ALKBH5 promotes inflammation and inhibits osteogenic differentiation by activating the TLR9/MAPK pathway in human periodontal ligament stem cells.

BACKGROUND: Periodontitis (PD), driven by dysregulated inflammation and bone loss, remains a therapeutic challenge. AlkB homolog 5, RNA demethylase (ALKBH5), a regulator of N6-methyladenosine (m6A) modification, influences immune responses and osteogenesis, yet its role in PD is unexplored. METHODS: An in vitro PD model was established by treating human periodontal ligament stem cells (hPDLSCs) using lipopolysaccharide (LPS). Western blotting assay was performed to detect the protein expression of ALKBH5, toll-like receptor 9 (TLR9), inflammatory cytokines (IL-6, IL-1β, TNF-α), and osteogenic markers (RUNX2, OPN, OCN). The mRNA levels of ALKBH5, TLR9, IL-6, IL-1β and TNF-α were detected by quantitative real-time polymerase chain reaction. Osteogenic capacity was assessed by alizarin red S staining and alkaline phosphatase (ALP) activity. Methylated RNA immunoprecipitation (MeRIP), RNA immunoprecipitation and actinomycin D assays were used to explore ALKBH5-TLR9 interactions and mRNA stability, respectively. RESULTS: Expression levels of both ALKBH5 and TLR9 were elevated in the gingival tissues of patients with PD as well as in hPDLSCs stimulated by LPS. LPS elevated IL-6, IL-1β, and TNF-α levels (mRNA/protein), but ALKBH5 knockdown reversed these effects. LPS suppressed mineralization, ALP activity, and the protein expression of RUNX2, OPN, and OCN, while ALKBH5 depletion restored these outcomes. Mechanistically, ALKBH5 silencing reduced TLR9 mRNA stability via m6A modification. Moreover, TLR9 overexpression relieved ALKBH5 knockdown-induced anti-inflammatory and pro-osteogenic effects in LPS-treated cells. Further, ALKBH5 silencing inactivated the MAPK pathway by regulating TLR9 in LPS-treated cells. CONCLUSION: ALKBH5 stabilized TLR9 to activate the MAPK pathway, further amplifying inflammation and inhibiting osteogenic differentiation in LPS-induced hPDLSCs. Targeting the ALKBH5-TLR9 axis might alleviate periodontal inflammation and enhance regeneration, offering a novel therapeutic avenue for PD management. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-025-00889-2.