Excessive Fluoride Stimulated Osteoclast Formation Through Up-Regulation Of Receptor Activator For Nuclear Factor-Kappa B Ligand (Rankl) In C57bl/6 Mice

Skeletal fluorosis is a metabolic bone disease caused by excessive consumption of fluoride. However, the pathogenesis of skeletal fluorosis remains unclear. Recent vitro studies have demonstrated that fluoride could increase RANKL mRNA expression in osteoblast. In this study, the relationship between RANKL and osteoclast formation was investigated in fluorosis mice. Twenty male C57BL/6 mice were randomly divided into two groups: control group (drinking distilled water) and 100 mg/L fluoride group (drinking distilled water with 100 mg/L fluoride ion). Mice were fed for 15 weeks and allowed to eat and drink freely. The occurrence of dental fluorosis in mice was observed during the fed period. Bone fluoride content was detected by ion selective electrode method. The effect of fluoride on bone tissue was assessed with hematoxylin and eosin (HE) staining. Tartrate-resistant acid phosphatase (TRAP) staining was used to evaluate osteoclast formation in bone tissue. The mRNA and protein expression and distribution of RANKL were separately detected using real-time quantitative PCR (RT-PCR), Western-blot and immunohistochemistry. By the end of the animal experiment, all mice in the fluoride group had dental fluorosis with different severities, while none dental fluorosis was found in the control group. Bone fluoride content in fluoride group was about 4.6 times higher than that in the control group. Compared with control group, the trabecular bone in experimental group was expanded and fused, which lead to an increase of bone mass in fluoride group. An increase of osteoclast number was found in bone tissue of fluorosis mice. The expression of RANKL mRNA and protein in bone tissue of fluorosis mice were significantly higher than that of control group, however, the distribution of RANKL between the two groups did not differ. Excessive fluoride could stimulate osteoclastogenesis in bone tissue of mice, and the molecular mechanism of fluoride action may be through up-regulation of RANKL expression. It is suggested that RANKL may play a vital role in the pathogenesis of skeletal fluorosis.