肥厚心肌缺血后适应时细胞外信号调节激酶的保护作用

目的 研究缺血后适应(IPost)在离体小鼠肥厚心肌缺血再灌注(L/R)损伤中的保护作用,探讨细胞外信号调节激酶(ERK1/2)在此保护中的作用机制.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,30 min全心缺血随后再灌注120 min.分为4组,I/R组、Ipost组(采取缺血10 s及再灌注10 s的3次IPost周期)、I/R+PD98059组(ERK1/2抑制剂)、Ipost+PD98059组,进行心脏血流动力学、心肌梗死范围检测,Western印迹方法检测细胞外信号调节激酶(ERK1/2)、Bcl-2、Bax、细胞色素C(Cyt.c)蛋白表达水平,脱氧核苷酸转移酶介导的生物素原位缺口末端标记(TUNEL)法检测心肌细胞的凋亡.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、左心室压力变化最大速率显著改善(85±4)mm Hg比(68±5)mm Hg,(3811±230)mm Hg比(2830±230)mm Hg,均P<0.05],IPost组心肌的ERK1/2磷酸化水平、Bcl-2、线粒体Cyt.C表达显著增加,Bax、胞质Cyt.C蛋白表达显著降低,凋亡指数显著降低,心肌梗死范围减小(均P<0.05).与I/R组比较,I/R+PD98059组上述指标差异均无统计学意义(均P>0.05).IPost+PD98059组显示在再灌注的最初15 min使用PD98059能消除IPost对肥厚心肌的上述保护作用并显著增加心肌梗死面积,与I/R组水平相同.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细胞信号途径参与IPost对缺血再灌注肥厚心肌保护作用并可能通过其抗凋亡的机制实现.

Ischemic postconditioning protects hypertrophic myocardium by ERK1/2 signaling pathway: experiment with mice

Objective To investigate the effects of ischemic postconditioning (IPest) in protecting hypertrophic myocardium subjected to ischemic-reperfusion (I/R) and to study the role of extracellular regulated protein kinase (ERK1/2) in mediating such protection. Methods Transverse aortic constriction (TAG) operation was performed on 12-week-old C57/BL mice to establish left ventricular hypertrophy models. Sixty-four isolated TAG mouse hearts were mounted onto the Langendorff perfusinn system and randomly divided into 4 equal group: (1) I/R group undergoing stable perfusion for 30 rain, ischemia for 30 min, and re-perfusion for 120 min (an I/R cycle) to cause hypertrophic myocardium I/R injury, (2) IPost group undergoing ischemia for 10s and reperfusion for 10s, totally 3 cycles (60s) before reperfusion for 120 rain, (3) I/R + PD98059 (an ERK1/2 inhibitor) group undergoing perfusion of Krebs-Henseleit(KH) buffer with PD98059 10-5>mol/L for 15 min and perfusion of KH buffer without PD98059 at the beginning of re-perfusion, and (4) IPost + PD98059 group undergoing 3 cycles of IPost and perfusion of KH buffer with PD98059 10-5mol/L for 15 min at the beginning of re-perfusion. Hemodynamic examination was conducted 120 min after re-perfusion to measure the left ventricular systolic pressure (LVSP), left ventricular end diastolic pressure (LVEDP), maximal uprising velocity of left ventricle pressure (dp/dtmax), and minimal uprising velocity of left ventricle pressure (dp/dtmin). After the I/R procedure the mvocardium of the left ventricle was isolated to detect the infarction size (IS). Weetem blotting was used to detect the protein expresson of extracellular signal-regulated kinase (ERK) 1/2, phospharylated ERK1/2, Bcl-2, Bax, and mitochondrial and cytosolic eytochrome (Cyt). C. TUNEL was used to detect the apoptotic rate. Results The LVSP and dp/dtmaxlevels of the IPost group were(85 ±4)mm Hg and (3811±230) mm Hg/s, both significantly higher than those of the I/R group (68 ±5)mm Hg and (2830±230) mm Hg/s respectively, both P < 0.05] .Compared with the I/R group, the protein levels of phosphorylated ERK1/2, Bcl-2, mitochondrial CytC of the IPost group were all significantly higher , the protein levels of Bax and cytosolic CytC, and apoptosis index were significantly lower (all p < 0. 05), and the IS was smaller (P < 0. 05). I/R + PD98059 showed no effects on the above-mentioned parameters. However, the results of the IPost + PD98059 group showed that in the first 15 min of reperfusion addition of PLY)8059 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 has protective effect in hypertrophic myocardium with L/R injury in vitro. ERK1/2 signaling pathway is involved in the protection of IPost by regulating the myocyte apoptosis.