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

Abstract

Fracture is a common type of traumas and alternative therapies to boost bone fracture healing is necessary. The aim of this study is to elucidate the role of sulfasalazine in bone fracture healing by using MC3T3-E1 cells in vitro and 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, histological staining, immunohistochemistry, immunofluorescence and Surface plasmon resonance analysis were performed in this study. Sulfasalazine failed to elicit ferroptosis in osteoblasts within acceptable dose manner while 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. Murine study showed sulfasalazine promotes fracture healing as evidenced by increased bone remodeling both histologically and radiologically. Overall, sulfasalazine accelerates osteogenic differentiation and promotes bone healing by direct binding to and thus inhibiting TNFα, which subsequently suppresses NF-κB signaling. Therefore, sulfasalazine shows a promising outcome for the treatment of bone fracture.