大黄素治疗糖尿病肾病的网络药理学

Network pharmacology of emodin in the treatment of diabetic kidney disease

摘要

目的 基于网络药理学、分子对接技术及体外细胞实验探究大黄素治疗糖尿病肾病的作用机制。方法 利用公共数据库获取大黄素和糖尿病肾病的交集靶点，建立蛋白-蛋白互作网络，对所得靶点进行基因本体(gene ontology, GO)、京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)功能富集分析，对大黄素与核心治疗靶点进行分子对接及分子动力学模拟。将小鼠肾足细胞(mouse podocyte clone-5, MPC-5)分为对照组、高糖组和大黄素干预组，大黄素干预组分别给予 5、10、20、40 μmol/L 的大黄素干预 24 h，检测细胞活力、铁离子、相关基因表达水平。进行非靶向代谢组学分析，筛选差异代谢物。结果 获得 80 个大黄素治疗糖尿病肾病的潜在作用靶点，其中核因子 E2 相关因子 2(nuclear factor erythroid 2-related factor 2, NRF2)是关键治疗靶点。GO 分析获得 190 条生物过程、80 条细胞组成、100 条分子功能。KEGG 富集分析得到铁死亡、脂质与动脉粥样硬化、白细胞介素-17(interleukin-17, IL-17)等信号通路。大黄素与 NRF2 之间存在强而稳定的相互作用，结合构象稳定。大黄素可显著改善高糖诱导的细胞损伤；10 μmol/L 大黄素干预后，细胞内铁离子水平、肿瘤坏死因子(tumor necrosis factor, TNF)、IL-17 的表达降低，铁死亡核心标志物谷胱甘肽过氧化物酶 4(glutathione peroxidase 4, GPX4)、NRF2 及其下游相关基因的 mRNA 表达升高(P<0.001)。代谢组学分析显示，大黄素干预后共鉴定出 29 种差异代谢物，其中 10 种上调、19 种下调。结论 大黄素通过关键靶点 NRF2 协同调控铁死亡、IL-17 等多条信号通路，发挥抗铁死亡、抗炎及代谢调节作用，从而实现对糖尿病肾病的肾脏保护作用。

Abstract

Objective To explore the mechanism of emodin treating diabetic kidney disease (DKD) based on network pharmacology, molecular docking technology, and in vitro cell experiments. Methods The intersection targets of emodin and DKD were retrieved from public databases. A protein-protein interaction network was established, and gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) functional enrichment analyses were conducted on the obtained targets. Molecular docking and molecular dynamics simulation of emodin and core therapeutic targets were carried out. Mouse podocyte clone-5 (MPC-5) cells were divided into a control group, a high-glucose group, and emodin intervention groups. The intervention groups were treated with 5, 10, 20, and 40 μmol/L emodin for 24 hours respectively. Untargeted metabolomics analysis was performed to screen differential metabolites. Results Eighty potential therapeutic targets of emodin for DKD were obtained, with nuclear factor erythroid 2-related factor 2 (NRF2) being the key therapeutic target. GO analysis identified 190 biological processes, 80 cellular components, and 100 molecular functions. KEGG enrichment analysis revealed signaling pathways, including ferroptosis, lipid and atherosclerosis, and interleukin-17 (IL-17). There was a strong interaction between emodin and NRF2, and the binding conformation was stable. Emodin could significantly alleviate cell damage induced by high glucose. After intervention with 10 μmol/L emodin, the iron levels in cells, the expression of tumor necrosis factor (TNF) and IL-17 were significantly reduced, while the expression of glutathione peroxidase 4 (GPX4), NRF2, and its downstream related genes was significantly increased (P<0.001). Metabonomics analysis showed that a total of 29 differential metabolites were identified after emodin intervention, with 10 up-regulated and 19 down-regulated. Conclusion Emodin exerts anti-ferroptosis, anti-inflammation and metabolic regulation effects by mediating multiple signaling pathways such as ferroptosis and IL-17 through the key target NRF2, thereby achieve a renoprotective effect on DKD.

关键词

大黄素 / 核因子 E2 相关因子 2 / 铁死亡信号通路 / 糖尿病肾病 / 网络药理学

Key words

Emodin / Nuclear factor erythroid 2-related factor 2 / Ferroptosis signaling pathway / Diabetic kidney disease / Network pharmacology

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基金资助

济宁市重点研发计划项目(2024YXNS245)