Effects of fluoride on fibronectin expression of rats osteoblasts / 中国地方病学杂志

ABSTRACT

Objective To observe the expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblast,and to study the role of fibronectin in pathogenesis of chronic fluorosis.Methods Male and female Wistar rats 144 were randomly divided into four groups,which were designated as the control group(normal diets,n =36),fluoride group(normal diets + 100 mg/L fluoride,n =36),lower calcium monophagia group (synthetic diets,n =36) and lower calcium monOphagia with fluoride group(synthetic diets + 100 mg/L fluoride,n =36).Rats were sacrificed 4 and 8 months after beginning of the experiment,respectively,and femur tissue was fixated and paraffin-embedded.The osteoblast isolated from calvaria of neonatal rats was treated with different dose of fluoride(0,1,2,4 mg/L fluoride,respectively) for 48 and 72 h,cell culture supernatant and cells were collected,respectively.The cranial osteoblasts were cultured in vitro and divided into four groups according to different concentration of fluoride added,which were 0(control group),0.01,1.00,and 10.00 mg/L groups.These cells were treated with mineralized induced medium at day 2 and cultured for 3 more weeks whereafter,and then the slides were fixed in alcohol.The expression of fibronectin in rat femur tissue was detected by immunohistochemistry (IHC),and fibronectin mRNA expression was determined by in situ hybridization; the fibronectin levels in supernatant of cultured osteoblast was examined by enzyme-linked immunosorbent assay(ELISA),and the expression of fibronectin mRNA in osteoblasts was detected with RT-PCR; skull mineralized nodule formation of osteoblasts was observed under a light microscopy after stained with 0.1％ red alizarin liquid.Results Little expression of fibronectin (brown granules under light microscope) could be seen in femur tissue of fluorosis rats of control group and lower calcium monophagia group; but abundantly expressed in fluoride group and lower calcium monophagia with fluoride group; the fibronectin was also expressed in osteoblasts,bone cells and bone marrow cells with less red particles in the control group and lower calcium monophagia group,but more in the fluoride group and lower calcium monophagia with fluoride group.The expression of fibronectin protein in supernatant of cultured osteoblasts was significantly increased in the group of 4 mg/L fluoride at 48 h(0.108 ± 0.042,t =0.764,P＜ 0.05) compared with control group(0.081 ± 0.010); the value was also significantly increased in 1,2,4 mg/L groups at 72 h(0.089 ± 0.010,0.087 ± 0.012,0.098 ± 0.023; t =0.765,0.704,0.996; all P ＜ 0.05) compared with control group (0.070 ± 0.014) ; the expression of fibronectin mRNA was much higher in 1,2,4 mg/L groups at 48 h (0.61 ±0.06,0.77 ± 0.07,0.77 ± 0.07) and 72 h(1.61 ± 0.14,2.54 ± 0.20,2.75 ± 0.22) compared with control group [0.48 ± 0.04(t =0.111,0.182,0.182,all P ＜ 0.05),0.97 ± 0.08(t =0.093,0.109,0.108,all P＜ 0.05) ].A lot of mineralized nodules could be seen under light microscope in 1.00 and 10.00 mg/L groups.Conclusions The expression of fibronectin in bone of fluorosis rats and in vitro cultured osteoblasts are increased,and fluoride also promotes the mineralization nodules formation of osteoblasts.These results suggest that fibronectin may regulate the process of bone mineralization,and possibly play a role in the development of skeletal fluorosis.