PI3K、ERK1／2、mito—KATP通道及mPTP在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用

Sevoflurane postconditioning-induced cardioprotection against ischemia-reperfusion injury to isolated rat hearts： the role of PI3K and ERK1/2, mito-KATP channel and mPTP

目的 评价磷脂酰肌醇-3-激酶（P13K）及细胞外信号调节激酶1／2（ERK1／2）、线粒体ATP敏感性钾（mito-KATP）通道及线粒体膜通透性转换孔（mPTP）在七氟醚后处理减轻大鼠离体心脏缺血再灌注损伤中的作用。方法雄性清洁级sD大鼠，周龄7-10周，体重250～300g，采用Langendorff法建立大鼠离体心脏灌注模型，取模型制备成功的心脏180个，随机分为12组（n=15），对照组（C组）：持续灌注90min；缺血再灌注组（IR组）：停止灌注K—H液30min，再灌注60min；IR＋LY组、IR＋PD组、IR＋ATR组、IR＋5-HD组和IR＋DMSO组：于再灌注即刻分别灌注P13K特异性抑制剂LY294002（LY）15μmol／L、ERK1／2特异性抑制剂PD98059（PD）20／Lmol／L、mPWP开放剂苍术甙（ATR）20／Lmol／L、mito—K。。通道抑制剂5-羟癸酸（5-HD）100μmol／L和溶剂二甲基亚砜（DMSO）0．02％15min；七氟醚后处理组（S组）：于再灌注即刻灌注经3％t氟醚饱和的K—H液15min，随后更换正常K-H液再灌注45min；S＋LY组、S＋PD组、S＋ATR组和s＋5-HD组：于再灌注即刻灌注经七氟醚饱和的K—H液同时分别灌注LY15μmol／L、PD20μmool/L、ATR20μmol／L、5-HD100μmol／L 15min，随后更换为正常K．H液再灌注45min。于平衡灌注20min、停灌前即刻、再灌注15、30和60min（T0-4）时测定冠状动脉流量（CF），记录心功能指标；C组、IR组和S组分别于R和L时收集冠状动脉流出液测定乳酸脱氢酶（LDH）、肌酸激酶同工酶（CK—MB）活性和肌钙蛋白1（cTnI）浓度；于T4时取左心室，测定心肌梗死面积，C组、IR组、IR＋ATR组、IR＋5-HD组、IR＋DMSO组、S组、S＋ATR组和S＋5-HD组检测细胞凋亡情况，计算凋亡指数（AI），C组、IR组、IR＋LY组、IR＋PD组、IR＋DMSO组、S组、S＋LY组和S＋PD组测定心肌炳酰胺腺嘌呤二核苷酸（NAD＋）含量。结果与c组比较，其余各组再灌注时心功能降低，CF降低，心肌梗死面积增大，IR组、IR＋ATR组、IR＋5-HD组、IR＋DMSO组、S组、s＋ATR组和S＋5-HD组AI升高，IR组、IR＋LY组、IR＋PD组、IR＋DMSO组、S组、S＋LY组和S＋PD组NAD＋含量降低，IR组和S组LDH、CK—MB活性和cTnI浓度升高（P〈0．05）；与IR组比较，S组再灌注时心功能提高，CF升高，心肌梗死面积减小，AI降低，NAD＋含量升高，LDH、CK-MB活性和cTnⅠ浓度降低（P〈0．05），其余组上述指标差异无统计学意义（P〉0．05）。结论七氟醚后处理可能通过激活P13K及ERKI／2、促进mito-KATP通道开放、抑制mPTP开放，从而减轻大鼠离体心脏缺血再灌注损伤。

Objective To evaluate the role of phosphatidylinositol-3-kinase （PI3K） and extracellular signal-regulated kinase 1/2 （ERK 1/2）, mitochondrial ATP-sensitive potassium （mito-KATP ） channel and mitochondrial permeability transition pore （mPTP） in cardioprotection induced by sevoflurane postconditioning against ischemia-reperfusion （IR） injury to isolated rat hearts. Methods Healthy male SD rats 7-10 weeks old weighing 250-300g were anesthetized with intraperitoneal 3 % pentobarbital 40 mg/kg. Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 36.5-37.5℃ . One hundred and nighty isolated rat hearts with t/R injury were randomly assigned to one of 12 groups （n = 15） after 20 min of equilibration： control group （group C）, IR group, IR ＋ LY group, IR ＋ PD group, IR ＋ ATR group, IR＋5-HD group, IR ＋ DMSO group, sevoflurane postconditioning group （group S）, S ＋ LY group, S ＋ PD group, S ＋ ATR group and S ＋ 5-HD group. Group C received continuous perfusion for another 90 min. Group IR was made ischemic for 30 min followed by 60 min of reperfusion. Group IR ＋ LY, IR ＋ PD, IR ＋ ATR, IR ＋ 5-HD and IR ＋ DMSO received perfusion with PI3K inhibitor LY294002 （LY） 15μmol/L, ERK 1/2 inhibitor PD98059 （PD） 20 μmol/L, mPTP opener atractyloside （ATR） 20μmol/L, mito-KATP charmel blocker 5-hydroxydecanoatc （5-HD） 100μmol/L and 0.02% the vehicle dimethyl sulfoxide （DMSO） respectively for 15 min immediately before reperfusion. Group S received perfusion with K-H solution saturated with 3% sevoflurane for 15 min and then with plain K-H solution for 45 min. Group S ＋ LY, S ＋ PD, S ＋ ATR and S ＋ 5- HD received perfusion with K-H solution saturated with 3 % sevoflurane and with LY 15μmol/L, PD 20 μmol/L, ATR 20 p_mol/L and 5-HD 100 μmol/L respectively at the same time for 15 min immediately before reperfusion, and then with plain K-H solution for 45 min. Coronary flow （CF） was measured and left ventricular developed pressure （LVDP）,± dp/dtmax, left ventricular end-diastolic pressure （LVEDP） and HR were recorded at 20 min of equilibration, immediately before suspension of perfusion and at 15, 30 and 60 min of reperfusion （T0-4）. Coronary effluent was collected at To and T4 in group C, IR and S for determination of lactate dehydrogenase （LDH） and creatine kinase-MB （CK-MB） activities and cardiac troponin I （cTnI） concentration. Left ventricle was taken at T4 for determination of infarct size （IS）. The apoptosis was detected using TUNEL and apoptosis index （AI） was calculated in group C, IR, IR ＋ ATR, IR ＋ 5-HD, IR ＋ DMSO, S, S ＋ ATR and S ＋ 5-HD. NAD＋ content in myocardium was determined in group C, IR, IR ＋ LY, IR ＋ PD, IR ＋ DMSO, S, S ＋ LY and S ＋ PD. Results Compared with group C, LVDP, ＋ dp/dt HR and CF were significantly decreased and LVEDP was significantly increased during reperfuion and IS was significantly increased in the other groups, AI was significantly increased in group IR, IR ＋ ATR, IR ＋ 5-HD, IR ＋ DMSO, S, S ＋ ATR and S ＋ 5-HD, NAD＋ content was significantly decreased in group IR, IR ＋ LY, IR ＋ PD, IR ＋ DMSO, S, S ＋ LY and S ＋ PD, and activities of LDH and CK-MB and cTnl concentration were significantly increased in group IR and S （ P 〈 0.05） . Compared with group IR, LVDP, ± dp/dt HR and CF were significantly increased and LVEDP was significantly decreased during reperfusion, IS and AI were significantly decreased, NAD ＋ content was significantly increased, and activities of LDH and CK-MB and cTnI concentration were significantly decreased in group S （ P 〈 0.05）, but there was no significant difference in the indices mentioned above between the left ten groups and group IR （ P 〉 0.05）. Conclusion Sevoflurane postconditioning may attenuate IR injury to isolated rat hearts through activating PI3K and ERK 1/2, promoting mito-KATP channel opening and inhibiting mPTP opening.