PFKFB3-Inhibitor 3PO-Mediated Glycolytic Reprogramming Promotes Inflammatory Dental Pulp Repair: An In Vitro and In Vivo Study.

Abstract

Aim: This study explores the role of PFKFB3 in pulpitis and its potential as a therapeutic target by modulating glycolytic reprogramming in dental pulp stem cells (DPSCs). Pulpitis, a common inflammatory disease, causes long-term damage to dental structures. Recent evidence suggests that metabolic reprogramming can modulate inflammatory responses and promote tissue repair. This study aims to investigate the anti-inflammatory and reparative effects of glycolysis inhibitors on inflamed dental pulp through in vivo and in vitro experiments.

Methodology: In vitro, hDPSCs were stimulated with lipopolysaccharide (LPS) (1 μg/mL, 3 h) to mimic pulpitis. The effects of the PFKFB3 inhibitor 3PO (10 μM) and siRNA targeting PFKFB3 (50 nM) on glycolysis were assessed using Seahorse analysis, while their impacts on inflammation were evaluated via ELISA and qRT-PCR. A co-culture of DPSCs and macrophages was used to study 3PO's effects on inflammation interactions and glycolytic reprogramming of the inflammatory microenvironment. The influences of 3PO on odontogenic differentiation were examined through qRT-PCR, Western blotting, ALP staining and ARS staining. The related signalling pathways were validated through Western blot (WB) experiments. An SD rat model was employed to validate the in vivo efficacy of 3PO@GelMA pulp capping, and HE staining and immunohistochemistry were used to evaluate the degree of pulp inflammation and reparative dentin formation.

Results: In vitro, LPS elevated glycolytic activity and inflammatory factors (IL-6, IL-1β, TNF-α) in DPSCs. 3PO and si-PFKFB3 mitigated these factors and promoted odontogenic differentiation, evidenced by increased DSPP and ALP expression. Metabolic reprogramming mediated by 3PO and si-PFKFB3, as assessed by Seahorse XF analysis, indicated a shift from glycolysis to oxidative phosphorylation. Additionally, 3PO induced metabolic reprogramming of the co-culture system, reduced the levels of pro-inflammatory cytokines, and promoted the polarisation of macrophages towards the M2 phenotype. PFKFB3 inhibition activated the AMPK/SIRT1/PGC-1α/NF-κB and AMPK/mTOR/NF-κB signalling pathways. In vivo, in the rat pulpitis model, 3PO@GelMA hydrogel application resulted in significantly decreased IL-6 and increased DSPP expression compared to the LPS group (p < 0.001), accompanied by reduced inflammation and enhanced reparative dentin formation.

Conclusion: The PFKFB3 inhibitor 3PO reduces inflammation and promotes reparative dentin formation in pulpitis via metabolic reprogramming and specific signalling pathways, offering a new therapeutic strategy.