UPLC-Q-TOF-MS结合网络药理学探讨南柴胡-赤芍药对抗肝癌作用机制研究  

Mechanism of Bupleurum scorzonerifolium and Paeonia lactiflora herbal pair against liver cancer:an exploration based on UPLC-Q-TOF-MS combined with network pharmacology

作　　者：邹翔[1] 隋洋 唐锡玉 张茹[2] 舒淇 宫甜 吴双 孙志伟 李文兰[1,2] 曲中原[2] ZOU Xiang;SUI Yang;TANG Xi-yu;ZHANG Ru;SHU Qi;GONG Tian;WU Shuang;SUN Zhi-wei;LI Wen-lan;QU Zhong-yuan(Engineering Research Center of Natural Anticancer Drugs,Ministry of Education,Harbin University of Commerce,Harbin 150076,China;Pharmaceutical Engineering Technology Research Center,College of Pharmacy,Harbin University of Commerce,Harbin 150076,China)

机构地区：[1]哈尔滨商业大学国家教育部抗肿瘤天然药物工程研究中心,黑龙江哈尔滨150076 [2]哈尔滨商业大学药学院药物工程技术研究中心,黑龙江哈尔滨150076

出　　处：《中国中药杂志》2022年第13期3597-3608,共12页China Journal of Chinese Materia Medica

基　　金：黑龙江省重点研发计划项目(GZ20210110);黑龙江省省属高等学校基本科研业务费项目(2020-KYYWF-0071);哈尔滨商业大学青年创新人才支持计划项目(2020CX11);哈尔滨商业大学研究生创新基金项目(YJSCX2020-681HSD)。

摘　　要：探究南柴胡-赤芍药对抗肝癌的药效物质基础和作用机制。采用MTT法和人肝癌HepG2细胞筛选南柴胡-赤芍药对体外抗肝癌的有效部位,并采用UPLC-Q-TOF-MS分析有效部位的化学成分;通过中药系统药理学数据库与分析平台(TCMSP)筛选出口服利用度(OB)≥30%的活性成分,运用TCMSP和SwissTargetPrediction数据库查找和预测成分靶点;采用GeneCards数据库和DisGeNET数据库获取肝癌的疾病靶点;运用Venny 2.1.0取交集靶点,并采用STRING数据库进行蛋白质-蛋白质互作网络(PPI)分析,得到核心靶点;采用DAVID数据库进行基因功能GO和KEGG通路富集分析;采用Western blot法验证药对有效组分对人肝癌HepG2细胞富集关键通路相关蛋白表达的影响。结果表明,与南柴胡-赤芍30%、50%、70%乙醇提取部位相比,95%乙醇提取后正丁醇萃取部位(CSYZ)抗肿瘤作用最为显著,UPLC-Q-TOF-MS解析出31个化学成分,其中OB≥30%的活性成分有14个;获得成分和疾病的交集靶点220个,根据degree中位数的2倍筛选获得核心靶点35个。GO富集分析获得215个P<0.05的条目,其中生物学过程条目154个,细胞组分条目22个,分子功能条目39个;KEGG富集分析获得显著影响通路95条,其中与南柴胡-赤芍药对抗肝癌密切相关的信号通路(P由小到大)包括PI3K/AKT信号通路(PI3K/AKT signaling pathway)、TNF信号通路(TNF signaling pathway)、MAPK信号通路(MAPK signaling pathway)、HIF-1信号通路(HIF-1 signaling pathway)、ErbB信号通路(ErbB signaling pathway)等。对涉及靶点最多的核心信号通路PI3K/AKT进行反推,发现该通路涉及15个核心靶点和8种有效成分。验证实验结果表明,南柴胡-赤芍药对有效组分可显著抑制p-PI3K和p-AKT的表达,与网络药理学预测结果一致。综上,南柴胡-赤芍抗肝癌的主要药效物质基础是柴胡皂苷a、柴胡皂苷d、芍药苷等14个成分,并可通过调控PI3K/AKT通路发挥抗肝癌作用。This study aimed to decipher the pharmacodynamic material basis and mechanism of herbal pair Bupleurum scorzonerifolium-Paeonia lactiflora(BS-PL)against liver cancer based on UPLC-Q-TOF-MS and network pharmacology.MTT assay and human hepatocellular carcinoma HepG2 cells were used to screen the effective part of BS-PL,the active components of which were further analyzed and identified by UPLC-Q-TOF-MS.Next,we applied Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)to screen the active ingredients with OB≥30%.Then TCMSP and SwissTargetPrediction were used to collect and predict component targets,followed by the search of liver cancer-related targets with GeneCards and DisGeNET.The intersection targets were obtained using Venny 2.1.0.Protein-protein interaction(PPI)network was constructed using STRING to uncover the core targets,which were subjected to Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis based on DAVID.Finally,the effects of active ingredients on the expression of main proteins enriched in the key pathways of HepG2 cells were verified by Western blot.The results indicated that compared with 30%,50%,and 70%ethanol extracts of BS-PL,the n-butanol extraction part(CSYZ)from 95%ethanol extract of BS-PL exhibited the best anti-tumor effect.UPLC-Q-TOF-MS revealed 31 ingredients,14 of which showed OB≥30%.A total of 220 intersection targets were obtained,from which 35 were selected as the key targets under the condition of two times the median of degree.Among the 215 items with P<0.05 obtained through GO enrichment analysis,154 were classified into biological processes,22 into cell components and 39 into molecular functions.KEGG enrichment analysis revealed 95 significantly affected signaling pathways,and the ones(sorted in a descending order by P value)closely related to the anti-liver cancer effect of herbal pair were PI3 K-AKT signaling pathway,TNF signaling pathway,MAPK signaling pathway,HIF-1 signaling pathway,and ErbB signaling

关 键 词：南柴胡 赤芍 肝癌 UPLC-Q-TOF-MS 网络药理学 实验验证 

分 类 号：R285[医药卫生—中药学]