液相色谱-质谱联用技术分析秦巴硒菇提取物活性成分及其治疗慢性粒细胞白血病的网络药理学研究

Analysis of effective components of Agaricus blazei Murill extract by LC-MSand network pharmacology analysis in treatment of chronic myeloid leukemia

目的 利用液相色谱质谱联用和网络药理学、分子对接技术探讨秦巴硒菇提取物治疗慢性粒细胞白血病(chronic myeloid leukemia, CML)的潜在活性靶点及相关信号通路,并通过体外实验进一步验证。方法 应用液相色谱质谱分析秦巴硒菇提取物的活性成分,通过Swiss Target Prediction数据库预测药物靶点;从GeneCards、DisGeNET数据库获取CML的疾病靶点。筛选出药物和CML的共同靶点,通过STRING数据库进行蛋白质互作分析,利用Cytoscape软件构建蛋白互作网络图,并通过DAVID数据库进行GO和KEGG通路富集分析。分别应用DockThor和Pymol进行分子对接及可视化处理。利用CCK-8法测定秦巴硒菇提取物对K562细胞的增殖抑制作用,Western blot验证其对相关蛋白及磷酸化水平的影响。结果 秦巴硒菇提取物活性成分共126种,药物和疾病共同靶点172个;KEGG通路分析结果显示PI3K/Akt/mTOR信号通路可能在治疗CML疾病中起到关键作用。秦巴硒菇提取物在K562细胞中24、48 h的IC50分别为1.86、1.48 g·L^(-1),秦巴硒菇提取物可抑制PI3K、Akt、mTOR蛋白磷酸化水平。结论 秦巴硒菇提取物可通过多成分、多通路、多靶点、多种方式协同作用治疗CML,其作用机制可能与调控PI3K/Akt/mTOR信号通路有关。

Aim To explore the potential targets and related signaling pathways of Agaricus blazei Murill(AbM)extract in the treatment of chronic myeloid leukemia(CML)based on liquid chromatography mass spectrometry(LC-MS),network pharmacology,molecular docking,and were further verified by experiments in vitro.Methods The active components of AbM extract were retrieved from LC-MS,Swiss Target Prediction database was used to predict related targets,and CML disease target genes were obtained from GeneCards and DisGeNET databases.After screening the common targets of drug and CML,the protein-protein interaction network of the common targets was performed by STRING,and GO and KEGG enrichment analysis were done by DAVID database.Cytoscape software was used to construct the network of target protein.Molecular docking was carried out by DockThor,and the Pymol software was used to make a visual picture.The inhibitory effect of AbM extract on leukemia cells K562 was determined by CCK-8 experiment,and the effect of AbM extract on the expression and phosphorylation level of related proteins was verified by Western blot.Results The prediction results showed that 126 active components of AbM extract,and 172 common targets were collected.KEGG pathway analysis results showed that PI3K/Akt/mTOR signaling pathway might play an important role in the treatment of CML disease.The IC 50 of AbM extract in K562 cells 24 and 48 hours was 1.86 and 1.48 mg·mL^(-1),respectively.The protein expression of p-PI3K,p-Akt,and p-mTOR was down-regulated.Conclusions AbM extract can perform synergistic effect in treatment of CML through multi-component,multi-channel,multi-target ways,and the mechanism may be related to the regulation of PI3K/Akt/mTOR signaling pathway.