摘要
目的探讨地榆皂苷Ⅱ对H2O2诱导的H9C2心肌细胞株凋亡的作用机制。方法采用不同浓度地榆皂苷Ⅱ(分别为5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL、100μg/mL)处理H9C2心肌细胞株,采用四甲基偶氮唑盐微量酶反应比色法(MTT)测定细胞活性,荧光显微镜测定活性氧(ROS)表达量,利用流式细胞技术检测细胞凋亡情况,通过蛋白免疫印迹法检测Bax、半胱氨酸蛋白酶-3(Caspase-3)、半胱氨酸蛋白酶-9(Caspase-9)等凋亡相关蛋白含量和细胞磷酸化的细胞外信号调节激酶(p-ERK1/2)、卵黄状黄斑病蛋白3(Bestrophin3)表达情况及应用细胞外信号调节激酶(ERK1/2)抑制剂后表达量的变化。结果MTT检测结果显示:地榆皂苷Ⅱ对心肌细胞株无毒副作用,可有效抑制H2O2对心肌细胞株的损伤。H2O2能明显提高凋亡相关蛋白Bax、Casepase-3及Casepase-9表达;地榆皂苷Ⅱ可减少心肌细胞株ROS产生,促进p-ERK1/2表达,并抑制Bax、Casepase-3及Casepase-9表达。应用ERK1/2抑制剂后结果发现,Bestrophin3表达量明显下降(P<0.05)。结论地榆皂苷Ⅱ可有效抑制心肌细胞株凋亡,原因可能通过促进依赖于ERK1/2表达的Bestrophin3,抑制心肌细胞株凋亡。
Objective To observe the protective mechanism of ziyuglycosideⅡon apoptosis of H9C2 cardiomyocytes induced by H2O2.Methods H9C2 cells were treated with different concentrations of ziyuglycosideⅡ(5μg/mL、10μg/mL、20μg/mL、40μg/mL、80μg/mL、100μg/mL)and apoptosis was detected by tetramethylazolium salt microenzyme reaction colorimetric method(MTT).H9C2 cells were divided into H9C2 group,H2O2 group,ziyuglycosideⅡgroup,and H2O2+ziyuglycosideⅡgroup.The expression of reaction oxygen species(ROS)was detected by fluorescence microscope.The apoptosis of H9C2 cells was detected by Flow cytometry.Apoptosis-related proteins like Bax,cysteine protease-3(Caspase-3),and cysteine protease-9(Caspase-9)were measured by Western Blot.The expressions of phosphorylation of extracellular signaling kinase(p-ERK1/2)and yolk macular disease protein 3(Bestrophin3)were detected by Western Blot.After using inhibitor of extracellular signaling kinase(ERK1/2),the levels of p-ERK1/2,and Bestrophin3 were detected by Western Blot.Results The results of MTT assay showed the different concentrations of ziyuglycosideⅡhad no cytotoxicity on H9C2 cells.Compared with the H2O2 group,the apoptosis rate of the H2O2+ziyuglycosideⅡgroup was decreased.H2O2 could significantly increased levels of Bax,Caspase-3,and Caspase-9.Compared with the H2O2 group,the generation of ROS in H9C2 cells of the H2O2+ziyuglycosideⅡgroup was more less.The expressions of p-ERK1/2 and Bestrophin3 were significantly up-regulated with the treatment of ziyuglycosideⅡ.After the inhibitor p-ERK1/2 was used,expression of Bestrophin3 was decreased effectively.Conclusion ZiyuglycosideⅡcould inhibit apoptosis of H9C2 cells induced by H2O2,which might promote relies on the expression of ERK1/2 Bestrophin3 inhibiting myocardial cell apoptosis.
作者
茹丹
苏立杰
姚轶立
宋玮
王肖龙
RU Dan;SU Lijie;YAO Yili;SONG Wei;WANG Xiaolong(Shuguang Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China)
出处
《中西医结合心脑血管病杂志》
2020年第8期1221-1226,共6页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
上海中医药大学预算内课题项目(No.2019LK013)。
