七氟醚预处理联合后处理对新生大鼠心肌细胞缺氧复氧损伤的影响

摘要

Objective To investigate the effect of sevoflurane preconditioning combined with postconditioning (Spost) on anoxia/reoxygenation (A/R) injury to neonatal rat cardiomyocytes.Methods Primary cultured neonatal rat cardiomyocytes were isolated from SD rats aged 1-3 days and cultured in DMEM liquid culture medium.The cells were seeded in 24-well plates (1 ml/hole),35 mm diameter dishes (5 ml/dish) or in 50 mm culture flasks (8 ml/flask) with a density of 3 × 105/ml and randomly divided into 9 groups (n =24 each):control group (group C),A/R group,Spre group (group S1),Spre + SB203580 group (group S1 + SB),sevoflurane postcon-ditioning (Spost) group (group S2),Spost + SB203580 group(group S2 + SB),Spre + Spost group (group S3),Spre + Spost + SB203580 group (group S3 + SB),and group SB203580 (group SB).The cells were cultured routinely for 160 min in group C and the cells were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 120 min followed by reoxygenation for 20 min in the other groups.The cells were incubated with 2.5 ％ sevoflurane for 20 min before anoxia in groups S1,S1 + SB,S3 and S3 + SB and in addition SB203580 (specific p38MAPK inhibitor) 5 μmol/L was added simultaneously in groups S1 + SB and S3 + SB.The cells were incubated with 2.5％ sevoflurane for 20 min after beginning of reoxygenation in groups S2,S2-SB,S3 and S3 + SB,and in addition SB203580 5 μmol/L was added simultaneously in groups S2 + SB and S3 + SB.The cells were incubated with SB203580 5 μmol/L for 20 min before anoxia and after beginning of reoxygenation in group SB.The lactate dehydrogenase (LDH) activity,cell survival rate and apoptotic rate were measured at the end of reoxygenation.The levels of phosphor-p38MAPK (p-p38MAPK) was detected at the end of Spre and Spost.Results Compared with group C,the LDH activity and apoptotic rate were significantly increased,while the cell survival rate was significantly decreased in the other groups (P ＜ 0.05).Compared with group A/R,the LDH activity and apoptotic rate were significantly decreased,while the cell survival rate was significantly increased in groups S1,S2 and S3 (P ＜ 0.05).There was no significant difference in the LDH activity,cell survival rate and apoptotic rate between groups S1,S2and S3 (P ＞ 0.05).The myocardial protective effect of Spre or Spost alone or in combination was eliminated by SB203580 (P ＜ 0.05).Spre or Spost alone up-regulated the expression of p-p38MAPK,Spre combined with Spost offered no additional benefit over Spre or Spost alone,and the up-regulative effect was eliminated by SB203580 (P ＜ 0.05).Conclusion Spre combined with Spost produces similar myocardial protective effect with that of either alone and it may because that both Spre and Spost attenuate A/R-induced injury to cardiomyocytes through p38MAPK signaling pathway.%目的 评价七氟醚预处理联合后处理对新生大鼠心肌细胞缺氧复氧损伤的影响.方法 健康新生SD大鼠,1～3d龄,处死后取心室肌组织,分离心肌细胞,将心肌细胞于DMEM培养液中培养,细胞密度3×105个/ml,接种于24孔培养板(1 ml/孔)、35 mm培养皿(5 ml/皿)或50 ml培养瓶(8ml/瓶),采用随机数字表法,将其随机分为9组(n=24):正常对照组(C组)常规培养160 min；缺氧复氧组(A/R组)、七氟醚预处理组(S1组)、七氟醚预处理+SB203580组(S1+SB组)、七氟醚后处理组(S2组)、七氟醚后处理+ SB203580组(S2+SB组)、七氟醚预处理+七氟醚后处理组(S3组)、七氟醚预处理+七氟醚后处理+ SB203580组(S3+SB组)和SB203580组(SB组)均进行缺氧120 min,复氧20 min.S1组、S1+ SB组、S3组和S3+SB组缺氧前用2.5％七氟醚孵育20 min,S1+SB组和S3+SB组七氟醚预处理同时加入5 μmol/L p38丝裂原激活蛋白激酶(p38MAPK)特异性抑制剂SB203580；S2组、S2+SB组、S3组和S3+ SB组复氧开始时采用2.5％七氟醚孵育20 min,S2+SB组和S3+SB组于七氟醚后处理同时加入5 μmol/L SB203580；SB组缺氧前20 min和复氧开始时均用5μmol/L SB203580孵育20 min.复氧结束时测定细胞培养液乳酸脱氢酶活性、细胞存活率和细胞凋亡率,分别于七氟醚预处理结束和后处理结束时测定磷酸化p38MAPK (p-p38MAPK)表达水平.结果 与C组比较,其余各组乳酸脱氢酶活性和细胞凋亡率升高,细胞存活率降低(P＜0.05)；与A/R组比较,S1组、S2组和S3组LDH活性和细胞凋亡率降低,细胞存活率升高(P＜0.05)；S1组、S2组和S3组间上述指标差异无统计学意义(P＞0.05)；SB203580可取消七氟醚预处理、后处理及二者联合应用时心肌保护效应(P＜0.05).七氟醚预处理和后处理均可上调p-p38MAPK表达,且二者联合应用时上调p-p38MAPK表达的效应并未增强,SB203580可取消其上调p-p38MAPK表达的效应(P＜0.05).结论 七氟醚预处理联合后处理减轻新生大鼠心肌细胞缺氧复氧损伤效应与单独一种措施的效应相似,其机制与二者均通过p38MAPK信号转导通路发挥心肌保护作用有关.