摘要
目的基于网络药理学预测益肺散结方治疗肺腺癌的潜在作用机制,并通过体外细胞实验对关键信号通路之一Janus蛋白酪氨酸激酶2(JAK2)/信号转导和转录激活子3(STAT3)/血管内皮生长因子(VEGF)信号通路进行实验验证。方法利用中药系统药理数据库(TCMSP)筛选益肺散结方的主要活性成分和潜在作用靶点。通过人类基因数据库(GeneCards)和在线人类孟德尔遗传数据库(OMIM)检索获取肺腺癌主要靶点。运用韦恩图获取两者的交集靶点,通过Cytoscape3.7.2进行拓扑学分析,并建立中药-活性成分-靶点网络图。采用蛋白质相互作用平台(STRING)与Cytoscape3.7.2软件构建蛋白质-蛋白质相互作用(PPI)网络。应用Metascape数据库进行基因本体(GO)功能富集分析和京都基因与基因组百科全书(KEGG)通路富集分析。借助Vina 1.2.3软件进行分子对接验证。通过CCK-8法检测益肺散结方对细胞活性的影响,细胞划痕实验观察对细胞迁移的影响,Western blot法检测p-STAT3、STAT3、p-JAK2、JAK2、VEGF-A表达。结果获取益肺散结方活性成分94种,相关药物靶点329个,肺腺癌靶点1358个,二者交集150个。PPI网络可视化分析显示,益肺散结方治疗肺腺癌的潜在关键靶点为:蛋白激酶B1(AKT1)、β-肌动蛋白(ACTB)、抑癌基因P53(TP53)、血清白蛋白(ALB)、caspase-3(CASP3)、血管内皮生长因子A(VEGFA)。KEGG富集分析筛选了138条相关信号通路,显示JAK/STAT信号通路可能在益肺散结方治疗肺腺癌的过程中起到关键作用。分子对接结果表明槲皮素、木犀草素、熊果酸与JAK2、STAT3之间均有较好的结合活性。细胞实验表明,与空白组比较,益肺散结方可显著抑制A549细胞活性(P<0.01),抑制A549细胞迁移(P<0.01),同时能降低p-JAK2/JAK2、p-STAT3/STAT3、VEGF-A蛋白表达(P<0.05),此外,JAK2/STAT3通路激活剂Colivelin可逆转益肺散结方对A549相关蛋白表达的影响(P<0.05)。结论益肺散结方治疗肺腺癌具有多成分、多靶点、多通路的特点,其作用机制可能与下调p-JAK2、p-STAT3、VEGF-A蛋白的表达,进而抑制细胞增殖和迁移有关。
Objective To predict the potential mechanism of Yifei Sanjie prescription in the treatment of lung adenocarcinoma based on network pharmacology,and to verify one of the key signal pathways,Janus protein tyrosine kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3),by cell experiments in vitro.Methods To screen the main active components and potential action targets of Yifei Sanjie prescription,with traditional Chinese medicine system pharmacological database(TCMSP).To search and retrieve the main targets of lung adenocarcinoma,with human genetic database(GeneCards)and online human Mendelian genetic database(OMIM).To obtain the intersection targets by screening and apply Wayne diagram,then analysis the topology and establish the traditional Chinese medicine-active compound-target network diagram by using of Cytoscape 3.7.2 software.To construct the protein-protein interaction(PPI)network,with the protein-protein interaction platform(STRING)and Cytoscape3.7.2 software.To analyze the functional enrichment of gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG),with the Metascape database.To carry out the molecular docking verification by using of Vina1.2.3 software.Using CCK-8 method to detect the effect of Yifei Sanjie prescription on cell activity.Using the cell scratch test to observe the effect on cell migration.And using Western blot method to test the expression of p-STAT3,STAT3,p-JAK2,JAK2 and VEGF-A.Results 94 active components,329 related drug targets and 1358 lung adenocarcinoma targets were obtained from Yifei Sanjie prescription,among which,150 of them intersected.PPI network visualization analysis shows that the potential key targets of Yifei Sanjie prescription in the treatment of lung adenocarcinoma are protein kinase B1(AKT1),β-actin(ACTB),tumor suppressor gene p53(TP53),serum albumin(ALB),caspase-3(CASP3)and vascular endothelial growth factor A(VEGFA).KEGG enrichment analysis screened 138 related signal pathways,indicating that JAK/STAT signaling pathway may play a key role in the treatment of lung adenocarcinoma with Yifei Sanjie prescription.Molecular docking results showed that quercetin,luteolin,and ursolic acid had good binding activities with JAK2 and STAT3.The cell experiment showed that compared with the blank group,Yifei Sanjie prescription could significantly inhibit the activity of A549 cells,inhibit the migration of A549 cells,and decrease the expression of p-JAK2/JAK2,p-STAT3/STAT3 and VEGF-A protein.In addition,Colivelin,an activator of JAK2/STAT3 pathway,could reverse the effect of Yifei Sanjie prescription on the expression of A549 related proteins.Conclusion Yifei Sanjie prescription has the characteristics of multi-component,multi target and multi pathway in the treatment of lung adenocarcinoma,and its mechanism may be related to the down-regulation of p-JAK2,p-STAT3 and VEGF-A protein expression,thereby inhibiting cell proliferation and migration.
作者
毛梦婷
董孟佳
许尤琪
李伟良
Mao Mengting;Dong Mengjia;Xu Youqi;Li Weiliang(Second Affiliated Hospital,Nanjing University of Chinese Medicine,Nanjing 210029,China)
出处
《世界科学技术-中医药现代化》
CSCD
北大核心
2023年第9期3049-3062,共14页
Modernization of Traditional Chinese Medicine and Materia Medica-World Science and Technology
基金
江苏省中医药管理局科技发展计划项目(YB2020023):益肺散结方调控PRMT6-YBX1-CDC25A通路抑制肺腺癌发生的作用研究,负责人:唐杰
江苏省教育厅研究生科研与实践创新项目(SJCX22_0818):益肺散结方通过JAK2-STAT3通路下调HIF-1α、VEGF抑制肺癌的机制研究,负责人:毛梦婷
南京中医药大学第二附属医院院内课题(SEZ202119):益肺散结方通过STAT3通路下调PD-L1抑制肺癌进展的相关机制研究,负责人:董孟佳。
