下肢骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响

目的:探讨在体情况下,骨骼肌缺血后处理对兔缺血/再灌注心肌坏死和凋亡的影响。方法:新西兰大白兔36只,随机分成3组(每组随机选取6只进行梗死范围的测定,另外6只进行凋亡测定):①假手术组(Sham组);②缺血/再灌注组(I/R组);③远端后处理组(RPostC组)。在缺血前、后及再灌注60 min、120 min分别抽血测定肌酸激酶(CK),乳酸脱氢酶(LDH)的活性。采用伊文思兰(evans blue)和三苯基氯化四氮唑(TTC)染色方法确定心肌缺血区范围以及心肌坏死区范围。用Tunel法检测兔心肌缺血区细胞凋亡情况,免疫组织化学方法检测心肌缺血区蛋白caspase-3、Bcl-2及Bax的表达。结果:RPostC组心肌坏死程度、再灌注末CK活性较I/R组明显减低。RPostC组缺血区心肌Tunel阳性指数显著低于I/R组(21.79%±1.07%vs35.81%±1.10%,P<0.05)。而RPostC组缺血区心肌细胞caspase-3阳性指数显著低于I/R组(25.03%±1.16%vs39%±2.43%,P<0.05)。与Sham组比较,I/R组及RPostC组Bax蛋白表达指数、Bcl-2蛋白表达指数均升高;但RPostC组的Bax/Bcl-2比值降低,而I/R组的Bax/Bcl-2比值升高。与I/R组相比较,RPostC组Bax蛋白表达指数及Bax/Bcl-2比值显著降低,Bcl-2表达指数显著升高,差异均有统计学意义。结论:远端后处理能够明显的减少缺血/再灌注心肌细胞的坏死和凋亡,其减轻心肌细胞凋亡的机制可能与抑制促凋亡基因caspase-3的活化及Bcl-2表达的上调有关。

Effects of rabbit limbs ischemia/reperfusion on myocardial necrosis and apoptosis

Objective:To investigate the effects of rabbit limbs ischemia/reperfusion on myocardial necrosis and apoptosis in vivo.Methods: Thirty-six healthy new zealand rabbits were randomly divided into 3 groups: ①Sham group;② I/R(Ischemia/reperfusion) group;③RPostC(remote postconditioning) group.The activity of blood serum creatine kinase(CK) and lactate dehydrogenase(LDH) were measured at baseline,the end of ischemia after 60 min and 120 min of reperfusion respectively.The extent of myocardial ischemia and the scope of myocardial infarction were assessed by evans blue and Triphenyl tetrazolium chloride(TTC).The myocardial cell’s apoptosis at the area of myocardial ischemia was estimated by Tunel.Protein expression of caspase-3、Bcl-2 and Bax in myocardial ischemic area were analyzed with the method of immunohistochemistry.Results: Compared with I/R group,the myocardial infarct size and the CK activity were significantly reduced in RPostC group.The Tunel positive index of RPostC group in ischemic myocardium was significantly lower than that in I/R group(21.79%±1.07% vs 35.81%±1.10%,P<0.05).Caspase-3 positive cells index was calculated with randomly selected five regions in each slide and then the positive cells in per hundred cells were calculated.The RPostC group of caspase-3 positive cells was significantly lower than that in I/R group(25.03%±1.16% vs 39%±2.43%,P<0.05).Compared with the sham group,the Bax protein expression index and the Bcl-2 protein expression index of I/R group and RPostC group were increased.The Bax/Bcl-2 ratio of RPostC group decreased,while it was increased in I/R.Compared with the I/R group,the Bax protein expression and Bax/Bcl-2 ratio of RPostC group significantly reduced,but the expression index of Bcl-2 ratio was signifitcantly increased,the differences were statistically significant.Conclusion: Limbs ischemia/postconditioning could significantly reduce necrosis and apoptosis of ischemia/reperfusion myocardium.The mechanism of reducing the myocardial cell apoptosis may have relation to inhibiting the activation of pro-apoptotic gene caspase-3 and increased expression of Bcl-2.