The effects of fluoride on proliferation and apoptosis of primary cultured rat ameloblast

Objective To investigate the effects of fluoride at different concentrations on proliferation and apoptosis of primary rat ameloblast in vitro. Methods Ameloblasts were isolated from tooth germ of 4 days SD rat maxillomandibular molar and cultured in vitro. Cells were treated with NaF at 0.0 (control group), 0.4, 0.8, 1.6, 3.2 and 6.4 mmol/L for 24, 48 and 72 h, respectively. Inverted microscope was used to observe cell morphology; immunochemistry method was used to identify ameloblasts; 3-(4, 5-dimethylthiazole-2)-2, 5-diphenyl tetrazolium bromide (MTT) assay was applied to measure cell viability at each time point. The cells were treated with 1.6 mmol/L NaF for 24 and 48 h, or after 50 mol/L caspase pan-inhibitor Z-VAD-FMK pretreatment 1 h, 1.6 mmol/L NaF treatment for 48 h. Cell apoptosis was then tested by flow cytometry. In addition, activation of caspase-3 and poly (ADP-ribose) polymerase (PARP) were assessed by Western blotting to explore potential involvement of caspase activation in NaF-induced apoptosis. All data analysis was performed using SigmaStat V 3.5 software. Results ① Primary rat ameloblasts were in polygonal shape at low density and appeared like paving stone at high density with obvious nucleus, showing typical morphological characteristics of cells with epithelial origin. ② The results of immunochemistry assay indicated that the cultured cells were positive in cytokeratin 14 (CK14) and ameloblastin (AMBN) staining, in accordance with the immunocytochemical characteristics of ameloblasts. ③The effects of NaF on ameloblast proliferation were in a dose- and time-dependent manner. For low dose NaF (0.4 and 0.8 mmol/L) groups, cells treated for 24 h had significantly higher cell proliferation rates than that of the control group (0.0 mmol/L, P 0.05); cell proliferation indexes at 72 h were significantly lower than that of the control group (0.87 ± 0.03 and 0.80 ± 0.04, P 0.05); while cell proliferation indexes at 48 and 72 h were obviously reduced than that of the control group (0.38 ± 0.03 and 0.26 ± 0.04, P < 0.01). For high NaF concentration (3.2 and 6.4 mmol/L) groups, cell proliferation indexes were significantly decreased at all time points compared with control cells, the rates for 3.2 mmol/L groups were 0.57 ± 0.14, 0.08 ± 0.03 and 0.00 ± 0.00, respectively, and the rates for 6.4 mmol/L groups were 0.11 ± 0.04, 0.00 ± 0.00 and 0.00 ± 0.00, respectively (P < 0.01). ④Flow cytometry was used to detect apoptosis. The results showed that treatment with 1.6 mmol/L NaF resulted in significantly increased apoptosis in ameloblasts at both 24 h [(5.80 ± 2.03)%] and 48 h [(17.45 ± 4.97)%] compared to the control group [(2.59 ± 0.95)%, P < 0.05]. In cells pre-treated with pan-caspase inhibitor Z-VAD-FMK, NaF-induced apoptosis was significantly lower than that of cells treated with only 1.6 mmol/L NaF [(9.43 ± 3.79)% vs (18.26 ± 3.39)%, P < 0.05]. ⑤Cleavage of caspase-3 and PARP was detected in ameloblasts treated with 1.6 mmol/L NaF for 48 h. Conclusion Overdose fluoride could inhibit proliferation and induce apoptosis via activation of caspase cascade in primary cultured rat ameloblasts. Key words: Fluoride; Ameloblast; Apoptosis; Caspase-3