环磷酸鸟苷/蛋白激酶G信号通路的激活与再灌注损伤挽救激酶信号通路的关系

Relationship between activation of cyclic guanosine monophosphate/protein kinase G signaling pathway and reperfusion injury saloage kinase signaling pathway

摘要:

目的：观察脑钠肽（brain natriuretic peptide，BNP）激活环磷酸鸟苷/蛋白激酶G（cyclic guanosine monophosphate/protein kinase G，cGMP/PKG）信号通路的心肌保护作用是否与细胞外调节蛋白激酶１/２（extracellular regulated protein kinase1/2，ERK1/2）、磷酸酰肌醇-３-激酶（phosphatidylinositol 3 kinase，PI3K）蛋白激酶的激活有关。方法：将84只新西兰兔随机分为7组（n=12）：假手术组；对照组；BNP组；BNP+LY294002组（LY294002为PI3K抑制剂）；LY294002组；BNP+PD98059组（PD98059为ERK1/2抑制剂）；PD98059组。除了假手术组只开胸不结扎冠状动脉外，其余6组均于结扎左回旋支45 min后恢复左回旋支血流，进行再灌注180 min。全程观察血流动力学及心电变化；实验终末，各组随机抽取6只兔子心脏做左室梗死面积测定，采用伊文思蓝和氯化三苯基四氮唑双染色法测定心肌梗死面积；每组另外6只兔子心脏取梗死边缘区组织，Western blot方法测定P-Akt/Akt、P-ERK1/2/ ERK1/2的表达。结果：心率（heart rate，HR）和平均动脉压（mean arterial blood pressure，MABP）在基线水平上无明显差异（P >0.05）。与基线水平相比，HR和MABP在缺血以及再灌注时期有明显下降（P<0.05），而在再灌注时期维持较稳定的水平。各组之间HR、MABP差异无统计学意义（P >0.05）；与对照组相比，BNP组心律失常发生率明显减少（P<0.003 125）。与BNP组比较，BNP+LY294002组、LY294002组、BNP+PD98059组及PD98059组心律失常明显增加（P<0.003 125），BNP+LY294002组和LY294002组，BNP+PD98059组和PD98059组差异无统计学意义（P >0.003 125）；假手术组无心肌梗死。与对照组比较，BNP组心肌梗死范围明显减少（P <0.05）。与BNP组比较，BNP+LY294002组、LY294002组、BNP+PD98059组及PD98059组梗死面积明显增加（P<0.05）。BNP+LY294002组和LY294002组，BNP+PD98059组和PD98059组差异无统计学意义（P >0.05）；与对照组比较，BNP组Akt、ERK1/2的磷酸化水平明显增加（P <0.05）。与BNP组相比，BNP+LY294002组Akt的磷酸化水平及BNP+PD98059组ERK1/2的磷酸化水平分别明显降低（P <0.05）。BNP+LY294002组和LY294002组Akt的磷酸化水平，BNP+PD98059组和PD98059组ERK1/2的磷酸化水平差异无统计学意义（P >0.05）。结论：BNP激活cGMP/PKG信号通路，对心肌缺血再灌注损伤产生保护作用，且该通路的激活与再灌注损伤挽救激酶通路的激活有关。

Abstract:

Objective：To observe whether myocardial protective effect of brain natriuretic peptide（BNP） which activate cyclic guano－sine monophosphate/protein kinase G（cGMP/PKG）signaling pathway is associated with the activation of extracellular regulated protein kinase1/2（ERK1/2） and phosphatidylinositol 3 kinase（PI3K） protein kinase. Methods：Totally 84 New Zealand white rabbits were randomly divided into seven groups（n=12）：sham group，control group，BNP group，BNP + LY294002 group（LY294002 is PI3K inhibitor），LY294002 group，BNP + PD98059 group（PD98059 is ERK1/2 inhibitor） and PD98059 group. Occlusion of the left circumflex artery for 45 min followed by 180 min reperfusion was performed on rabbits in all groups except those in sham group. Rabbits in sham group were underwent open thoracotomy without ligating the left circumflex artery. Hemodynamic，electrocardiograph monitor were obtained. At the end of experiment，6 rabbit hearts were stained by Evan’s blue/triphenyltetrazolium chloride methods to test the area of infarction. Expressions of P-Akt/Akt and P-ERK1/2/ERK1/2 proteins in the rest hearts were analyzed by Western blot assay. Results：There was no significant difference in heart rate（HR） and mean arterial blood pressure（MABP） at baseline among seven groups（P >0.05）. Compared with the baseline，HR and MABP were significantly decreased during ischemia and reperfusion（I/R） period（P<0.05），HR and MABP remained relatively stable during the reperfusion period. There was no significant difference in the HR and MABP among seven groups during I/R period（P >0.05）. Rate of arrhythmia was significantly reduced in BNP group than in control group（P<0.003 125）. Rate of arrhythmia was significantly increased in BNP + LY294002 group，LY294002 group，BNP + PD98059 group and PD98059 group than in BNP group（P<0.003 125）. Differences in arrhythmia between BNP + LY294002 group and LY294002 group as well as BNP + PD98059 group and PD98059 group were not statistically significant（P >0.003 125）. Myocardial infarction did not occur in sham group. Myocardial infarction size was significantly smaller in BNP group than in control group（P<0.05）. Myocardial infarction size was significantly increased in BNP + LY294002 group，LY294002 group，BNP + PD98059 group and PD98059 group than in BNP group（P<0.05）. Differences in myocardial infarction size between BNP + LY294002 group and LY294002 group as well as BNP + PD98059 group and PD98059 group were not statistically significant（P >0.05）. Level of Akt and ERK1/2 phosphorylation was increased significantly in BNP group than in control group（P<0.05）. Level of Akt phosphorylation in BNP + LY294002 group and level of ERK1/2 phosphorylation were significantly lower in BNP + PD98059 group than in BNP group（P<0.05）. Difference in Akt phosphorylation between BNP + LY294002 group and LY294002 group was not statistically significant（P >0.05）. There was also no difference in ERK1/2 phosphorylation between BNP + PD98059 group and PD98059 group（P >0.05）. Conclusions：Activation of cGMP/PKG signaling pathway by BNP exerts protective effect on myocardial I/R injury，which is associated with activation of reperfusion injury salvage kinase signaling pathway.