组(IPR组)、自噬抑制剂处理组(3-MA组)。采用夹闭双侧股动脉缺血4 h后恢复灌注4 h的方法建立肢体缺血再灌注模型;采用股动脉夹闭前30 min实施缺血5 min,再灌注5 min,循环3次后建立肢体缺血预处理模型;3-MA组于缺血预处理前30 min腹腔注射3-甲基腺嘌呤1.5 ml/kg。再灌注4 h后处死大鼠取心肌组织,电镜下观察自噬小体形成情况和病理学结果,采用HE染色和TUNEL法检测心肌细胞凋亡情况,采用Western blot法检测LC3-Ⅱ的表达情况。结果与S组相比,IR组、IPR组、3-MA组心肌细胞凋亡比例明显升高,LC3-Ⅱ/LC3-Ⅰ表达上调(P<0.05);与IR组相比,IPR组细胞凋亡降低、LC3-Ⅱ/LC3-Ⅰ表达下调(P<0.05);与IPR组相比,3-MA组细胞凋亡比例升高、LC3-Ⅱ/LC3-Ⅰ表达下调(P<0.05)。结论自噬参与了肢体缺血预处理减轻大鼠肢体缺血再灌注心肌损伤的作用。

Objective To investigate the role of autophagy in the reduction of myocardial injury induced by ischemia and reperfusion in rats. Methods Twenty-eight SD male rats （ 8 weeks old, weight 200 - 250 g） were divided into 4 groups u- sing random number table method （ n = 7） ： sham operation group （ group S）, limb ischemia reperfusion group （ group IR） limb ischemia reperfusion preconditioning group （group IPR）, Autophagy inhibitor treatment group（ group 3-MA）. A limb ischemia reperfusion model was established by using the method of reperfusion 4 h after clamping bilateral femoral artery ischemia 4 hours. A limb ischemia reperfusion model was implementing Ischemia for 5 minutes and reperfusion for 5 mi- nutes, after 3 cycles remaining, before closing femoral artery clamp 30 minutes. Group 3-MA was given intraperitoneal in- jection of 10% 3-MA 1.5 ml/kg before ischemic preconditioning 30 minutes. After reperfusion 4 h, rats were killed and took the apical tissue, observed the formation and pathological changes of the cell bodies under electron microscope, detec- ted apoptosis by HE staining and TUNEL assay, measured the expression of LC3 by western blot. Results Compared with group S, the proportion of myocardial cell apoptosis was significantly increased, the expression of LC3-11/LC3- I was up regulation on group IR, group IPR and group 3-MA （P 〈 0.05 ）. Conclusion with the group IR, the proportion of myocar- dial cell apoptosis was significantly reduced, the expression of LC3- II/LC3- I was down regulation on group IPR （P 〈 0.05）. Compared with the group IPR,the proportion of myocardial cell apoptosis was significantly increased, the expres- sion of LC3- II/LC3- I was down regulation on group 3-MA （P 〈 0.05 ）.