亚低温对心脏停搏复苏猪心肌损伤时需肌醇酶1信号通路的影响

Effect of mild hypothermia on inositol requiring enzyme 1 signaling pathway during myocardial injury after cardiac arrest and resuscitation in swine

目的评价亚低温对心脏停搏复苏猪心肌损伤时需肌醇酶1(IRE1)信号通路的影响。方法健康雄性白猪21头,体重33~41kg,采用随机数字表法将其分为3组:假手术组(S组,n=5)、心脏停搏复苏组(CA-CPR组,n=8)和亚低温组(MH组,n=8)。制备心脏停搏复苏模型,左股动脉、右颈内静脉分别置管连接PiCCO监测仪,右颈外静脉置入诱颤电极至右心室。经电极释放1mA交流电诱发心室颤动,室颤诱发成功后,停止机械通气8min,然后开始心肺复苏术。复苏2.5min时,静脉注射肾上腺素20μg/kg,此后每3min重复1次。复苏5min时,予电除颤,判断自主循环。若自主循环未恢复,立即心肺复苏2min,然后电除颤1次。复苏成功后,继续机械通气30h。S组只进行动物准备,不制备模型。MH组在复苏成功后5min时,利用降温毯将动物体温降到33℃,并维持至复苏后24h,后以1℃/h复温5h。余2组使用体表冰毯维持体温37.5~38.5℃。于复苏后1、6、12、24和30h(T1-5)时记录每搏输出量(SV)和全心射血分数(GEF),并经股静脉采血2ml,采用ELISA法检测血清cTnI浓度,采用免疫抑制法检测血清CK-MB活性。于复苏后30h时处死猪,迅速取左室心尖部组织,采用免疫组化染色法检测caspase-3表达,TUNEL法确定心肌细胞凋亡指数(AI),Westernblot法检测IRE1和caspase-12表达。结果与S组比较,CA-CPR组和MH组T1-5时SV和GEF降低,血清CK-MB活性升高,T2-5时血清cTnI浓度升高,心肌IRE1、caspase-12和caspase-3表达上调,AI升高(P<0.05);与CA-CPR组比较,MH组T2-5时SV和GEF升高,血清cTnI浓度降低,T3-5时血清CK-MB活性降低,心肌IRE1、caspase-12和caspase-3表达下调,AI降低(P<0.05)。结论亚低温减轻心脏停搏复苏猪心肌损伤的机制可能与抑制IRE1信号通路有关。

Objective To evaluate the effect of mild hypothermia on inositol-requiring enzyme l(IRE1) signaling pathway during myocardial injury after cardiac arrest and resuscitation in swine. Methods Twenty-one healthy male white swine, weighing 33-41 kg, were divided into 3 groups using a random number table method: sham operation group(group S, n=5), cardiac arrest-cardiopulmonary resuscitation group(group CA-CPR, n=8), and mild hypothermia group(group MH, n=8). The model of cardiac arrest and resuscitation was established based on the previously reported method.The catheters placed in the left femoral artery and right internal jugular vein were connected to the PiCCO Monitor system, and another pacing catheter was advanced from the right external jugular vein into the right ventricle.Ventricular fibrillation was induced by using a 1 mA alternating current through the pacing catheter.Once ventricular fibrillation was successfully induced, mechanical ventilation was discontinued for 8 min, and then cardiopulmonary resuscitation was initiated.Epinephrine 20 μg/kg was administered at 2.5 min of resuscitation followed by repetition every 3 min.Defibrillation was delivered at 5 min of resuscitation, and then spontaneous circulation was evaluated.If return of spontaneous circulation was not achieved, cardiopulmonary resuscitation was immediately resumed for 2 min and then defibrillation was delivered again.Mechanical ventilation was continued for 30 h after successful resuscitation.Animals in group S only underwent surgical preparation without experiencing cardiac arrest and resuscitation.At 5 min after successful resuscitation, body temperature was cooled down to 33 ℃ by using a cooling blanket, and then maintained at this level until 24 h after resuscitation, followed by 5 h of re-warming at a rate of 1 ℃/h in group MH.The temperature was maintained at 37.5-38.5 ℃ with the aid of surface cooling blanket in the other two groups.At 1, 6, 12, 24 and 30 h after resuscitation(T1-5), the values of stroke volume(SV) and global ejection fraction(GEF) were recorded, and meanwhile 2 ml of blood samples was obtained via the femoral vein to measure the concentration of serum cardiac troponin I(cTnI)(by enzyme-linked immunosorbent assay) and activity of serum creatine kinase-MB(CK-MB)(by immunosuppression). The swine were sacrificed at 30 h after resuscitation, and then myocardial specimens from the left ventricle were obtained for determination of the expression of caspase-3(by immunohistochemistry), cell apoptosis(by TUNEL), and expression of IRE1 and casepase-12(by Western blot). Apoptosis index was calculated. Results Compared with group S, SV and GEF were significantly decreased and the serum CK-MB activity was increased at T1-5, the concentration of serum cTnI was increased at T2-5, the expression of IRE1, caspase-12 and caspase-3 in myocardium was up-regulated, and apoptosis index was increased in CA-CPR and MH groups(P<0.05). Compared with group CA-CPR, the SV and GEF were significantly increased and the concentration of serum cTnI was decreased at T2-5, the activity of serum CK-MB was decreased at T3-5, the expression of IRE1, caspase-12 and caspase-3 in myocardium was down-regulated, and apoptosis index was decreased in group MH(P<0.05). Conclusion The mechanism by which mild hypothermia mitigates myocardial injury after cardiac arrest and resuscitation may be related to inhibiting IRE1 signaling pathway in swine.