人心肌细胞缺血再灌注损伤后ERK、Akt的表达以及黄芪多糖的干预作用

Study on the expression of ERK and Akt on HCMEC of myocardial ischemia-reperfusion injury and the effects of astragalus polysaccharides

目的观察人心肌细胞在缺血再灌注损伤过程中蛋白激酶B(protein kinase B,PKB)又称Akt,与细胞外信号调节激酶1/2(extracellar signal-regulated kiase,ERK1/2)的表达规律及其与细胞凋亡的关系,以探索黄芪多糖产生心肌保护作用的机制。方法培养原代人心脏微血管内皮细胞(human cardiac microvascular endothelial cells,HCMEC),通过缺氧再复氧刺激细胞损伤,模拟缺血再灌注损伤的实验技术平台,建立缺血再灌注损伤模型,继而采用黄芪多糖进行干预,随机分为:HCMEC正常对照组(对照组)、HCMEC损伤组(损伤组)、HCMEC损伤组+低浓度的药物(低药物组)、HCMEC损伤组+中浓度的药物(中药物组)、HCMEC损伤组+高浓度的药物(高药物组),进而采用Western Blot方法检测ERK、p-ERK、Akt蛋白表达情况,采用流式细胞仪检测细胞周期及细胞凋亡情况。结果从流式细胞仪结果显示:对照组为5.43%;损伤组细胞凋亡程度增高最明显,为92.88%;高药物组次之,为29.78%;低药物组及中药物组细胞凋亡程度较轻,生存状态较佳,分别为12.15%和6.08%。Western Blot结果显示:与对照相比较,p-ERK、ERK及Akt磷酸化水平在各组中表达均显著下降(P<0.05);与损伤组相比,低药物组、中药物组和高药物组三组p-ERK、ERK及Akt磷酸化水平升高(P<0.05);与低药物组相比,中药物组中p-ERK、ERK及Akt磷酸化水平升高(P<0.05)。结论黄芪多糖可提高人心肌细胞缺血再灌注损伤后ERK、Akt的活性,减少细胞凋亡。

Objective To investigate observe the expression of protein kinase B(PKB or Akt)and extracellular signal-regulated kinase1/2(ERK1/2)in human cardiomyocytes during ischemia-reperfusion injury and its relationship with cell apoptosis,so as to explore the protective mechanism of Astragalus Polysaccharides on myocardial cells.Methods The human cardiac microvascular endothelial cells(HCMEC)were cultured.The myocardial ischemia-reperfusion injury model was established by the hypoxia and reoxygenation to stimulate cellular damage.Then APS was used to intervene.The model rats were divided into HCMEC normal control group(control group),HCMEC injury group(injury group),HCMEC injury group+low concentration drug(low drug group),HCMEC injury group+medium concentration drug(medium group),HCMEC injury group+high concentration drug(high drug group).The expressions of ERK,p-ERK,Akt were detected by western blot,flow cytometry was used to analyze cell cycle and cell apoptosis.Results Flow cytometry results showed that the apoptosis rate of control group was5.43%;the apoptosis rate of injury group was92.88%which was the most obviously;apoptosis rate of high drug group was29.78%;apoptosis rate of low drug group and medium drug group was12.15%and6.08%respectively.Western Blot results showed that:compared with the control,the phosphorylation levels of p-ERK,ERK,and Akt were significantly decreased in each group(P<0.05);compared with the injury group,the phosphorylation levels of p-ERK,ERK,and Akt in low drug group,medium drug group and high drug group were increased(P<0.05);compared with low drug,the phosphorylation levels of p-ERK,ERK,and Akt in the medium group were increased(P<0.05).Conclusion Astragalus polysaccharides can improve the activity of ERK and Akt after ischemia reperfusion injury in human heart muscle cells and reduce apoptosis.