ABSTRACT
PURPOSE: To explore the effects of high-concentration fluoride(F) on apoptosis of human periodontal ligament stem cells (PDLSCs). METHODS: PDLSCs were isolated from periodontal ligament tissues of extracted third molars, and treated with different concentrations (0-40 ppm F) of NaF for indicated period of time. CCK-8 assay was performed to detect cell viability. After stained with Annexin V-PI and JC-1, cell apoptosis and mitochondrial membrane potential were analyzed by flow cytometry. Immunofluorescence staining and confocal microscopic assay were used to detect the protein expression level of cyt-c, cleaved-caspase-9 and -3. The mRNA level of caspase -9 and -3 were examined by RT-PCR. The protein expression level of total and phosphate-ERK, JNK and p38 were analyzed by Western blot. SPSS 13.0 software package was used for statistical analysis. RESULTS: Fluoride treatment inhibited cell viability (CCK-8 assay) and induced apoptosis of PDLSCs (Annexin V-PI staining) in a dose- and time-dependent manner. Immunofluorescence assay showed that fluoride with a dose ≥10 ppm significantly induced release of cyt-c from the mitochondria to cytosol, and up-regulation of expression of cleaved-caspase -9 and -3. RT-PCR confirmed that the mRNA level of caspase-9 and -3 increased with the dose of fluoride. Western blot assay confirmed that fluoride induced up-regulation of p-ERK, but not that of p-JNK and p-p38, and specifically blocking ERK pathway with U0126 could partially rescue the fluoride-induced cell apoptosis. CONCLUSIONS: High concentrations of fluoride induces apoptosis of PDLSCs via intrinsic mitochondrial pathway, and phosphation of MAPK/ERK is involved in the F-induce cell apoptosis.
