【作者】 田毅；

【导师】 徐军美；

【作者基本信息】 中南大学 ， 麻醉学， 2010， 博士

【摘要】 目的建立缺血再灌注模型,观察异氟醚、卡托普利联合预处理对兔缺血再灌注的心肌保护作用。方法健康新西兰大白兔,采用结扎冠状动脉左前降支法制备心肌缺血再灌注模型。随机分成6组：假手术组(S组)：仅开胸并分离冠状动脉左前降支,但不阻断血流；缺血再灌注组(I/R组)：缺血30min,再灌注120 mim缺血预处理组(IPC组)：缺血5 min,再灌注5 min,重复3次后同I/R处理；异氟醚预处理组(I组)：给予1.1%异氟醚持续吸入30min,洗脱15min,之后处理同I/R组；卡托普利预处理组(C组)：给予喂饲卡托普利片25 mg-kg-1,24h后处理同I/R组；异氟醚、卡托普利联合预处理组(I+C组)：给予喂饲卡托普利片25 mg·kg-1,24h后处理同I组。记录缺血再灌注前后不同时间点血流动力学指标：阻断前(T0),阻断30min(T1),再灌注30min(T2),再灌注60min(T3),再灌注120min(T4)；记录心律失常发生率；取动脉血检测乳酸脱氢酶(LDH)、肌酸磷酸激酶(CK)、肌酸磷酸激酶同工酶(CK-MB)、心肌肌钙蛋白(cTnI)浓度；再灌注结束后检测血清丙二醛(MDA)浓度及超氧化物歧化酶(SOD)活性；观察各组心肌细胞超微结构变化。结果1.T0-T2 I组与I+C组HR相对高于其它各组,T0-T4 I组、C组与I+C组MAP与RPP相对较低并依次递减(P<0.05,或P<0.01),IPC组、I组、C组再灌注期间心律失常发生率(37.5%、50%、50%)低于I/R组(62.5%),但高于I+C组(25%,P<0.05)。2.心肌酶变化：与S组比,各组心肌酶学指标从T1至T4均较T0有所升高(P<0.05或P<0.01),但IPC组、I组、C组和I+C组增高程度低于I/R(P<0.05),IPC组和I+C组又较I组和C组低(P<0.05)。3.生化指标变化：与S组比,各组MDA均增高,SOD降低(P<0.01),但与I/R组比,各处理组MDA降低,SOD较高(P<0.01)。IPC组和I+C组MDA和SOD分别较I组和C组降低和升高(P<0.05)。4.心肌超微结构变化：预处理组心肌细胞损伤轻于I/R组。结论异氟醚、卡托普利联合预处理对缺血再灌注兔心肌有保护作用,较单独应用异氟醚、卡托普利预处理的保护作用增强,其效果与缺血预处理相似。目的建立缺血再灌注模型,观察异氟醚、卡托普利联合预处理对兔心肌细胞凋亡的影响。方法健康新西兰大白兔,采用结扎冠状动脉左前降支法制备心肌缺血再灌注模型。随机分成6组：假手术组(S组)：仅开胸并分离冠状动脉左前降支,但不阻断血流；缺血再灌注组(I/R组)：缺血30min,再灌注120 min；缺血预处理组(IPC组)：缺血5 min,再灌注5 min,重复3次后处理同I/R组；异氟醚预处理组(I组)：给予1.1%异氟醚持续吸入30min,洗脱15min,之后处理同I/R组；卡托普利预处理组(C组)：给予喂饲卡托普利片25 mg·kg-1,24h后处理同I/R组；异氟醚、卡托普利联合预处理组(I+C组)：给予喂饲卡托普利片25 mg·kg-1,24h后处理同I组。再灌注结束后采用伊文思蓝和TTC染色法检测心肌梗死面积,流式细胞仪检测细胞凋亡的情况。结果1.心肌梗死面积：IPC组、I组、C组和I+C组心肌梗死面积(33.6±3.8%、39.8±5.7%、39.6±3.4%、33.9±5.9%)均小于I/R组(60.7±6.0)(P<0.01),IPC、I+C组心肌梗死面积均小于I组、C组(P<0.05)。2.细胞凋亡：与S组比较各组凋亡指数明显增高(P＜0.01)；与I/R组相比各预处理组细胞凋亡指数均有所降低(P<0.01)；I组和C组较IPC组和I+C组细胞凋亡指数有所增加(P<0.05)。但IPC组和I+C组之间,差异无统计学意义(P＜0.05)。结论1.异氟醚、卡托普利联合预处理可明显减少心肌缺血再灌注的梗死面积以及降低细胞凋亡,较单独药物预处理组作用增强,与缺血预处理的作用相似。2.异氟醚、卡托普利联合预处理对心肌缺血再灌注损伤产生了协同的抗细胞凋亡保护作用。目的建立缺血再灌注模型,初步探讨异氟醚、卡托普利联合预处理对凋亡相关基因的影响,以及对凋亡通路的作用机制。方法健康新西兰大白兔,采用结扎冠状动脉左前降支法制备心肌缺血再灌注模型。随机分成6组：假手术组(S组)：仅开胸并分离冠状动脉左前降支,但不阻断血流；缺血再灌注组(I/R组)：缺血30min,再灌注120 mim缺血预处理组(IPC组)：缺血5 min,再灌注5 min,重复3次后处理同I/R组；异氟醚预处理组(I组)：给予1.1%异氟醚持续吸入30min,洗脱15min,之后处理同I/R组；卡托普利预处理组(C组)：给予喂饲卡托普利片25mg·kg-1,24h后处理同I/R组；异氟醚、卡托普利联合预处理组(I+C组)：给予喂饲卡托普利片25mg·kg-1,24h后处理同I组。再灌注结束后采用免疫印迹法(Western-Blot)检测各组心肌凋亡相关基因Bcl-2、Bax和凋亡通路蛋白Caspase-3、Caspase-8和Caspase-9的表达,流式细胞仪检测细胞凋亡的情况。结果1.各组心肌Bcl-2、Bax蛋白表达的变化：与I/R组比较,各预处理组Bcl-2表达明显增加,Bax表达降低,Bcl-2/Bax比值明显增加,差异有显著统计学意义(P<0.01)；与I组和C组相比,IPC组和I+C组Bcl-2/Bax比值明显增高(P<0.01),IPC组和I+C组之间比值没有差异(P>0.05)。2.各组心肌凋亡通路蛋白Caspase-3、Caspase-8和Caspase-9蛋白表达的变化：与I/R组比较各预处理组Caspase-3、Caspase-8和Caspase-9蛋白表达明显降低,差异有显著统计学意义(P<0.01)；与I组和C组相比,IPC组Caspase-3蛋白表达较低(P<0.01),但与I+C组相比差别无统计学意义(P>0.05)；各药物预处理组Caspase-8蛋白表达与IPC组相比均较低(P<0.05或P<0.01)；I+C组Caspase-8蛋白的表达量也低于I组和C组(P<0.01)；与各药物预处理组相比,IPC组的Caspase-9蛋白表达较低(P<0.01)；I+C组与I组和C组相比,Caspase-9蛋白的表达量也有差异(P<0.05)。3.细胞凋亡：与I/R组相比,各预处理组细胞凋亡指数均有所降低(P<0.01),I+C组凋亡指数低于I组和C组(P<0.05),但与IPC组无差异(P>0.05)结论1.异氟醚、卡托普利联合预处理组通过上调Bcl-2蛋白表达,下调Bax蛋白表达,增加Bcl-2/Bax比值,对缺血再灌注兔心肌产生保护作用。2.异氟醚、卡托普利联合预处理组降低心肌缺血再灌注后Caspase-3、Caspase-8和Caspase-9的蛋白表达,抑制凋亡通路信号转导,减少心肌细胞的凋亡。3.异氟醚、卡托普利联合预处理对凋亡的死亡受体通路和线粒体通路具有抑制作用,但主要是通过死亡受体通路发挥抗凋亡作用。更多还原

【Abstract】 Objective:To investigate the cardioprotective effect of the combina-tion of isoflurane and captopril preconditioning on myocardial ischemia reperfusion injury in rabbit.Methods:All healthy New Zealand white rabbits were randomly assigned to six groups (n=8 each):(1) Sham-operated group(group S), rabbits were opened chest without ligating the left anterior descending coronary artery (LAD); (2) Ischemia/reperfusion group (group I/R) group; (3) Ischemic preconditioning group (group IPC),5min ischemia-5min reperfusion for three cycles before the long time ischemia; (4) Isoflurane group (groupⅠ), rabbits were pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout; (5) Captopril group(group C), rabbits were feeded captopril tablets (25 mg·kg-1) before 24h; (6) Combination of isoflurane and captopril preconditioning group(group I+C), rabbits were feeded captopril tablets (25mg·kg-1) before 24h and pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout after 24h. After preconditioning, all rabitts were treated 30min of ischemia and 2h of reperfusion except for group S. Hemodynamic parameters and incidence rate of arrhythmia were recorded at before ischemia (To),30min after ischemia (T1),30min after reperfusion(T2),60min after reperfusion(T3) and 120min after reperfusion(T4). Blood samples were taken from arterial line for determination of plasma CK, CK-MB, LDH and cTnI levels. At the end of the reperfusion, the plasma concentration of MDA and activity of SOD were examined and myocardial structure was observed under light and electron microscope.Results:Heart Rate(HR) of group I+C was significantly higher than other groups and Mean Arterial Pressure (MAP) was lower (P<0.05 or P＜0.01). And incidence rate of arrhythmia was lower in group I+C (P＜0.05). The CK, CKMB, LDH and cTnI levels of group IPC and group I+C were significantly lower than that of groupⅠ, group C and group I/R (P＜0.05), but were higher than group S (P＜0.05). The injury of group I/R was worse than the others from the changes of the cellular structure under light and electron microscope. Group IPC and group I+C had lower concentration of MDA and also had higher activity of SOD than other groups except for group S.Conclusions:The combination of isoflurane and captopril pre-conditioning induces cardioprotection against ischemia reperfusion injury in rabbits. The cardioprotection was better than that of solitary preconditioning and similar with that of group IPC.Objective:To investigate the effect of the combination of isoflurane and captopril preconditioning on myocardial infarct size(IS) and apoptosis during ischemia-reperfusion in rabbit.Methods:All healthy New Zealand white rabbits were randomly assigned to six groups(n=8 each):(1) Sham-operated group(group S), rabbits were opened chest without ligating the left anterior descending coronary artery; (2) Ischemia/reperfusion group(group I/R); (3) Ischemic preconditioning group(group IPC),5min ischemia-5min reperfusion for three cycles before the long time ischemia; (4) Isoflurane group (groupⅠ), rabbits were pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout; (5) Captopril group(group C), rabbits were feeded captopril tablets (25mg·kg-1) before 24h; (6) Combination of isoflurane and captopril preconditioning group(group I+C), rabbits were feeded captopril tablets (25mg·kg-1) before 24h and pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout after 24h. After preconditioning, all rabitts were treated 30min of ischemia and 2h of reperfusion except for group S. At the end of the reperfusion, infarct size(IS) and area at risk(AAR) were accessde by Evans Blue and TTC staining. Apotosis index were detected by flow cytometry.Results:Myocardial infarct size of group IPC, groupⅠ, group C and group I+C (33.6±3.8%,39.8±5.7%,39.6±3.4%,33.9±5.9%) were less than that of group I/R (60.7±6.0, P<0.01). Myocardial infarct size of group IPC and group I+C were less than that of groupⅠand group C (P<0.05), but distinction between group IPC and group I+C was not significantly (P>0.05). Apoptotic index of group S was the lowest in eath group (P＜0.01). Apoptotic index of group I/R was less than that of group IPC, group I, group C and group I+C(P<0.01). Apoptotic index of group I+C was less than that of groupⅠand group C, but not different with that of group IPC (P＜0.05).Conclusions:The combination of isoflurane and captopril preconditioning can reduce myocardial infarct size and the incidence rate of apoptotic during ischemia reperfusion in rabbits. The effect was better than that of solitary preconditioning and similar with that of group IPC. The combination of isoflurane and captopril preconditioning produced a synergistic protective effect of anti-apoptosis.Objective:To investigate the effect of the combination of isoflurane and captopril preconditioning on apoptosis-related gene and apoptosis pathway during myocardial ischemia and reperfusion in rabbit.Methods:All healthy New Zealand white rabbits were randomly assigned to six groups (n=8 each):(1) Sham-operated group(group S), rabbits were opened chest without ligating the left anterior descending coronary artery; (2) Ischemia/reperfusion group(group I/R); (3) Ischemic preconditioning group (group IPC),5min ischemia-5min reperfusion for three cycles before the long time ischemia; (4) Isoflurane group (groupⅠ), rabbits were pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout; (5) Captopril group (group C), rabbits were feeded captopril tablets (25 mg·kg-1) before 24h; (6) Combination of isoflurane and captopril preconditioning group (group I+C), rabbits were feeded captopril tablets (25 mg·kg-1) before 24h and pretreated with 30min end-tidal 1.1% isoflurane and 15min of washout after 24h. After preconditioning, all rabitts were treated 30min of ischemia and 2h of reperfusion except for group S. At the end of the reperfusion, the heart was harvested and the activity of apoptosis-related gene (Bcl-2 and Bax) and apoptosis pathway proteins (caspase-3, caspase-8 and caspase-9) were determined by Western Blot analysis, apotosis index was detected by flow cytometry.Results:Bcl-2/Bax ratio of group I+C and group IPC was higher than that of groupⅠand group C (P<0.01). But Bcl-2/Bax ratio of group I+C was the same high as that of group IPC. The activity of caspase-3, caspase-8 and caspase-9 of every preconditioning group were lower than that of group I/R. The activity of caspase-8 of group I+C was lower than that of other preconditioning group. The activity of caspase-9 of group IPC was lower than that of drug preconditioning group. Apotosis index of every preconditioning group was lower that of group I/R(P<0.01). Apotosis index of group I+C was lower that that of group I and group C, but ot different with that of group IPC (P>0.05).Conclusions:The combination of isoflurane and captopril preconditioning can promote the activity of Bcl-2, inhibit the activity of Bax and increase Bcl-2/Bax ratio in myocardium during ischemia reperfusion, which maybe one of molecular mechanisms of the combina-tion of isoflurane and captopril preconditioning on cardioprotection. The combination of isoflurane and captopril preconditioning can inhibit the activity of caspase-3, caspase-8 and caspase-9 and signal transduction of apoptosis pathway, which indicate that the combination of isoflurane and captopril preconditioning can inhibit mitochondrion pathway and death receptor pathway, but mainly the latter.更多还原