Y-27632预处理对心肌缺血再灌注损伤过程中心肌细胞凋亡的影响研究

Y-27632 attenuates myocardial ischemia-reperfusion injury in rats

目的探讨Rho激酶抑制剂Y-27623对心肌缺血再灌注损伤(MIRI)中细胞凋亡的影响,以及对丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)和凋亡相关蛋白表达水平的变化和意义.方法成年雄性SD大鼠60只,应用随机数字表法分为4组,每组15只:正常对照组(Sham组)、缺血再灌注组(ischemia-reperfusion,I/R组)、Dil组(Diltiazem,地尔硫卓组)和Y-27632组.Dil组每日给予地尔硫卓(10 mg/kg)灌胃,Y-27632组每日给予Y-27632(5 mg/kg),其余两组给予等体积清水.给药5天后Sham组只穿线,不结扎冠状动脉左前降支,I/R组、Dil组和Y-27632组均建立MIRI模型.TUNEL法检测各组心肌凋亡,计算心肌细胞凋亡指数(AI),western blotting法检测心肌组织中MAPK信号传导途径相关蛋白(p-JNK/ERK/P38)和凋亡相关蛋白(Bcl-2、Bax、Caspase-3和Caspase-9)的表达.结果相对于Sham组,I/R组AI明显增加(35.15±3.12比1.15±0.12,P<0.01),MAPK信号传导途径(p-JNK:0.792±0.071比0.344±0.055;p-ERK:0.809±0.087比0.273±0.055;p-P38:0.781±0.049比0.158±0.071)和心肌促凋亡相关蛋白(Bax:1.127±0.060比0.47±0.054;Caspase-3:0.843±0.092比0.289±0.068;Caspase-9:0.831±0.068比0.273±0.048)表达显著增加(P均<0.05),心肌抗凋亡蛋白Bcl-2表达显著减少(0.255±0.060比0.809±0.056,P<0.05);相对于I/R组,Y-27632治疗组AI明显下降(20.05±1.75比35.15±3.12,P<0.01),与Dil治疗组差异无统计学意义(20.05±1.75比22.12±2.01,P>0.05),Y-27632治疗组MAPK信号传导途径相关蛋白(p-JNK:0.443±0.027比0.792±0.344;p-ERK:0.284±0.038比0.809±0.087;p-P38:0.301±0.049比0.781±0.049)和Bax(0.807±0.072比1.127±0.060)、Caspase-3(0.329±0.040比0.843±0.092)和Caspase-9(0.454±0.048比0.831±0.068)表达均显著降低(P<0.05),Bcl-2表达显著增加(0.582±0.048比0.255±0.060,P<0.05),与Dil治疗组差异无统计学意义(p-JNK:0.443±0.027比0.552±0.044;p-ERK:0.284±0.038比0.273±0.049;p-P38:0.301±0.049比0.339±0.038;Bax:0.807±0.072比0.645±0.108;Caspase-3:0.329±0.040比0.378±0.096;Caspase-9:0.454±0.048比0.434±0.032;Bcl-2:0.582±0.048比0.614±0.06,P均>0.05).结论Y-27632通过抑制JNK/ERK/P38的磷酸化,抑制Bax、Caspase-3和Caspase-9的表达,加强Bcl-2的表达,减少心肌细胞的凋亡,从而减轻心肌缺血再灌注损伤.

Objective To investigate the effects of Rho kinase inhibitor Y-27623 on apoptosis in myocardial ischemia reperfusion injury(MIRI)and the changes of expression levels of mitogen-activated protein kinase(MAPK)and apoptosis related proteins.Methods 60 adult male SD rats were randomly divided into 4 groups(n=15):Sham group.ischemia reperfusion group(I/R group),Dil group(Diltiazem group)and Y-27632 group.Dil group reccived daily gavage of diltiazem(10 mg/kg).Y-27632 group received daily gavage of Y-27632(5 mg/kg)and the other two groups received equal volume of water.Five days afier the administration.the Sham group was only threaded and the left anterior descending coronary artery was not ligated.The MIRI model was established in the I/R group,Dil group and Y-27632 group.Myocardial apoptosis was detected by TUNEL method and myocardial cell apoptosis index(Al)was calculated.Expressions of MAPK signaling pathway related proteins(p-JNK/ERK/P38)and apoptosis-related proteins(Bcl-2.Bax.Caspase-3 and Caspase-9)in myocardial tissues were detected by western blotting method,Results Compared with Sham group,AI in I/R group was significantly increased(35.15±3.12 vs.1.15±0.12.P<0.01);the expression of MAPK signal transduction pathway(p-JNK:0.792±0.071 vs.0.344±0.055;p-ERK:0.809±0.087 vs.0.273±0.055;p-P38:0.781±0.049 vs.0.158±0.071)and myocardial pro-apoptosis-related proteins(Bax:1.127±0.060 vs.0.470±0.054;Caspase-3:0.843±0.092 vs.0.289±0.068;Caspase-9:0.831±0.068 vs.0.273±0.048)was significantly increased(all P<0.05)and the expression of myocardial anti-apoptotic protein Becl-2 was significantly decreased(0.255±0.060 vs.0.809±0.056.P<0.05).Compared with the I/R group,AI in the Y-27632 treatment group dereased significantly(20.05±1.75 Vs.35.15±3.12.P<0.01),the expression of MAPK signaling pathway(p-JNK:0.443±0.027 vs.0.792±0.344:p-ERK:0.284±0.038 vs.0.809±0.087;p-P38:0.301±0.049 vs.0.781±0.049),Bax(0.807±0.072 vs.1.127±0.060),Caspase-3(0.329±0.040 vs.0.843±0.092)and Caspasc-9(0.454±0.048 vs.0.831±0.068)in the Y-27632 treatment group was significantly reduced(P<0.05)and the expression of Bc1-2 was significantly increased(0.582±0.048 Vs.0.255±0.060.P<0.05);there were no significant difference between the Dil treatment group and the Y-27632 treatment group(p-JNK:0.443±0.027 vs.0.552±0.044;p-ERK:0.284±0.038 vs.0.273±0.049;p-P38:0.301±0.049 vs.0.339±0.038;Bax:0.807±0.072 vs.0.645±0.108;Caspase-3:0.32910.040 vs.0.378±0.096;Caspase-9:0.454±0.048 vs.0.434±0.032;Bcl-2:0.582±0.048 vs.0.614±0.060;all P>0.05).Conclusion By inhibiting the phosphorylation of JNK/ERK/P38 and then the expression of Bax.Caspasc-3 and Caspase-9 and enhancing the expression of Bcl-2 and Y-27632 can reduce the apoptosis of cardiac myocytes.thus reducing the myocardial ischemia-reperfusion injury.