再灌注抢救激酶在肥厚心肌缺血后适应中的作用

Ischemic postconditioning protects hypertrophic myocardium by reperfusion injury salvage kinase in mice

目的探讨再灌注抢救激酶细胞外信号调节激酶1/2(ERK1/2)和磷脂酰肌醇3激酶-蛋白激酶B(PI3K-Akt)信号通路在缺血后适应(IPost)对减轻肥厚心肌缺血再灌注(I/R)损伤中的作用。方法 12周龄C57/BL小鼠通过主动脉弓缩窄4周建立心肌肥厚模型,利用Langendorff灌流装置建立小鼠肥厚心肌I/R模型,30min全心缺血随后再灌注15、120min。分为缺血再灌注组(I/R组)、后适应组(IPost组,采取缺血10s及再灌注10s的3次IPost周期)、I/R+抑制剂组[分别加入ERK1/2特异性抑制剂PD98058,Akt特异性抑制剂渥曼青霉素(wortmannin)]、IPost+抑制剂组,进行心脏血流动力学检测,采用三苯基氯化四氮唑染色的方法确定心肌梗死范围,免疫印迹方法检测ERK1/2、p70s6k、Akt、糖原合成酶3β(GSK-3β)总蛋白及磷酸化蛋白表达水平。结果与I/R组比较,IPost组小鼠心脏血流动力学指标左心室收缩压、左室压力上升最大速度显著改善[(67±5)比(86±6),(2720±210)比(3678±330)mmHg,均P<0.05],心肌梗死范围减小(均P<0.05)。与I/R组比较,IPost组在再灌注15、120min心肌的ERK1/2、p70s6k蛋白磷酸化水平表达显著增加;与I/R组比较,I/R+PD98058组上述指标差异无统计学意义(均P>0.05);IPost+PD98058组显示在再灌注的最初15min使用PD98059能消除IPost对肥厚心肌的上述保护作用。与I/R组比较,IPost组再灌注15、120min心肌的Akt、GSK-3β蛋白磷酸化水平表达差异无统计学意义(均P>0.05);I/R+wortmannin组、IPost+wortmannin组上述指标差异亦无统计学意义(均P>0.05)。结论 IPost能有效地减轻离体小鼠肥厚心肌缺血再灌注损伤,ERK1/2细胞信号途径是IPost对缺血再灌注肥厚心肌保护作用的重要信号通路;而PI3K-Akt信号通路未参与IPost上述保护作用。

Objective To determine the effect of ischemic postconditioning（IPost）protection in hypertrophic myocardium subjected to ischemic-reperfusion（I/R）injury and to study the role of reperfusion injury salvage kinase（RISK）in mediating such protection.Methods Transversing aortic constriction（TAC）was induced for 4 weeks in 12 weeks old C57/BL mice to establish left ventricular hypertrophy models.Hypertrophic myocardium I/R injury was then induced by 30 min globe ischemia,followed by 15 min or 120 min reperfusion in Langendorff-perfused isolated models,which were randomly divided into 4 groups：（1）I/R group undergoing 30 min global ischemia and 120 min reperfusion;（2）IPost group undergoing ischemia for 10s and reperfusion for 10s.totally 3 cycles after 30 min global ischemia and 15 min or 120 min reperfusion;（3）I/R＋inhibitor（ERK1/2 inhibitor PD98059 or Akt inhibitor wortmannin）group undergoing 15 min reperfusion of Krebs-Henseleit（KH）buffer with inhibitor PD98059 or wortmannin after 30 min global ischemia,and others same as I/R group;（4）IPost＋inhibit or group undergoing ischemia for 10 s and reperfusion for 10 s,totally 3 cycles of IPost after 30 min global ischemia and others same as I/R＋inhibitor group.At the end of reperfusion,hemodynamic examination was carried out while the effects of IPost on infarct size（IS）detected by TTC staining.Protein levels of Extracellular regulated protein kinase（ERK1/2）,ribosomal protein s6 kinases（P70S6K）,Akt and Glycogen synthase kinase 3β（GSK-3β）,as well as phosphorylated proteins,were determined by Western blot after 15 min reperfusion or at the end of reperfusion.Results Compared with the I/R group＇s heart models,IS was significantly reduced in those of the IPost group.The IPost group hadhigher LVSP,dp/dtmax（all P0.05）.Myocardial function was equally improved compared with the I/R group（all P0.05）.ERK1/2 and P70S6K phosphorylation of myocardium was significantly increased in the IPost group after 15 min and 120 min reperfusion（all P0.05）.Compared with the I/R group,no significant changes were found with respect to phosphatidylinositol-3-kinase-Akt（PI3K-Akt）and GSK-3β phosphorylation of myocardium in the IPost group after 15 min and 120 min reperfusion（all P0.05）.However,in IPost＋PD98059 group,in the first 15 min of reperfusion the addition of ERK1/2 inhibitor PD98059 reversed all changes observed in the IPost group and eliminated the IPost protection by increasing IS to a level similar to that in the I/R group.Conclusion IPost plays a pivotal role in reducing ischemia/reperfusion injury in hypertrophic myocardium in vitro.ERK1/2 signaling pathway is involved in the protection of IPost,while PI3K-Akt signaling pathway is not.