基于网络药理学结合体内实验研究探讨四物汤的植物雌激素样效应及其分子机制

Phytoestrogen-like effect of Siwu Decoction and molecular mechanism:based on network pharmacology and in vivo experiment

基于网络药理学结合体内实验研究探讨四物汤靶向生殖系统的植物雌激素样效应及其雌激素受体(estrogen receptor,ER)亚型调节与介导的分子机制。首先利用中药系统药理学技术平台(TCMSP),收集四物汤的活性成分及靶点。通过GeneCards和OMIM数据库收集“雌激素”的相关基因。借助STRING网站和Cytoscape软件构建蛋白相互作用(PPI)网络及“化学成分-靶点-疾病”网络,筛选核心靶点。利用R软件对核心靶点进行GO(Gene Ontology)、KEGG(Kyoto Encyclopedia of Genes and Genomes)富集分析。体内实验选取22 d龄SD雌鼠,四物汤灌胃4 d。HE染色观测子宫形态学变化,RT-PCR方法检测子宫、卵巢ER亚型及其介导的EGFR/MAPK下游传导通路关键调控因子的mRNA表达量。结果显示,四物汤发挥植物雌激素样效应的相关靶点共74个;KEGG通路富集分析表明,其发挥效应的机制与雌激素信号通路密切相关。体内实验结果发现,四物汤组大鼠子宫内膜显著增厚;卵巢组织ERα、ERβ、GPER mRNA表达量均显著下降,子宫组织ERα、ERβmRNA表达量显著下降,GPER mRNA表达量显著上升。卵巢、子宫组织EGFR mRNA表达量均明显增加,p38 MAPK、MEK1/2、ERK1/2 mRNA表达量均明显减少。该研究通过网络药理学分析及体内实验揭示了四物汤靶向生殖系统发挥雌激素样效应的分子机制,为四物汤妇科疗效机制的全面揭示提供了依据。

This study explored the phytoestrogen-like effect of Siwu Decoction(SWD)and the estrogen receptor(ER)-mediated molecular mechanism based on network pharmacology and in vivo experiment.The active components and targets of SWD were retrieved from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),and related targets of"estrogen"from GeneCards and Online Mendelian Inheritance in Man(OMIM).Cytoscape and STRING were employed to construct the protein-protein interaction(PPI)network and"chemical component-target-disease"network and core targets were identified,followed by Gene Ontology(GO)term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment of the core targets by R software.For the in vivo experiment,the 22-day-old SD female rats were treated(ig)with SWD for 4 days.Via hematoxylin-eosin(HE)staining,the morphological changes of rat uterus were observed.Reverse transcriptase-polymerase chain reaction(RT-PCR)was performed to detect mRNA expression of ER subtypes,estrogen-related targets,and the main regulatory factors in the estrogen signaling pathway.The results indicated 74 targets of SWD exerted phytoestrogen-like effect.KEGG pathway enrichment result suggested that estrogen signaling pathway was closely related to the phytoestrogen-like effect of SWD.Rats in SWD group demonstrated significantly thickened endometrium and significantly decreased expression of ERα,ERβ,and G protein-coupled estrogen receptor(GPER)mRNA in ovarian tissue.In addition,significant lowering of ERαand ERβmRNA expression and significant rise of GPER mRNA expression in uterine tissue were observed in the SWD group.The expression of mitogen-activated protein kinase(MAPK)p38,MEK1/2 and extracellular signal-regulated kinase(ERK)1/2 mRNA was significantly low while that of epidermal growth factor receptor(EGFR)mRNA was significantly high in both ovarian and uterine tissues of SWD group compared with those in the control group.In conclusion,the phytoestrogen-like effect of SWD is closely related to the estrogen signaling pathway.The result lays a basis for revealing molecular mechanism of SWD in the treatment of gynecological diseases.