N-acetylcysteine restores osteogenesis in human periodontal ligament stem cells by attenuating HIF-1α-driven oxidative stress and glycolytic reprogramming.

BACKGROUND/PURPOSE: Periodontitis-associated hypoxia signaling and oxidative stress severely impair the regenerative capacity of human periodontal ligament stem cells (hPDLSCs). This study investigated whether antioxidant intervention rescues hPDLSC osteogenesis under CoCl2-induced HIF-1α stabilization by modulating PI3K/Akt/HIF-1α/HO-1 signaling and glycolytic reprogramming.

MATERIALS AND

METHODS

Primary hPDLSCs isolated from healthy premolars were characterized by flow cytometry and multilineage differentiation. To simulate periodontitis-associated stress, a CoCl2-induced HIF-1α-stabilizing condition was established. Cells were treated with the antioxidant N-acetylcysteine (NAC), the glycolysis inhibitor 2-deoxy-d-glucose (2-DG), or the PI3K inhibitor LY294002. Redox balance (DCFH-DA, MDA, GPX activity), glycolysis-related markers (HK2, GLUT1, lactate), and osteogenic-related markers (RUNX2, ALP, OCN) were quantified via qRT-PCR, Western blot, and biochemical assays, alongside an analysis of the PI3K/ Akt/HIF-1α/HO-1 signaling axis.

RESULTS

CoCl2 exposure triggered profound redox imbalance and significantly impaired hPDLSC osteogenesis. Crucially, NAC effectively reversed these damages and restored osteogenic capacity. Mechanistically, CoCl2-induced HIF-1α stabilization activated PI3K/Akt/HO-1 signaling, a pathological cascade attenuated by either NAC or targeted PI3K inhibition. Furthermore, CoCl2 forced a pronounced glycolytic shift characterized by upregulated HK2, GLUT1, and lactate production, which was directly intercepted by ROS clearance. Functionally, targeted glycolysis inhibition via 2-DG also successfully rescued the osteogenic potential of HIF-1α-stabilized hPDLSCs.

CONCLUSION

HIF-1α stabilization suppresses hPDLSC osteogenesis by inducing both oxidative stress and pathological glycolytic reprogramming. Targeted antioxidant intervention effectively mitigates these impairments, highlighting the ROS/PI3K/Akt/HIF-1α axis as a key link between redox imbalance and metabolic dysfunction, and suggesting a potential therapeutic target for periodontal regeneration.