基于网络药理学和体外实验探讨大白桩菇抗乳腺癌的作用机制

Molecular mechanism of Leucopaxillus giganteus against breast cancer based on network pharmacology and in vitro experiments

目的 利用网络药理学预测和分子生物学验证探讨大白桩菇抗乳腺癌的活性成分及作用机制。方法 采用溶剂法提取,硅胶柱色谱等方法分离纯化,根据波谱数据进行结构鉴定;通过MTT法检测抗肿瘤活性;通过TCMSP和PharmMaper平台进行靶点预测;在Genecards数据库筛选疾病基因;对核心靶点进行GO和KEGG富集分析;利用iGEMDOCK软件进行分子对接;采用Western blot验证大白桩菇成分绿原酸对MDA-MB-231中CHEK2和CASP3靶点的调控作用。结果 从大白桩菇分离纯化到绿原酸等13个化合物;大白桩菇乙醇提取物(200 mg·L^(-1))对MDA-MB-231的抑制率为87.35%;13个化合物主要作用在CHEK2和CASP3等21个核心靶点,通过p53和FOXO等多条信号通路发挥抗乳腺癌作用;Western blot发现绿原酸可作用于靶点CHEK2和CASP3,促进乳腺癌细胞凋亡,证明了网络药理学分析的可靠性。结论 初步阐明了大白桩菇的化学成分及潜在作用机制,为大白桩菇抗乳腺癌的进一步研究提供了参考。

Aim To study the mechanism of the ethanol extract from Leucopaxillus giganteus(LGEE)in treating breast cancer based on network pharmacology and molecular experimental validation.Methods Some chromatographic methods were used to isolate the chemical constituents of LGEE,and their structures were elucidated based on spectral data.The antitumor activities of LGEE were determined by MTT assay.The predicted targets of LGEE were selected by TCMSP and Pharmmaper,and Genecards database was used to screen the targets.GO and KEGG analysis of target genes were performed.Molecular docking was used to test the binding of active components to core targets.Western blotting was used to validate the regulating function of chlorogenic acid on CHEK2 and CASP3 targets of MDA-MB-231 cells.Results Thirteen compounds were identified including clitocine,chlorogenic acid and so on.LGEE displayed anticancer activities against MDA-MB-231 with the inhibition percent(87.35±1.55)%,at the concentration of 200 mg·L^(-1).A total of 203 drug targets were obtained from the 13 active compounds,78 targets intersected with disease targets for breast cancer were screened out,and 21 core targets including CHEK2 and CASP3 were obtained.The results of KEGG analysis showed that it might treat breast cancer through signaling pathways,such as p53 signaling pathway,FOXO signaling pathway and so on.Chlorogenic acid from LGEE was validated to regulate CHEK2 and CASP3 targets,which proved the reliability of results of pharmacology.Conclusions Based on the network pharmacology,the possible targets and signaling pathways for treating breast cancer by LGEE can be preliminary explored,which may provide a reference for the study of the mechanism of the LEGG.