基于均匀设计法优化菟丝子方对卵巢储备功能影响的组方配比及机制  

Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method

作　　者：李源 杜寒倩 李佳珊 郭丽[2] 李泽辉 林娜[2] 徐颖 LI Yuan;DU Hanqian;LI Jiashan;GUO Li;LI Zehui;LIN Na;XU Ying(Institute of Chinese Materia Medica,Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]上海中医药大学中药研究所,上海201203 [2]中国中医科学院中药研究所,北京100700

出　　处：《中国实验方剂学杂志》2025年第7期53-62,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：中国中医科学院科技创新工程项目(CI2021A03802,CI2023E001TS);国家自然科学基金项目(81974537)。

摘　　要：目的:通过均匀设计法结合体外实验,优化菟丝子方对卵巢储备功能影响的组方配比并探讨其作用机制。方法:采用均匀设计法对菟丝子方水提物进行5因素11水平的实验设计,以细胞增殖与活性检测(CCK-8)实验测定菟丝子方水提物1~11处理的人卵巢颗粒细胞(KGN细胞)活力为指标,进行多元回归分析得出最优组方配比;在中药系统药理学数据库与分析平台(TCMSP)、中药百科全书数据库(ETCM)中查询活性成分潜在靶点,与疾病靶点取交集获得共同靶点,分别将共同靶点导入基因/蛋白质相互作用检索工具数据库(STRING)构建蛋白质-蛋白质相互作用(PPI)网络及基因功能注释数据库(DAVID)进行基因本体(GO)分析;以CCK-8实验测定金丝桃苷对卵巢生殖干细胞活力的影响;以CCK-8、细胞增殖检测试剂盒(EdU)、细胞凋亡检测试剂盒(TUNEL)、酶联免疫吸附测定法(ELISA)实验对均匀设计得出最优组方和菟丝子方水提物11(菟丝子-枸杞子-山药-茯苓-莲子4∶4∶2∶1∶1)在影响KGN细胞增殖、凋亡、雌二醇(E2)分泌方面进行对比研究,以期得出对卵巢储备功能影响的最优组方配比及初探其机制。结果:菟丝子方的278个药物靶点和1721个疾病靶点取交集,获得共同靶点147个;GO分析显示主要涉及细胞对外源性化合物刺激的反应、凋亡过程的负调控、正向调控细胞增殖等194个生物学过程,细胞膜、线粒体、神经元细胞体等84个细胞组成,酶结合、雌激素应答元件结合、核雌激素受体结合等144个分子功能;多元回归分析得出菟丝子方各药材按照菟丝子-枸杞子-山药-茯苓-莲子27∶30∶17∶12∶14组方时增强KGN细胞活力具有最佳效果;CCK-8结果表明,与正常组比较,金丝桃苷组卵巢生殖干细胞活力显著提高(P<0.01);CCK-8和EdU结果表明,与正常组比较,均匀设计筛选出的最优组方和菟丝子方水提物11组KGN细胞增殖能力明显提高(P<0.05Objective:To optimize Tusizi prescription for ovarian reserve function based on the uniform design method combined with in vitro experiments and explore the underlying mechanisms of this prescription.Methods:The uniform design method was adopted to design a 5-factor 11-level experiment on the water extract of Tusizi prescription.The cell-counting kit-8(CCK-8)assay was employed to measure the viability of human ovarian granulosa cells(KGN cells)treated with Tusizi prescription extracts 1-11,and multivariate regression analysis was performed to determine the optimal herb ratio in this prescription.The potential targets of active ingredients in the prescription were retrieved from traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)and encyclopedia of traditional Chinese medicine(ETCM).The common targets shared by Tusizi prescription and diminished ovarian reserve(DOR)were selected and imported into search tool for the retrieval of interacting genes/proteins(STRING)to construct a protein-protein interaction(PPI)network and into gene function annotation database(DAVID)for gene ontology(GO)analysis.The CCK-8 assay was used to measure the viability of ovarian germline stem cells treated with hyperoside.The CCK-8 assay,5-ethynyl-2'-deoxyuridine(EdU)staining,terminaldeoxynucleoitidyl transferase mediated nick-end labeling(TUNEL),and enzyme-linked immunosorbent assay(ELISA)were employed to examine the proliferation,apoptosis,and estradiol(E2)secretion of KGN cells treated with the water extract 11 of Tusizi prescription(Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 4∶4∶2∶1∶1)and the optimal prescription screened by uniform design.On this basis,the optimal prescription composition for maximizing the effect on ovarian reserve function was determined and preliminary insights into the underlying mechanisms of this prescription were gained.Results:A total of 147 common targets were obtained from 278 targets of Tusizi prescription and 1721 targets of DOR.GO ana

关 键 词：菟丝子方 卵巢储备功能 均匀设计 卵巢颗粒细胞 卵巢生殖干细胞 

分 类 号：R242[医药卫生—中医临床基础] R285[医药卫生—中医学] R271