UPLC-Q-TOF-MS结合网络药理学及实验验证研究深绿卷柏富含双黄酮部位治疗喉癌的作用机制

UPLC-Q-TOF-MS combined with network pharmacology and experimental validation to study the mechanism of action of Selaginella doederleinii Hieron.rich in biflavonoids in the treatment of laryngeal cancer

目的:研究深绿卷柏富含双黄酮部位治疗喉癌的作用机制。方法:运用UPLC-Q-TOF-MS对深绿卷柏富含双黄酮部位(SD-BFRE)进行化学成分分析并通过PharmMapper数据库进行靶点预测,通过检索OMIM、GeneCards和DrugBank数据库收集喉癌相关靶点,利用Venny 2.1.0在线平台将二者取交集获得潜在靶点。使用STRNG平台对潜在靶点进行蛋白相互作用分析,通过DAVID数据库进行GO功能分析和KEGG通路分析,使用Cytoscape 3.8.0软件构建PPI网络和“成分-靶点-通路”网络。通过SYBYL-X 2.0软件对所有成分和核心靶点进行分子对接验证并通过Discovery Studio 4.5软件进行可视化。采用MTT法测定细胞活力和Western blot法验证SD-BFRE对喉癌细胞系富集关键通路相关蛋白表达的影响。结果:鉴定化合物14个,药物靶点72个,喉癌相关靶点2366个,潜在靶点49个。GO分析得到181个P≤0.05的条目,KEGG分析共得到74条P≤0.05的通路,主要涉及IL-17、FoxO、TNF和PI3K-Akt信号通路等,分子对接结果显示92.9%有较好的对接活性。细胞实验结果表明,SD-BFRE可显著抑制p-Akt和p-Bad的表达,与网络药理学预测结果基本一致。结论:运用UPLC-Q-TOF-MS结合网络药理学及实验验证分析了SD-BFRE治疗喉癌的作用机制,为进一步深入研究提供参考。

OBJECTIVE To study on the mechanism of the treatment of laryngeal cancer by a biflavonoid-rich extract from Selaginella doederleinii Hieron..METHODS UPLC-Q-TOF-MS was used to analyze the chemical constituents of the bisflavonoid-rich extract(SD-BFRE)of S.doederleinii and target prediction by PharmMapper database,collect laryngeal cancer-related targets by searching OMIM,GeneCards and DrugBank databases,use the Venny 2.1.0 online platform to intersect the two to obtain potential targets.Protein interaction analysis of potential targets using the STRNG platform,GO functional analysis and KEGG pathway analysis by DAVID database,PPI network and"component-target-pathway"network were constructed using Cytoscape 3.8.0 software.All components and core targets were molecular docking verification by SYBYL-X 2.0 software and visualized by Discovery Studio 4.5 software.Determination of cell viability by MTT assay and Western blot method was used to verify the effect of SD-BFRE on the expression of key pathway-related proteins enriched in laryngeal carcinoma cell line.RESULTS UPLC-Q-TOF-MS identified 14 compounds,72 drug targets,2366 laryngeal cancer-related targets,and 49 potential targets.GO analysis yielded 181 entries with P≤0.05,and KEGG analysis yielded 74 pathways with P≤0.05,mainly involving IL-17,FoxO,TNF and PI3K-Akt signaling pathways.Molecular docking results showed that 92.9%had good docking activity.The results of cellular expriments showed that SD-BFRE significantly inhibited the expression of p-Akt and p-Bad,which was in general agg agreenment with the network pharmacological predictions.CONCLUSION The mechanism of action of SD-BFRE in the treatment of laryngeal cancer was analyzed by using UPLC-Q-TOF-MS combined with network pharmacology and experimental verification,providing reference for further research.