二陈汤通过调节类固醇生物合成途径改善多囊卵巢综合征大鼠模型

IF 6.7 1区医学 Q1 CHEMISTRY, MEDICINAL

Phytomedicine Pub Date : 2025-05-20 DOI:10.1016/j.phymed.2025.156852

Jinrong Zhang , Hongming Huang , Min Xiao , Xiaocui Jiang , Yong Yang , Min Huang , Shang Wang , Biran Zhu , Min Zhao

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It may cause infertility, menstrual irregularities, and increase the likelihood of developing type 2 diabetes, immune system disturbances, and cardiovascular conditions.</div></div><div><h3>Objective</h3><div>To identify the key components, underlying mechanisms, and therapeutic targets of Erchen decoction (ECD) for treating PCOS.</div></div><div><h3>Methods</h3><div>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.</div></div><div><h3>Results</h3><div>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 (<em>p</em> < 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) (<em>p</em> < 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 (<em>p</em> < 0.05). E2 levels recovered significantly (<em>p</em> < 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 (<em>p</em> < 0.01) and a significant increase in AKR1D1 and HSD11B2 expression (<em>p</em> < 0.01) in the Con, Abi, and Tcm groups compared to that in the DHT group.</div></div><div><h3>Conclusion</h3><div>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. 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引用次数: 0

摘要

背景：多囊卵巢综合征（PCOS）是一种常见的内分泌和代谢疾病，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。它可能会导致不孕、月经不规律，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。目的探讨二陈汤治疗多囊卵巢综合征的关键成分、作用机制及治疗靶点。方法英文缩写：Abi组、阿比特龙组；醛酮还原酶；抗<s:1>勒氏杆菌激素；公元前,中间状态;BP，生物过程；牛血清白蛋白；CC，细胞成分；CCK-8细胞计数试剂盒；对照组，对照组；DC，度中心性；DDA，数据依赖型采集；脱氢表雄酮、脱氢表雄酮;脱氢表雄酮，硫酸脱氢表雄酮；二氢睾酮、二氢睾酮;二辰汤ECD；Ecd组、二辰汤组；ELISA，酶联免疫吸附试验；E2,雌二醇;促卵泡激素；GO，基因本体；HE、苏木精和伊红；H-Ecd组、二辰汤高剂量组；HFD，高脂肪饮食；羟类固醇脱氢酶；KEGG，京都基因基因组百科全书；KGN，人颗粒样肿瘤细胞；LAC，本地平均连通性；l-Ecd组、二辰汤低剂量组；LH：黄体生成素；Met组、二甲双胍（Glucophage）组；MF，分子功能；MIS: <s:1>勒氏抑制物质；MST：微尺度热泳；PCOS模型组、PCOS组；主成分分析；PCOS，多囊卵巢综合征；PPI，蛋白质-蛋白质相互作用；T,睾酮;中医，传统中医；中医组，中药组；采用超高效液相色谱生物信息学方法预测ECD治疗PCOS的作用靶点。将sd - dawley大鼠分为对照组（Con）和PCOS模型组。后者通过来曲唑（Femara）灌胃（1 mg/kg）结合高脂饲料诱导。多囊卵巢综合征组再进行28天的干预。记录体重，苏木精和伊红染色评价卵巢形态，酶联免疫吸附法测定血清激素。利用免疫荧光、western blotting和RT-qPCR验证了蛋白质组学分析的潜在机制和潜在靶点。在二氢睾酮（DHT）处理的人颗粒KGN细胞中证实了关键ECD成分的药理作用。结果与PCOS组相比，Con和高剂量ECD （H-Ecd）组大鼠体重下降。H-Ecd和二甲双胍（Glucophage）组的睾酮(T)、黄体生成素、抗<s:1>勒氏激素（AMH）和促卵泡激素(p &lt；0.05)，而低剂量Ecd组（l-Ecd）无显著变化。确定了13种血液进入成分和168种PCOS的潜在治疗靶点。京都基因和基因组百科全书和基因本体分析表明类固醇生物合成途径的参与。主成分分析显示Con、H-Ecd和PCOS组之间存在显著差异。微尺度热电泳（MST）验证了甘草素、甘草酸、槲皮素和染料木素对细胞色素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17.6 μM。免疫荧光、western blotting和RT-qPCR分析显示CYP17A1和11β-羟基类固醇脱氢酶1型（HSD11B1）的蛋白表达显著降低(p &lt；与PCOS组相比，Con、Met和H-Ecd组aldo-酮还原酶家族1成员D1 （AKR1D1）和HSD11B2的表达显著升高，而l-Ecd组差异无统计学意义。细胞计数试剂盒-8检测显示，阿比特龙（100 μM）、DHT （100 nM）、甘草酸（50 μM）、甘草素（600 μM）、染料木素（300 μM）和黄芪素（100 μM）显著降低T和AMH水平(p &lt；0.05)。E2水平显著恢复(p &lt；对照组、阿比特龙组（Abi）、中药组（Tcm）与DHT组比较差异无统计学意义（0.05）。Western blot分析显示CYP17A1和HSD11B1蛋白表达显著降低(p &lt；0.01)， AKR1D1和HSD11B2表达显著升高(p &lt；Con、Abi、Tcm组与DHT组比较，差异均为0.01)。结论：我们试图确定ECD治疗PCOS的有效成分，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，从而改善激素水平和卵泡发育，从而增强卵巢功能。这些结果强调了ecd介导的多囊卵巢综合征治疗作用的潜在机制。分子对接验证了甘草素、甘草酸、槲皮素和染料木素与CYP17A1、HSD11B1、AKR1D1和HSD11B2的相互作用。这些相互作用在体外用MST进一步证实，并在细胞水平上得到验证。本研究结果为ECD在PCOS治疗中的应用提供了科学依据。

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Erchen Decoction ameliorates the rat model of polycystic ovary syndrome by regulating the steroid biosynthesis pathway

Background

: 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.

Objective

To identify the key components, underlying mechanisms, and therapeutic targets of Erchen decoction (ECD) for treating PCOS.

Methods

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.

Results

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.

Conclusion

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.

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来源期刊

Phytomedicine 医学-药学

CiteScore

10.30

自引率

5.10%

发文量

670

审稿时长

91 days

期刊介绍： Phytomedicine is a therapy-oriented journal that publishes innovative studies on the efficacy, safety, quality, and mechanisms of action of specified plant extracts, phytopharmaceuticals, and their isolated constituents. This includes clinical, pharmacological, pharmacokinetic, and toxicological studies of herbal medicinal products, preparations, and purified compounds with defined and consistent quality, ensuring reproducible pharmacological activity. Founded in 1994, Phytomedicine aims to focus and stimulate research in this field and establish internationally accepted scientific standards for pharmacological studies, proof of clinical efficacy, and safety of phytomedicines.