Effect Of Fluoride On Insulin Secretion And Skeletal Pathologensis As Well As Their Relationship

Background:It is a valuable research question that how osteoblasts were activated in chronicfluorosis. Researchers still studied bone tissue and insulin regulation by fluorine andinvestigated the pathological processes in the pathogenesis of endemicfluorosis.Through culturing osteoblasts and animal exposed to varying dose offluordie to investigate the changes of oxidative stress (SOD, MDA, GPX, etc.) fromfluorosis, and detected fluctuation of glucose, lipids, serum calcium and serum insulinin Wistar rats treated by fluoride. We also analyzed the relationshio between thesedata and index of bone formation. Thus we detected the changes of osteoblastproliferation and differentiation-related gene expression and regulatory factors. Thecompletion of this study was helpful to understand the intrinsic correlation betweenfluoride as well as the interaction of bone and insulin.Methods:Animal experiments in vivo:100Wistar rats were dividedevenly into control group, low-fluoride group and high-fluoride group, accordingto the weight. Fluoride was given by gavage to rats, with NaF (2.21kg/L) dissolved indistilled water, equal to100mg/mL fluoride. Rats in low-fluoride group were givenfluoride by gavage, with10mg F-/kg per day, while high-fluoride group20mg F-/kgper day, and control group gavaged by distilled water. After administration offluoride for1,2and3month, respectively, then rats were treated with ether to collectblood for biochemical testing. One femur was treated with10%EDTA,conventional dehydration, embedded in paraffin, and made intopathological section. The other femur was used to conduct Real time-PCR to test theexpression of related genes.Detection for cell viability of MC3T3-E1cells in vitro: cells were plated into96-well plates, and each period was divided into control group and0.1,1.0,2.0,4.0, 8.0,16.0mg F-/L group, using CCK-8for cell viability assay. According to the trendof cell activity, we selected2,8and20mgF-/L as representative concentrations. Theexperimental groups were added50nmol insulin/L/well combined administration withfluorine. Each group was set up eight wells. They represented low, medium and highdoses of fluoride condition to interfe osteoblast. Cells were implanted into24-wellplates and divided into control group,2,8and20mgF-/L group for Anti-insulinreceptor immunofluorescence staining and real time PCR.Results:Fluorosis animal models through gavage means accurately developedcharacteristic osteopathology of skeletal fluorosis. Using morphological,immunofluorescence and RT-PCR, we observed insulin, transcription factors,biochemical factors and cytokines related skeletal fluorosis, which were involved inthe process of changes of osteoblast function induced by different concentrations offluoride. Results of bone pathology and osteogenesis related genes expressionconfirmed pathological changes in skeletal fluorosis that bone resorption dominatedearly, bone formation/bone resorption actived metaphase and the former dominatedfinally.Detection of CCK-8to MC3T3-E1cells in vitro activity: fluoride had dual effecton osteoblast that low-dose fluoride induced cell activity, while high-dose fluoridesuppressed cell activity. The low-dose fluoride stimulated osteogenic factorsexpression in osteoblast while high-dose fluoride inhibited them, which changesindicated dual effect of fluoride on osteoblastic activity.Conclusions：The expression of ALP and Runx2was enhanced, besides the expression ofinsulin receptor and IGF-Ⅰ is similar to the change of ALP and Runx2, suggestingthat insulin receptors and IGF-Ⅰ are involved in the osteogenic mechanism offluoride.Fluoride did not cause significant oxidative stress early, and caused mild decompensation of oxidative stress in the later phase indicating oxidative stress wasnot involved in the early process of fluoride-induced bone turnover. Rats withabnormal secretion of insulin showed metabolic indicators, such as glucose and lipid,were in the compensatory range, indicating the influence of fluorine on the islets didnot cause the decompensation of body’s metabolism.Experiments in vitro confirmed that the combinated treatment of insulin andfluoride caused obviously osteoblast proliferation, low and medium dose fluoridedirectly induced expression of insulin receptor in osteoblasts, which suggested thepossible role of insulin signaling pathway in fluoride-induced osteogenic mechanisms.The fact that fluorine stimulated osteogenesis accompanied by the enhancing ofinsulin secretion and the increased expression of insuliln receptor in bone tissue invivo or osteoblastic cells in vitro indicated that enhancing of insulin signal perhapsplayed an important role in mechanism of fluoride-induced osteogenesis.