七氟醚后处理对慢性心肌梗死大鼠离体心脏缺血再灌注损伤的影响

Effects of sevoflurane postconditioning on ischemia-reperfusion injury in chronically-infarcted rat hearts

目的评价七氟醚后处理对慢性心肌梗死大鼠离体心脏缺血再灌注损伤的影响。方法清洁级雄性sD大鼠，在体结扎左冠状动脉前降支建立心肌梗死模型，6周后开胸取心脏，建立离体心脏灌注模型。取模型制备成功的心脏80个，采用随机数字表法，将心脏随机分为8组（n=10），l组K．H液持续灌注90min；Ⅱ组全心缺血30min，再灌注60min；Ⅲ组～Ⅵ组全心缺血30min，再灌注最初15min灌注含磷脂酰肌醇一3．激酶（P13K）抑制剂LY29400215／μmol／L、丝裂原活化蛋白激酶激酶（MEKl／2）抑制剂PD9805920tμmol／L、0．02％二甲基亚砜及经3％七氟醚饱和的K—H液，随后更换为K—H液再灌注45min；Ⅶ组和Ⅷ组全心缺血30min，再灌注最初15min灌注3％七氟醚饱和的K．H液，同时分别给予LY29400215／μmol／L和PD9805920μmol／L，随后更换为K—H液再灌注45min。于平衡灌注20min、缺血前即刻、再灌注15、30和60rain（T0.4）时测定冠状动脉流量（cF）、记录左室发展压（LVDP）、左室最大收缩速率（＋dp／dt）、左室最大舒张速率（-dp／dt）、左室舒张末期压力（LVEDP）及心率（HR）；于T0.4时收集冠状动脉流出液，测定乳酸脱氢酶（LDH）和肌酸激酶同工酶（cK-MB）活性；于T4时测定急性心肌梗死面积，于T2时取左心室，测定蛋白激酶B（PKB／Akt）及细胞外信号调节激酶1／2（ERKI／2）的磷酸化表达水平及心肌细胞mPTP的开放程度。结果与I组比较，Ⅱ组～Ⅷ组再灌注时LVDP、±dp／dt、HR和cF降低，LVEDP升高，急性心肌梗死面积增大，冠状动脉流出液LDH和cK．MB活性升高，心肌细胞mPTP开放程度增加（P〈0．05）；与Ⅱ组比较，Ⅵ组再灌注时LVDP、±dp／dt、HR和cF升高，LVEDP降低，急性心肌梗死面积减小，冠状动脉流出液LDH和CK—MB活性降低，心肌PKB／Akt和ERK1／2磷酸化水平增强，心肌细胞mPTP开放程度降低（P〈0．05），其余组上述指标差异无统计学意义（P〉0．05）。结论3％七氟醚后处理可减轻慢性心肌梗死大鼠心肌缺血再灌注损伤，其机制与激活P13K—PKB／Akt和MEK1／2．ERKl／2，从而抑制心肌细胞mPTP开放有关。

Objective To evaluate the effects of sevoflurane postconditioning on ischemia-reperfusion injury in chronically-infarcted rat hearts. Methods Left anterior descending coronary artery was figated to induce myocardial infarction in male Sperague-Dawley rats. Six weeks later, chronically-infarcted hearts were isolated and passively perfused in a Langendorff apparatus. Eighty chronically-infarcted hearts were randomized into 8 groups （n = 10 each）： Ⅰ -Ⅵgroups. In group Ⅰ, hearts were continously perfused with Krebs-Henseleit （K-H） solution for 90 rain. In group Ⅱ , hearts were subjected to 30 min of global ischemia, followed by 60 min of reperfusion. In groups Ⅲ to Ⅵ, hearts were exposed to 30 rain of global ischemia, specific phosphatidylinositol-3-kinase（PI3K） inhibitor LY294002 15/μmol/L and mitogen-activated extracellular regulated kinase 1/2 （MEK1/2） inhibitor PD98059 20 μmol/L, 0.02% dimethyl sulfoxide, and K-H solution saturated with 3% sevoflurane were administered, respectively, during the first 15 min of reperfusion, followed by perfusion with plain K-H solution for 45 min. In groups and , hearts were exposed to 30 min of global ischemia, K-H solution saturated with 3% sevoflurane was given during the first 15 min of reperfusion-,- LY294002 15 /μmol/L and PD98059 20 μmol/L were simultaneously administered, respectively, followed by perfusion with plain K-H solution for 45 min. Coronary flow （CF）, left ventricular developed pressure （LVDP）, ± dp/dt, left ventricular end-diastolic pressure （LVEDP） and heart rate （HR） were recorded after 20 min of equilibration （baseline, TO ）, immediately before ischemia （Tt）, and at 15, 30 and 60 min of reperfusion （T2.4） The concentrations of lactate dehydrogenase （LDH） and creatine kinase-MB （CK-MB） in the collected coronary effluent were determined at To and T4 Acute myocardial infarct size was determined at T4 Left ventricular tissue samples were collected at T2 to measure the phosphorylation of protein kinase B/Akt （PKB/Akt）, and extracellular regulated kinase 1/2 （ERK1/2） and degree of mitoehondfial perme- ability transition pore （mPTP） opening. Results Compared with group , LVDP, ＋ dp/dt, HR and CF were significantly decreased, LVEDP was increased, the acute myocardial infarct size was enlarged, and the concentrations of LDH and CK-MB in the coronary effluent and degree of mPTP opening were increased during reperfusion in groups Ⅱ-Ⅵ（P 〈 0.05）. LVDP, ± dp/dt, HR and CF were significantly higher, LVEDP was lower, the acute myocardial infarct size was smaller, the concentrations of LDH and CK-MB in the coronary effluent were lower, the phosphorylation of PKB/Akt and ERK1/2 was higher, and the degree of mPTP opening was lower during reperfusion in group Ⅵ than in group Ⅱ （ P 〈 0.05）. Conclusion Sevoflurane postconditioning protects chronically-infarcted rat hearts against ischemia-reperfusion injury by activating PI3K-PKB/Akt and MEK1/2-ERK1/2 and inhibiting mPTP opening.