Combined effects of fluoride and arsenic exposure on proliferation, differentiation and beta-catenin expression in rat osteoblasts / 中华地方病学杂志

ABSTRACT

Objective To investigate the combined effects of fluoride (NaF) and arsenate (NaAsO2) exposure on proliferation,differentiation and bata-catenin expression in SD rat osteoblasts.Methods Osteoblasts were isolated from calvarias of twelve SD rats born in 1-3 days and cultured.The method was divided into 9 groups [F0.0As0.0 (control group),F0.5As0.0,F4.0As0.0,F0.0As0.1,F0.0As10.0,F0.5As0.1,F0.5As10.0,F4.0As0.1,F4.0As10.0] by factorial experiment design (3 factors and 2 levels).Osteoblasts were exposed to NaF (F-0.0,0.5,4.0 mmol/L,F0.0,F0.5,F4.0),NaAsO2 (As3+0.0,0.1,10.0 μmol/L,As0.0,As0.1,Asi10.0) and cultured for 72 hours.The proliferation and alkaline phosphatase (ALP) was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) and Enzyme-linked immunosorbent assay (ELISA).The expression of beta-catenin was analyzed by quantitative real-time PCR (qPCR) and Western blotting after 72 hours of experiment.Results ①There was significant difference in cell proliferation and the activity of ALP among groups after 72 hours (F =14.022,14.425,all P ＜ 0.05).Compared with control group,the proliferation and the activity of ALP were significantly induced in F0.5 treated osteoblasts groups (0.313 ±0.023 vs0.455 ± 0.152,4.46 ± 0.72 vs 6.09 ± 0.68,all P ＜ 0.05),and the proliferation was significantly suppressed in F4.0As0.0 group (0.029 ± 0.014,P ＜ 0.05),the activity of ALP was significantly induced in F0.0As10.0 group (0.156 ± 0.010,6.29 ± 0.67) and the proliferation was significantly suppressed in F0.0As0.1 group (0.370 ± 0.029,3.68 ± 0.45,all P ＜ 0.05).②There was statistical difference in beta-catenin mRNA and protein expressions among groups with F-(F =7.782,559.455,all P ＜ 0.05) at As0.0 condition.There was significant difference in the expression of betacatenin mRNA and beta-catenin protein in F0.5As0.0 group compared with control group (1.00 ± 0.32 vs 1.99 ± 0.14,3.56 ± 0.15 vs 5.11 ± 0.26,all P ＜ 0.05),the beta-catenin protein was significantly suppressed in F4.0As0.0 group (1.10 ± 0.02,P ＜ 0.05).There was significant difference in the expression of beta-catenin protein of all groups with As3+ (F =154.736,P ＜ 0.05) at F0.0 condition.③Factorial analysis showed that fluoride or arsenic alone could affect the proliferation and the expression level of beta-catenin mRNA and protein (F =82.081,11.991,514.741;19.302,8.753,523.698,all P ＜ 0.05),the effect of arsenic on ALP activity of osteoblasts was also the main effect (F =17.444,P ＜ 0.05);and there was an interaction between fluoride or arsenic to cell proliferation and the activity of ALP and the expression of beta-catenin mRNA and protein (F =13.085,18.157,4.936,426.036,all P ＜ 0.05).Conclusions A biphasic pattern of fluoride or arsenic on proliferation and differentiation has been induced in SD rat osteoblasts.Fluoride or arsenic can affect bone metabolic by beta-catenin.