基于网络药理学-分子对接及实验研究探讨高良姜-香附药对治疗原发性痛经的作用机制

Mechanism of Alpinia officinarum-Cyperus rotundus in the treatment of primary dysmenorrhea based on network pharmacology,molecular docking and experimental study

目的采用网络药理学、分子对接和实验验证研究高良姜-香附药对(简称“良附”)治疗原发性痛经(primary dysmenorrhea,PD)的作用机制。方法通过TCSMP及文献检索,获得高良姜和香附的活性成分,由TCSMP与Swiss Target Prediction平台获得相应成分及靶点;以“primary dysmenorrhea”“dysmenorrhea”为检索词,经GeneCard、DrugBank、DisGenet、TTD和OMIM数据库获取疾病靶点。利用STRING数据库进行PPI网络分析,由Metascape平台进行KEGG和GO富集分析,取高良姜-香附药对交集靶点、活性成分和相关通路构建“成分-靶点-通路”网络图。选取“成分-靶点-通路”网络和PPI网络的核心成分与靶点通过AutoDock进行对接。采用苯甲酸雌二醇和缩宫素联合制备小鼠原发性痛经模型,观察扭体反应;HE染色观察小鼠子宫病理变化;ELISA法检测PD小鼠血清中前列腺素E2(PGE2)、前列腺素F2α(PGF2α)和β-内啡肽(β-EP)的水平,Western blot法和RT-qPCR法验证蛋白和mRNA的表达。结果良附共获得29种主要活性成分,核心靶点为STAT3、PIK3CA、AKT1、ESR1、TP53、PTGS2等;分子对接结果显示,关键成分与核心靶点都有较强的结合能力。动物实验结果表明,良附组可缓解PD小鼠子宫组织水肿、充血和炎症,降低小鼠血清中PGF2α的水平,升高PGE2和β-EP的水平,抑制COX-2、p38、PI3K、和p-AKT的蛋白表达,mRNA的表达与蛋白表达趋势一致。结论良附能够缓解PD引起的疼痛,其机制可能是通过PI3K/AKT、MAPK和PTGS2等多途径发挥药效。

Objective To explore the mechanism of Alpinia officinarum-Cyperus rotundus(Referred to as"Liangfu")in the treatment of primary dysmenorrhea(PD)based on network pharmacology,molecular docking and experimental study.Methods The active components of Alpinia officinarum and Cyperus rotundus were obtained by TCSSMP and literature search,and the corresponding targets were obtained from TCSMP and the Swiss Target Prediction platform."Primary dysmenorrhea"and"dysmenorrhea"were used as the keywords to search the corresponding targets in the GeneCard,DrugBank,DisGenet,TTD,and OMIM databases.STRING database was used to build a PPI network.Enrichment analysis of KEGG and GO was carried out by Metascape platform.The"component-target-pathway"network was constructed by taking Alpinia officinarum and Cyperus rotundus intersection target,corresponding active components,and related pathways.Then the main compounds and Core target in the"component-target-pathway"and PPI networks were used for docking.PD mouse model was established by treatment with combining estradiol benzoate and oxytocin,and the writhing reaction was observed.HE staining was used to observe the pathological changes in the mouse uterus.The content of prostaglandin E2(PGE2),prostaglandin F2α(PGF2α),andβ-endorphin(β-EP)in serum was detected by ELISA.The expression of protein and mRNA was verified by western blot and RT-qPCR.Results Liangfu contained 29 active ingredients,and the key targets included STAT3,PIK3CA,AKT1,ESR1,TP53,and PTGS2.The molecular docking showed that the key components and core targets had strong binding capacity.The results of animal experiments showed that the Liangfu group alleviated the edema,congestion,and inflammation of uterine tissues in PD mice,reduced the level of PGF2αin serum,increased the level of PGE2 andβ-EP,and inhibited the expression of COX-2,p38,PI3K,and p-AKT mRNA and protein.Conclusion Liangfu can relieve the pain caused by PD,which may be related to modulation of PI3K/AKT,MAPK,and PTGS2 pathways.