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

LI Yuan; DU Hanqian; LI Jiashan; GUO Li; LI Zehui; LIN Na; XU Ying

doi:10.13422/j.cnki.syfjx.20242039

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

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- Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method
  增强出版
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 31, Issue 7, Pages: 53-62(2025)
- 作者机构：
  
  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
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20242039
  CLC： R242;R285;R271
- Received：30 August 2024，
  
  Accepted：15 November 2024，
  
  Published Online：22 November 2024，
  
  Published：05 April 2025
- 稿件说明：
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李源,杜寒倩,李佳珊等.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[J].中国实验方剂学杂志,2025,31(07):53-62.

LI Yuan,DU Hanqian,LI Jiashan,et al.Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method[J].Chinese Journal of Experimental Traditional Medical Formulae,2025,31(07):53-62.
李源,杜寒倩,李佳珊等.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[J].中国实验方剂学杂志,2025,31(07):53-62. DOI： 10.13422/j.cnki.syfjx.20242039.

LI Yuan,DU Hanqian,LI Jiashan,et al.Optimization and Mechanism Exploration of Tusizi Prescription for Ovarian Reserve Function Based on Uniform Design Method[J].Chinese Journal of Experimental Traditional Medical Formulae,2025,31(07):53-62. DOI： 10.13422/j.cnki.syfjx.20242039.

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（E

）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1 721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（

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（

0.05，

0.01）；TUNEL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组明显减少KGN细胞凋亡（

0.05）；ELISA结果表明，与正常组比较，菟丝子方水提物11组显著促进KGN细胞E

的分泌（

0.05），而均匀设计得出的最优组方对KGN细胞E

的分泌差异无统计学意义。

结论

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）与均匀设计筛选出的组方（菟丝子-枸杞子-山药-茯苓-莲子27∶30∶17∶12∶14）均可能是菟丝子方改善卵巢储备功能的最佳组方配比，但尤以组方11配比效果最佳。菟丝子方可通过Hyperoside等活性成分，正向调控细胞增殖、抑制细胞凋亡等生物学过程和影响酶结合、雌激素应答元件结合等分子功能，从而促进KGN细胞增殖、E

分泌、抑制凋亡，进而发挥对卵巢储备功能的保护作用。

Abstract

Objective

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， terminal-deoxynucleoitidyl transferase mediated nick-end labeling （TUNEL）， and enzyme-linked immunosorbent assay （ELISA） were employed to examine the proliferation， apoptosis， and estradiol （E

） 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 1 721 targets of DOR. GO analysis revealed 194 biological processes， primarily involving cellular responses to exogenous compound stimuli， negative regulation of apoptotic process， and positive regulation of cell proliferation. It identified 84 cellular components， including cell membrane， mitochondria， and neuronal cell body， as well as 144 molec

ular functions such as enzyme binding， estrogen response element binding， and nuclear estrogen receptor binding. The multivariate regression analysis revealed that when Tusizi prescription was composed of Cuscutae Semen， Lycii Fructus， Dioscoreae Rhizoma， Poria， and Nelumbinis Semen in a ratio of 27∶30∶17∶12∶14， the water extract of Tusizi prescription had the best effect of enhancing the viability of KGN cells. CCK-8 results showed that compared with the normal group， the hyperoside group demonstrated increased viability of ovarian germline stem cells （

0.01）. The CCK-8， EdU， and ELISA results showed that compared with the normal group， the optimal prescription screened by uniform design and the water extract 11 of Tusizi prescription increased the proliferation and reduced the apoptosis of KGN cells （

0.05，

0.01）. ELISA results showed that compared with the normal group， the water extract 11 of Tusizi prescription promoted the E

secretion of KGN cells （

0.05）， while the optimal prescription screened by uniform design had no significant effect on the E

secretion.

Conclusion

Both 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 （Cuscutae Semen-Lycii Fructus-Dioscoreae Rhizoma-Poria-Nelumbinis Semen 27∶30∶17∶12∶14） can improve the ovarian reserve function， and the former has better effect. Tusizi prescription can modulate biological processes （such as cell proliferation and apoptosis） and molecular functions （such as enzyme binding and estrogen response element binding） through active components like hyperoside to promote the proliferation and E

secretion and inhibit the apoptosis of KGN cells， thereby protecting the ovarian reserve function.

关键词

Keywords

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