Over the past two decades, fluoride effects on osteoclasts have been evaluated; however, its molecular mechanisms remain unclear. In this study, we investigated the effect of fluoride on osteoclast formation, function, and regulation using osteoclasts formed from mice bone marrow macrophages treated with the receptor activator of NF-κB ligand and macrophage colony-stimulating factor. Our data showed that fluoride levels ≤ 8 mg/L had no effect on osteoclast formation; however, it significantly reduced osteoclast resorption at 0.5 mg/L. Fluoride activity on bone resorption occurred through the inhibition of nuclear factor of active T cells (NFAT) c1 expression. Furthermore, the expression of its downstream genes, including the dendritic cell-specific transmembrane protein, c-Src, the d2 isoform of vacuolar (H+) ATPase v0 domain, matrix metalloproteinase 9, and cathepsin K were decreased, leading to impaired osteoclast acidification, reduced secretion of proteolytic enzymes, and decreased bone resorption. In summary, our results suggested that fluoride has different roles in osteoclast formation and function. Fluoride ≤ 8 mg/L did not impact osteoclast formation; however, it significantly decreased the resorption activity of newly formed osteoclasts. The molecular mechanism of fluoride action may involve inhibition of NFATc1 and its downstream genes.

The BMP/Smad signaling pathway plays an important role in the viability and differentiation of osteoblast; however, it is not clear whether this pathway is involved in the fluoride-induced osteoblast differentiation. In this study, we investigated the role of BMP/Smad signaling pathway in fluoride-induced osteoblast-like Saos-2 cells differentiation. Cells were exposed to fluoride of different concentrations (0, 0.1, 0.2, 0.4, 0.8, and 1.6 mM), and cell proliferation was determined using WST assays. The expression of osteoblast marker genes such as osteocalcin (BGP) and bone alkaline phosphatase (BALP) were detected by qRT-PCR. We found that fluoride enhanced the proliferation of Saos-2 cells in a dose-dependent manner and 0.2 mM of fluoride resulted in a higher expression of osteoblast marker genes. In addition, immunofluorescence analysis showed that the promotion effects of 0.2 mM of fluoride on Saos-2 cells differentiation were associated with the activation of the BMP/Smad pathway. Expression of phosphorylated Smad1/5(p-Smad1/5) was higher in cells exposed to 0.2 mM of fluoride. Plasmid expression vectors encoding the short hairpin RNA (shRNA) targeting Smad4 gene were used to block the BMP/Smad pathway, which resulted in a significantly reduced expression of BGP and BALP as well as their corresponding mRNA. The mRNA levels after transfection remained low even in the presence of fluoride. The present results reveal that BMP/Smad signaling pathway was altered during the period of osteogenesis, and that the activities of p-Smad1/5 were required for Saos-2 cells viability and differentiation induced by fluoride.

Oxidative stress and selenoprotein deficiency are thought to be associated with the pathogenesis of Keshan disease (KD). However, to our knowledge, the level of oxidative stress and expression of selenoproteins have not been investigated in the myocardium of patients with KD. In this study, 8-hydroxy-2-deoxy guanosine (8-OH-dG), a marker of oxidative stress, was used to assess the level of oxidative stress, and thioredoxin reductase 1 (TrxR1) and glutathione peroxidase 1 (GPx1) were assessed to reflect the level of selenoproteins. Myocardial samples from 8 patients with KD and 9 non-KD patients (controls) were immunohistochemically stained for 8-OH-dG, TrxR1, and GPx1. The staining intensities were subsequently quantified using Olympus Image-Pro Plus 6.0 software. The data showed that the positive rate of 8-OH-dG expression in myocardial nuclei was higher in the KD group (68.6%) than that in the control group (2.4%). In addition, a positive correlation between the positive rate of 8-OH-dG and the degree of myocardial damage was observed in the KD group. The distribution of TrxR1 and GPx-1 was not associated with the distribution of myocardial damage. The expression of these two selenoproteins was higher in the control group than that in the KD group. Our study represents the first report on the expression profiles of oxidative stress and selenoproteins in the myocardium of patients with KD. The level of oxidative stress significantly increased and was positively correlated with the degree of myocardial damage in patients with KD. The selenoproteins, TrxR1 and GPx1, may have a role in the pathogenesis of KD.