DS96432529 enhances osteogenic differentiation and mitigates inflammatory damage in periodontal ligament stem cells involving mitophagy-related processes

Abstract Aim Periodontitis can impair the osteogenic function of periodontal ligament stem cells (PDLSCs), thereby compromising their capacity for periodontal tissue regeneration. In this study, we explored the impact of a synthetic small molecule, DS96432529 (DS), on the osteogenic differentiation potential of PDLSCs and its underlying mechanism. Methods The viability of DS was assessed by cell proliferation assays and apoptosis analysis. Osteogenic potential was evaluated through alkaline phosphatase (ALP) activity staining and Alizarin Red S (ARS) staining for mineralized nodule formation. Inflammatory injury was induced using recombinant tumor necrosis factor-alpha (TNF-α). RNA sequencing analyzed signaling pathways involved in DS-enhanced osteogenic differentiation. Western blotting quantified key pathway protein expression. Specific small molecule inhibitors and agonists modulated relevant signaling pathways. Therapeutic efficacy was evaluated in a ligature-induced rat periodontitis model. Results DS inhibited cell proliferation at lower concentrations but did not induce significant apoptosis at concentrations up to 250 nM. Across tested concentrations, DS significantly enhanced ALP activity and accelerated mineralized nodule formation in PDLSCs. DS upregulated mitophagy-related protein expression under both inflammatory and non-inflammatory conditions. Additionally, DS restored TNF-α-inhibited ALP activity and attenuated TNF-α-induced activation of the RIG-I-like receptor (RLR) signaling pathway. The RIG-I activator Poly(I: C) counteracted DS-mediated repair of inflammatory injury during osteogenesis. Mitophagy inhibition diminished DS’s beneficial effects on osteogenic differentiation under inflammation and reduced its suppression of RIG-I expression. DS alleviated ligation-induced alveolar bone loss in rats with periodontitis. Conclusions DS enhances the osteogenic potential of PDLSCs in association with the activation of mitophagy-related processes. It mitigates inflammation-impaired osteogenesis, potentially via modulation of the RIG-I-mediated RLR signaling pathway, in association with increased mitophagy-related activity. DS represents a potent therapeutic small molecule for ameliorating periodontitis-induced bone loss.