Effects On Osteogenic Activity And MiR-23a In Bone Tissue Of Rat Treated With Fluoride

Objective: To establish a rat model of sodium fluoride exposure,and observe the effects of different fluorine concentrations and different times with treat fluorine on the osteogenic activity markers and mir-23 a in rat bone tissue.Methods:1.Establishment and verification of rat model poisoned by sodium fluoride:96 SD rats were randomLy divided into control group(0mg/L),low fluoride group(50mg/L),medium fluoride group(150mg/L)and high fluoride group(250mg/L)according to body weight.We take the drinking water poisoning means and eight SD rats in each group were executed in February,April and June,then the contents of urinary fluorine,blood fluorine,bone fluorine and dental fluorine in rats were detected by fluoride ion selective electrode method.Observe the occurrence of dental fluorosis regularly and take photos.2.Rats' right lower extremity were fixed,decalcified,rinsed,soaked in wax,embedded and sectioning in the epiphyseal region of the femoral shaft.Hematoxylin-eosin(HE)staining was performed and the bone histological changes were observed by light microscopy.3.Total RNA was extracted from the left forelimb of rats,and the mRNA expressions of ALP,Osx,CoL I,Runx2,OC,bmp-2and mir-23 a mRNA were determined by RT-PCR.4.Total protein was extracted from the right forelimb of rats,and the expressions of ALP,Osx,CoL I,Runx2 and bmp-2proteins were determined by Western Blot.Results: 1.The contents of urinary fluorine,blood fluorine,bone fluorine and dental fluorine increased with the increase of fluorine dose and Compared with the control group,the occurrence rate of dental fluorosis was higher in each dose group at different times.With the increase of the fluoride dose and the extension of the exposure time,the occurrence rate of dental fluorosis in rats showed an rising trend,and the degree of damage tended to beserious.2.With the increase of the fluoride dose,the thickness of trabecular bone decreased,the degree of atrophy increased,and the connection became more loose.Epiphyseal plate thickness decreased;Osteoblasts are decreasing;The trabecular area and epiphyseal plate area of rats in the medium and high fluorine groups and in each dose group was larger than that of the control group at different stages,while that of the low fluorine group was lower than that of the control group.Moreover,with the increase of fluorine exposure dose,the area of bone trabecular and epiphyseal plates showed an rising trend.The number of osteoblasts in each times was lower than that of the control group,and showed a decreasing trend with the increase of dose.With the prolongation of fluorination time,the area of bone trabeculae,epiphyseal plate and the number of osteoblasts in each group showed a decreasing trend.3.(1)The ALP mRNA expression levels in all the dose groups were higher than control group at different times,except the low fluorine group at 6 months.The expression of ALP mRNA decreased in the low fluoride group and the middle fluoride group,but increased in the high fluoride group.Osx mRNA levels in the groups with fluorine exposure at 2 and 4 months,with high fluorine exposure at 6months and with low fluorine exposure at 6 months were all higher than the control group,while those with high fluorine exposure at 6 months were lower than those in the control group.Osx mRNA expression increased in February,while decreased in April and June from mid-fluorine group.The mRNA expression levels of CoL I and Runx2 in all the dose groups were lower than those in the control group at different times,except for the low fluorine group at 2 and 4 months,which showed a decreasing trend with the increase of the exposure dose.The expression of OC mRNA in the low fluorine group at different times was higher than the control group.With the extension of time,OC mRNA expression showed an rising trend in the middle fluorine group,while decreased in the high fluorine group.The mRNA expression levels of bmp-2 in all the dose groups were higher than the control group at different periods,except the low fluoride group and the middle fluoride group at 6 months.The expression of bmp-2 mRNA decreased in all groups after fluorine exposure to 6months.(2)After fluorination for 2,4 and 6 months,the mirna-23 a mRNA expressionlevels of rats in each fluorination group were higher than the control group.At 6months of fluorination group,the mirna-23 a expression level of rats in each fluorination group was higher than that at 2 months and 4 months,showing an rising trend.4.The expression of bmp-2 protein in each dose group was higher than that in the control group when fluorinated for 2 months,showing an rising trend.At 4months,the group with low fluoride was higher than the control group,and the group with medium fluoride and high fluoride were lower than the control group,showing a downward trend.At 6 months,the low fluoride group was lower than the control group,and the high fluoride group was higher than the other groups.With the exception of the 4 month low fluorine group,the ALP protein expression in different times was higher than the control group,and the ALP protein expression in different times showed an rising trend with the increase of the exposure dose.CoL I protein expression in different times showed a decreasing trend with the increase of the fluorine dose.The Osx protein expression showed an rising trend in all dose groups compared with the control group at 2 months after fluorine exposure,and first increased to medium fluorine group and then decreased to high fluorine group at 4and 6 months after fluorine exposure.Runx2 protein expression was higher in the low fluorine group than in the other experimental groups at different times,and was lower in the high fluorine group than in the control group at 4 and 6 months,except for 2months.Conclusion: 1.The rat model of sodium fluoride exposure was successfully established.3.Sodium fluoride exposure has a complex effect on the osteogenic active markers in rat bone tissue.The expression of the osteogenic active markers is related to the fluorine dose and time of fluorine exposure,which has a significant bidirectional regulation.4.The change trend of mirna-23 a expression was associated with fluorine dose and fragments change in rats.It was speculated that mirna-23 a was involved in the process of abnormal bone transformation in rats induced by fluorosis,and mirna-23 a had a regulatory effect on a large number of physiological diseases,suggesting a potential correlation between mirna-23 a and fluorine-induced systemic harm.