Abstract
Excess consumption of fluoride during the development of tooth enamel will cause dental fluorosis, but the exact molecular mechanisms remain to be elucidated. Circadian rhythm is implicated in many physiological processes and various diseases. There is increasing evidence indicates that ameloblast differentiation is under the control of clock genes. However, it has not been reported whether fluoride regulates ameloblast differentiation through clock genes and the downstream PPARγ. To explore the effect of fluoride on ameloblast differentiation and the underlying regulatory mechanism, we used both rat dental fluorosis model and an ameloblast cell line LS8 to conduct a series of experiments. Our results showed that fluoride significantly reduced the expression of PCNA, RUNX2 and MMP9 in rat ameloblasts and LS8 cells (P < 0.05). Fluoride increased nuclear translocation of β-catenin in vivo and in vitro, and 0.1 μg/ml Dkk1 pretreatment ameliorated the decreased expression of CXXC5, RUNX2 and MMP9 induced by fluoride. Furthermore, we found fluoride significantly inhibited the expression of Clock, Bmal1, Per2 and PPARγ in rat mandibular ameloblasts and LS8 cells by immunostaining, qPCR and Western blot (P < 0.05). Flow cytometry analysis showed that fluoride promoted ROS generation. Remarkably, 50 μM resveratrol significantly ameliorated the inhibitory effect of fluoride on ameloblast differentiation markers, clock genes and PPARγ, and inhibited the Wnt/β-catenin signaling (P < 0.05). Taken together, these findings suggested that excessive fluoride promoted ROS generation, leading to the inhibition of clock genes, which resulted in reduced PPARγ and activated Wnt/β-catenin signaling pathway, thus inhibiting ameloblast differentiation and matrix degradation. This study provides a better understanding of the molecular mechanism of enamel defects in dental fluorosis.