摘要
目的:基于网络药理学预测丹参治疗膀胱癌(BC)的潜在作用靶点及可能相关的信号通路,并通过体外细胞实验验证其潜在的分子机制。方法:应用中药系统药理数据库(TCMSP)筛选中药丹参活性成分;运用Gene Cards和在线人类孟德尔遗传数据库(OMIM)获取BC疾病靶点;通过Venny 2.1工具整合丹参治疗BC的潜在靶点并绘制韦恩图;STRING数据库构建蛋白质-蛋白质相互作用(PPI)网络;DAVID数据库进行基因本体(GO)富集分析及京都基因与基因组百科全书(KEGG)富集分析;细胞增殖与活性检测-8(CCK-8)法检测丹参酮Ⅱ_(A)(TanⅡ_(A)),隐丹参酮(CPT),木犀草素(LUT)分别以不同浓度(0、1、2、4、8、16、32μmol·L^(-1))对膀胱癌T24、5637细胞的增殖抑制活性;碘化吡啶(PI)染色法分析TanⅡ_(A)、CPT及LUT(0、4、8μmol·L^(-1))诱导5637细胞凋亡;蛋白免疫印迹法(Western blot)检测TanⅡ_(A)(0、4、8、16μmol·L^(-1))对关键靶点蛋白表达的调控。结果:筛选结果显示丹参65个活性成分,39个丹参-BC共同作用靶点,KEGG通路富集分析主要包含神经元-配体-受体的相互作用、磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)信号传导途径、表皮生长因子受体酪氨酸激酶抑制剂抗性、缺氧诱导因子(HIF)-1信号通路等。CCK-8结果表明,与空白组比较,TanⅡ_(A)、CPT及LUT均能明显抑制BC细胞T24和5637细胞的增殖(P<0.05),且3个药物均对5637细胞的增殖抑制作用更为显著;同时在5637细胞系中,TanⅡ_(A)组的半抑制浓度(IC_(50))明显低于CPT及LUT组(P<0.05)。PI染色结果显示,与空白组比较,TanⅡ_(A)、CPT及LUT均能诱导5637细胞凋亡,诱导凋亡程度由高到低依次为TanⅡ_(A)、CPT、LUT(P<0.05)。Western blot实验表明TanⅡ_(A)作用于5637细胞后能降低表皮生长因子受体(EGFR)、磷酸化磷脂酰肌醇3-激酶(p-PI3K),磷酸化蛋白激酶B(p-Akt)蛋白表达水平,且呈浓度依赖性。结论:丹参治疗BC具有多成分、多靶点
Objective:To predict the potential targets and possible related signaling pathways of Salviae Miltiorrhizae Radix et Rhizoma against bladder cancer(BC)based on network pharmacology and verify the potential molecular mechanism through in vitro cell experiment.Method:Active components of Salviae Miltiorrhizae Radix et Rhizoma were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and BC-related targets were searched from Gene Cards and Online Mendelian Inheritance in Man(OMIM).Via Venny 2.1,the potential targets of Salviae Miltiorrhizae Radix et Rhizoma against BC were screened out and the Venn diagram was plotted.Protein-protein interaction(PPI)network was constructed by STRING,followed by Gene Ontology (GO) term enrichment and Kyoto Encyclopedia of Genes and Gnomes(KEGG)pathway enrichment with DAVID.Cell Counting Kit-8(CCK-8)assay was employed to detect the inhibitory effect of tanshinoneⅡ_(A)(TanⅡ_(A)),cryptotanshinone(CPT),and luteolin(LUT)at different concentration(0,1,2,4,8,16,32μmol·L^(-1))on the proliferation of BC T24 and 5637 cells,propidium iodide(PI)staining to analyze the apoptosis of 5637 cells induced by TanⅡ_(A),CPT,and LUT(0,4,8μmol·L^(-1)),and Western blotting to detect the regulatory effect of TanⅡ_(A)(0,4,8,16μmol·L^(-1))on the expression of key target proteins.Result:A total of 65 active components and 39 anti-BC targets of Salviae Miltiorrhizae Radix et Rhizoma were screened out.The anti-BC targets were mainly involved in the KEGG pathways of neuron-ligand-receptor interaction,phosphatidylinositol 3-kinases(PI3K)/protein kinase B(Akt)signaling pathway,epidermal growth factor receptor(EGFR)tyrosine kinase inhibitor resistance,and hypoxia inducible factor(HIF)-1 signaling pathway.As for the CCK-8 assay,compared with the blank group,TanⅡ_(A),CPT,and LUT significantly inhibited the proliferation of T24 and 5637 cells,particularly the 5637cells.The half maximal inhibitory concentration(IC_(50))of TanⅡ_(A)on 5637 cells was signifi
作者
白婕
王伟峰
毛广敏
张军
杨波
董昌盛
龚华
BAI Jie;WANG Weifeng;MAO Guangmin;YANG Bo;ZHANG Jun;DONG Changsheng;GONG Hua(Graduate School of Shanghai University of Traditional Chinese Medicine(TCM),Shanghai 201203,China;Zhoupu Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201318,China;Longhua Hospital Shanghai University of TCM,Shanghai 200032,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第15期153-161,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81774064)
上海中医药大学杏林中青年人才培养体系—杏林青年学者项目(RC-2017-02-02)
浦东新区卫生系统领先人才培养项目(PWRI2021-10)。
关键词
丹参
膀胱癌(BC)
网络药理学
丹参酮Ⅱ_(A)
体外细胞实验
Salviae Miltiorrhizae Radix et Rhizoma
bladder cancer(BC)
network pharmacology
tanshinoneⅡ_(A)(TanⅡ_(A))
molecular docking
in vitro cell experiment
