低pH液复灌对兔缺血再灌注心肌内质网应激与细胞凋亡的影响研究

Effects of acidic reperfusion fluid on myocardial endoplasmic reticulum stress and apoptosis after ischemia/reperfusion in rabbits

目的探讨体外循环(CPB)下再灌注初短暂低pH液复灌对兔缺血再灌注(I/R)心肌内质网应激(ERS)与细胞凋亡的影响。方法健康成年新西兰大白兔24只,雌雄不拘,体重2.5～3.0kg,随机分为4组(n=6)。对照组(C组)不阻断主动脉,单纯转机3h;其余各组心脏停搏缺血40min,再灌注2h。pH7.4组与pH6.9组再灌注前2min分别给予pH7.4、6.9的Krebs-Henseleit(K-H)液持续灌注至主动脉开放;缺血后处理组(I-postC组)于再灌注前2min给予10s再灌/10s缺血共6次的缺血后处理。实验结束后,ELISA法测定肌钙蛋白I(cTnI)浓度,TUNEL法检测心肌细胞凋亡情况,RT-PCR检测葡萄糖调节蛋白78(GRP78)、钙网蛋白(CRT)mRNA的表达,免疫组化检测caspase-12、C/EBP同源蛋白(CHOP)表达。结果与C组比较,其余各组cTnI浓度、心肌细胞凋亡率、GRP78和CRTmRNA表达、caspase-12和CHOP蛋白表达均明显增加(P<0.05);与pH7.4组比较,各检测指标在pH6.9组及I-postC组均降低(P<0.05),而后两者比较差异无统计学意义(P>0.05)。结论再灌注初短暂低pH液复灌可调控ERS反应程度,抑制I/R导致的过度ERS,减轻内质网凋亡信号介导的细胞凋亡的发生,从而产生心肌保护作用。

Objective To determine the effect of acidic repeffusion fluid in cardiopulmonary bypass （CPB） on the myocardial endoplasmic reticulum stress （ERS） and apoptosis after ischernia/reperfusion in rabbits. Methods Twenty-four adult New Zealand white rabbits of both sexes weighing 2. 5-3.0 kg were randomly divided into 4 groups （6 each）. Control group （C） ： CPB for 3 hours without aortic clamping; pH7. 4 group and pH6. 9 group： hearts were subjected to ischemia for 40min followed by 2 hours of reperfusion, and then perfused with Krebs-Henseleit bicarbonate buffer solution, respectively, at pH7. 4 and pH6. 9 in the first 2rain of reperfusion; ischemic postconditioning group （I-postC）： 40rain after aortic clamping, 6 cycles of 10-second reperfusion/10-second occlusion were given in the first 2min of 2-hour reperfusion. Cardiac troponin I （cTnI） concentration was determined with ELISA, apoptosis of myocardial cell was detected by TUNEI., the expressions of glucose-regulated protein 78（GRP78）and calreticulin （CRT） mRNA were detected by RT-PCR, and the expressions of caspase-12 and C/EBP homologous protein （CHOP） were assayed by immunohistochemistry. Results Compared with the C group, the cTnI concentration, apoptotic rate, expressions of GRP78, CRT mRNA, caspase-12 and CHOP of the other groups increased significantly （P〈0. 05）. Compared with the pH7.4 group, all the indexes were significantly lower in pH6. 9 group and I-postC group （P〈0. 05）, but no significant difference was found between the latter two groups （P〈0. 05）. Conclusion Acidic reperfusion may protect the heart against I/R injury, regulate ERS response, inhibit excessive ERS, and alleviate ERS-mediated apoptosis during I/R.