Zhang Jinrong, Huang Hongming, Xiao Min, Jiang Xiaocui, Yang Yong, Huang Min, Wang Shang, Zhu Biran, Zhao Min
School of Basic Medical Sciences, Hubei University of Chinese Medicine, Wuhan 430065, China.
Experimental Center of Traditional Chinese Medicine, Hubei University of Chinese Medicine, Wuhan 430065, China; Hubei Shizhen Laboratory, Wuhan 430061, China.
Phytomedicine. 2025 Jul 25;143:156852. doi: 10.1016/j.phymed.2025.156852. Epub 2025 May 20.
Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder associated with chronic inflammation and metabolic issues, including insulin resistance, weight gain, and lipid imbalance. It may cause infertility, menstrual irregularities, and increase the likelihood of developing type 2 diabetes, immune system disturbances, and cardiovascular conditions.
To identify the key components, underlying mechanisms, and therapeutic targets of Erchen decoction (ECD) for treating PCOS.
Abbreviations: Abi group, abiraterone group; AKR, aldo-keto reductase; AMH, anti-Müllerian hormone; BC, betweenness; BP, biological process; BSA, bovine serum albumin; CC, cellular component; CCK-8, cell counting kit-8; Con group, control group; DC, degree centrality; DDA, data-dependent acquisition; DHEA, dehydroepiandrosterone; DHEAS, DHEA sulfate; DHT, dihydrotestosterone; ECD, Erchen decoction; Ecd group, Erchen decoction group; ELISA, enzyme-linked immunosorbent assay; E2, estradiol; FSH, follicle-stimulating hormone; GO, gene ontology; HE, hematoxylin and eosin; H-Ecd group, high-dose Erchen decoction group; HFD, high-fat diet; HSD, hydroxysteroid dehydrogenases; KEGG, Kyoto encyclopedia of genes and genomes; KGN, human granulosa-like tumor cells; LAC, local average connectivity; l-Ecd group, low-dose Erchen decoction group; LH, luteinizing hormone; Met group, metformin (Glucophage) group; MF, molecular function; MIS, Müllerian inhibiting substance; MST, microscale thermophoresis; PCOS model group, PCOS group; PCA, principal component analysis; PCOS, polycystic ovary syndrome; PPI, protein-protein interaction; T, testosterone; TCM, traditional Chinese medicine; Tcm group, traditional Chinese medicine group; UHPLC, ultra-high-performance liquid chromatography Bioinformatics was used to predict the targets of ECD components for treating PCOS. Sprague-Dawley rats were assigned to control (Con) and PCOS model groups. The latter was induced via letrozole (Femara) gavage (1 mg/kg) combined with a high-fat diet. The PCOS group was then subdivided for 28 days of intervention. Body weight was recorded, ovarian morphology was assessed through hematoxylin and eosin staining, and serum hormones were quantified using enzyme-linked immunosorbent assay. Proteomic analyses were performed to examine the underlying mechanisms and potential targets, which were validated using immunofluorescence, western blotting, and RT-qPCR. The pharmacological effects of the key ECD components were confirmed in dihydrotestosterone (DHT)-treated human granulosa KGN cells.
The body weight of the rats in the Con and high-dose ECD (H-Ecd) groups decreased compared with that in the PCOS group. The H-Ecd and metformin (Glucophage) groups had significantly elevated levels of testosterone (T), luteinizing hormone, anti-Müllerian hormone (AMH), and follicle-stimulating hormone (p < 0.05), whereas the low-dose Ecd (l-Ecd) group showed no significant change. Thirteen blood-entering components and 168 potential therapeutic targets for PCOS were identified. Kyoto encyclopedia of genes and genomes and gene ontology analyses indicated the involvement of the steroid biosynthesis pathway. Principal component analysis revealed notable differences among the Con, H-Ecd, and PCOS groups. Microscale thermophoresis (MST) validated the binding affinities of liquiritin, glycyrrhizic acid, esculetin, and genistein to cytochrome P450 family 17 subfamily A member 1 (CYP17A1) as 5.4, 12.55, 32.8, and 17.6 μM, respectively. Immunofluorescence, western blotting, and RT-qPCR analyses revealed significantly decreased protein expression of CYP17A1 and 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) (p < 0.01) and significantly increased the expression of aldo-keto reductase family 1 member D1 (AKR1D1) and HSD11B2 in the Con, Met, and H-Ecd groups relative to that in the PCOS group, whereas the l-Ecd group showed no significant difference. The cell counting kit-8 assay demonstrated that abiraterone (100 μM), DHT (100 nM), glycyrrhizic acid (50 μM), liquiritin (600 μM), genistein (300 μM), and esculetin (100 μM) significantly decreased T and AMH levels (p < 0.05). E2 levels recovered significantly (p < 0.05) in the Con, abiraterone (Abi), and traditional Chinese medicine (Tcm) groups compared to that in the DHT group. Western blot analysis indicated a significant reduction in CYP17A1 and HSD11B1 protein expression (p < 0.01) and a significant increase in AKR1D1 and HSD11B2 expression (p < 0.01) in the Con, Abi, and Tcm groups compared to that in the DHT group.
We sought to identify the active constituents of ECD for treating PCOS and found that ECD enhances ovarian function by modulating the expression of CYP17A1, HSD11B1, AKR1D1, and HSD11B2 in the steroid hormone biosynthesis pathway resulting in improved hormone levels and follicular development. These results highlight the potential mechanism underlying ECD-mediated effects for PCOS therapy. Molecular docking validated the interaction of liquiritin, glycyrrhizic acid, esculetin, and genistein with CYP17A1, HSD11B1, AKR1D1, and HSD11B2. These interactions were further confirmed in vitro using MST and validated at the cellular level. Our results offer a scientific foundation for the therapeutic application of ECD in PCOS treatment.
多囊卵巢综合征（PCOS）是一种常见的内分泌和代谢紊乱疾病，与慢性炎症和代谢问题相关，包括胰岛素抵抗、体重增加和脂质失衡。它可能导致不孕、月经不调，并增加患2型糖尿病、免疫系统紊乱和心血管疾病的可能性。
确定二陈汤（ECD）治疗PCOS的关键成分、潜在机制和治疗靶点。
缩写：阿比特龙组（Abi组）；醛糖酮还原酶（AKR）；抗苗勒管激素（AMH）；介数中心性（BC）；生物过程（BP）；牛血清白蛋白（BSA）；细胞成分（CC）；细胞计数试剂盒-8（CCK-8）；对照组（Con组）；度中心性（DC）；数据依赖采集（DDA）；脱氢表雄酮（DHEA）；硫酸脱氢表雄酮（DHEAS）；二氢睾酮（DHT）；二陈汤（ECD）；二陈汤组（Ecd组）；酶联免疫吸附测定（ELISA）；雌二醇（E2）；促卵泡生成素（FSH）；基因本体论（GO）；苏木精-伊红染色（HE）；高剂量二陈汤组（H-Ecd组）；高脂饮食（HFD）；羟类固醇脱氢酶（HSD）；京都基因与基因组百科全书（KEGG）；人颗粒细胞瘤细胞（KGN）；局部平均连通性（LAC）；低剂量二陈汤组（l-Ecd组）；黄体生成素（LH）；二甲双胍（格华止）组（Met组）；分子功能（MF）；苗勒管抑制物质（MIS）；微量热泳动（MST）；PCOS模型组（PCOS组）；主成分分析（PCA）；多囊卵巢综合征（PCOS）；蛋白质-蛋白质相互作用（PPI）；睾酮（T）；传统中药（TCM）；中药组（Tcm组）；超高效液相色谱法（UHPLC） 采用生物信息学方法预测ECD成分治疗PCOS的靶点。将Sprague-Dawley大鼠分为对照组（Con）和PCOS模型组。后者通过来曲唑（法乐通）灌胃（1mg/kg）联合高脂饮食诱导。然后将PCOS组再细分进行28天的干预。记录体重，通过苏木精-伊红染色评估卵巢形态，使用酶联免疫吸附测定法定量血清激素。进行蛋白质组学分析以研究潜在机制和潜在靶点，并通过免疫荧光、蛋白质印迹和RT-qPCR进行验证。在二氢睾酮（DHT）处理的人颗粒KGN细胞中证实了ECD关键成分的药理作用。
与PCOS组相比，Con组和高剂量ECD（H-Ecd）组大鼠体重下降。H-Ecd组和二甲双胍（格华止）组的睾酮（T）、黄体生成素、抗苗勒管激素（AMH）和促卵泡生成素水平显著升高（P<0.05），而低剂量Ecd（l-Ecd）组无显著变化。确定了13种入血成分和168个PCOS潜在治疗靶点。京都基因与基因组百科全书和基因本体论分析表明类固醇生物合成途径参与其中。主成分分析显示Con组、H-Ecd组和PCOS组之间存在显著差异。微量热泳动（MST）验证了甘草苷、甘草酸、七叶亭和染料木黄酮与细胞色素P450家族17亚家族A成员1（CYP17A1）的结合亲和力分别为5.4、12.55、32.8和17.6μM。免疫荧光、蛋白质印迹和RT-qPCR分析显示，与PCOS组相比，Con组、Met组和H-Ecd组中CYP17A1和Ⅰ型11β-羟类固醇脱氢酶（HSD11B1）的蛋白表达显著降低（P<0.01），醛糖酮还原酶家族1成员D1（AKR1D1）和HSD11B2的表达显著增加，而l-Ecd组无显著差异。细胞计数试剂盒-8检测表明，阿比特龙（100μM）、DHT（100nM）、甘草酸（50μM）、甘草苷（600μM）、染料木黄酮（300μM）和七叶亭（100μM）显著降低T和AMH水平（P<0.05）。与DHT组相比，Con组、阿比特龙（Abi）组和中药（Tcm）组的E2水平显著恢复（P<0.05）。蛋白质印迹分析表明，与DHT组相比，Con组、Abi组和Tcm组中CYP17A1和HSD11B1蛋白表达显著降低（P<0.01），AKR1D1和HSD11B2表达显著增加（P<0.01）。
我们试图确定ECD治疗PCOS的活性成分，发现ECD通过调节类固醇激素生物合成途径中CYP17A1、HSD11B1、AKR1D1和HSD11B2的表达来增强卵巢功能，从而改善激素水平和卵泡发育。这些结果突出了ECD介导的PCOS治疗作用的潜在机制。分子对接验证了甘草苷、甘草酸、七叶亭和染料木黄酮与CYP17A1、HSD11B1、AKR1D1和HSD11B2的相互作用。这些相互作用在体外通过MST进一步得到证实，并在细胞水平上得到验证。我们的结果为ECD在PCOS治疗中的应用提供了科学依据。
