MFG-E8 prevents M1 macrophage polarization to alleviate osteoarthritis by inhibiting NLRP3 inflammasome.

Macrophage polarization represents a potential therapeutic target for ameliorating osteoarthritis (OA). Milk fat globule EGF and factor V/VIII domain containing (MFG-E8) can modulate macrophage polarization; however, its underlying mechanism in OA remains elusive. This study aims to investigate MFG-E8's effects on macrophage polarization in OA. Nine healthy volunteers and twenty-nine patients with knee OA were recruited to collect knee synovial fluid for detecting interleukin (IL)-1β, tumor necrosis factor (TNF)-α, transforming growth factor-β1 (TGF-β1), and MFG-E8. Lipopolysaccharide (LPS) was used to induce M1 polarization in RAW264.7 cells, with interventions of recombinant MFG-E8 (500 ng/mL) and the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) agonist nigericin (10 µmol/L). Enzyme-linked immunosorbent assay, flow cytometry, immunofluorescence, quantitative real-time polymerase chain reaction, and western blotting were used to analyze M1 macrophage biomarkers and the NLRP3 signaling pathway. IL-1β and TNF-α levels were elevated in OA patients, and positively correlated with Kellgren Lawrance grading, whereas TGF-β1 and MFG-E8 showed the opposite trend. M1/M2 macrophages were increased in OA patients and inversely correlated with MFG-E8 levels. In LPS-stimulated macrophages, mRNA and protein levels of IL-6, IL-1β, TNF-α, NLRP3, and IL-18 were upregulated, along with increased expressions of inducible Nitric Oxide Synthase, cleaved-caspase 1, and N-terminal domain of Gasdermin-D; these were reduced by MFG-E8 addition. Nigericin reversed the effect of rmMFG-E8 on LPS-stimulated macrophages. This study proves that MFG-E8 inhibits NLRP3 to suppress M1 macrophage polarization, laying a foundation for the application of MFG-E8 in OA treatment. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-026-00936-6.