芒柄花黄素通过雌激素受体抑制前列腺增生

doi:10.5246/jcps.2024.03.017

摘要/Abstract

摘要：

本研究通过网络药理学的方法预测核心靶点, 再进行分子对接和动物实验来探讨芒柄花黄素的抗良性前列腺增生(BPH)作用。从Swiss ADME数据库筛选有效活性成分并进行药物靶点预测。收集TTD、GeneCards和DrugBank数据库中收录的良性前列腺增生的疾病靶点。将药物靶点与疾病靶点取交集靶点, 使用交集靶点构建PPI网络并进行GO和KEGG富集分析, 利用AutodockVina软件进行分子对接芒柄花黄素的雌激素活性。建立小鼠良性前列腺增生模型, 分为: 对照组、增生组、2.5 mg/kg、5 mg/kg、10 mg/kg、5 mg/kg + fulvestrant (20 mg/kg/week)、10 mg/kg + fulvestrant (20 mg/kg/week)组。HE染色和Masson染色观察病理变化。QPCR检测雌激素受体和细胞周期基因表达量。芒柄花黄素与良性前列腺增生存在35个潜在的蛋白靶点, 核心靶点有ER-α、EGFR、ER-β、CYP19A1、AChE和PPARA。GO和KEGG富集分析表明, 芒柄花黄素的作用靶点主要涉及细胞对雌二醇刺激的反应、雌激素生物合成、类固醇激素受体活性和类固醇结合等过程相关过程, 以及类固醇激素的生物合成和雌激素信号通路。分子对接显示, 与ER-α相比, 芒柄花黄素与ER-β有更好的亲和力。前列腺增生模型小鼠用芒柄花黄素处理后病理学切片结果显示, 芒柄花黄素能明显地抑制小鼠良性前列腺增生, 而雌激素受体抑制剂(氟维司群)显著抑制芒柄花黄素的治疗效果。QPCR结果表明, 芒柄花黄素可以降低ER-β的表达, 并提高前列腺组织的ER-α。同时, 芒柄花黄素处理后, 与细胞周期相关的CDK1、cyclin A2、CDK2和 cyclin B1的表达水平出现下降, 而雌激素受体抑制剂处理后, 这一现象被抑制。综上所述, 网络药理学分析和动物实验结果发现芒柄花黄素通过雌激素受体阻滞细胞周期进程抑制前列腺增生发展。

关键词: 芒柄花黄素, 良性前列腺增生, 网络药理学, 分子对接, 雌激素受体

Abstract:

The primary objective of this study was to forecast the core targets of formononetin through network pharmacology and to assess its potential anti-benign prostatic hyperplasia (BPH) effects using molecular docking and animal experimentation. Active compounds were filtered from the Swiss ADME database, while drug targets were extrapolated from TTD, GeneCards, and DrugBank databases. Targets overlapping between drug and disease were pinpointed, and PPI networks were subsequently developed. Both GO and KEGG enrichment analyses were executed. The AutodockVina software facilitated molecular docking to examine formononetin’s estrogen activity. Mice diagnosed with BPH were grouped into control, TP, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, 5 mg/kg + fulvestrant (20 mg/kg/week), and 10 mg/kg + fulvestrant (20 mg/kg/week) groups. Pathological deviations were discerned through H&E and Masson staining. Estrogen receptor and cell cycle gene expressions were quantified by qPCR. Our findings pinpointed 35 potential protein targets bridging formononetin and BPH, with ER-α, EGFR, ER-β, CYP19A1, AChE, and PPARA being the focal targets. The GO and KEGG enrichment analyses indicated that formononetin's targets predominantly revolved around cellular responses to estradiol stimulation, estrogen biosynthesis, steroid hormone receptor activity, steroid binding, and other related processes. These extended to pathways like steroid hormone biosynthesis and estrogen signaling. Molecular docking deduced a stronger affinity of formononetin for ER-β than for ER-α. Examination of pathological sections in formononetin-treated BPH mice demonstrated its significant inhibitory effect on BPH. Concurrently, the estrogen receptor inhibitor, fulvestrant, markedly dampened formononetin’s therapeutic efficacy. qPCR assessments revealed that formononetin effectively curtailed ER-β expression within hyperplastic prostate tissues. Moreover, post-formononetin treatment led to a decline in expression levels of cell cycle-related genes, such as CDK1, cyclin A2, CDK2, and cyclin B1. However, this trend was notably stunted with the introduction of an estrogen receptor inhibitor. In summation, through the combined insights of network pharmacology and animal testing, we ascertained that formononetin mitigated cell cycle progression via the estrogen receptor when addressing prostatic hyperplasia.

Key words: Formononetin, Benign prostatic hyperplasia, Network pharmacology, Molecular docking, Estrogen receptor

Supporting:

王晓航, 王乐, 肖銮娟, 芦春斌. 芒柄花黄素通过雌激素受体抑制前列腺增生[J]. 中国药学(英文版), 2024, 33(3): 216-229.

Xiaohang Wang, Le Wang, Luanjuan Xiao, Chunbin Lu. Formononetin inhibits benign prostatic hyperplasia through estrogen receptors[J]. Journal of Chinese Pharmaceutical Sciences, 2024, 33(3): 216-229.

图/表 13

Table 1. Primer sequences.

Figure 1. Venn diagram of formononetin and the intersection target of prostatic hyperplasia.

Figure 2. Gene target network of formononetin active ingredient.

Figure 3. PPI network diagram.

Figure 4. GO enrichment analysis.

Figure 5. KEGG pathway enrichment analysis.

Figure 6. Formononetin or estradiol docking with the molecules of ER-β or ER-α. (A) The structure of estradiol; (B) The structure of formononetin; (C) Molecular docking of formononetin with ER-β; (D) Molecular docking of formononetin with ER-α; (E) Molecular docking of estradiol with ER-β; (F) Molecular docking of estradiol with ER-α.

Table 2. Amino residues and binding energy of molecular docking.

Figure 7. Prostatic index. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the TP group; %P < 0.05, %%P < 0.01; nsP > 0.05.

Figure 8. Prostate H&E staining.

Figure 9. Masson staining of the prostate.

Figure 10. The mRNA expression of ER-α and ER-β. (A) mRNA expression of ER-α; (B) mRNA expression of ER-β. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group; #P < 0.05, ##P < 0.01, and ##P < 0.001 compared to the TP group; nsP > 0.05 compared to the TP group.

Figure 11. The mRNA expression of CDK2, CDK1, cyclin B1 and cyclin A2. (A) mRNA expression of CDK2; (B) mRNA expression of CDK1; (C) mRNA expression of cyclin B1; (D) mRNA expression of cyclin A2. *P < 0.05, **P < 0.01, and ***P < 0.001 compared to the control group; #P < 0.05, ##P < 0.01, and ###P < 0.001 compared to the TP group; &P < 0.05, &&P < 0.01; nsP > 0.05 compared to the TP group.

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