Abstract
Excessive fluoride intake can cause dental fluorosis during teeth development and growth. However, the mechanisms underlying fluoride-induced enamel damage are still not fully elucidated. Previously, we observed fluoride-induced autophagy in ameloblasts, but the effects of fluoride on autophagy flux in ameloblasts remain unclear. Hence, this study aimed to clarify the effects of fluoride and rapamycin, an autophagy activator, on autophagy flux in ameloblasts. This in vitro study used the murine ameloblast-derived cell line LS8. Cells were treated with different concentrations of sodium fluoride (NaF) to evaluate NaF-induced cytotoxicity. Using transmission electron microscopy, we observed an increase in the number of autophagosomes with increasing fluoride concentrations. Western blot analyses showed increases in microtubule-associated protein 1 light chain 3 (LC3) and SQSTM1 (p62) expression after NaF treatment and an increase in LC3II expression after bafilomycin A1 administration. Together with changes in RFP-GFP-LC3 lentivirus expression, this demonstrated that fluoride impaired autophagy flux. Furthermore, we evaluated whether rapamycin can alleviate fluoride-induced cytotoxicity by restoring autophagy flux. Compared to the NaF-treated group, LS8 cells cotreated with NaF and rapamycin grew considerably better and had significantly decreased p62 expression. Taken together, these data suggest that fluoride-induced impaired autophagosome degradation may damage ameloblasts. This provides experimental in vitro evidence and an explanation for the observed NaF-induced toxicity of ameloblasts. Rapamycin probably alleviates this impairment by decreasing the expression of p62, thereby preventing autophagy defects.