RESUMO
Objective To investigate the effects of chronic fluorosis on the expressions of matrix metalloproteinase-9 (MMP-9) mRNA and protein and the differentiation and maturation process of bone cell in the osteoclast of bone tissue of rats.Methods According to body weight,thirty-six healthy SD rats(body mass 100-120 g) were divided into three groups by random number table,twelve in each group,half male and half female.The rats of control group were given tap water(NaF ＜ 1 mg/L),and rats of low-fluorine and high-fluorine groups were fed with tap water containing 5 and 50 mg/L NaF to establish chronic fluorosis model.Rats were sacrificed after eight months; the contents of urinary fluoride in 24 hours and bone fluoride were analyzed by fluoride selective electrode.Serum content of tartrate resistant acid phosphatase 5b(TRACP5b)was detected by enzyme-linked immunosorbent assay (ELISA).The paraffin section of bone tissue was stained by hematoxylin-eosin (HE) and pathological morphometry was observed under optical microscope.The protein and mRNA levels of MMP-9 in the osteoclast of bones were detected by immunohistochemistry (IHC) and in situ hybridization (ISH),respectively.Results The differences of fluoride contents of urine and bone in rats were statistically significant between groups(F =400.612,48.229,all P ＜ 0.05).Fluoride contents of urine and bone were increased in lowfluorine and high-fluorine groups[(6.09 + 0.56),(7.69 + 0.64)mg/L,(12.65 ± 3.07),(26.53 + 5.88)mg/kg] compared to the control groups[(1.36 ± 0.51)mg/L,(0.67 ± 0.16)mg/kg,all P ＜ 0.05],and the fluoride contents of urine and bone were gradually increased with increasing fluoride doses(all P ＜ 0.05).The difference of TRACP5b content in serum was statistically significant between groups (F =9.607,P ＜ 0.05),in low-fluorine and high-fluorine groups,the TRACP5b contents[(1.86 ± 0.13),(1.92 ± 0.22)U/L] were higher than that of control group [(1.57 + 0.20)U/L,all P ＜ 0.05].The pathological examination showed osteosclerosis in fluoride exposed groups.The differences of MMP-9 mRNA and protein expressions were statistically significant between groups (F =365.727,331.382,all P ＜ 0.05).Compared to the control groups(97.22 ± 2.24,78.51 ± 1.16),the expressions of MMP-9 protein(108.18 ± 1.97,119.28 ± 1.76) and mRNA(89.44 ± 2.86,102.14 ± 2.39) were increased(all P ＜ 0.05),and the expressions of MMP-9 mRNA and protein were gradually increased with increasing fluoride doses (all P ＜ 0.05).Conclusions Chronic fluorosis might influence osteoclast differentiation and maturation process through regulating the expression levels of MMP-9 protein and mRNA.