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目的探讨缺血后处理对心肌缺血再灌注损伤(myocardial ischemia-reperfusion injury,MIRI)中细胞凋亡及特异性内质网应激损伤相关蛋白葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)和半胱氨酸蛋白酶12(cysterine protease-12,caspase-12)表达水平的影响和意义。方法 Wistar大鼠24只随机分为假手术组、缺血再灌注组和缺血后处理组,每组8只。采用改良Pferfer MA推管法制备大鼠缺血再灌注模型,假手术组于左冠状动脉前降支下穿线、套管,不结扎,旷置220min;缺血再灌注组结扎左冠状动脉40min后完全开放,再灌注180min;缺血后处理组结扎左冠状动脉40min后,再灌注缺血开始前连续实施3个循环的30s/30s的缺血再灌注后处理,随后完全开放左冠状动脉再灌注180min。采用Evans blue与TTC双染法测定心肌梗死面积百分比和缺血区面积百分比,采用TUNEL法检测心肌细胞凋亡指数,采用Western blot法检测心肌组织caspase-12、GRP78蛋白表达水平。结果假手术组心肌缺血区面积百分比(0)和梗死区面积百分比(0)明显低于缺血后处理组[(46.46±2.13)%、(41.02±2.93)%]和缺血再灌注组[(53.31±3.87)%、(52.19±3.44)%](P<0.01),心肌细胞凋亡指数[(6.70±2.25)%]、心肌组织caspase-12蛋白(0.11±0.01)和GRP78蛋白(0.13±0.03)表达水平明显低于缺血后处理组[(20.54±3.05)%、0.35±0.06、1.17±0.14]和缺血再灌注组[(26.92±1.91)%、0.41±0.06、1.04±0.16](P<0.01);缺血后处理组心肌缺血区面积百分比、梗死区面积百分比、心肌凋亡指数、心肌组织caspase-12蛋白表达水平低于缺血再灌注组(P<0.01),心肌组织GRP78蛋白表达水平高于缺血再灌注组(P<0.05)。结论缺血后处理可减轻心肌细胞凋亡,而内质网应激激活参与了大鼠MIRI过程,推测缺血后处理在大鼠MIRI过程中可能通过调节内质网应激途径抑制细胞凋亡,改善MIRI。
Objective To investigate the effect of ischemic postconditioning on the apoptosis and the expression of glucose regulated protein 78 (GRP78) in myocardial ischemia-reperfusion injury (MIRI) and endoplasmic reticulum Effect and significance of cysterine protease-12 (caspase-12) expression. Methods Twenty-four Wistar rats were randomly divided into sham-operated group, ischemia-reperfusion group and ischemic postconditioning group, with 8 rats in each group. The model of ischemia-reperfusion was established by the improved Pferfer MA push-tube method. The sham-operated group was perfused with anterior descending branch of the left coronary artery, cannulated without ligation and exclusion for 220 min, and the ischemia-reperfusion group was ligated with the left coronary artery for 40 min Completely open and reperfused for 180min. After ischemic postconditioning, the left coronary artery was ligated for 40min, and three cycles of 30s / 30s of ischemia-reperfusion were performed before reperfusion ischemia followed by complete reoperation of left coronary artery 180min. The percentage of myocardial infarction area and the percentage of ischemic area were determined by Evan’s blue and TTC double staining. The apoptosis index of cardiomyocytes was detected by TUNEL method. The protein expression of caspase-12 and GRP78 in myocardium was detected by Western blot. Results The percentage of myocardial ischemic area (0) and infarct area (0) in sham operation group was significantly lower than that in ischemic postconditioning group [(46.46 ± 2.13)%, (41.02 ± 2.93)%] and ischemia reperfusion group (53.31 ± 3.87)%, (52.19 ± 3.44)%, P <0.01). The apoptosis index of cardiomyocytes was (6.70 ± 2.25)%, that of caspase-12 protein 0.13 ± 0.03) were significantly lower than those in ischemic postconditioning group [(20.54 ± 3.05)%, 0.35 ± 0.06, 1.17 ± 0.14] and ischemia reperfusion group [(26.92 ± 1.91)%, 0.41 ± 0.06, 1.04 ± 0.16] (P <0.01). The area of myocardial ischemic area, the percentage of infarct area, the index of myocardial apoptosis and the expression of caspase-12 protein in myocardium of ischemic postconditioning group were lower than that of ischemia reperfusion group (P <0.01) , And the GRP78 protein expression in myocardium was higher than that in ischemia-reperfusion group (P <0.05). Conclusion Ischemic postconditioning can reduce cardiomyocyte apoptosis, while endoplasmic reticulum stress activation is involved in the process of MIRI. It is speculated that ischemic postconditioning may inhibit apoptosis by regulating the endoplasmic reticulum stress pathway during MIRI in rats , Improve MIRI.