ABSTRACT
Objective To explore the effect of excessive fluoride on expression of mRNA and protein of Wnt3a and β-catenin in rats' osteoblasts and its correlation with pathogenic mechanism of fluorosis.Methods Thirty-six healthy SD rats,weighting 100-120 g and according to body mass,were randomly divided into three groups(twelve in each group).The rats of control were fed wich tap water(fluoride ＜ 1 mg/L) and the experimental rats were exposed to NaF(low-fluoride group:5 mg/L,high-fluoride group:50 mg/L) added to the drinking water to establish the chronic fluorosis model.After fed for eight morth,all rats were killed and metaphysic of femoral was collected.Rat dental fluorosis was observed and bone fluorine was detected by ashing-fluorin ion selective electrode method.The content of bone alkaline phosphatase (BALP) and tartrate-resistant acid phosphatase 5b(TRACP 5b) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA).The morphologic changes of the bone were observed by microscopy.The expression of mRNA and protein of Wnt3a and β-catenin in osteoblasts of rats was analyzed with gray scale by hybridization in situ and immunohistochemistry methods,respectively.Results Detection rate of dental fluorosis,fluoride contents of urine and bone were significantly increased [control group:0.0％,(1.26 + 0.17)mg/L,(305.58 ± 91.26)mg/kg; low-fluoride group:66.7％,(2.06 ± 0.64)mg/L,(632.33 ±123.21)mg/kg; high-fluoride group:91.7％,(7.69 ± 1.96)mg/L,(1088.75 ± 156.16) mg/kg] in the rats treated with fluoride,the difference between groups was statistically significant(χ2 =21.6; F =36.57,467.02; all P ＜0.05).The contents of BALP and TRACP-5b in rats' serum were significantly different between groups(F =89.57,7.68; all P ＜ 0.05).Compared with control group[(16.24 + 1.57)U/L],the contents of BALP in rats' serum of the low-fluoride and high-fluoride groups[(31.47 ± 5.30) and (54.61 ± 2.27)U/L] were increased gradually(all P ＜0.05).Compared with the low-fluoride group,the value in the high-fluoride group decreased significantly (P ＜ 0.05).The contents of TRACP-5b in rats' serum of low-fluoride group[(3.45 ± 1.85)U/L] were elevated significantly(all P ＜ 0.05) compared with the control group[(1.26 ± 0.23)U/L] and the high-fluoride group[(2.74 ± 1.85)U/L].The bone cortices were thickened and the bone trabecula was broadened,arranged closely together in chronic fluorosis rats with significant difference compared with the control group.In the low-fluoride and high-fluoride groups,the expression levels of Wnt3a and β-catenin mRNA (low-fluoride group:132.87 ± 5.72 and 132.57 ± 9.56; highfluoride group:135.60 ± 6.64 and 137.87 ± 9.16) were markedly elevated with significant difference,respectively (F =12.47,5.96; all P ＜ 0.05) compared with those in control groups(119.86 ± 5.04 and 120.58 ± 7.84) by hybridization in situ(P ＜ 0.05),but there was no statistical significance (P ＞ 0.05) of the level of Wnt3a and β-catenin mRNA between low-fluoride and high-fluoride groups.In the low-fluoride and high-fluoride groups,the protein expression of Wnt3a and β-catenin (low-fluoride group:137.50 ± 4.32 and 140.85 + 3.54; high-fluoride group:142.65 ± 11.84 and 152.52 ± 4.64) were markedly elevated with significant difference,respectively (F =10.07,53.82; all P ＜ 0.05) compared with those in control group (124.01 ± 2.63 and 126.75 ± 4.65) by immunohistochemistry(all P＜ 0.05),Wnt3a protein production in the low-fluoride group was increased without statistical significance compared with the high-fluoride group (P ＞ 0.05).But the protein production of β-catenin in the lowfluoride group was elevated with significant difference compared with the high-fluoride group(P ＜ 0.05).The mRNA and protein production of Wnt3a were positively correlated with the mRNA and protein production of β-catenin (r =0.731,0.658; all P ＜ 0.05).Conclusions Rat bone tissue lesions caused by excessive fluoride may be associated with an increased expression of Wnt3a and β-catenin mRNA and protein in osteoblasts.In chronic fluorosis,fluoride stimulates the overexpression of Wnt3a and β-catenin in the Wnt signal transduction pathway,enhances bone osteogenesis and causes skeletal fluorosis.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the changes of mRNA and protein expression of CaN in the bone of rats with chronic fluorosis, and the mechanism of skeletal fluorosis.</p><p><b>METHODS</b>Thirty-six SD rats were divided into three groups (12 in each group, half male and half female selected according to body weight): control, low-dose and high-dose fluorosis groups. Controls were fed tap water (NaF < 0.5 mg/L), experimental animals in the low- or high-dose groups were fed water containing NaF of 5.0 and 50.0 mg/L, respectively. The rats were sacrificed after 6 months of treatment with fluoride. The serum was kept for testing bone metabolic marker bone gla protein (BGP) by enzyme-linked immunosorbent assay (ELISA), the protein and mRNA levels of CaN in distal femur of the rats with chronic flurosis were assessed by immunohistochemistry and in-situ hybridization.</p><p><b>RESULTS</b>The levels of BGP (1.99 ± 0.62, 2.38 ± 0.16)µg/L in the low- or high-dose fluorosis groups were higher than that in the control group (0.15 ± 0.03) µg/L; and the high fluorosis group showed higher level than the low fluorosis group (all P < 0.05). Compared to the control group (131.11 ± 1.95, 111.82 ± 2.39), the protein and mRNA levels of CaN were higher in the low- or high-dose fluorosis groups (142.69 ± 1.17, 157.54 ± 1.88 and 121.28 ± 3.27, 134.63 ± 3.19, respectively), and the high fluorosis group showed higher levels than the low fluorosis group (all P < 0.05).</p><p><b>CONCLUSIONS</b>BGP content could be used as a bone metabolic index in endemic fluorosis disease. Fluoride might up-regulate the mRNA and protein expression of CaN, and the changes in CaN level may be involved in the increase of the bone turnover and could be one of the pathogenetic factors in fluorosis.</p>