Glutathione Primes Mitochondrial Autophagy to Guide Pulp-Dentine Complex Repair

Pulp-dentine complex repair following injury is fundamental to preserving tooth vitality and function. Despite advances in understanding reparative dentinogenesis, the molecular mechanisms orchestrating odontogenic differentiation of human dental pulp stem cells (hDPSCs) remain fragmented, lacking systematic integration across transcriptomic, proteomic and metabolomic levels. This study employs integrated multi-omics approaches to identify key regulatory pathways during odontogenic differentiation and elucidate the underlying mechanisms controlling pulp-dentine complex repair.

Single-cell RNA sequencing was performed on human carious and healthy pulp tissues. Temporal proteomic and metabolomic profiling was conducted during hDPSCs' odontogenic differentiation. Based on multi-omics integration revealing glutathione metabolism as a central regulatory node, hDPSCs were treated with L-buthionine-sulfoximine (BSO) or exogenous glutathione (GSH). Intracellular reactive oxygen species (ROS), GSH levels, mitochondrial membrane potential and mitophagy markers (PINK1/Parkin) were quantified. siRNA-mediated PINK1 knockdown was performed to assess the involvement of PINK1 in GSH-regulated odontogenic differentiation. A mouse molar injury model evaluated GSH effects on tertiary dentine formation in vivo.

Single-cell transcriptomic analysis revealed enrichment of oxidative stress response pathways in mesenchymal stem cells from injured pulp. Multi-omics integration identified glutathione metabolism as a central regulatory pathway, with glutamate-cysteine ligase catalytic subunit (GCLC) progressively upregulated during differentiation. BSO treatment depleted intracellular GSH, elevated ROS levels, suppressed PINK1/Parkin-mediated mitophagy and impaired odontogenic differentiation. GSH supplementation restored mitophagy activity and rescued differentiation capacity in vitro. PINK1 depletion impaired odontogenic differentiation and markedly attenuated the pro-differentiation effects of exogenous GSH. In vivo, GSH administration enhanced tertiary dentine formation and activated mitophagy pathways in injured dental pulp.

Our findings establish that the GCLC-mediated GSH antioxidant system orchestrates the odontogenic differentiation of dental pulp cells and pulp-dentine complex repair via the PINK1/Parkin-mitophagy axis, revealing a novel metabolic-oxideative signalling circuit as a potential therapeutic target for regenerative endodontics.