Melatonin enhances hydrogen peroxide-induced apoptosis in human dental pulp cells

Melatonin, at physiological concentrations, was previously found to inhibit proliferation and promote odontogenic differentiation in human dental pulp cells (hDPCs), but its effect on apoptosis is unclear. Our study aimed to investigate the effect of melatonin on the H₂O₂-mediated viability reduction and apoptosis in hDPCs.

hDPCs were treated with H₂O₂ (0, 250, 500, 1000 μmol/L), melatonin (0, 10^-12, 10^-10, 10^-8 mol/L), and melatonin with H₂O₂ for 24 h. CCK-8 assays were performed to evaluate cell viability. Apoptosis was measured by DAPI and Annexin V/propidium iodide staining. Intracellular reactive oxygen species (ROS) were measured by CellROX® staining and mitochondrial membrane potential (ΔΨm) was examined by JC-1 staining.

H₂O₂ obviously decreased the viability of hDPCs in a concentration-dependent manner and melatonin alone also reduced viability by 16-20%. Melatonin was also found to enhance H₂O₂-induced toxicity in a concentration-dependent manner, and the highest physiological concentration of melatonin (10^-8 mol/L) had the most obvious effect (P < 0.001). Treating H₂O₂-exposed hDPCs with melatonin significantly increased the ratio of apoptotic cells with condensed and deformed nuclei (P < 0.001), as well as the percentage of Annexin V-positive cells (P < 0.01). Furthermore, melatonin significantly increased intracellular ROS levels and induced the loss of ΔΨm in H₂O₂-exposed cells (P < 0.05).

Our results indicate that melatonin, at physiological concentrations, can enhance H₂O₂-induced apoptosis in hDPCs and increase H₂O₂-mediated ROS production and ΔΨm loss. Further studies are needed to investigate whether melatonin targets the mitochondrial death pathway during the process.