基于网络药理学和分子对接探讨积雪草酸抑制炎症巨噬细胞铁死亡的作用机制及初步验证

Mechanism study and preliminary validation of asiatic acid inhibiting ferroptosis on inflammatory macrophages based on network pharmacology and molecular docking

目的:采用网络药理学和分子对接方法探讨积雪草酸(AA)调控铁死亡的作用机制,并在脂多糖(LPS)诱导的炎症巨噬细胞模型上进行初步验证。方法:检索SwissTargetPrediction、SEA Search Server、HERB等9个数据库获得AA相关靶点,通过FerrDb 2.0、GeneCards 5.14、KEGG、NCBI等数据库获取铁死亡相关靶点,利用Venny 2.1.0在线工具映射获得AA和铁死亡的交集靶点。采用DAVID 6.9数据库对交集靶点进行GO和KEGG富集分析,String 11.5数据库和Cytoscape 3.9.1软件构建蛋白质相互作用(PPI)网络,并采用CytoHubba插件筛选出核心靶点。最后采用Autodock Tool 1.5.7软件将AA与核心靶点进行分子对接,Pymol 4.2软件对分子对接结果可视化。构建LPS诱导的RAW264.7小鼠巨噬细胞炎症模型,激光共聚焦法检测AA对炎症巨噬细胞内脂质活性氧(lipid ROS)荧光强度的影响;细胞免疫荧光法检测AA和Akt激活剂SC79对谷胱甘肽过氧化物酶4(GPX4)、p-Akt表达的影响。结果:网络药理学分析共筛选出203个AA与铁死亡交集靶点。富集分析结果显示,AA可能通过AKT1等核心靶点介导PI3K/Akt等信号通路调控铁死亡;分子对接结果显示,AA与AKT1等核心靶点均能稳定结合。在LPS诱导的巨噬细胞,AA明显降低巨噬细胞lipid ROS荧光强度,增强GPX4而减弱p-Akt荧光表达(与LPS组比较,P<0.01);SC79使AA增强GPX4表达的作用明显减弱(与单用AA比较,P<0.01)。结论:AA抑制炎症巨噬细胞铁死亡,机制可能与负调控Akt活性有关。

Objective:To explore the mechanism of asiatic acid(AA)against ferroptosis based on network pharmacology and molecular docking,and to preliminarily validate it in the inflammatory macrophage model induced by lipopolysaccharide(LPS).Methods:AA-related targets were obtained by searching 9 databases such as SwissTargetPrediction,SEA Search Server and HERB databases;the ferroptosis-related targets were obtained through FerrDb 2.0,GeneCards 5.14,KEGG and NCBI databases.The intersection targets of AA and ferroptosis were obtained by online tool Venny 2.1.0.DAVID 6.9 database was used to perform GO and KEGG enrichment analysis of the intersection targets,String 11.5 database and Cytoscape 3.9.1 software were used to construct the proteinprotein interaction(PPI)network,and the CytoHubba plug-in was used to screen out the core targets.Finally,Autodock Tool 1.5.7 software was used to perform molecular docking between AA and the core targets,and the docking results were visualized by Pymol 4.2 software.Once the LPS-induced RAW264.7 mouse inflammatory macrophage model was established,the influence of AA on the fluorescence intensity of lipid reactive oxygen species(lipid ROS)was monitored under laser confocal microscopy,and the influences of AA in combination with Akt activator SC79 on the expressions of glutathione peroxidase 4(GPX4)and p-Akt were detected by cell immunofluorescence assay.Results:A total of 203 intersection targets of AA and ferroptosis were screened out by network pharmacology analysis.The enrichment analysis results showed that AA might regulate ferroptosis via the core targets including AKT1 mediating PI3K/Akt signaling pathway.Molecular docking results showed that AA could stably bind to the core targets.In LPS-induced inflammatory macrophages,AA significantly reduced the fluorescence intensity of lipid ROS,increased the expression of GPX4 but decreased that of p-Akt(compared with LPS group,P<0.01);the up-regulation effect of AA on GPX4 expression was significantly attenuated by SC79(compared with AA group,P<0.01).Conclusion:AA can inhibit ferroptosis in inflammatory macrophages,and its mechanism may be related to the negative regulation of Akt activity.