Additionally, bone-destructive factors like receptor activator of nuclear factor kappa-B ligand (RANKL) and matrix metalloproteinase-9 (MMP-9) were significantly downregulated in treated rats.<h4>Conclusion</h4>The conditioned medium derived from estradiol-treated macrophages, enriched with anti-inflammatory and regulatory components, presents a promising cell-free therapeutic strategy for immunotherapy in RA. Here, MMP9 is linked to rheumatoid arthritis.