基于网络药理学和分子对接技术探究红花逍遥片治疗卵巢早衰的作用机制及实验验证
Exploration on the mechanism and experimental verification of Honghua Xiaoyao Tablets in the treatment of premature ovarian failure based on network pharmacology and molecular docking technology
陈玥 1佘婧瑶 2王静 3叶宇齐 3梁春云 2卢燕 3钟伟萍3
作者信息
- 1. 江苏省中西医结合医院妇产科,南京 210028;南京中医药大学第三临床医学院,南京 210023
- 2. 南京中医药大学第三临床医学院,南京 210023
- 3. 江苏省中西医结合医院妇产科,南京 210028
- 折叠
摘要
目的 运用网络药理学方法和分子对接技术探究红花逍遥片治疗卵巢早衰的作用机制,并进一步实验验证.方法 应用TCMSP、PubChem数据库获得红花逍遥片活性成分及相关靶点,应用GeneCards数据库获得卵巢早衰相关靶点,利用Venn在线工具筛选两者交集基因,采用STRING数据库构建交集靶点PPI网络,通过Metescape数据库对核心靶点进行GO功能富集分析和KEGG通路富集分析,采用Cytoscape 3.7.1软件构建"活性成分-靶点"网络和"中药-活性成分-靶点-关键通路-疾病"网络,并进行分子对接验证.采用随机数字表法将小鼠分为空白组、模型组及红花逍遥片组,每组8只.除空白组外,其余各组小鼠采用透明带多肽3(ZP3)和弗式完全佐剂(FCA)建立免疫性卵巢早衰小鼠模型.红花逍遥片组灌胃红花逍遥片溶液0.56 g/kg,空白组、模型组灌胃等体积生理盐水,连续灌胃4周.采用HE染色观察小鼠卵巢组织病理变化.采用免疫组化染色检测ESR1表达,采用Western blot检测Akt、p-Akt表达.结果 获得红花逍遥片与卵巢早衰交集靶点80个,PPI网络包含核心靶点44个,度值排名前5位的活性成分分别为芒柄花素、槲皮素、白桦脂酸、羟基红花黄A和黄芩苷,排名前5位的靶点分别为PPARG、ESR1、AR、AKT 1和IL6.核心靶点分子功能主要为受体配体活性,生物过程主要涉及细胞迁移的正向调节,细胞组分主要包括膜筏,主要涉及信号通路有癌症信号通路、癌症中的蛋白聚糖、PI3K-Akt信号通路及HIF-1信号通路等."中药-成分-靶点-通路-疾病"网络显示,红花逍遥片处方中8味药具有重要核心成分,其中甘草和红花涉及重要核心成分最多.分子对接结果显示,活性成分与核心靶点亲和力良好.实验验证结果显示,红花逍遥片可促进卵泡发育,提高卵巢组织中ESR1表达,并上调PI3K-Akt信号通路关键因子Akt磷酸化水平.结论 红花逍遥片多种活性成分可能通过作用于PPARG、ESR1等多个靶点,并通过PI3K-Akt、HIF-1等信号通路治疗卵巢早衰,其潜在活性成分主要为芒柄花素、槲皮素等.
Abstract
Objective To explore the mechanism of Honghua Xiaoyao Tablets in the treatment of premature ovarian failure(POF)using network pharmacology and molecular docking technology;To conduct further experimental verification.Methods The active components and related targets of Honghua Xiaoyao Tablets were obtained using TCMSP and PubChem databases,and the related targets of POF were obtained by using GeneCards database.The Venn online tool was used to screen the intersection genes,and the STRING database was used to construct the PPI network.Then,GO function enrichment analysis and KEGG pathway enrichment analysis were performed on the intersecting genes through the Metescape database,and Cytoscape 3.7.1 software was used to construct the"active component-target"network and"Chinese materia medica-active component-target-key pathway-disease"network.Finally,molecular docking verification was carried out.Mice were divided into blank group,model group,and Honghua Xiaoyao Tablets group using a random number table method,with 8 mice in each group.Except for the blank group,mice in each group were treated with zona pellucida polypeptide 3(ZP3)and Freund's Complete Adjuvant(FCA)to establish a mouse model of immune POF.Mice in the Honghua Xiaoyao Tablets group received Honghua Xiaoyao Tablets solution 0.56 g/kg for gavage,and the blank control group and the model group received saline for gavage for consecutive 4 weeks.The histopathological changes of the mouse ovary were observed by HE staining.Immunohistochemical staining was used to observe the expression of ESR1,and Western blot was used to detect the expressions of Akt and p-Akt.Results A total of 80 intersection targets between Honghua Xiaoyao Tablets and POF were obtained,and the PPI network contained 44 core targets.The top 5 compounds in the topological analysis were formononetin,quercetin,Betulinic acid,Hydroxysafflor Yellow A and Baicalin,and the top 5 targets were PPARG,ESR1,AR,AKT1 and IL6.The molecular function of core genes was mainly receptor ligand activity,and its biological process mainly involved the positive regulation of cell migration.The cellular components mainly included membrane rafts,which were involved in signaling pathways such as cancer signaling pathway,proteoglycans in cancer,PI3K-Akt signaling pathway,and HIF-1 signaling pathway."Chinese materia medica-component-target-pathway-disease"network showed that 8 kinds of Chinese materia medica in the Honghua Xiaoyao Tablets had important core components,among which Glycyrrhizae Radix et Rhizoma and Carthami Flos involved the most important core components.Molecular docking results showed that the active components had a good affinity with the core target.The experimental verification confirmed that Honghua Xiaoyao Tablets promoted follicular development,increased the expression of ESR1 in ovarian tissues and up-regulated the expression level of the key factor of Akt phosphorylation in the PI3K-Akt signaling pathway.Conclusions The various active components of Honghua Xiaoyao Tablets may act on PPARG,ESR1 and other targets through multiple signaling pathways such as PI3K-Akt and HIF-1 to treat POF.The potential active components are mainly formononetin,quercetin,etc.
关键词
卵巢/早衰/红花逍遥片/网络药理学/分子对接/PI3K-Akt信号通路Key words
Ovary/Progeria/Honghua Xiaoyao Tablets/Network pharmacology/Molecular docking/PI3K-Akt signaling pathway引用本文复制引用
基金项目
国家自然科学基金(81903998)
中国中西医结合妇产科专项研究基金普正制药专项(FCK-PZ-09)
王佩娟全国名老中医药专家传承工作室项目(国中医药人教函[2022]75号)
出版年
2024
NETL
NSTL
万方数据