摘要
目的 应用网络药理学方法预测去氢骆驼蓬碱(HM)治疗细粒棘球蚴病的作用机制。方法 从中药系统药理学数据库(TCMSP)、瑞士靶点预测数据库(SwissTargetPrediction)、药物银行数据库(Drugbank)获取HM相关基因,在基因卡数据库(GeneCards)、DISGENET数据库、DrugBank数据库获取细粒棘球蚴病的作用靶点,将HM的预测靶点和细粒棘球蚴病相关基因相互映射,得到HM作用细粒棘球蚴病的靶点基因,导入String数据库进行网络拓扑属性分析,筛选重要靶点蛋白,构建蛋白质-蛋白质互作(PPI)和成分-疾病-靶点网络。采用DAVID数据库进行基因本体论(GO)功能分析和Kyoto京都基因与基因组百科全书(KEGG)通路富集分析,筛选与DNA损伤相关的基因靶点。采用Autodock Vina软件及Pymol软件对相关靶点进行分子对接验证和展示。将活力不低于98%的细粒棘球蚴随机分为空白对照组、1%DMSO组、25μmol/L HM组、50μmol/L HM组、100μmol/L HM组,采用实时荧光定量逆转录聚合酶链式反应(qRTPCR)法、免疫印迹(Western blot)法体外验证HM干预细粒棘球蚴肿瘤蛋白P53(TP53)、拓扑异构酶Ⅰ(TopoⅠ)、染色体组蛋白H2A的变异体(H2AX)、共济失调毛细血管扩张突变基因(ATM)mRNA和蛋白表达水平。结果 共获得HM靶点105个,细粒棘球蚴病相关基因88个,二者共有靶点为2个。HM干预细粒棘球蚴病的作用靶点主要有促分裂原活化蛋白激酶3(MAPK3)、MAPK1、TP53、热休克蛋白90α家族A类成员1(HSPAA1)、MAPK14、Jun,机制可能涉及MAPK信号通路、受体酪氨酸激酶(ERBB)信号通路、DNA损伤与DNA修复通路、细胞凋亡等过程。体外验证结果显示,与空白对照组相比,25μmol/L HM组、50μmol/L HM组、100μmol/L HM组的TP53,TopoⅠmRNA和蛋白表达均呈下调趋势(P <0.05),H2AX,ATM mRNA和蛋白表达均呈上调趋势(P <0.05)。结论 HM可通过多种生物学过程和相关信号途径治疗细粒棘球蚴病,HM参与细粒棘球蚴病DNA损伤与DNA修复信号通路的调控作用,TP53,TopoⅠ,H2AX,ATM均可能是其作用靶点。该研究为进一步阐明HM治疗细粒棘球蚴病的分子机制奠定了基础。
Objective To predict and analyze the mechanism of dehydrocamptothecin(HM)in the treatment of granulosa(EG)based on the network pharmacology method.Methods HM-related genes were obtained from the Chinese Medicine System Pharmacology Database and Analysis Platform(TCMSP),SwissTargetPrediction,and Drugbank databases.The target genes of EG were obtained from GeneCards,DISGENET database,and Drugbank databases.The predicted targets of HM and genes related to EG were mapped to obtain the target genes of HM acting on EG.The network topology attribute analysis was performed by importing them into the String database to screening important target proteins to construct protein-protein interaction(PPI)network and component-disease-target network.DAVID database was used for gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis to screen gene targets related to DNA damage.Autodock Vina and Pymol softwares were used to validate and display molecular docking of relevant targets.Echinococcus granulosus with activity not less than 98%was randomly divided into the blank control group,1%DMSO group,25μmol/L HM group,50μmol/L HM group,and 100μmol/L HM group through in vitro experiments.Quantitative reverse transcription-polymerase chain reaction(qRT-PCR)method and Western blot method were selected to verify the intervention effect of HM on the expression levels of TP53,TopoⅠ,H2AX,and ATM mRNA and proteins.Results A total of 105 targets of HM and 88 genes related to EG were obtained,with two intersecting targets.The main targets of HM acting on EG were MAPK3,MAPK1,TP53,HSPAA1,MAPK14,Jun,and the mechanism might involve MAPK signaling pathway,ERBB signaling pathway,DNA damage pathway and DNA repair pathway,cell apoptosis,and other processes.In vitro validation method showed that compared with those in the blank control group,the expression levels of TP53 and TopoⅠmRNA and proteins showed a downward trend,while the expression levels of H2AX and ATM mRNA and proteins showed an upward trend in the 25μmol/L HM group,50μmol/L HM group,and 100μmol/L HM group(P<0.05).Conclusion HM can treat EG through various biological processes and related signaling pathways.HM is involved in the regulation of DNA damage pathway and DNA repair signaling pathway in the treatment of EG,and TP53,TopoⅠ,H2AX,and ATM may be the targets.This study lays a foundation for further elucidating the molecular mechanism of HM in the treatment of EG.
作者
林玉霞
巩月红
赵一聪
潘美驰
王建华
LIN Yuxia;GONG Yuehong;ZHAO Yicong;PAN Meichi;WANG Jianhua(School of Pharmacy,Xinjiang Medical University,Urumqi,Xinjiang,China 830011;The First Affiliated Hospital of Xinjiang Medical University,Urumqi,Xinjiang,China 830054;State Key Laboratory of Pathogenesis,Prevention and Treatment of High Incidence Diseases in Central Asia,Xinjiang Medical University,Urumqi,Xinjiang,China 830000)
出处
《中国药业》
CAS
2024年第11期42-48,共7页
China Pharmaceuticals
基金
新疆维吾尔自治区自然科学基金[2020D01C240,2021D01D15]。
