雌马酚对DN的保护作用及潜在靶点

doi:10.6040/j.issn.1671-7554.0.2024.0556

摘要/Abstract

摘要： 目的通过网络药理学方法探讨雌马酚(equol, Eq)对糖尿病肾病(diabetic nephropathy, DN)的潜在作用靶点,对相关保护作用靶点进行实验验证。方法利用有机小分子生物活性数据库(PubChem)、小分子药物靶点预测在线平台(SwissTargetPrediction)和药效预测靶点数据库(PharmMapper)筛选出Eq的潜在靶点,随后与DisGeNET基因疾病关联数据库、人类基因组数据库(GeneCards)和在线人类孟德尔遗传病数据库(OMIM)共同选取的糖尿病肾病靶点取交集,然后通过STRING平台进行蛋白相互作用(protein-protein interaction, PPI)研究、通过对关键靶点进行基因本体(Gene Ontology, GO)分析,以及与京都基因和基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)通路富集研究,利用Cytoscape3.8.0软件,构建出“药物-靶点-疾病”网络图,并通过分子对接方法,验证了潜在的保护作用靶点。在体外培养MPC5细胞并分别用不同浓度的Eq处理48 h。通过CCK-8检测各组细胞存活率,以确定雌马酚干预的最佳浓度梯度,最终实验分组设置为:对照(Control)组、模型(Model)组(30 mmol/L高糖培养基)、低剂量Eq组(模型+1×10^-8mol/L Eq)、中剂量Eq组(模型组+1×10^-7mol/L Eq)、高剂量Eq组(模型+1×10^-6mol/L Eq);通过流式细胞仪来分析各组的凋亡状况;Western blotting检测各组细胞中EGFR、P-EGFR、Bcl-2、Bax和Cleaved Caspase-3蛋白表达水平。结果通过对Eq与DN的交集靶点进行分析,共发现128个目标,其中核心靶点包括PIK3CB、PIK3CA、AKT2、MAPK1、HRAS、RAF1、MAP2K1和EGFR等。流式实验结果表明,Eq干预后细胞存活率相较于模型组明显提高、凋亡率明显下降(P<0.05),Westem blotting实验显示,雌马酚干预后抗凋亡蛋白Bcl-2表达水平较模型组显著升高(P<0.05),而P-EGFR/EGFR、Bax和Cleaved Caspase-3蛋白表达水平显著降低(P<0.05)。结论 Eq对DN发生发展保护作用具有多个靶点和通路调控途径,特别是Eq干预可以缓解肾脏足细胞凋亡现象,其机制可能与EGFR通路有关。

关键词: 雌马酚, 糖尿病肾病, 网络药理学, 分子对接, 凋亡

Abstract: Objective To study the potential role of equol in(diabetic nephropathy, DN)through network pharmacology methods, and validate the target for the protective effect of equol on DN. Methods The potential targets of Eq were identified through screening in databases such as PubChem, SwissTargetPrediction, and PharmMapper, intersecting with DN targets identified in DisGeNET, GeneCards, and OMIM. Subsequently, protein-protein interaction(PPI)analysis was conducted using the STRING platform. Enrichment analyses of Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were performed for key targets, and the “component target disease” network diagram was constructed by Cytoscape 3.8.0 software. Molecular docking was used to validate possible targets. Cultivate MPC5 cells in vitro and treat them with varying concentrations of Eq for 48 hours.The cell viability of each group was measured using the CCK-8 assay to determine the optimal concentration gradient for equol intervention. The final experimental group settings were as follows: Control group(GLU, 5.5 mmol/L), Model group(GLU, 30 mmol/L), LEq group(model+1×10^-8mol/L Eq), MEq group(model +1×10^-7mol/L Eq), and HEq group(model+1×10^-6mol/L Eq). Apoptosis in each group was analyzed using flow cytometry. Western bloting was used to detect the protein expression levels of EGFR, P-EGFR, Bcl-2, Bax and Cleaved Caspase-3 in each group. Results A total of 128 intersection targets of Eq and DN were obtained. The core targets were PIK3CB、PIK3CA、AKT2、MAPK1、HRAS、RAF1、MAP2K1 and EGFR. The findings from the flow cytometry experiments indicated that, in comparison to the model group, the cell survival rate and apoptosis rate were notably elevated following Eq intervention. Western bloting experiment indicated that the expression level of anti-apoptotic protein Bcl-2 was significantly higher than that of the model group after equol intervention(P<0.05). The protein expression levels of P-EGFR/EGFR, Bax and Cleaved Caspase-3 were significantly reduced(P<0.05). Conclusion Equol exhibits multi-target and pathway regulatory effects against DN. Intervention with equol can alleviate the phenomenon of apoptosis in renal podocytes, and its mechanism may be related to the EGFR signaling pathway.

Key words: Equol, Diabetic nephropathy, Network pharmacology, Molecular docking, Apoptosis

中图分类号:

R459.3

杜学识,倪向敏,梁馨予,白倩,朱文艺,王建. 雌马酚对DN的保护作用及潜在靶点[J]. 山东大学学报 (医学版), 2024, 62(8): 49-58.

DU Xueshi, NI Xiangmin, LIANG Xinyu, BAI Qian, ZHU Wenyi, WANG Jian. The protect effect of equol and its potential targets in the context of diabetic nephropathy[J]. Journal of Shandong University (Health Sciences), 2024, 62(8): 49-58.

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