Abstract:
Objective To investigate the effects of treatment protocols using fluoride, arsenic, and fluoride-arsenic on the expressions of tumor necrosis factor-related receptor factor 6 (TRAF-6), nuclear factor kappa B1 (NF-κB1), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), and tartrate-resistant acid phosphatase (TRAP) mRNA in a co-culture system of mouse precranial osteoblasts MC3T3-E1 and mouse macrophages RAW 264.7.
Methods After the induction of osteogenic inducer, mouse precranial osteoblasts MC3T3-E1 and monocytes Raw 264.7 were established as a co-culture system. After seven days of culture, osteoclasts were identified by TRAP staining. The effects of different concentrations of the drugs on cell proliferation were determined by CCK-8 method to determine the final experimental concentration. In co-culture system, the cells were cultured for 24 h with sodium fluoride (0, 0.1, 0.4, and 1.6 mmol/L NaF, designated as F0, F0.1, F0.4, and F1.6), sodium arsenite (0, 0.5, 2.5, and 12.5 μmol/L NaAsO2, designated as As0, As0.5, As2.5, and As12.5), and combined fluoride-arsenite (combinations of 2 doses from previous 2 groups), respectively. The expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in each group were detected by real-time fluorescence quantitative PCR.
Results After co-culturing osteoblasts with RAW 264.7 cells for 7 days, the TRAP staining showed that the cells were fused, larger, and multinucleate, indicating that RAW 264.7 cells had differentiated into osteoclasts with multiple nuclei. As to only fluoride exposure, the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA in the F1.6 group (1.74±0.16, 2.04±0.09, 1.33±0.06, and 3.24±0.29, respectively) were statistically higher than those in the control group (1.00±0.08, 1.00±0.04, 1.00±0.02, and 1.00±0.19, respectively) (P < 0.05). As to only arsenic exposure, the TRAF-6 and TRAP mRNA expressions in the As12.5 group (1.24±0.06 and 1.26±0.08) were higher than those in the control group (1.00±0.08 and 1.00±0.19), but the NF-κB1 and NFATc1 mRNA expressions in the As12.5 group (0.61±0.05 and 0.60±0.06) were significantly lower than those in the control group (1.00±0.04 and 1.00±0.02) (P < 0.05). As to combined exposure, compared with the F0.1 group (0.80±0.02 and 0.59±0.03), the expression of NF-κB1 and NFATc1 increased in the F0.1As12.5 group (1.11±0.02 and 1.07±0.05, P < 0.05), but they were lower than the sum of fluoride and arsenic exposure alone; compared with the F0.4 group (2.85±0.13, 1.11±0.04, and 1.09±0.03), the expressions of TRAF-6, NF-κB1 and NFATc1 decreased in the F0.4As12.5 group (1.38±0.11, 0.87±0.03, and 0.44±0.03, P < 0.05); compared with the F1.6 group (2.04±0.09 and 3.24±0.29), the NF-κB1 and TRAP mRNA expressions decreased in the F1.6As12.5 group (1.24±0.07 and 2.09±0.22, P < 0.05); compared with the As0.5 and As12.5 group, the TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions increased in the F1.6As0.5 group and the F1.6As12.5 group (P < 0.05), but lower than the sum of fluoride and arsenic exposure alone. The main effects of fluoride and arsenic on the expressions of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA were statistically significant (F=437.22, 657.76, 321.89, and 187.11; F=123.08, 170.53, 78.99, and 74.08, Ps < 0.05), while the combination of fluoride and arsenic also showed significant interactions on TRAF-6, NF-κB1, NFATc1, and TRAP mRNA expressions (F=153.76, 97.14, 100.31, and 46.78, Ps < 0.05).
Conclusion In the co-culture system, high fluoride can up-regulate the expression levels of TRAF-6, NF-κB1, NFATc1, and TRAP mRNA, thereby enhance the differentiation, maturation, and bone resorption function of osteoclasts. Fluoride or arsenic alone have adverse effects on osteoclasts. The interaction of fluoride and arsenic with the expressions of osteoclast-related genes is mainly the antagonism of arsenic tofluoride.
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