摘要
目的:该研究通过网络药理学的方法探讨归肾丸治疗薄型子宫内膜的关键核心靶点及相关信号通路等作用机制,进一步运用动物实验加以验证所得靶点,验证归肾丸对薄型子宫内膜的影响。方法:利用中药系统药理学数据库与分析平台(TCMSP)数据库检索归肾丸中8味中药组成的有效化学成分、活性成分靶点和靶点简称;运用GeneCards数据库和在线人类孟德尔遗传数据库(OMIM)检索薄型子宫内膜相关的靶点基因;运用韦恩软件对归肾丸的药物靶点和薄型子宫内膜的疾病靶点进行取交集操作,将交集靶点导入STRING数据库及Cytoscape 3.7.2软件进行可视化分析获得"药物-疾病"的蛋白质-蛋白质相互作用(PPI)网络;再将交集靶点输入Enrichr数据库和David数据库分别进行基因本体(GO)富集分析及京都基因与基因组百科全书(KEGG)富集分析。以得到的可能核心关键靶点作为理论基础,建立大鼠薄型子宫内膜模型,归肾丸及雌激素干预21 d后,通过苏木素-伊红(HE)染色法观察大鼠的内膜厚度;通过蛋白免疫印迹法(Western blot)和实时荧光定量聚合酶链式反应(Real-time PCR)检测表皮生长因子受体(EGFR),基质金属蛋白酶9(MMP9),白细胞介素1β(IL^(-1)β),丝裂原激活蛋白激酶14(MAPK14)这4个核心关键靶点的蛋白和mRNA的表达水平。结果:通过韦恩软件共获得130个交集靶点;将130个交集靶点导入STRING数据库和Cytoscape软件中,得到包括33个节点,107条边的蛋PPI网络;DAVID 6.8数据库进行GO分析中的功能注释分析,共涉及167个生物过程(BP),22个细胞组分(CC),39个分子功能(MF);DAVID 6.8数据库进行KEGG富集分析,与薄型子宫内膜相关的通路共34条;Western blot和Real-time PCR分别对6组大鼠子宫内膜组织中的EGFR,MMP9,IL^(-1)β,MAPK14蛋白和mRNA的表达结果进行分析,归肾丸能够增强EGFR,MMP9,IL^(-1)β,MAPK14蛋白和mRNA在薄型子宫内膜上的表达。结论:通过网络药理学的理论分析及动物实验的研究结果发现,归肾丸能够有效改善薄型子宫内膜的相关指标,并通过增加EGFR,MMP9,IL^(-1)β,MAPK14蛋白和mRNA表达水平,促进内膜组织增殖,改善内膜偏薄的症状。
Objective:This study explores the key core targets of Guishenwan in the treatment of thin endometrium and related signaling pathways through the method of network pharmacology,and further uses animal experiments to verify the obtained targets and verify that Guishenwan are effective for thin endometrium.Method: Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)was used to retrieve the effective chemical components,active component targets and target abbreviations of the eight Chinese medicines in Guishenwan,the GeneCards database and Online Mendelian Inheritance in Man(OMIM)database were used to retrieve thin endometrial related targets gene.Use Wayne software to take the intersection of the drug target of Guishenwan and the disease target of the thin endometrium,and import the intersection target into the STRING database and Cytoscape 3.7.2 software for visual analysis to obtain the "drug-disease" protein protein interaction(PPI) network,then input the intersection target into Enrichr database and DAVID database for gene ontology(GO)enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis. Using the obtained possible core and key targets as the theoretical basis,a thin endometrial model in rats was established. After Guishenwan and estrogen intervention for 21 days,the endometrial thickness of rats was observed by hematoxylin-eosin staining(HE) staining. Western blot and quantitative real time polymerase chain reaction(Real-time PCR)detect the protein and mRNA expression levels of the four core key targets of epidermal growth factor receptor(EGFR),matrix metalloproteinase 9(MMP9),interleukin-1 beita(IL^(-1)β)and mitogen activated protein kinase 14(MAPK14). Result: The Venn software obtained 130 intersection targets in total,imported 130 intersection targets into the STRING database and Cytoscape database,and obtained a protein interaction network diagram including 33 nodes and 107 edges. DAVID 6.8 database for GO analysis. The function annotation analysis involving 167 biological processes(BP),22 cell components(CC),39 molecular functions(MF). DAVID 6.8 database for KEGG enrichment analysis,and thin endometrium related A total of 34 pathways. Western blot and Real-time PCR were used to analyze the expression results of EGFR,MMP9,IL^(-1)β,MAPK14 protein and genes in the endometrial tissues of the 6 groups of rats. Guishenwan can enhance the expression of EGFR,MMP9,IL^(-1)β,MAPK14 protein and mRNA on the thin endometrium.Conclusion: According to the theoretical analysis of network pharmacology and the results of animal experiments,it is found that Guishenwan can effectively improve the related indicators of thin endometrium,and promote the expression of EGFR,MMP9,IL^(-1)β,MAPK14 protein and genes. The intimal tissue proliferates and improves the symptoms of thin intima.
作者
邵芷若
关永格
宋阳
刘燕
李玥
吕妍
SHAO Zhi-ruo;GUAN Yong-ge;SONG Yang;LIU Yan;LI Yue;LYU Yan(The Third Clinical Medical College of Guangzhou University of Chinese Medicine,Guangzhou 510006,China;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510000,China;School of Nursing of Guangzhou University of Chinese Medicine,Guangzhou 510006,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2021年第17期168-177,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
广东省自然科学基金项目(2021A1515011435,2020A151501757)
国家自然科学基金项目(81873331)
广东省中医药局科研项目(20203003)。
关键词
归肾丸
薄型子宫内膜
网络药理学
作用机制
Guishenwan
thin endometrium
network pharmacology
mechanism of action
