环黄芪醇改善阿奇霉素诱导药物性肝损伤的作用机制研究  

Mechanisms of cycloastragenol in ameliorating azithromycin-induced drug-induced liver injury

作　　者：张翠锋 钱海溢 何溢宸 王佳音 解心怡 徐启祥 郭文俊[5] ZHANG Cuifeng;QIAN Haiyi;HE Yichen;WANG Jiayin;XIE Xinyi;XU Qixiang;GUO Wenjun(School of Anesthesiology,Wannan Medical College,Wuhu 241000,China;School of Pharmacology,Wannan Medical College;Wuhu Perioperative Monitoring and Prognostic Technology Research and Development Center;Wuhu Anesthesia Organ Protection Basic and Clinical Research Technology Research and Development Center;Yijishan Hospital,Wannan Medical College)

机构地区：[1]皖南医学院麻醉学院,安徽芜湖241000 [2]皖南医学院药学院 [3]芜湖市围术期监测与预后技术研发中心 [4]芜湖市麻醉器官保护基础与临床研究技术研发中心 [5]皖南医学院附属弋矶山医院

出　　处：《沈阳医学院学报》2025年第2期141-148,共8页Journal of Shenyang Medical College

基　　金：安徽省高校自然科学研究项目重点项目(No.2023AH051762,No.2022AH051229);皖南医学院校中青年科研基金项目(No.WK2023ZQNZ08);国家级大学生创新创业训练计划项目(No.202210368052,No.202310368014,No.202410368019);安徽省大学生创新创业项目(No.S202310368095,No.S202310368087)。

摘　　要：目的:通过网络药理学和体外实验验证的方法探讨环黄芪醇改善阿奇霉素诱导药物性肝损伤(DILI)可能的作用靶点及机制。方法:通过数据库检索、预测环黄芪醇和DILI的潜在靶点,提取、筛选共有和关键靶点,并进行GO及KEGG富集分析以及分子对接验证。分离C57BL/6小鼠原代肝细胞,通过细胞活性检测试剂盒(CCK8)和氧自由基检测试剂盒(ROS)探索阿奇霉素诱导小鼠原代肝细胞DILI的最佳浓度和时间,采用RT-qPCR、Western blot检测NF-κB p65、p-NF-κB p65、AMPKα、p-AMPKα等基因和蛋白的表达,评估环黄芪醇(10~50μmol/L)的干预效果。结果:网络药理学筛选出关键基因10个,包括热休克蛋白90AA1 (HSP90AA1)、基质金属蛋白酶2 (MMP2)等。GO富集分析显示,环黄芪醇主要可能通过调节膜电位和化学突触传递等生物学过程,以及影响神经元胞体和远端轴突等细胞组分和相关激酶活性发挥作用。KEGG富集分析显示其主要通过神经信号传递通路、IL-17信号通路等发挥作用。分子对接结果显示,环黄芪醇与HSP90AA1、MMP2以及核因子激活的B细胞的κ-轻链增强p65 (NF-κB p65)、腺苷酸活化蛋白激酶(AMPKα)、核因子红细胞来源2样2 (Nrf2)、血红素加氧酶1 (HO-1)、 NAD (P) H:醌氧化还原酶-1 (NQO1)等靶点良好结合,尤其与Nrf2的结合能≤-5.0 kcal/mol。细胞实验显示,阿奇霉素(50μmol/L,12 h)显著降低肝细胞活力并升高ROS水平(P<0.01)。环黄芪醇可明显改善肝细胞活性和缓解氧自由基的生成,下调NF-κB p65的磷酸化水平,上调AMPKα、Nrf2、HO-1、NQO1的mRNA表达水平以及相关蛋白表达水平(P<0.05)。结论:环黄芪醇可通过抑制NF-κB磷酸化、激活AMPK/Nrf2/HO-1/NQO1通路,改善阿奇霉素诱导的肝细胞氧化应激和炎症反应,其作用机制可能与靶向Nrf2密切相关。然而,DILI的复杂病理机制可能涉及其他未被验证的通路,需进一步在动物模型中验证环黄芪醇的体内Objective:To investigate the targets and mechanisms of cycloastragenol in ameliorating azithromycin-induced drug-induced liver injury(DILI)based on network pharmacology and in vitro experiment validation.Methods:Potential targets of cycloastragenol and DILI were predicted using databases.The common and key targets were screened and subjected to GO and KEGG enrichment analyses,as well as molecular docking validation.Primary hepatocytes from C57BL/6 mice were isolated.The optimal concentration and time for azithromycin-induced DILI in mouse primary hepatocytes were determined using CCK8 and ROS assays.The expression of genes and proteins such as NF-κB p65,p-NF-κB p65,AMPKα,and p-AMPKαwas assessed using RT-qPCR and Western blot to evaluate the intervention effect of cycloastragenol(10-50μmol/L).Results:Network pharmacology analysis identified 10 key genes related to cycloastragenol's improvement of DILI,including heat shock protein 90AA1(HSP90AA1),matrix metalloproteinase 2(MMP2),etc.GO enrichment analysis suggested that cycloastragenol primarily regulates biological processes such as membrane potential and chemical synaptic transmission,and affects cellular components such as neuronal cell bodies and distal axons,and related kinase activities.KEGG enrichment analysis showed that it mainly exerts intervention effects through neuro-signaling pathways and IL-17 signaling pathways.Molecular docking demonstrated strong binding of cycloastragenol to HSP90AA1,MMP2,NF-κB p65,AMPKα,nuclear factor erythroid 2-related factor 2(Nrf2),heme oxygenase 1(HO-1),and NAD(P)H:quinone oxidoreductase 1(NQO1),with a binding energy≤-5.0 kcal/mol for Nrf2.In vitro experiments showed that azithromycin(50μmol/L,12 h)significantly reduced hepatocyte viability and increased ROS levels(P<0.01).Different concentrations of cycloastragenol significantly improved the activity of mouse primary hepatocytes,reduced the generation of intracellular ROS,downregulated the phosphorylation level of NF-κB p65,and upregulated the mRNA and protein l

关 键 词：环黄芪醇 网络药理学 分子对接 药物性肝损伤 

分 类 号：R285.5[医药卫生—中药学]