蓝萼甲素对H2O2诱导H9c2心肌细胞凋亡的保护作用及机制研究  

Protective effects and potential mechanisms of glaucocalyxin A on H2O2-induced H9c2 cardiomyocytes

作　　者：李津 李羽[1] 刘明洁[2] Li Jin;Li Yu;Liu Mingjie(Department of Anesthesiology,West China Hospital,Sichuan University,Chengdu 610041,China;Medical College,Inner Mongolia University for the Nationalities,Tongliao 028000,China)

机构地区：[1]四川大学华西医院麻醉科,成都610041 [2]内蒙古民族大学医学院,通辽028000

出　　处：《国际中医中药杂志》2020年第12期1137-1144,共8页International Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金(81560590)。

摘　　要：目的观察蓝萼甲素预处理对H2O2诱导H9c2心肌细胞损伤模型的保护作用,并采用网络药理学方法预测蓝萼甲素抗H9c2心肌细胞凋亡的作用机制。方法将H9c2心肌细胞按随机数字表法分为空白对照组、模型组及蓝萼甲素低、中、高剂量组。空白对照组使用无血清培养液处理。模型组采用无血清培养4 h后,加入250 μmol/L H2O2干预6 h。蓝萼甲素低、中、高剂量组分别给予蓝萼甲素0.125、0.250、0.500 μg/ml预处理4 h后,加入浓度为250 μmol/L H2O2干预6 h。采用MTT法检测心肌细胞活力,观察细胞形态,采用Annexin Ⅴ-FITC/PI荧光双染及Hoechst 33258荧光染色检测细胞凋亡情况。通过Pubchem数据库获得蓝萼甲素的化学结构式,使用Swiss Target Prediction和Pharmmapper平台预测蓝萼甲素潜在靶点,利用GeneCards数据库筛选蓝萼甲素抗H9c2心肌细胞凋亡的作用靶点。采用Cytoscape软件构建蓝萼甲素作用的靶点网络,通过String数据库和Cytoscape软件绘制蛋白相互作用网络,并用Metascape数据库对靶点进行GO及KEGG通路富集分析。结果与模型组比较,蓝萼甲素低、中、高剂量组H9c2心肌细胞活力[(66.56±6.51)%、(79.21±6.89)%、(94.06±5.19)%比(51.75±4.14)%]升高(P<0.01),细胞凋亡率[(24.12±4.71)%、(17.42±4.39)%、(7.65±1.56)%比(36.73±5.65)%]降低(P<0.01)。网络药理学分析结果提示,蓝萼甲素有22个与抗H9c2心肌细胞凋亡相关的靶点,主要调节氧化应激、细胞迁移、激酶结合活性等,可调节MAPK1、VEGFA、MMP9、NOS3、MMP2、MAPK14等靶点通路,发挥抗H9c2心肌细胞凋亡作用。结论蓝萼甲素预处理对H2O2诱导H9c2心肌细胞损伤有保护作用,其机制可能通过多靶点-多途径发挥抗氧化应激和减少细胞凋亡的作用。Objective To observe the cardioprotective effects of glaucocalyxin A pretreatment on H2O2-induced H9c2 cell injury in vitro and predict its mechanisms on apoptosis of H9c2 cardiomyocytes by network pharmacology.Methods H9c2 cardiomyocytes were randomly divided into blank control group,model group,low-dose group,medium-dose group and high-dose group.The model group was cultured in serum-free medium for 4 h and then intervened with 250 pmol/L H2O2 for 6 h.H9c2 cells in the low-dose group,medium dose group and high-dose group were pretreated with 0.125,0.250,0.500 pg/ml glaucocalyxin A for 4 hours and then exposed to 250|imol/L H2O2 incubation for 6 hours.The cell viability was measured by MTT assay to determine the effects of glaucocalyxin A.Cell morphology was observed by Hoechst 33258 assay.Flow cytometry analysis of Annexin V-FITC/PI staining was used to assess the effects of glaucocalyxin A on H2O2~induced H9c2 cell apoptosis.The chemical structure of glaucocalyxin A was obtained by Pubchemistry,its potential targets were predicted by Swiss Target Prediction and Pharmmapper,and its candidate targets against apoptosis in H9c2 cells were screened by GeneCards database.Cytoscape software was used to construct the target network of glaucocalyxin A,meanwhile String database and Cytoscape software were used to plot the protein interaction network,and Metascape database was used for enrichment analysis of GO and KEGG pathways of the target.Results Compared with the model group,the H9c2 cell viability(66.56%±6.51%,79.21%±6.89%,94.06%±5.19%vs.51.75%±4.14%)in the the low-,medium-,high-dose group significantly increased(P<0.01),the apoptosis rate(24.12%±4.71%,17.42%±4.39%,7.65%±1.56%w.36.73%±5.65%)significantly decreased(P<0.01).The results of network phannacology analysis indicated that 22 targets was related to apoptosis of H9c2 cells,and showed that glaucocalyxin A was mainly regulated biological processes of oxidative stress and cell migration,and kinase binding activity.Meanwhile,glaucocalyxin A could regulat

关 键 词：细胞凋亡 H9C2心肌细胞 蓝萼甲素 氧化性应激 网络药理学 

分 类 号：R285[医药卫生—中药学]