Targeting dipeptidyl peptidase-۸/۹ to combat inflammation-induced osteoclastogenesis in RAW۲۶۴.۷ macrophages and analysis of anti-osteoclastogenesis potential of chrysin

Objective(s): Osteoclasts drive bone resorption under inflammation, with cytokines promoting osteoclastogenesis. The role of proline enzymes like dipeptidyl peptidase-۸ and ۹ (DPP-۸/۹) in this process remains unclear. This study aimed to explore the DPP-۸/۹ involvement in inflammation-driven osteoclastogenesis using the RAW۲۶۴.۷ macrophage model.Materials and Methods: Receptor activator of nuclear factor-κB ligand (RANKL) and lipopolysaccharide (LPS) induced osteoclastogenesis, raising interleukin-۶ (IL-۶), tumor necrosis factor (TNF-α), and IL-۲۳ levels. Using RAW۲۶۴.۷ cells, DPP-۸/۹ protein and tartrate-resistant acid phosphatase (TRAPc) were assayed. Antibodies for cluster of differentiation (CD۸۶ and CD۲۰۶) were used to analyze macrophage polarization, while molecular docking was used to assess flavonoid binding to DPP-۸/۹. Western blot confirmed DPP-۸/۹ expression in treated macrophages.Results: Administering RANKL and LPS increased IL-۶ and TNF-α levels, significantly promoting osteoclastogenesis in RAW۲۶۴.۷ macrophages. This treatment also elevated the levels of the inflammatory macrophage marker IL-۲۳. Osteoclast formation was confirmed by measuring TRAPc levels in the culture. Analysis of the cell supernatant revealed elevated DPP-۸/۹ levels in the RANKL+LPS group. Inhibition of DPP-۸/۹ with ۱G۲۴۴ decreased inflammatory cytokines and TRAPc levels in the cell culture. Molecular docking analysis of various flavonoids identified chrysin as a potential molecule with sufficient binding energy against DPP-۸/۹, a finding confirmed by blotting assay.Conclusion: This study emphasizes the involvement of DPP-۸/۹ in inflammatory osteoclastogenesis in RAW۲۶۴.۷ macrophages. Inhibition of DPP-۸/۹ reduced osteoclastogenesis markers and inflammatory cytokines levels, indicating decreased osteoclast formation. Additionally, chrysin demonstrated potential as an anti-DPP-۸/۹ agent, highlighting its possible role in future therapeutic strategies targeting inflammation-induced osteoclastogenesis.

کلیدواژه ها:

Chrysin ، Dipeptidyl peptidase ۸ ، Dipeptidyl peptidase ۹ ، Inflammatory - osteoclastogenesis ، M۱ macrophage ، RAW۲۶۴.۷ cells