Role Of Cell Autophagy In Aupoptosis Induced By Fluoride In Ameloblasts

Objective: The aim is to investigate the effect of excessive fluoride on the biological characteristics of ameloblastoma(proliferation and apoptosis)and autophagy in vitro.Methods: The cell line of mouse ameloblastoma(LS8)was cultured in vitro.1.the cells were treated with Na F(0 m M?0.25 m M?0.5 m M?1 m M?2 m M?4 m M).After the administration of 24 h,48 h and 72 h,the effect of Na F on the proliferation of LS8 cells was detected by CCK8 kit.2.The cells were treated with Na F(0 m M?0.8 m M?1.2 m M?1.6 m M?3.2 m M).After the administration of 24 h,the following experiments were carried out: ?1 After DAPI staining,the nucleus shape changes were observed by fluorescence microscope.?2 The apoptosis rate of cells was detected by flow cytometry.3.The cells were treated with Na F(0 m M?0.8 m M?1.2 m M?1.6 m M?3.2 m M): ?1 The expression of cell autophagy-related genes LC3 II,Beclin-1 were detected by q RT-PCR and Western blot,respectively.?2 The subcellular locationg of LC3 II was tested by immunofluorescence methods.4.The cells were divided into five groups: control group,Na F(1.6 m M)group,Na F(1.6 m M)+ 3-MA(5 m M)group,Na F(1.6 m M)+ 3-MA(10m M)group and Na F(1.6 m M)+ 3-MA(15 m M)group.After the administration of 24 h,the following experiments were carried out: ?1 The levels of LC3 II,Beclin-1 were detected by q RT-PCR and Western blot respectively.?2 After DAPI staining,the nucleus shape changes were observed by fluorescence microscope.?3 The apoptosis rate of cells was detected by flow cytometry.Results: 1.NaF could inhibit proliferation of LS8 cells,and the IC50 was 1.59 mmol/L.With the increase of drug concentration,the inhibitory effect on cell proliferation gradually increased,and the inhibitory effect increased with the increase of action time.2.The nuclear changes: the nuclei in the control group were intact without dense staining.Some nuclei was pyknosis,incomplete and dense staining after being treated with Na F.When the concentration of Na F was raised,the degree of pyknosis and fragmentation of nucleus increased significantly,and was not homogeneous.The apoptosis rate of LS8 cells were(2.267 ± 0.27)%,(6 ± 0.5)%,(15.7 ± 2.4)%,(43.8 ± 0.58)%,(64.63 ± 0.38)%after being treated with Na F(0 m M?0.8 m M?1.2 m M?1.6 m M?3.2m M).The apoptosis rate in the high concentration Na F groups were higer significantly than that of low concentration Na F groups(P<0.01).3.Expression of LC3 II and Beclin-1: The level of LC3 II,Beclin-1 m RNA and protein in Na F(1.6 m M)group was increased compare with control groups and Na F(0.8 m M?1.2m M)groups,and decreased compare with Na F(3.2 m M)group.Immunofluorescence results showed that: In the control group,the expression of LC3 II was low level,and showed a homogeneous weak green fluorescence.The number and intensity of LC3 II fluorescence spots in the cytoplasm of Na F(1.6 m M)group were significantly stronger than those in the control group,and the aggregated fluorescence spots appeared in the cytoplasm.4.Expression of LC3 II and Beclin-1: The levels of LC3 II,Beclin-1 m RNA and protein in Na F + 3-MA groups were decreased compare with Na F(1.6 m M)group.The nuclear changes: the nuclei in the control group were intact without dense staining.Some nuclei was pyknosis,incomplete and dense staining after the effect of Na F.In combination with Na F and 3-MA group,the degree of pyknosis and fragmentation of nucleus was increased and unhomogeneous significantly.The apoptosis rate of LS8 cells were(2.267 ±0.27)%,(43.8 ± 0.58)%,(55.17 ± 0.22)%,(61.3 ± 0.53)%,(78.4 ± 0.85)% after being treated with different concentration of Na F(1.6 m M),Na F+3-MA(5 m M/10 m M/15 m M).Moreover,the apoptosis of rate of Na F+3-MA groups were higer significantly than that of Na F group(P<0.01).Conclusion: Excess fluoride could inhibit the proliferation and promote apoptosis in ameloblasts,LS8 cells,and this effect was related to the level of cell autophagy.The cell autophagy was inhibited but the apoptosis increased when Na F exceeded a certain concentration or 3-MA was used in the cells.The results show that autophagy in ameloblasts has an anti-apoptotic effect and cell autophagy plays a protective role in the damage of fluoride to ameloblasts.