基于生物信息学和分子动力学的中药治疗原发性骨髓纤维化作用机制研究

Mechanism of Action of Chinese Medicinal Herbs in the Treatment of Primary Myelofibrosis based on Bioinformatics and Molecular Dynamics

目的 利用生物信息学和分子动力学方法探讨中药治疗原发性骨髓纤维化(PMF)的分子机制。方法 通过检索中国知网数据库(CNKI)、中文科技期刊数据库(CCD)和中国学术期刊数据库(CSPD)相关文献,发掘整理1985—2024年间用于PMF治疗的高频中药,利用TCMSP、Swiss Target Prediction数据库及相关文献检索高频中药的主要活性成分及其相关靶点;GEO数据库下载PMF的基因表达谱数据集GSE44426和GSE124281,并运用R软件进行数据归一化处理和差异表达基因筛选;运用加权基因共表达网络分析(WGCNA)获取关键模块枢纽基因。取中药活性成分靶点、差异表达基因和关键模块枢纽基因的共同交集基因,运用Cytoscape3.9.2软件生成目标网络,通过拓扑分析生成核心靶点网络,通过GO和KEGG通路富集分析选取关键通路,并从String数据库获得蛋白相互作用关系,构建药物-成分-靶点网络和蛋白质互作网络(PPI)关系图。利用Discovery Studio 2020软件进行分子对接,GROMACS程序进行分子动力学模拟。结果 共搜集21首处方,涉及121种中药,用药频次≥10次的中药有9种,由高到低排序依次为丹参、黄芪、白术、当归、党参、甘草、白芍、茯苓、熟地黄。9种高频中药共获得98种活性成分和1125个潜在靶点。GSE44426和GSE124281数据集共筛选24个基因样本,其中健康对照组(14例)和PMF组(10例),共鉴定出319个PMF组与健康对照组之间的差异表达基因,包含122个上调基因,197个下调基因。WGCNA共筛选24个共表达模块基因,发现与PMF发病密切相关的5个模块分别为MEpink、MEdarkred、MEblack、MEgrey、MEturquoise,包含7112个关键模块枢纽基因。GO和KEGG通路富集分析显示,上述高频中药治疗PMF主要涉及脂质和动脉粥样硬化通路,包含热休克蛋白90家族(HSP90AA1、HSP90AB1)、酪氨酸激酶(SRC)、丝裂原活化蛋白激酶(MAPK1)、白细胞介素10 (IL-10)和白细胞介素1β (IL-1β) 6个核心靶点。从药物-成分-靶点网络找到靶向于这6个核心靶点的中药成分共7种活性成分,包括毛蕊花糖苷、毛蕊异黄酮、山柰酚、木犀草素、柚皮素、槲皮素和茯苓酸。分子对接和分子动力学分析显示,关键中药为熟地黄、黄芪、白芍、丹参、甘草和茯苓,7个活性成分中毛蕊异黄酮与HSP90AB1结合最稳定。结论 治疗PMF的主要中药可能为熟地黄、黄芪、白芍、丹参、甘草和茯苓,有效成分包括毛蕊花糖苷、毛蕊异黄酮、山柰酚、木犀草素、柚皮素、槲皮素、茯苓酸,相关靶点为HSP90AA1、HSP90AB1、SRC、MAPK1、IL-10、IL-1β,最关键通路为脂质和动脉粥样硬化通路。

Objective To explore the molecular mechanism implicated in the treatment of primary myelofibrosis(PMF)using Chinese medicinal herbs(CMH)by bioinformatics and molecular dynamics.Methods Data mining was performed to find the high-frequency CMH in treating PMF between the year of 1985 and 2024 by searching CNKI,Chinese Science and Technology Journal Database(CCD),and China Academic Journal Database(CSPD).TCMSP,SwissTargetPrediction and related reports were used to collect the main active ingredients of high-frequency CMH and their targets.The PMF datasets GSE44426 and GSE124281 were downloaded from GEO database,and R software was used for data normalization and differentially expressed genes(DEGs)screening.Key module hub genes were obtained by weighted gene co-expression network analysis(WGCNA)analysis.The common intersection genes of active ingredient targets,DEGs and key module hub genes of CMH were selected,and the target network was generated using Cytoscape 3.9.2 software.The core target network was generated by topological analysis,while key pathways were selected by GO and KEGG pathway enrichment analysis,and protein interaction relationships were obtained from the String database,so as to construct drug-ingredient-target network and protein interaction network(PPI)relationship diagrams.Discovery Studio 2020 software was used to perform molecular docking,and the GROMACS program was used to perform molecular dynamics simulation.Results A total of 21 prescriptions were collected involving 121 herbs.There were 9 herbs with a frequency≥10 times,which were Danshen(Radix et Rhizoma Salviae Miltiorrhizae),Huangqi(Radix Astragali),Baizhu(Rhizoma Atractylodis Macrocephalae),Danggui(Radix Angelicae Sinensis),Dangshen(Radix Codonopsis),Gancao(Radix et Rhizoma Glycyrrhizae),Baishao(Radix Paeoniae Alba),Fuling(Poria)and Shudihuang(Radix Rehmanniae Praeparata)from high-to low-frequency.A total of 98 active ingredients and 1125 potential targets were obtained from 9 high-frequency CMH.GSE44426 and GSE124281 data sets screened out 24 gene samples,including 14 of the healthy control group and 10 of the PMF group,and identified 319 DEGs between the two groups,including 122 up-regulated genes and 197 down-regulated genes.WGCNA screened out 24 co-expression module genes and found that the five modules closely related to the onset of PMF were MEpink,MEdarkred,MEblack,MEgrey,and MEturquoise,involving 7112 key module hub genes.The GO and KEGG enrichment analyses indicated that lipids and the atherosclerosis pathways were mainly involved in the mechanism of above high-frequency CMH in treating PMF,which included six hub protein targets:HSP90AA1,HSP90AB1,SRC,MAPK1,IL1B and IL10.From the drug-ingredient-target network,seven active ingredients of CMH targeting at these six hub targets were found,including verbascoside,verbascos isoflavone,kaempferol,luteolin,naringenin,quercetin and pachymic acid.The molecular docking and molecular dynamics analyses showed that the key CMH were Shudihuang,Huangqi,Baishao,Danshen,Gancao and Fuling,and among the seven active ingredients,calycosin had the highest binding affinity with HSP90AB1.Conclusion The main CMH for the treatment of PMF may be Shudihuang,Huangqi,Baishao,Danshen,Gancao and Fuling,and the active ingredients include verbascoside,verbascos isoflavones,kaempferol,luteolin,naringenin,quercetin and pachymic acid.The relevant targets are HSP90AA1,HSP90AB1,SRC,MAPK1,IL-10,and IL-1β,and the most critical pathways are lipid and atherosclerosis pathways.