Sulfasalazine disrupts the interaction between TNFα and TNFR1 thus inhibiting NF-kB signaling activation to promote bone fracture healing

Fracture is one of the most common types of traumas. It is of great necessity to explore alternative therapies to boost bone fracture healing process. The aim of this study is to elucidate the role of sulfasalazine in bone fracture healing process. The role that sulfasalazine played in osteogenesis was investigated using MC3T3-E1 in vitro and in murine femoral fracture model in vivo. Western blotting, flow cytometry, RNA sequencing, Calcein AM/PI staining, Alizarin Red S staining, ALP activity assay, transmission electron microscope (TEM), H&E staining, immunohistochemistry (IHC), immunofluorescence (IF) and Surface plasmon resonance (SPR) analysis were performed in this study. Sulfasalazine failed to elicit ferroptosis and could antagonize apoptosis in osteoblasts within acceptable dose manner. Moreover, sulfasalazine promoted osteogenic differentiation. Furthermore, sulfasalazine was identified to inhibit inflammation by declination of inflammatory biomarkers. Besides, TNFα was verified as a potential downstream target for sulfasalazine and the adverse effect of TNFα on osteogenic differentiation could be largely salvaged by sulfasalazine due to direct binding between these two molecules. RNA-seq further implied decreased transcription of genes related to NF-κB pathway in sulfasalazine-treated osteoblasts. This was further verified by dampened p65 nuclear translocation. In vivo study showed sulfasalazine could promote fracture healing as evidenced by increased callus formation and better bone remodeling both histologically and radiologically. In conclusion, sulfasalazine accelerates osteogenic differentiation in vitro and promotes bone healing in vivo, instead of inducing cell death or vitiating fracture repair process. Sulfasalazine achieves this via direct binding to and thus inhibiting TNFα, which subsequently suppresses NF-κB p65 nuclear translocation. Therefore, sulfasalazine shows a promising outcome for the treatment of bone fracture.