基于网络药理学分析铁皮石斛花总黄酮抑制结肠癌作用机制及体外实验验证

Anti-colon cancer mechanism of total flavonoids of Dendrobium candidum flower based on network pharmacology and in vitro experimental verification

目的运用网络药理学和体外生物学实验对铁皮石斛花总黄酮治疗结肠癌的活性成分、潜在作用靶点和信号通路进行研究,探索其治疗结肠癌的可能作用机制。方法利用LC-MS/MS对铁皮石斛花黄酮类代谢物进行成分分析。通过TCMSP筛选出铁皮石斛总黄酮的活性成分及靶点基因,GeneCards数据库及DisGeNET数据库筛选出结肠癌的疾病靶点基因,构建“药物-有效成分-靶基因-疾病”调控网络图,STRING数据库构建蛋白互作网络图(PPI),对关键靶点进行GO富集分析和KEGG通路富集分析。采用MTT法检测铁皮石斛花总黄酮对HCT116细胞的增殖抑制活性,流式细胞术检测细胞凋亡情况,细胞划痕实验观察铁皮石斛花总黄酮对HCT116细胞迁移能力的影响;Western blot检测相关凋亡蛋白(Bax、Bcl-2、caspase-3、cleaved-caspase-3)和基质蛋白(MMP-12、MMP-3)的表达。结果代谢组分析获得52个黄酮类化合物,经过筛选得到27个活性成分,通过相关数据库平台预测得到作用靶点178个,PPI网络核心蛋白为丝氨酸-苏氨酸蛋白激酶1(AKT1)、血管内皮细胞生长因子受体2(VEGFR2)、肿瘤蛋白P53(TP53)、转录因子AP-1(JUN)、肿瘤坏死因子(TNF)、雷帕霉素靶蛋白(MTOR)等;GO富集分析显示铁皮石斛花总黄酮会影响信号转导、细胞凋亡和增殖调控等;KEGG通路富集分析显示铁皮石斛花总黄酮在细胞增殖、凋亡、PI3K-Akt及肿瘤等信号通路中发挥作用。实验结果表明,铁皮石斛花总黄酮能抑制HCT116细胞的增殖,促进细胞的凋亡,抑制细胞的迁移;还能上调Bax/Bcl-2、caspase-3、cleaved-caspase-3蛋白的表达,诱导细胞凋亡。结论铁皮石斛花总黄酮可通过多靶点、多途径发挥抗结肠癌的作用,其作用机制可能与影响凋亡基因Bax及Bcl-2的表达有关。

Objective To determine the main active components,the potential targets and molecular pathways of total flavonoids in Dendrobium candidum flower(DOPTF)for colon cancer by network pharmacology-experimental verification in vitro,and to explore the mechanism of DOPTF treating on colon cancer.Methods The compositions of DOPTF were analyzed by HPLC-MS/MS.The main active components and the gene targets were screened with TCMSP.Colon cancer target genes were screened by the GeneCards database and DisGeNET database.Cytoscape software was used to construct regulatory network diagram of“Chinese herb-active ingredient-target gene-disease”.The STRING database was used to construct protein-protein interaction(PPI).GO enrichment and KEGG pathway analysis were conducted.MTT assay was used to detect the proliferation inhibition activity of total flavonoids in DOPTF on HCT116 cells,the flow cytometry was used to detect the cell apoptosis,and the cell scratch assay was used to observe the effect of DOPTF on migration of HCT116 cells.Western blot was used to detect the expression of related apoptotic proteins Bax,Bcl-2,caspase-3,cleaved-caspase-3,MMP-12 and MMP-3.Results Totally 52 DOPTF components were obtained by HPLC-MS/MS,and 27 active components were obtained after screening.Totally 178 DOPTF intervention colon cancer related targets were constructed through the relevant database platform.The core genes in PPI included AKT1,VEGFR2,TP53,JUN,TNF,MTOR and so on.GO analysis showed that flavonoid active ingredients affected the signal transduction,apoptosis and proliferation regulation of colon cancer cells.KEGG pathway analysis also showed that flavonoid active ingredients acted on the cell proliferation,apoptosis,PI3K-Akt,and tumor signaling pathways of colon cancer cells.DOPTF significantly inhibited the proliferation of HCT116 cells,promoted cell apoptosis,and inhibited cell migration.DOPTF also significantly up-regulated the expression of Bax/Bcl-2,caspase-3,cleaved-caspase-3,and induced cell apoptosis.Conclusion DOPTF can intervene colon cancer through multiple targets and multiple pathways,whose mechanism may be related to affecting the expression of apoptosis-related genes Bax and Bcl-2.