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

Objective:To explore the mechanism of asiatic acid(AA)against ferroptosis based on network phar-macology 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 Swis-sTargetPrediction, 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 ob-tained 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 confo-cal microscopy, and the influences of AA in combina-tion with Akt activator SC79 on the expressions of glutathione peroxidase 4 (GPX4) and p-Akt were de-tected 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 re-sults showed that AA could stably bind to the core targets.In LPS-induced inflammatory macrophages, AA signif-icantly 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 inflammato-ry macrophages, and its mechanism may be related to the negative regulation of Akt activity.