摘要
目的:基于网络药理学与分子对接技术预测青黄散治疗急性髓系白血病(AML)的潜在活性靶点与信号通路,通过体外细胞实验进一步验证其作用机制。方法:基于中药系统药理学数据库与分析平台(TCMSP)、中医药综合数据库(TCMID)、TargetNet、SwissTargetPrediction数据库获取青黄散活性成分和作用靶点;通过DrugBank、DisGeNET、GeneCards、在线人类孟德尔遗传数据库(OMIM)获取AML疾病相关靶点。筛选出青黄散与AML的共同靶点后,使用STRING数据库进行蛋白质-蛋白质相互作用网络分析,并通过RStudio软件使用R语言及clusterProfiler、Bioconductor等包进行基因本体论(GO)和京都基因和基因组百科全书(KEGG)通路富集分析。同时应用Cytoscape软件进行“疾病-药物成分-靶点”与“化合物-靶点-通路”网络的构建。选取青黄散活性成分与“化合物-靶点-通路”网络排名前8个靶点进行分子对接。利用体外细胞实验及蛋白免疫印迹法(Western blot)进一步验证青黄散抗AML作用。结果:预测结果显示青黄散主要活性成分11个,收集青黄散与AML的共同靶点22个;KEGG通路分析结果显示磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)和丝裂原活化蛋白激酶(MAPK)信号通路可能在青黄散治疗AML疾病中起到关键作用。“化合物-靶点-通路”网络分析显示排名前8的靶点包括Akt1、磷脂酰肌醇3-激酶催化亚基α(PIK3CA)、双特异性丝裂原活化蛋白激酶1(MAP2K1)、凋亡相关基因(TP53)、原癌基因丝苏氨酸蛋白激酶1(RAF1)、B细胞淋巴瘤-2基因(Bcl-2)、胱天蛋白酶-9(Caspase-9)和原癌基因(JUN)。分子对接结果表明3-吲哚基-β-D-吡喃葡萄糖苷与MAP2K1、异牡荆素与PIK3CA、靛玉红与Bcl-2对接最优。细胞实验表明3-吲哚基-β-D-吡喃葡萄糖苷、异牡荆素和靛玉红可有效抑制AML细胞增殖,并调控MAPK/PI3K信号通路,抑制Bcl-2蛋白的表达。结论:青黄散可通过多成分-多靶点-多途径方式协同作用治疗AML,其作用机制可能与调控MAPK信号通路及PI3K/Akt信号通路有关。
Objective:To predict the therapeutic target genes and related signaling pathways of Qinghuangsan(QHP)in the treatment of acute myeloid leukemia(AML)by network pharmacology,molecular docking,and further clarify its mechanisms through in vitro cell experiment.Method:The active components and targets of QHP were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP),traditional Chinese medicine integrated database(TCMID),TargetNet and SwissTargetPrediction databases,and AML-related target genes were obtained by GeneCards and online mendelian inheritance in man(OMIM)databases.After screening the common targets of QHP and AML,the protein-protein interaction(PPI)network of the common targets was constructed with STRING,followed by gene ontology(GO)term and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis based on RStudio software and clusterProfiler,Bioconductor packages.At the same time,Cytoscape software is used to construct the network of"disease-component-target"and"compound-target-pathway".Select the active ingredients of QHP for molecular docking with the top 8 targets in the"compound-target-pathway"network.In vitro cell experiment and Western blot were used to further verify the anti-AML effect of QHP.Result:The prediction results show that there are 11 main active components of QHP,and 22 common targets of QHP and AML are collected.KEGG pathway analysis results show that phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt)and mitogen-activated protein kinase(MAPK)signaling pathways may play a key role in the treatment of AML disease by QHP."Compound-target-pathway"network analysis showed that the top 8 targets include Akt1,phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha(PIK3CA),mitogenactivated protein kinase kinase 1(MAP2K1),TP53,serine/threonine kinase(RAF1),B cell lymphoma(Bcl)-2,cysteine aspartic acid specific protease(Caspase)-9 and JUN.Molecular docking results showed that 3-indolyl-β-D-glucopyranoside was optimally docked with MAP2K1,isovitexin docked with PIK3CA,and indirubin docked with Bcl-2.Cell experiments show that 3-indolyl-β-D-glucopyranoside,isovitexin and indirubin can effectively inhibit the proliferation of AML cells,regulate the MAPK/PI3K signaling pathway,and inhibit the expression of Bcl-2 protein.Conclusion:QHP can treat AML through"multi-component,multi-target,multi-pathway"synergistic treatment,and its mechanism of pharmacology may be related to the regulation of MAPK signaling pathway and PI3K/Akt signaling pathway.
作者
伍振辉
张欢
吴欣平
周露
曾英坚
WU Zhen-hui;ZHANG Huan;WU Xin-ping;ZHOU Lu;ZENG Ying-jian(The Affiliated Hospital of Jiangxi University of Chinese Medicine,Nanchang 330006,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第7期179-189,共11页
Chinese Journal of Experimental Traditional Medical Formulae
基金
江西省自然科学基金面上项目(20192BAB205100)
江西省中医药管理局临床研究基地建设项目(赣中医药科教字〔2021〕3号)
江西省卫生健康委科技计划项目(202210758)
江西省中医药管理局科技计划项目(2020A0184、2021B651)。
关键词
青黄散
急性髓系白血病
网络药理学
分子对接
细胞实验
Qinghuangsan
acute myeloid leukemia
network pharmacology
molecular docking
cell experiment
