2019 年第 9 期 第 14 卷

人参皂苷Rb1通过腺苷酸活化蛋白激酶通路改善乳鼠缺氧心肌细胞自噬能力分析

Ginsenoside-Rb1 improves autophagy ability of hypoxia cardiomyocytes of neonatal rats by adenosine monophosphate-activated protein kinase pathway

作者：宋丽杰孔宏亮蒋玉昆黄华廷

英文作者：

单位：110016沈阳，辽宁省人民医院中国医科大学人民医院心脏中心

英文单位：

关键词：心肌细胞；缺氧；人参皂苷Rb1；腺苷酸活化蛋白激酶；自噬流

英文关键词：

• 摘要：

• 【摘要】目的    探讨人参皂苷Rb1(Gs-Rb1)能否改善氯化钴(CoCl2)诱导的心肌细胞自噬能力以及该效应是否通过腺苷酸活化蛋白激酶（AMPK）通路介导。方法    将1～3 d Wistar乳鼠心肌细胞在Dulbecco改良培养基（含10%胎牛血清）中同步孵育3 d后随机分为对照组、缺氧组（氯化钴）、Gs-Rb1组(Gs-Rb1+氯化钴)、Ara-A组(Ara-A+氯化钴，Ara-A为AMPK活性抑制剂)、Ara-A+Gs-Rb1组（Ara-A+Gs-Rb1+氯化钴)、AICAR组（AICAR+氯化钴，AICAR为AMPK激活剂）和AICAR+Gs-Rb1组(AICAR+Gs-Rb1+氯化钴)。氯化钴、Gs-Rb1、Ara-A和AICAR干预浓度分别为500 μmol/L、200 μmol/L、500 μmol/L和1 mmol/L。分别干预12 h后，噻唑蓝法检测细胞存活率，酶联免疫吸附试验（ELISA）法检测心肌肌钙蛋白I(cTnI)水平，DCFH-DA ELISA法检测AMPK活性，蛋白质印迹法测定心肌细胞自噬标记蛋白Atg4B、Atg5、Beclin1、Atg7、LC3BⅡ、LC3BⅠ、P62表达。结果    缺氧组心肌细胞存活率明显低于对照组［（0.63±0.03）比（0.98±0.03）］，Gs-Rb1组、AICAR组和AICAR+Gs-Rb1组心肌细胞存活率［（0.82±0.02）、（0.79±0.01）、（0.83±0.02）］均明显高于缺氧组，Ara-A+Gs-Rb1组心肌细胞存活率［（0.74±0.01）］明显低于Gs-Rb1组（均P＜0.05）。Gs-Rb1组、AICAR组和AICAR+Gs-Rb1组cTnI水平均明显低于缺氧组［（118±30）、（116±10）、（97±11）ng/L比（188±45）ng/L］，Gs-Rb1组cTnI水平明显低于Ara-A+Gs-Rb1组［（161±20）ng/L］（均P＜0.05）。缺氧组、Ara-A组和Ara-A+Gs-Rb1组心肌细胞AMPK活性明显低于对照组［（2.09±0.05）、（0.07±0.00）、（0.09±0.00）比（7.11±0.03）］，Gs-Rb1组、AICAR组、AICAR+Gs-Rb1组AMPK活性［（4.38±0.03）、（4.09±0.01）、（4.47±0.01）］明显高于缺氧组(均P＜0.05)。缺氧组Atg4B、Atg5、Beclin-1、Atg7、LC3BⅡ和P62的表达及LC3BⅡ/LC3BⅠ比值均明显高于对照组(均P＜0.05)。与缺氧组相比，Gs-Rb1组、AICAR组和AICAR+Gs-Rb1组的Atg4B、Atg5、Beclin1、Atg7和P62的表达及LC3BⅡ/LC3BⅠ比值均明显降低(均P＜0.05)，且3组间各指标比较差异均无统计学意义（均P＞0.05）。结论    Gs-Rb1可能通过改善缺氧心肌细胞自噬能力进而提高缺氧心肌细胞生存能力，此效应可被AMPK通路介导。

• 【Abstract】Objective    To investigate the mechanisms of ginsenoside-Rb1(Gs-Rb1) improving cobalt chloride(CoCl2)-induced autophagy of cardiomyocytes via the adenosine monophosphate-activated protein kinase(AMPK) pathway. Methods    Cardiomyocytes from 1-3-day-old Wistar neonatal rats were incubated in Dulbecco modified culture-medium supplemented with 10% fetal bovine serum for 3 days and randomly divided into 7 groups: control group, hypoxia group(CoCl2), Gs-Rb1 group(Gs-Rb1+ CoCl2), Ara-A group(AMPK inhibitor Ara-A+CoCl2), Ara-A+Gs-Rb1 group(Ara-A+Gs-Rb1+ CoCl2), AICAR group(AMPK activator AICAR+CoCl2) group and AICAR+Gs-Rb1 group(AICAR+Gs-Rb1+CoCl2). Concentrations of CoCl2, Gs-Rb1, Ara A and AICAR were 500 μmol/L, 200 μmol/L, 500 μmol/L and 1 mmol/L, respectively. Cells were treated for 12 hours. Cell viability was determined by MTT assay. Cardiac troponin I(cTnI) expression was detected by enzyme linked immunosorbent assay（ELISA）. AMPK activity was assessed by DCFH-DA ELISA assay. Expressions of autophagy marker protein Atg4B, Atg5, Beclin1, Atg7, LC3BⅡ, LC3BⅠ and P62 were detected by western blotting. Results    Survival rate of myocardial cells in hypoxia group was significantly lower than that in control group［（0.63±0.03） vs （0.98±0.03）］; survival rates of myocardial cells in Gs-Rb1 group, AICAR group and AICAR+Gs-Rb1 group［（0.82±0.02）,（0.79±0.01）,（0.83±0.02）］ were significantly higher than that in hypoxia group; survival rate of myocardial cells in Ara-A+Gs-Rb1 group［（0.74±0.01）］ was significantly lower than that in Gs-Rb1 group(all P＜0.05). Levels of cTnI in Gs-Rb1 group, AICAR group and AICAR+Gs-Rb1 group were significantly lower than that in hypoxia group［（118±30）,（116±10）,（97±11）ng/L vs （188±45）ng/L］; cTnI level in Gs-Rb1 group was significantly lower than that in Ara-A+Gs-Rb1 group［（161±20）ng/L］(all P＜0.05). AMPK activities in hypoxia group, Ara-A group and Ara-A+Gs-Rb1 group were significantly lower than that in control group［（2.09±0.05）,（0.07±0.00）,（0.09±0.00） vs （7.11±0.03）］; AMPK activities in Gs-Rb1 group, AICAR group and AI+Gs-Rb1 group［（4.38±0.03）,（4.09±0.01）,（4.47±0.01）］ were significantly higher than that in hypoxia group(all P＜0.05). Expression levels of Atg4B, Atg5, Beclin-1, Atg7, LC3BⅡ and P62 and LC3BⅡ/LC3BⅠ ratio in hypoxia group were significantly higher than those in control group(all P＜0.05). Expression levels of Atg4B, Atg5, Beclin1, Atg7 and P62 and LC3BⅡ/LC3BⅠ ratio in Gs-Rb1 group, AICAR group and AICAR+Gs-Rb1 group were significantly lower than those in hypoxia group(all P＜0.05); there was no significant difference of the expression levels among Gs-Rb1 group, AICAR group and AICAR+Gs-Rb1 group(all P＞0.05). Conclusion    Gs-Rb1 may improve the viability of hypoxia cardiomyocytes by ameliorating cell autophagy via the up-regulation of AMPK pathway.