Abstract: BACKGROUND: The polarization state of macrophages is closely related to the occurrence and development of knee osteoarthritis. Luteolin can regulate the nuclear transcription factor κB (NF-κB) signaling pathway to affect the polarization process of macrophages, but the specific mechanism remains unclear. 
OBJECTIVE: To investigate the mechanism of luteolin in the treatment of knee osteoarthritis. 
METHODS: (1) Intersectional targets of luteolin, macrophage polarization, and knee osteoarthritis were retrieved via network pharmacology. A protein-protein interaction network was constructed, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, and molecular docking. (2) After knocking out nuclear factor κB-light-chain-enhancer of activated B cells p65 subunit (NF-κB p65), RAW264.7 cells were induced to polarize toward M1 and M2 phenotypes, and polarization trends were detected by flow cytometry. Different concentrations of luteolin were applied to RAW264.7 cells, and the suitable concentration for subsequent experiments was selected using the cell counting kit-8 assay. RAW264.7 cells were induced to polarize toward M1 and M2 phenotypes with simultaneous luteolin intervention, and polarization trends were detected by flow cytometry. RAW264.7 cells were induced toward M1 polarization with simultaneous luteolin intervention. The levels of tumor necrosis factor-α, interleukin-6, and interleukin-10 in the cell supernatant were measured by ELISA. Nuclear translocation of NF-κB p65/p50 was detected by immunofluorescence staining. The expression and phosphorylation of NF-κB p65 and NF-κB inhibitor α (IκBα) were detected by western blot.
RESULTS AND CONCLUSION: (1) A total of 137 intersection genes were screened, among which 135 target genes participated in the construction of the protein-protein interaction network. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses showed that the NF-κB complex was enriched in the protein-protein interaction network. Molecular docking indicated that luteolin bound well to IκBα, NF-κB p50, and NF-κB p65 proteins. (2) After NF-κB p65 knockout, the tendency of RAW264.7 cells to polarize toward the M1 phenotype was inhibited, and spontaneous polarization toward the M2 phenotype occurred. Luteolin intervention inhibited RAW264.7 cell polarization toward the M1 phenotype and promoted polarization toward the M2 phenotype. During the induction of RAW264.7 cells toward M1 polarization, luteolin intervention inhibited the release of pro-inflammatory factors and promoted the release of anti-inflammatory factors, suppressed nuclear translocation of NF-κB p65/p50, promoted IκBα expression while inhibiting its phosphorylation, and inhibited NF-κB p65 expression and its phosphorylation. These findings indicate that luteolin can regulate macrophage polarization through the NF-κB signaling pathway, showing potential therapeutic effects on knee osteoarthritis.

Key words: luteolin, macrophage, nuclear factor-κB signaling pathway, network pharmacology, knee osteoarthritis, macrophage polarization