The effects of fluorine on expression of osteoprotegerin and receptor activator of NF-κB ligand in cultured rat osteoblasts

Objective To study the influence of fluorine on signaling pathway of osteoprotegerin(OPG)/ receptor activator of NF-κB ligand(RANKL) in cultured rat osteoblasts.Methods Osteoblasts were isolated from skull of neonatal rats(＜ 24 hours) by enzyme digestion,and fluorine of different concentrations [0 (control),1 × 10-3,1 × 10-4,1 × 10-5,1 × l0-6 and 1 × 10-7 mol/L] were added into the culture medium of second generation of osteoblasts.The expressions of OPG and RANKL mRNA were determined using real-time PCR 24 and 48 hours after culturing.The expressions of OPG and RANKL protein were measured by Western blotting.Results ① After exposed to fluorine for 24 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =30.95,22.62,all P ＜ 0.01),the expression of RANKL mRNA(5.99 ± 0.39) in the 1 × 10-5 mol/L group and the expressions of OPG mRNA(3.52 ± 0.09,4.81 ± 0.15,3.68 ± 0.04) in the 1 × 10-4,1 × 10-5 and 1 × 10-6 mol/L groups were higher than those of the control group(3.20 ± 0.19,3.09 ± 0.58,all P ＜ 0.05),but in the 1 × 10-3 mol/L group,RANKL mRNA(2.29 ± 0.18) was lower than that of the control group(P ＜ 0.05).After exposed to fluorine for 48 hours,the differences of RANKL and OPG mRNA expression had statistical significance between groups(F =26.62,5.72,all P ＜ 0.01),the expressions of RANKL and OPG mRNA(6.67 ± 0.49 and 5.05 ± 0.51) in the 1 × 10-5 mol/L group were higher than those of the control group(4.29 ± 0.07 and 4.34 ± 0.12,all P ＜ 0.05),and in the 1 × 10-3 mol/L group the expression of OPG mRNA(3.63 ± 0.49) was lower than that of the control group(P ＜ 0.05).② The expression of RANKL protein was not statistically significant between 24 hours and 48 hours groups (F =0.07,0.49,all P ＞ 0.05) ; the differences of OPG protein expression had statistical significance between groups(F =3.26,P ＜ 0.05),the expression of OPG protein in the 1 × 10-5 mol/L group(1.45 ± 0.10) was higher than that of the control group(1.05 ± 0.06,P ＜ 0.05) at the 24 hours.After 48 hours,the expression of OPG protein was not statistically significant(F =0.44,P ＞ 0.05).Conclusions At lower fluorine concentrations,bone formation is the main activity.But when fluorine concentration increased and time prolonged,the osteoclast differentiation and maturation are promoted,and the bone resorption is the main thing. Key words: Fluorine;  Osteoblasts ;  Osteoprotegerin ;  Receptor activator of NF-κB ligand