吴忠秀;陆定艳;杨畅;何彬;李勇军;王永林;刘亭;
WU Zhongxiu;LU Dingyan;YANG Chang;HE Bin;LI Yongjun;WANG Yonglin;LIU Ting;Guizhou Provincial Key Laboratory of Pharmaceutics & State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University;School of Pharmacy,Guizhou Medical University;State Key Laboratory of Functions and Applications of Medicinal Plants & Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,Guizhou Medical University;

• 1:贵州医科大学贵州省药物制剂重点实验室&省部共建药用植物功效与利用国家重点实验室
• 2:贵州医科大学药学院
• 3:贵州医科大学民族药与中药开发应用教育部工程研究中心&省部共建药用植物功效与利用国家重点实验室

摘要(Abstract)：

目的运用网络药理学技术结合细胞体外实验,探究参芎葡萄糖注射液(SGI)的抗细胞凋亡途径。方法采用中药系统药理学平台(TCMSP)和SwissTarget Prediction数据库检索SGI可能的作用靶点,并运用DAVID数据库(DAVID)平台进行京都基因与基因组百科全书(KEGG)通路富集、基因本体(GO)功能分析,预测SGI抗凋亡机制;体外培养H9c2细胞,将细胞分为空白组(Con组)、模型组(H_2O_2组)和SGI低、中、高浓度组,Con组加入完全培养基,H_2O_2组用300μmol/L H_2O_2处理0.5 h,SGI低、中、高浓度组分别用100、200、400μmol/L SGI预处理6 h后,用300μmol/L H_2O_2处理0.5 h;利用H_2O_2建立H9c2细胞凋亡模型,流式细胞术检测线粒体膜电位(MMP)水平,蛋白免疫印迹法(Western blot)检测H9c2细胞中细胞色素C(Cyt-c)的表达水平。结果网络药理学分析结果显示,SGI的12个成分涉及142个靶点,KEGG通路富集涉及丝裂原活化蛋白激酶(MAPK)信号通路、磷脂酰肌醇3-激酶/蛋白激酶B(PI3K-AKT)信号通路等,GO功能分析主要富集在线粒体外膜和线粒体,其生物过程与线粒体膜电位的调控有关;流式细胞术和Western blot实验结果显示,与H_2O_2组比较,SGI低、中、高浓度组均显著增加H_2O_2诱导H9c2细胞中的MMP(P<0.001),并显著降低Cyt-c的释放(P<0.001)。结论SGI可能通过线粒体途径拮抗H_2O_2诱导的H9c2细胞凋亡。
Objective To explore the anti-apoptotic pathway of shenxiong glucose injection(SGI)based on network pharmacology and H9 c2 cell apoptosis model induced by H_2 O_2 Methods The possible SGI targets were detected by the Traditional Chinese Medicine System Pharmacology Platform(TCMSP) and Swiss Target Prediction database;and the Database for Annotation,Visualizationand Integrated Discovery(DAVID) platform was used for Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis and Gene Ontology(GO) function analysis to detect the antiapoptotic mechanism of SGI.H9 c2 cells were cultured in vitro and divided into the control group(Con group),the model group(H_2 O_2 group) and SGI low,medium,and high concentration groups.Con group was added to complete medium.H_2 O_2 group was treated with 300 μmol/L H_2 O_2 for 0.5 h,while SGI low,medium and high concentration groups were pretreated with 100,200 and 400 μmol/L SGI for 6 h and then treated with 300 μmol/L H_2 O_2 for 0.5 h.H9 c2 cell apoptosis model was established induced by H_2 O_2;flow cytometry was used to detect mitochondrial membrane potential(MMP) level;Western blot was used to detect the expression level of Cytochrome c(Cyt-c) in H9 c2 cells.Results The network pharmacology analysis showed that the 12 components of SGI involved 142 targets;KEGG pathway enrichment involved the Mitogen-Activated Protein Kinase(MAPK) signaling pathway,Phosphoinositide 3-Kinases/Protein Kinase B(PI3 K-AKT) signaling pathway,etc.Through GO function analysis,the cell components were mitochondrial outer membrane and mitochondria,and the biological process was related to the regulation of mitochondrial membrane potential.The flow cytometry and Western blot showed that H_2 O_2-induced MMP in H9 c2 cells significantly increased(P <0.001),and the release of Cyt-c were significantly reduced(P <0.001)in SGI low,medium and high concentration groups,compared with those in H202 group.Conclusion SGI possesses an anti-apoptotic function by increasing MMP and reducing the release of Cyt-c,which suggests that SGI may antagonize H_2 O_2-induced H9 c2 cell apoptosis through the mitochondrial pathway.

关键词(KeyWords)： 细胞凋亡;参芎葡萄糖注射液;H9c2细胞;网络药理学;线粒体途径;线粒体膜电位;细胞色素C
cell apoptosis;shenxiong glucose injection(SGI);H9c2 cells;network pharmacology;mitochondrial pathway;mitochondrial membrane potential(MMP);cytochrome c(Cyt-c)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（81760699,U1812403-5）;; 贵州省科技计划项目[黔科合基础（2019）1280号];; 贵州省普通高等学校科技拔尖人才项目[黔教合KY字（2021）033]

作者(Authors): 吴忠秀;陆定艳;杨畅;何彬;李勇军;王永林;刘亭;
WU Zhongxiu;LU Dingyan;YANG Chang;HE Bin;LI Yongjun;WANG Yonglin;LIU Ting;Guizhou Provincial Key Laboratory of Pharmaceutics & State Key Laboratory of Functions and Applications of Medicinal Plants,Guizhou Medical University;School of Pharmacy,Guizhou Medical University;State Key Laboratory of Functions and Applications of Medicinal Plants & Engineering Research Center for the Development and Application of Ethnic Medicine and TCM of Ministry of Education,Guizhou Medical University;

DOI: 10.19367/j.cnki.2096-8388.2021.11.006

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