Apoptosis In OLC Odontoblast-like Cells Induced By Fluoride

Fluoride plays an important role in preventing dentin hypersensitivity, remineralization of caries and mechanism of dental fluorosis. It also has been widely used in clinical and oral hygiene. The mineralization of dentin has been found affected by Fluoride exposure related to gene expression changes. For sake of odontoblasts developing function, they keep to converted pulp chamber through human lives, External pressure and temperature stimulations will induced the apoptosis of pulp cells and stimulate migration and differentiation, resulting in reparative dentin.Some studie shows that 3mmol / L of fluoride could not induce apoptosis of odontoblasts . Can fluoride induce apoptosis in odontoblasts ? What is the appropriate time and concentration? Through which molecular pathways did the fluoride-induced apoptosis regulated by? Some studies show that the chloride ion channel was affected by fluoride exposure with an increase of sulfur transfer. It may be associated with the expression, regulation and modification of some mineralization moleculars. what is the relationship between apoptosis-related protein and mineralization-related molecules in odontoblasts ?This research include four experiments:Experiment 1: Cell viability and cell proliferationTo study cell proliferation, OLC cells were exposed to increasing concentrations of NaF at 0-6mmol/L for 12 to 48 hours after in serum-free medium for 12 hours. Cell viability was evaluated using a MTT assay. Results: Incubation of OLC cells with NaF for 12, 24,48 hrs induced a dose-dependent decrease in cell viability .Following exposure to 4 mM NaF for 24 hrs, cell viability was reduced by approximately 50%.Experiment 2: Measurement of apoptosisTo detect the apoptosis induced by fluoride, we use flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) to check membrane alteration and DNA fragmentation. we also found the ultrastructural alterations by Scanning electron microscopy. Representative immunoblot obtained with caspase-3 and actin antibodies. Full-length and cleaved forms of caspase-3 were detected .Experiment 3:Involvement of MAPKs in NaF-induced ApoptosisTime-course of NaF-induced accumulation of phosphorylated JNK and phosphorylated ERK in OLC cells. In OLC cells exposed to 4 mM NaF for 24 hrs, treatment with the JNK inhibitor, SP600125 suppressed JNK phosphorylation, while treatment with the ERK inhibitor, PD98059 suppressed ERK phosphorylation partially.Experiment 4: Involvement of mitochondrion in NaF-induced Apoptosis we observe the mitochondrial dynamic morphology by cell stained with MitoTracker Deep Red 633 and Bcl-2 expression increased after flouride exposure.It shows that apoptosis induced by flouride is through the mitochondrial pathway.