摘要
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.
出处
《中华麻醉学杂志》
CAS
CSCD
北大核心
2009年第10期915-922,共8页
Chinese Journal of Anesthesiology