基于网络药理学分析小檗碱在免疫微环境中对糖尿病视网膜病变的作用及实验验证

doi:10.6040/j.issn.1673-3770.0.2021.481

摘要/Abstract

摘要： 目的通过网络药理学和动物实验方法研究小檗碱(BBR)治疗糖尿病视网膜病变(DR)的免疫作用机制。方法利用中药数据库(HERB)获得BBR化学成分及其潜在靶点;DisGeNET和GeneCards数据库判断与DR发病相关的靶基因;随后使用String数据库与Cytoscape软件相结合绘制出蛋白相互作用网络,网络拓扑学分析筛选出BBR作用于DR的靶点;然后利用Metascape生物信息学数据库对这些作用靶点进行GO细胞组分、生物学过程、分子功能分析和KEGG通路分析;接着用Cytoscape构建并分析BBR“靶点-通路”网络图;借助Pymol软件对药物配体和靶点进行分子对接的结合能力预测;最后构建糖尿病小鼠模型,用BBR 100 mg/(kg·d)灌胃治疗8周后,取视网膜组织切片染色检测病变程度。流式细胞术检测糖尿病小鼠眼和淋巴结CD4⁺T细胞、IL-17⁺T细胞的比例。结果从中药数据库筛选出67个BBR与DR共有的靶点,其中INS、IL-6、CASP3、TNF、VEGFA等是BBR作用DR的核心靶点。GO分析结果表明,BBR能通过氧化应激和细胞凋亡信号通路在DR治疗中发挥作用。KEGG通路富集分析显示,BBR的抑制免疫炎症的潜在靶点通路主要是IL-17信号通路和Th17分化通路且核心靶点为RELA、MAPK1、IL-6、NFKBIA、TNF。分子对接结果表明BBR对RELA、MAPK1、IL-6有较强结合力。组织病理学检测发现BBR治疗后糖尿病小鼠视网膜病变减轻,且淋巴结和眼组织CD4⁺T、IL-17⁺T细胞数量明显低于治疗组。结论 BBR可通过调控Th17/IL-17信号通路,抑制DR的炎症反应,揭示BBR治疗DR的新的免疫机制,为深入探究BBR治疗DR的药理靶点及机制提供了理论依据。

关键词: 糖尿病视网膜病变, 小檗碱, 免疫微环境, 网络药理学, 分子对接

Abstract: Objective To study the immune mechanism of berberine(BBR)in the treatment of diabetic retinopathy(DR)using network pharmacology and animal model experiments. Methods Chemical constituents and potential BBR targets were obtained from a traditional Chinese medicine database(HERB), target genes related to DR pathogenesis were identified in the DisGeNET and GeneCards databases, and the protein interaction network was drawn using a combination of the String database and Cytoscape software. The BBR target(s)acting on DR were screened out by network topology analysis and the GO cell composition, biological processes, molecular function, and KEGG pathways of these targets were analyzed using the Metascape bioinformatics database. Following this, a BBR “target-path” network diagram was constructed and analyzed with Cytoscape, the molecular docking ability of drug ligands and targets was predicted with Pymol, and, finally, a diabetic mouse model was established, treated with BBR 100 mg/(kg·d)for 8 weeks, and then had retinal tissue sections taken and stained to detect the degree of pathological change. The proportion of CD4⁺ and IL-17⁺ T cells in the eyes and lymph nodes of diabetic mice was determined by flow cytometry. Results 67 common BBR and DR targets were screened from the traditional Chinese medicine database, including INS, IL-6, CASP3, TNF and VEGFA as the core BBR targets which affect DR. The GO analysis suggested that BBR could play a role in the treatment of DR through response to oxidative stress and regulation of apoptotic signaling pathway. KEGG pathway enrichment analysis identified the IL-17 signaling and Th17 differentiation pathways as potential target pathways for BBR suppression of immune inflammation. The core targets identified were RELA, MAPK1, IL-6, NFKBIA, and TNF. Molecular docking showed a strong binding force between BBR and RELA, MAPK1, and IL-6. The tissue sections from diabetic mice revealed alleviated retinal pathology following BBR treatment, and the number of CD4⁺ and IL-17⁺ T cells were significantly lower in the treatment group. Conclusion BBR can inhibit the DR inflammatory response by regulating the Th17/IL-17 signaling pathway. This is a newly identified immune mechanism of BBR, and provides a theoretical basis for further exploration of the pharmacological targets and mechanisms of BBR in the treatment of DR.

Key words: Diabetic retinopathy, Berberine, Immune microenvironment, Network pharmacology, Molecular docking

中图分类号:

R774.1

刘通,林玮,冯萌,杨依,刘婷婷,张敏. 基于网络药理学分析小檗碱在免疫微环境中对糖尿病视网膜病变的作用及实验验证[J]. 山东大学耳鼻喉眼学报, 2023, 37(1): 94-104.

LIU Tong, LIN Wei, FENG Meng, YANG Yi, LIU Tingting, ZHANG Min. Analysis of the effect of berberine on diabetic retinopathy in the immune microenvironment based on network pharmacology and experimental verification[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2023, 37(1): 94-104.

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