[IGFBP-4 improves synovitis by reducing M1 polarization of macrophages via the Wnt/β-catenin pathway]

Objective To investigate the mechanism by which insulin-like growth factor binding protein 4 (IGFBP-4) ameliorates synovial inflammation in osteoarthritis (OA) by inhibiting M1-type polarization of synovial macrophages via the Wnt/β-catenin signaling pathway. Methods The OA model was established in male C57BL/6 mice by intra-articular injection of sodium iodoacetate. Adenoviral vectors carrying IGFBP-4 overexpression or knockdown sequences were injected into the knee joint cavity for in vivo intervention. Micro-computed tomography (Micro-CT) was used to observe the subchondral bone changes in the knee joint. Hematoxylin-eosin (HE) staining was used to observe the pathological morphology of knee synovial tissues. ELISA was applied to detect serum levels of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) in mice. Immunofluorescence staining was used to evaluate M1 polarization level of the synovium macrophages. Quantitative real-time PCR and Western blot were used to measure the expression of IGFBP-4, β-catenin, cyclooxygenase 2 (COX2), inducible nitric oxide synthase (iNOS), and CD86 in synovial tissue. In vitro, a RAW264.7 macrophage inflammatory model was induced by lipopolysaccharide (LPS), and cells were treated with recombinant IGFBP-4 protein and the specific Wnt/β-catenin pathway agonist SKL2001. Cell viability was measured by CCK-8 assay. Flow cytometry was used to quantify M1 macrophage polarization. ELISA was used to measure levels of TNF-α and IL-6 in cell culture supernatants, and Western blot was performed to analyze the expression of key proteins involved in the Wnt/β-catenin signaling pathway. Results Micro-CT and pathological staining revealed subchondral bone surface defects and marked synovial hyperplasia and thickening in the OA group. These pathological changes were alleviated in the IGFBP-4 overexpression group and aggravated in the IGFBP-4 knockdown group. Immunofluorescence results demonstrated that the co-localization of F4/80 and iNOS in the synovium was significantly higher in the OA group than that in the Sham group. Compared with the OA group, the co-localization of F4/80 and iNOS in the IGFBP-4 overexpression group decreased, while that increased in the IGFBP-4 knockdown group. Consistently, compared with the Sham group, the OA group showed elevated serum levels of TNF-α and IL-1β, as well as increased expression of β-catenin, COX2, iNOS and CD86 in the synovium. Compared with the OA group, the IGFBP-4 overexpression group showed significant decreases in both serum levels of TNF-α and IL-1β and synovial expressions of β-catenin, COX2, iNOS and CD86, whereas these factors increased significantly in the IGFBP-4 knockdown group. Compared with the Control group, the LPS-treated group cells showed significantly increased expression of β-catenin and COX2, promoted M1 polarization, and elevated levels of TNF-α and IL-6. Compared with both the LPS-treated group and the LPS combined with IGFBP-4 and SKL2001 group, the LPS combined with IGFBP-4 group showed decreased β-catenin and COX2 expressions, reduced M1 polarization, and lower levels of TNF-α and IL-6. Conversely, the LPS combined with SKL2001 group exhibited increased levels of these parameters compared with the LPS combined with IGFBP-4 and SKL2001 group. Conclusion IGFBP-4 can effectively inhibit M1 polarization of synovial macrophages and alleviates synovial inflammation, and its protective mechanism is closely associated with the negative regulation of the Wnt/β-catenin signaling pathway.