Mechanism by which luteolin regulates macrophage polarization in the treatment of knee osteoarthritis

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 screened by network pharmacology, a protein-protein interaction network was constructed, and GO, KEGG enrichment analysis and molecular docking were performed. (2) After knocking out NF-κB p65, RAW264.7 cells were induced to polarize to M1 and M2 types, and flow cytometry was used to detect cell polarization tendency. Different concentrations of luteolin were used to intervene in RAW264.7 cells, and the appropriate concentration of luteolin was screened by CCK-8 method for subsequent experiments. RAW264.7 cells were induced to polarize to M1 and M2 types, and luteolin was added for intervention, and flow cytometry was used to detect cell polarization tendency. RAW264.7 cells were induced to polarize to M1 type, and luteolin was added for intervention. ELISA was used to detect the levels of tumor necrosis factor α, interleukin-6, and interleukin-10 in the cell supernatant, immunofluorescence staining was used to detect the nuclear translocation of NF-κB p65/p50, and western blot was used to detect the expression and phosphorylation of NF-κB p65 and NF-κB inhibitor protein α. RESULTS AND CONCLUSION: (1) A total of 137 intersection genes were screened, of which 135 target genes were involved in the construction of the protein-protein interaction network. GO and KEGG analysis showed that the NF-κB complex was enriched in the protein-protein interaction network. Molecular docking showed that luteolin docked well with NF-κB inhibitor protein α and NF-κB p50/p65 proteins. (2) After NF-κB p65 knockout, the polarization tendency of RAW264.7 cells to M1 type was inhibited, and spontaneous M2 polarization tendency appeared. Luteolin intervention could inhibit the polarization of RAW264.7 cells to M1 type and promote the polarization to M2 type. During the induction of RAW264.7 cells to M1 polarization, luteolin intervention could inhibit the release of pro-inflammatory factors, promote the release of anti-inflammatory factors, inhibit the nuclear translocation of NF-κB p65/p50, promote the expression of NF-κB inhibitor protein α and inhibit its phosphorylation, and inhibit the expression and phosphorylation of NF-κB p65. The results indicate that luteolin can regulate macrophage polarization through the NF-κB signaling pathway and has a potential therapeutic effect on knee osteoarthritis.

1. Introduction

Knee osteoarthritis is a common degenerative disease of the knee joint. In recent years, due to the acceleration of population aging, the number of patients with knee osteoarthritis has been increasing globally. The causes of knee osteoarthritis include internal factors such as aging and obesity, as well as external factors such as trauma and excessive exercise. The pathogenesis of knee osteoarthritis is a complex pathological process, with progressive degeneration of articular cartilage as the core, accompanied by multi-tissue synergistic lesions. Under abnormal mechanical load and stimulation by inflammatory factors such as interleukin-1 and tumor necrosis factor-α, the synthesis and degradation of chondrocytes are imbalanced, and the activity of matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs is enhanced, leading to the rupture of type II collagen network, loss of proteoglycan, and the appearance of fissures and erosion on the cartilage surface. Subchondral bone remodeling occurs due to abnormal load transmission, manifesting as osteosclerosis, cystic changes, and marginal osteophyte formation, further aggravating cartilage damage. Chronic synovitis releases inflammatory mediators, which aggravate cartilage degeneration through circulation or direct diffusion, while the concentration of hyaluronic acid in joint fluid decreases, weakening lubrication and protection functions. Therefore, studying the pathogenesis of knee osteoarthritis is of great significance for its prevention and treatment.

Macrophages are widely distributed in the body and are important components of the immune system, playing key roles in inflammatory responses, metabolic homeostasis, and various disease processes. When stimulated by inflammatory factors such as lipopolysaccharide or interferon-γ, macrophages transform into M1 type, secreting a large number of pro-inflammatory cytokines and aggravating the inflammatory response. When stimulated by interleukin-4 or interleukin-13, macrophages transform into M2 type, secreting anti-inflammatory and repair factors, reducing inflammation and promoting tissue repair. The polarization state of macrophages plays an important regulatory role in the occurrence and development of knee osteoarthritis: inhibiting M1 polarization of macrophages in the synovial tissue of the knee can reduce the release of pro-inflammatory factors such as interleukin-6 and tumor necrosis factor-α, thereby reducing the inflammatory response of the synovial tissue and avoiding the progression of knee osteoarthritis.