It has been reported that sodium fluoride (NaF) suppresses the proliferation and induces apoptosis of chondrocytes. However, the cellular and molecular mechanisms of the effect have not been elucidated. Therefore, the aim of this study was to evaluate the mechanisms of the effects of NaF on primary cultured rat chondrocytes in vitro. Chondrocytes were treated with NaF at concentrations of 0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 mM. Cell viability decreased and the rate of apoptotic cells increased significantly with the gradient concentration of NaF in a time- and dose-dependent manner. Electron microscopy revealed cytoplasmic, organelle and nuclear alterations in the ultrastructure of chondrocytes exposed to various NaF concentrations. The cell cycle distribution was analyzed by flow cytometry, and the results indicated that NaF induced G2 cell cycle arrest. Western blotting was used to detect the apoptotic pathways. Downregulation of the Bcl-2 protein and upregulation of Bax, cleaved caspase-9, -12 and -3 proteins suggested that NaF was capable of inducing apoptosis through the mitochondrial and endoplasmic reticulum pathways. The results also showed that the levels of hypoxia-inducible factor 1a (HIF-1?), sex determining region Y box gene 9 (Sox9) and the collagen II (Col II) protein of the NaF groups were lower compared to those of the control groups. Thus, NaF may induce apoptosis through the downregulation of HIF-1a and disrupt the synthesis of extracellular matrix (ECM) through the downregulation of HIF-1a via the Sox9 pathway in primary cultured rat chondrocytes.
Results of previous studies have shown that fluoride has a higher and special affinity for calcium in bone tissue, which could result in osteofluorosis (19,20). It has been reported that osteofluorosis may increase the severity of osteoarthritis (OA) characterized by progressive degeneration of articular cartilage (21,22). Chondrocyte apoptosis may be involved in the onset and development of osteoarthritic cartilage degeneration (23). Previously, chondrocytes were found to be responsible for the synthesis of cartilage extracellular matrix (ECM) (24,25), while ECM is considered crucial to the survival of chondrocytes (26,27). Subsequently, there is a close correlation between chondrocyte apoptosis and the synthesis of ECM. Additionally, fluoride may induce OA by promoting chondrocytes apoptosis and disrupting the synthesis of ECM in cartilage; however, the molecular mechanism involved remains to be determined.
Hypoxia-inducible factor 1a (HIF-1a) is important in the maintenance of the survival of chondrocytes. HIF-1a may inhibit the apoptosis of chondrocytes and regulate the synthesis of ECM (28-30). This synthesis may be mediated by transactivation of sex determining region Y box gene 9 (Sox9), a key transcription factor for chondrocyte-specific genes such as collagen II (Col II) which encode the Col II protein (31,32). Thus, we hypothesized that sodium fluoride (NaF) would induce chondrocytes apoptosis through the downregulation of HIF-1a and cause matrix disruption through the downregulation of HIF-1a via the Sox9 pathway.
The purpose of this study was to demonstrate the molecular mechanism of NaF on apoptosis in primary cultured rat chondrocytes.