基于网络药理学及动物实验探讨温经汤治疗多囊卵巢综合征的作用机制

Mechanism of Wenjing Decoction(温经汤)in Treating Polycystic Ovarian Syndrome Based on Network Pharmacology and Animal Experiments

目的:基于网络药理学与动物实验,探讨温经汤治疗多囊卵巢综合征(Polycystic Ovarian Syndrome,PCOS)的作用机制。方法:在TCMSP、BATMAN-TCM、Gene Cards、Dis Genet数据库分别检索温经汤的主要活性成分与靶点,PCOS疾病靶点,并将药物靶点与疾病靶点取交集。运用STRING数据库进行蛋白质相互作用分析,借助Metascape平台进行基因本体GO功能富集分析和京都基因与基因组百科全书KEGG通路富集分析。通过来曲唑联合高脂饲料诱导PCOS小鼠模型,给予不同剂量温经汤干预,观察各组小鼠体质量变化,空腹血糖水平及卵巢组织病理形态;采用ELISA法与免疫组化法检测小鼠血液与卵巢组织中核心靶点和核心通路的表达。结果:温经汤筛选出活性成分253个,主要包括苯代南蛇碱、甘草酚、芍药素酮、吴茱萸碱、等,与536个疾病靶点取交集后得到118个共同靶点,核心靶点包括AKT1、TP53、STAT3、TNF、VEGFA、IL6和EGFR等;GO富集在BP中主要包括激素反应、多肽反应、促进蛋白质磷酸化、等;MF主要包括蛋白质同源二聚体活性、血红素结合蛋白和四吡咯结合等;CC主要包括核膜、侧膜及膜筏等;KEGG富集发现HIF-1、PI3K/AKT及TNF等信号通路能在温经汤治疗PCOS中起关键作用。动物实验结果显示,与正常对照组比较,模型对照组体质量变化量显著增加(P<0.01),空腹血糖含量明显增加(P<0.05),血清中IL-6、TNF-α含量显著升高(P<0.01),卵巢组织中VEGFA表达显著上调,AKT、HIF-1α表达显著下调(P<0.01),卵巢组织中原始卵泡数量增多,卵泡颗粒细胞层数减少,卵巢指数显著降低(P<0.05);与模型对照组相比,温经汤10.14、20.28、40.56 g/kg组小鼠体质量变化量明显降低(P<0.05),血清中IL-6、TNF-α含量显著降低(P<0.01),卵巢组织中VEGFA表达显著下调,AKT、HIF-1α表达明显上调(P<0.05或P<0.01),卵泡颗粒细胞层厚度增加,黄体数量增多,可见各发育时期的卵泡。结论:温经汤可干预卵巢组织PI3K/AKT/HIF-1α/VEGF信号通路相关蛋白表达,减轻机体炎性反应,可能是其治疗PCOS的作用机制之一。

Objective:To explore the mechanism of Wenjing Decoction(温经汤)in treating polycystic ovarian syndrome(PCOS)based on network pharmacology and animal experiments.Methods:The main active components and targets of Wenjing Decoction and the disease targets of PCOS were searched in TCMSP,BATMAN-TCM,Gene Cards,and Dis Genet databases,respectively,and the intersection of drug targets and disease targets was taken.Protein-protein interaction analysis was performed using the STRING database.Gene ontology(GO)function enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were performed using the Metascape platform.The PCOS mice model was induced by letrozole combined with high-fat diet,and different doses of Wenjing Decoction were administered to observe the changes in body weight,fasting blood glucose level,and pathological morphology of ovarian tissue of rats.Enzyme-linked immunosorbent assay(ELISA)and immunohistochemistry were used to detect the expression of the core targets and core pathways in the blood and ovarian tissue of rats.Results:A total of 253 active ingredients were screened in Wenjing Decoction,mainly including Celabenzine,Glycyrol,paeoniflorgenone,evodiamine,etc.118 common targets were obtained after intersected with 536 disease targets,and core targets included protein kinase Bα(AKT1),TP53,signal transducer and activator of transcription 3(STAT3),tumor necrosis factor(TNF),vascular endothelial growth factor A(VECFA),interleukin-6(IL-6),epidermal growth factor receptor(EGFR),etc.GO enrichment in BP mainly included hormone response,peptide reaction,protein phosphorylation,etc.MF mainly included protein homodimer activity,heme-binding protein,tetrapyrrole binding,etc.CC mainly includes nuclear membrane,lateral membrane,membrane raft,etc.KEGG enrichment discovered that signaling pathways such as hypoxia-inducible factor-lα(HIF-lα),phosphoinositide 3-kinase(PI3K)/protein kinase B(AKT),and TNF could play a key role in the treatment of PCOS by Wenjing Decoction.The results of animal experiments showed that compared with the normal control group,the body weight change was significantly increased in the model control group(P<0.01),the fasting blood glucose level was increased significantly(P<0.05),the serum levels of IL-6 and TNF-αwere significantly increased(P<0.01),the expression of VECFA in ovarian tissue was significantly up-regulated,while the expressions of AKT and HIF-1αwere significantly down-regulated(P<0.O1).The number of primordial follicles in ovarian tissue was increased,the number of cell layers of follicular granules was decreased,and the ovarian index was significantly reduced(P<0.05).Compared with the model control group,the body weight change of rats was significantly reduced in Wenjing Decoction with doses of 10.14,20.28,and 40.56 g/kg(P<0.05),the serum levels of IL-6 and TNF-αwere significantly reduced(P<0.O1),the expression of VECFA in ovarian tissue was significantly down-regulated,while the expressions of AKT and HIF-lαwere significantly up-regulated(P<0.01 or P<0.05).The thickness of the cell layer of follicular granules was increased,the number of corpus luteum was increased,and follicles at various developmental stages were observed.Conclusion:Wenjing Decoction can interfere with the expression of PI3K/AKT/HIF-1α/VEGF signal pathway-related protein in ovarian tissue and reduce inflammatory reaction,which may be one of the mechanisms of Wenjing Decoction in the treatment of PCOS.