Study On Damage Mechanism Of Cultured Pc12Cells Exposed To Fluoride

Fluoride is an essential trace element to organism which is widely distributed in nature. But long-term excessive exposure to fluoride can accumulate in the body and cause chronic fluorosis, seriously damaged the body’s phrenology and non phrenology organs.The mechanism of injury may associated with oxidative stress induced by fluoride,but the exact mechanism remains unknown. Cell apoptosis may be one of the key links.In recent years, it has become a hot spot that endoplasmic reticulum stress induced apoptosis on the research of the body damaged by fluoride.However, these studies mostly focused on the study of ER stress induced by fluoride in phrenology organ damage, while few in non-phrenology organs.Malondialdehyde and superoxide dismutase, which are the most commonly used indicators of this theory, exhibit low specificity and sensitivity. Results from other studies also vary, which is possibly due to many factors. It has a very important significance to further explore the molecular mechanism of apoptosis caused by fluoride which direct determined the change in intracellular ROS concentration caused by fluoride exposure.PC12cells derived from rat adrenal medulla pheochromocytoma, which could proliferate the cells with the characteristics of sympathetic neurons induced by the nerve growth factor, and widely used in ex-vitro studies of neurological disorders.PC12cells were treated with different fluorine concentrations (0.5,1.0,1.5and2.0mM). Cell activity was detected using a Cell Counting Kit-8at different time periods (2,4,6,8,12,24and48h). Intracellular reactive oxygen species (ROS) levels were detected with DCF mark after2 h of fluoride exposure.After8h of fluoride exposure,mitochondrial membrane potential changes were detected with Rhodamine123and JC-1,and cell apoptosis was detected using Hoechst33342after.After24h of fluoride exposure, morphological changes were observed using an inverted microscope,the expressions of apoptosis-related proteins PARP, p-elFand endoplasmic reticulum stress-related protein XBP-1were detected by western blot.Results:1.Cell viability:Compared with the control group, after2h of fluoride exposure, cell viability in the2.0mM groups significantly decreased (p<0.05). After4h of fluoride exposure, cell viability in the1.5and2.0mM groups significantly or highly decreased (p<0.05or P<0.01). After treatment with fluoride for8and48h, the cell survival rate of each group significantly decreased (p<0.01). After12h of fluoride exposure, the survival rate significantly decreased in1.0and1.5mM group(p<0.05).After12and24h of fluoride exposure, the survival rate of the0.005mM groups significantly increased (p<0.05), whereas that of the2.0group significantly decreased (p<0.01).After treatment with fluoride for24h, the survival rate significantly decreased in0.5and1.0mM group(p<0.05),and highly decreased in1.5and2.0mM groups(P<0.01).2.Cell morphology:Normal cells were fusiform with more than two synapses. After24h of fluoride exposure, cell density decreased and some cells became round. The number of apoptotic or dead cells increased with increasing fluoride concentration. Furthermore, apoptotic vesicles and cell debris were clearly observed.3. Intracellular ROS concentration:After2h of fluoride exposure,the intracellular reactive oxygen concentration increased gradually with fluoride concentration. Compared with the control group, the intracellular ROS concentration significantly increased in the1.5and2.0mM group (p<0.01).4.Mitochondrial membrane potential:Exposed to fluoride for8h,the results of Rho-123labeled showed that the mitochondrial membrane potential decreased with the increase of fluoride concentration. Compared with the control group, mitochondrial membrane potential significantly decreased in2.0mM group (P<0.01).The fluorescence gradually turned from red to green in the cells with JC-1labeled,described mitochondrial membrane depolarization gradually increased, maening the mitochondrial membrane potentia continuously reduceed, indicating the occurrence of apoptosis.5.Cell apoptosis:Normal nuclei were oblong or oval, but they became round and karyopyknotic after apoptosis. In addition, their colour deepened, and the fluorescence intensity increased after apoptosis. The fluorescence intensity of the fluoride groups was no significant difference than that of the control group (P>0.05).The fluorescence intensity slightly increased with the fluoride concentration except in0.5mM group,indicating that the number of apoptotic cells also increased slightly.6.Western blot analysis:Compared with the control group, protein level of XBP-1and PARP were significantly increased in1.OmM group(P<0.05).While protein level of PARP,the cut tape of PARP were increased significantly(P<0.01), protein level of other groups were floated slightly, but not significantly different (P>0.05). Suggesting that PARP may be one of the key proteins of fluoride-induced apoptosis in PC12.XBP-1was probably one of the key proteins in the endoplasmic reticulum stress-mediated apoptosis.7.Conclusion:Fluoride exposure resulted in a reduction in cell activity, which was negatively correlated with exposure duration,and was also basically negative correlated with fluorine exposure dose. During a certain duration of fluoride exposure, the levels of intracellular ROS, cell damage, cell apoptosis and mitochondrial membrane damage showed a substantial linear relationship with the level of fluoride exposure. This finding suggests that intracellular oxidative stress induced by fluoride exposure was one of the causes of fluoride-induced apoptosis, and XBP-1was probably one of the key proteins in the endoplasmic reticulum stress-mediated apoptosis, which further provided proof for the theory of fluoride-induced free radical damage.