摘要
目的:基于网络药理学分析佛手散治疗薄型子宫内膜的作用机制,以分子对接及动物实验验证。方法:通过中药系统药理学数据库与分析平台(TCMSP)和UniProt数据库筛选佛手散的有效活性成分和药物靶点。利用GeneCards数据库、NCBI基因数据库以及OMIM数据库收集薄型子宫内膜相关的疾病靶点。利用Venny 2.1.0和NetworkAnalyzer工具筛选药物治疗疾病的关键靶点。利用STRING数据库进行基因本体(GO)分析及京都基因与基因组百科全书(KEGG)通路富集分析。利用Cytoscape 3.8.0软件构建PPI网络图。分子对接验证活性成分与关键靶点的结合效能。采用95%无水乙醇宫腔注射构建薄型子宫内膜大鼠模型,造模同时进行灌胃干预,连续14 d, HE染色比较各组大鼠子宫内膜形态学变化、厚度和腺体数量;免疫组化观察各组大鼠子宫内膜角蛋白、波形蛋白变化;Western blot法检测大鼠子宫组织中雌激素受体α(ER-α)、JUN、p-JUN、血管内皮生长因子(VEGF)蛋白表达情况。结果:网络药理学分析结果表明,共筛选出佛手散的9种活性成分,即β-谷甾醇、杨梅酮、豆甾醇、等。药物-疾病共有靶点有41个,包括Caspase-3(CASP3)、JUN、ER-α、等。GO功能涉及到806条生物过程(BP),主要与类固醇激素反应、生殖结构/系统发育、雌激素反应有关;80项分子功能(MF),主要与细胞核受体和配体的激活,DNA-结合转录因子结合有关;19项细胞组分(CC),主要与膜筏、膜微区、膜区有关。KEGG主要涉及雌激素信号通路、内分泌抵抗、VEGF信号通路、P53信号通路、细胞凋亡相关通路、等。分子对接结果显示主要活性成分与关键靶点均有较好的结合能。HE和免疫组化染色结果显示,与正常对照组相比,模型对照组大鼠子宫内膜厚度、腺体数量和子宫内膜容受性明显下降,上皮细胞和基质细胞排列紊乱(P<0.01);与模型对照组相比,佛手散2.5 g/kg组可通过上调ER-α蛋白表达及JUN的磷酸化表达,促血管生成,增加薄型子宫内膜的厚度和容受性(P<0.01)。结论:佛手散通过多通路、多靶点治疗薄型子宫内膜,其作用机制可能与调控ER-α、p-JUN、VEGF表达有关。
Objective:To investigate the mechanism of Foshou San(佛手散)in the treatment of thin endometrium based on network pharmacology and verify the mechanism by molecular docking and animal experiments.Methods:Active ingredients and targets of Foshou San were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)and Uniprot databases.Disease targets related to thin endometrium were obtained from GeneCards database,NCBI gene database,and OMIM database.Venny 2.1.0 and Net-workAnalyzer tools were used to retrieve the key targets of drugs for the treatment of diseases.Gene ontology(GO)analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis were performed by using STRING database.The protein-protein interaction(PPI)network diagram was constructed with Cytoscape 3.8.0 software.Molecular docking was used to verify the binding efficacy of active ingredients and key targets.The thin endometrium model of rats was built by injecting 95%absolute ethyl alcohol into the rats’uterine cavities.After the gavage intervention was performed simultaneously for 14 days,HE staining was used to compare the endometrial morphology changes,thickness,and gland number in each group of rats.Immunohistochemistry was used to observe the changes in endometrial keratin and vimentin in each group.The protein expressions of estrogen receptorα(ER-α),JUN,p-JUN,and vascular endothelial growth factor(VEGF)in rats’uterine tissues were detected by Western blot method.Results:The results of network pharmacology analysis showed that a total of 9 active components of Foshou San were screened,includingβ-sitosterol,myricetone,stigmasterol,etc.A total of 41 common targets of drugs and diseases were obtained,including caspase-3(CASP3),JUN,ER-α,etc.The GO function was involved in the 806 biological processes(BP)of steroid hormone response,reproductive structure/system development,and estradiol response,80 molecular functions(MF)of nuclear receptor and ligand activation and DNA binding transcription factor binding,and 19 cellular components(CC)of membrane raft,membrane microdomain,and membrane region.KEGG pathway enrichment analysis mainly involved estrogen signal pathway,endocrine resistance,VEGF signal pathway,p53 signal pathway,cell apoptosis-related pathways,etc.Molecular docking showed that the main active ingredients of Foshou San had good binding rates with key targets.HE and immunohistochemical staining results showed that compared with those in the normal control group,the endometrial thickness,gland number,and endometrial receptivity were significantly decreased in the model group,while the epithelial cells and stromal cells were in disorder(P<0.01).Compared with the model group,Foshou San improved the thickness and receptivity of thin endometrium by activating ER-α,up-regulating phosphorylation expression of JUN,and promoting angiogenesis(P<0.01).Conclusion:Foshou San exerts a therapeutic effect on thin endometrium through multiple targets and multiple pathways,and the mechanism may be related to ER-α,p-JUN,and VEGF.
作者
韩瑞芳
梁杏珊
关永格
宋阳
HAN Ruifang;LIANG Xingshan;GUAN Yongge;SONG Yang(School of Nursing,Guangzhou University of Chinese Medicine,Guangzhou 510006;The Third Affiliated Hospital of Guangzhou University of Chinese Medicine,Guangzhou 510000)
出处
《中药药理与临床》
CSCD
北大核心
2023年第12期24-31,共8页
Pharmacology and Clinics of Chinese Materia Medica
基金
广东省自然科学基金(编号:2021A1515011435)
广州市荔湾区科技计划项目(编号:202201018)。
关键词
佛手散
网络药理学
分子对接
薄型子宫内膜
促血管生成
雌激素受体
Foshou San(佛手散)
Network pharmacology
Molecular docking
Thin endometrium
Promote angiogenesis
Estrogen receptor
