p38MAPK信号转导通路在七氟烷后处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用

The role of p38 mitogen-activated protein kinase pathway in protective effect of sevoflurane postconditioning on cultured neonatal rat cardiomyocytes against anoxia/reoxygenation injury

目的评价p38丝裂原活化蛋白激酶（p38MAPK）信号转导通路在七氟烷后处理减轻乳鼠心肌细胞缺氧复氧损伤中的作用。方法健康新生SD大鼠，日龄1～3d，处死后取心室肌组织，培养心肌细胞，随机分为7组：对照组（C组）于CO2培养箱中持续培养3h；缺氧复氧组（AR组）细胞缺氧2h，复氧1h；七氟烷后处理组（SP组）细胞缺氧2h，复氧开始即刻更换为3％七氟烷饱和的DMEM培养液，孵育20min，再更换为无血清DMEM培养液，继续复氧40min；七氟烷后处理＋SB203580组（SP＋SB组）于七氟烷后处理同时加入SB203580（p38MAPK特异性抑制剂）至5μmol／L，孵育20min；七氟烷后处理＋二甲亚砜组（SP＋DMSO组）于七氟烷后处理同时加入0．1％DMSO，孵育20min；SB203580组（SB组）于复氧开始时加入SB203580至5μmol／L，孵育20min；二甲亚砜组（DMSO组）于复氧开始即刻加入0．1％DMSO，孵育20min。各组细胞分别接种于24孔培养板（1ml／孔）、35mm培养皿（5ml／皿）和50ml培养瓶（8ml／瓶）中，每组12孔、6皿和6瓶。于复氧结束后，采用比色法测定细胞培养液乳酸脱氢酶（LDH）活性；采用台盼蓝排斥实验测定细胞存活率；采用流式细胞仪测定细胞凋亡率；采用Western blot法测定磷酸化p38MAPK（p-p38MAPK）表达水平。结果与C组比较，其余各组LDH活性升高，细胞存活率降低，细胞凋亡率升高，AR组、SP组、SP＋SB组、SP＋DMSO组和DMSO组p-p38MAPK表达上调（P〈0．05）；与AR组比较，SP组、SP＋SB组和SP＋DMSO组LDH活性降低，细胞存活率升高，细胞凋亡率降低，p-p38MAPK表达上调（P〈0．05）；与SP组比较，SP＋SB组、SB组和DMSO组LDH活性升高，细胞存活率降低，细胞凋亡率升高，p-p38MAPK表达下调（P〈0．05）。结论七氟烷后处理可通过激活p38MAPK信号转导通路减轻乳鼠心肌细胞缺氧复氧损伤。

Objective To evaluate the role of p38 mitogen-activated protein kinase （p38MAPK） signal pathway in the protective effect of sevoflurane postconditioning （S-Postcon） on cultured neonatal rat cardiomyocytes against anoxia/reoxygenation （A/R） injury. Methods Primary cultured neonatal rat cardiomyocytes were randomly divided into 7 groups： group Ⅰ normal control （C）; group Ⅱ A/R; group Ⅲ S-Postcon ＋ A/R; group Ⅳ S-Postcon ＋ SB203580 ＋ A/R; groupV S-Postcon ＋ DMSO ＋ A/R; group Ⅵ SB203580 ＋ A/R and group Ⅶ DMSO ＋ A/R. Group Ⅱ-Ⅶ were exposed to 2 h anoxia （95% N2-5% CO2 ） followed by 1 h reoxygenation. In group Ⅲ , Ⅳ and Ⅴ the cultured myocytes were postconditioned with 20 min 3 % sevoflurane in 97 % 02 alone （in group Ⅲ ） or in conjunction with 5 μmol/L SB203580 （a specific p38 MAPK inhibitor） （in group Ⅳ ） or 0.1% DMSO （in group Ⅴ ） followed by 40 min reoxygenation. The cardiomyocytes were reoxygenated in the presence of 5 μmol/L SB203580 in group Ⅵ or 0.1% DMSO in group Ⅶ . The LDH activity, cell survival rate and apoptotic rate were measured at the end of the experiment. The levels of phosphor-p38MAPK （p-p38MAPK） was detected by Western blotting.Results S-Postcon reduced LDH activity and apoptofic rate and increased cell survival rate and the level of p-p38MAPK as compared with group A/R （group Ⅱ ）. The myocardial protective effect of S-Postcon was eliminated by p38MAPK inhibitor-SB203580 and p-p38MAPK level was also decreased at the same time. Conclusion Sevofluranc postconditioning can attenuate anoxia/rcoxygenation induced cardiomyocyte injury through activation of p38MAPK signal pathway.