To explore the potential anti-inflammatory targets of Hedyotis diffusa based on network pharmacology and experiment verification.Methods:The active components and the corresponding targets of the herb were searched against the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP).The common targets shared by the active components and inflammation were identified and the protein-protein interaction(PPI) network between targets was constructed.Metascape was employed to perform gene ontology(GO) annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment for the key targets.AutoDock was employed to simulate the docking between key components and targets.Pharmacodynamics observation was carried out with mouse models of ear edema and writhing.Enzyme-linked immunosorbent assay(ELISA) was employed to determine the expression of some key targets predicted based on network pharmacology.Results:A total of 6 active components were screened out,which corresponded to 173 targets,and 99 targets shared by the active components and inflammation were identified.The potential therapeutic targets included AKT serine/threonine kinase 1(AKT1),interleukin-6(IL-6),tumor necrosis factor(TNF),vascular endothelial growth factor A(VEGFA),and prostaglandin-endoperoxide synthase 2(PTGS2),which mainly participated in the TNF signaling pathway to play an anti-inflammatory role.The molecular docking results showed that β-sitosterol,poriferasterol,2-methoxy-3-methyl-9,10-anthraquinone,and stigmasterol had strong binding ability with the targets.The animal experiments verified that H.diffusa alleviated the ear edema and the number of writhing and lowered the levels of TNF-α and IL-6 in the serum of mice.Conclusion:H.diffusa mainly exerts the anti-inflammatory effect by inhibiting the expression of TNF-α,IL-6 and other cytokines.The results of network pharmacology indicate that β-sitosterol,poriferasterol,2-methoxy-3-methyl-9,10-anthraquinone,and stigmasterol in H.diffusa may regulate the TNF signaling pathway via AKT1,IL-6,TNF,VEGFA and other key targets to play the anti-inflammatory role.