Kaempferol combats the osteogenic differentiation damage of periodontal ligament stem cells in periodontitis via regulating EphrinB2-mediated PI3K/Akt and P38 pathways

Background: The osteogenic differentiation of periodontal ligament stem cells (PDLSCs) plays a fundamental role in endogenous bone regeneration during periodontitis treatment, yet achieving consistent differentiation under inflammatory conditions remains clinically challenging. Kaempferol, a phytochemical flavonol, has demonstrated osteoprotective efficacy in osteoporosis and bone repair models. However, whether kaempferol exerts pro-osteogenic effects on PDLSCs within the pathologically complex microenvironment of periodontitis, and through what molecular mechanisms, remains unexplored. Purpose: This study aimed to systematically characterize the therapeutic efficacy of kaempferol in restoring osteogenic differentiation of human PDLSCs under inflammatory stress, and promoting bone regeneration in a mice periodontitis model, and elucidate novel molecular targets and downstream mechanisms mediating these regenerative actions. Methods: An in vitro inflammatory microenvironment was established using lipopolysaccharide (LPS)-stimulated human PDLSCs to mimic periodontitis-induced osteogenic impairment. Osteogenic recovery was assessed through alkaline phosphatase (ALP), alizarin red S staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis of osteogenesis-related markers (ALP, RUNX2, OSX, OPN). The bioinformatics, network pharmacology and siRNA transfection were performed to identify EphrinB2 as kaempferol's putative cellular target. Downstream PI3K/Akt and p38 MAPK pathway activation was evaluated through phosphoprotein analysis. In vivo validation employed micro-CT quantification of alveolar bone loss and immunohistochemical profiling of pathways key proteins in a mice periodontitis model. Results: Kaempferol dose-dependently rescued LPS-impaired osteogenic differentiation in human PDLSCs, especially at 10 μM, where kaempferol significantly reversed suppressed ALP activity, mineralized nodule formation, and transcriptional and protein expression of osteogenic markers (ALP, RUNX2, OSX, OPN). Mechanistically, kaempferol upregulated the key target EphrinB2 under inflammatory stress, thereby reactivating the downstream PI3K/Akt and p38 pathways. In periodontitis mice, kaempferol administration (10 mg/kg) significantly promoted the periodontal expression of OPN and EphrinB2, restored the phosphorylation of PI3K, AKT, and P38, attenuating alveolar bone loss by 63.8 % (BV/TV: 72.4 % ± 2.07 vs. 44.2 % ± 3.19 in CON). Conclusion: Kaempferol could rescue PDLSCs’ osteogenic differentiation and mitigates bone loss in periodontitis microenvironments by targeting EphrinB2 to activate PI3K/Akt and P38 pathways. This work underscores kaempferol's potential as a natural therapeutic for reversing pathological bone resorption and promoting periodontal regeneration.