质谱分析联合网络药理学探析固本方对三阴性乳腺癌的作用机制

Exploration on the Mechanism of Guben Prescription on Triple-Negative Breast Cancer by Mass Spectrometry Analysis Combined with Network Pharmacology

【目的】基于质谱分析和网络药理学探析固本方(党参、黄芪、灵芝等组成)对三阴性乳腺癌的作用机制,并进行实验验证。【方法】采用液质联用质谱分析固本方冻干粉化学成分。运用中药系统药理学数据库和分析平台(TCMSP)、PubChem数据库获得固本方化学成分的SMILE结构,SwissTargetPrediction数据库预测其潜在作用靶点,检索TTD、Drugbank、OMIM、京都基因与基因组百科全书(KEGG)、PharmGkb、GeneCard、DisGeNET等数据库得到疾病靶点,运用Venny 2.1数据库得到固本方与三阴性乳腺癌交集的靶点,利用STRING数据库构建交集靶点的蛋白-蛋白互作(PPI)网络,应用DAVID工具进行基因本体论(GO)和KEGG富集分析。以人乳腺癌MDA-MB-231细胞为研究对象,分别用固本方、卡培他滨干预,采用四甲基偶氮唑盐(MTT)法检测药物对细胞增殖能力的抑制率,Western Blot法检测细胞蛋白激酶B(Akt)、磷酸化Akt(p-Akt)、磷酸化磷脂酰肌醇-3激酶(p-PI3K)蛋白表达水平。【结果】质谱分析结果显示,固本方主要化学成分为党参炔苷、灵芝酸B、毛蕊异黄酮葡萄糖苷、柴胡皂苷A、芒柄花苷、澳洲茄碱、贝母素甲、毛蕊异黄酮、熊果酸、齐墩果酸、芒柄花素、紫杉醇。PIK3CA、PIK3R1、Akt1等为固本方治疗三阴性乳腺癌的潜在核心靶点,PI3K-Akt信号通路富集结果显著且靶点数最多。细胞实验结果显示,固本方呈时间和浓度依赖性抑制MDA-MB-231细胞的增殖,固本方下调MDA-MB-231细胞Akt、p-Akt、p-PI3K蛋白的表达。【结论】通过质谱分析获得固本方主要化学成分12种,PIK3CA、PIK3R1、Akt1等为固本方治疗三阴性乳腺癌的潜在核心靶点,PI3K/Akt信号通路发挥主要作用。固本方可抑制MDA-MB-231细胞增殖,并调控其PI3K/Akt通路。

Objective To explore the mechanism of action of Guben Prescription(composed of Codonopsis Radix,Astragali Radix and Ganoderma,etc.)against triple-negative breast cancer based on mass spectrometry and network pharmacology,and to conduct experimental validation.Methods The chemical composition of lyophilized powder of Guben Prescription was analyzed by liquid mass spectrometry.The SMILE structure of chemical components in Guben Prescription was obtained by using TCMSP and PubChem database,and their potential action targets were searched by SwissTargetPrediction database.TTD,Drugbank,OMIM,Kyoto Encyclopedia of Genes and Genomes(KEGG),PharmGkb,GeneCard,DisGeNET and other databases were searched to obtain disease targets.The Venny 2.1 database was used to obtain targets of Guben Prescription intersected with triplenegative breast cancer,and the STRING database was used to construct protein-protein interaction(PPI)networks for the intersected targets,and the DAVID tool was used to perform gene ontology(GO)and KEGG enrichment analysis.Human breast cancer MDA-MB-231 cell was used as the object of the study,and was intervened with Guben Prescription and Capecitabine,respectively.The inhibition rate of drug on cell proliferation ability was detected by methyl thiazolyl tetrazolium(MTT)assay,and the protein expression levels of cellular protein kinase B(Akt),phosphorylated Akt(p-Akt)and phosphatidylinositol-3 kinase(p-PI3K)were detected by Western Blot.Results The results of mass spectrometry analysis showed that the main chemical components of Guben Prescription were lobetyolin ginsenoside,ganoderic acid B,calycosin-7-glucoside,saikosonin A,ononin,solasonine,verticine,calycosin,ursolic acid,oleanolic acid,formononetin,paclitaxel,etc..PIK3CA,PIK3R1 and Akt1 were the potential core targets of Guben Prescription for the treatment of triple-negative breast cancer,and PI3K-Akt signaling pathway showed significant enrichment results and the highest number of targets.The results of the cellular assay showed that Guben Prescription inhibited the proliferation of MDA-MB-231 cells in a time-and concentration-dependent manner,and Guben Prescription down-regulated the protein expressions of Akt,p-Akt and p-PI3K in MDA-MB-231 cells.Conclusion The 12 main chemical components of Guben Prescription were obtained by mass spectrometry,and PIK3CA,PIK3R1 and Akt1 were the potential core targets of Guben Prescription for the treatment of triple-negative breast cancer,with the PI3K/Akt signaling pathway playing a major role.This prescription inhibits the proliferation of MDA-MB-231 cells and regulates the PI3K/Akt pathway in them.