| Object:According to epidemiological data and our previous experimental observation,it’s confirmed that activated osteoblast and accelerated bone turnover are importantfeatures of skeletal fluorosis and they are the pathological basis of bone diversity.Besides, we found that low calcium diet is the main precipitating and aggravatingfactors of bone disease in skeletal fluorosis, and the overall low calcium-parathyroidhormone (PTH) secretion increased-intracellular Ca2+increase involved in thepathogenesis of skeletal fluorosis, which can be called as "calcium contradiction"(Calcium paradox). However, exact mechanism of above action is not clear.Parathyroid hormone related peptide (PTH-rp) has similar activities as PTH, and itplay an important role in human bone microenvironment. Additionally, it has animportant role in bone development of innate and acquired bone metabolism, and canregulate proliferation and differentiation of target cell in physiological andpathological conditions. Osteoblasts can synthesize PTH-rp and its receptor in anautocrine and paracrine manner to play an important role in bone formation. So far,studies of skeletal fluorosis have not yet involved in the pathogenesis of PTH-rp, andthus it is the one focus of our study. Calcium-sensing receptor (calcium sensingreceptor, CaSR) belongs to G protein-coupled receptor family, which is widelydistributed in the parathyroid glands, thyroid C cells, kidneys, bones and intestinesand other tissues in the body to maintain calcium homeostasis, regulating cell growth,as well as play a role in terms of differentiation and apoptosis. Experiments showedthat the CaSR have an important role on osteoblast differentiation and parathyroidfunction. During the study of mechanism of enhanced bone turnover in skeletalfluorosis, role of CaSR is worthy to be focused, but there is no related report on thisaspect by now. Therefore, we focuses on the content in this study.In our study, calcium ion of serum and cell culture supernatants, PTH, PTH-rpgene and protein expression are tested in vitro and in vivo. We try to explore molecular mechanisms of PTH, PTH-rp in promoting osteoblast function and boneturnover enhancement, and to observe changes of CaSR in fluoride-exposedosteoblasts and to investigate its possible role.Methods:In vivo, we replicated animal models of skeletal fluorosis by application ofsodium fluoride (NaF) and dietary calcium for2months, then serum calciumconcentration, gene and protein expression of PTH, PTH-rp, CaSR, OPG, RANKLin rat bones were tested.In vitro, the osteoblast cell line (MC3T3-E1) was treated by different fluorineconcentration (2mg/L F-,5mg/L F-,10mg/L F-) for48h, then proliferationactivity of cells were tested by CCK-8, and calcium ion concentration of cellsupernatant, mRNA and protein expression of PTH, PTH-rp, CaSR were tested byRealtime-PCR and Western Blot.Result:1. the in vivo experiments:1) Compared to the calcium-rich balance control (ctrl) group, serum calciumconcentration of the calcium-rich balance and fluoride group (G1group) reduced,PTH mRNA expression was significantly decreased while PTH protein expressionwas significantly increased (P<0.01). In addition, PTH-rp mRNA and protein weresignificantly lower (P<0.05), and CaSR mRNA and protein expression wassignificantly increased (P<0.05); mRNA expression of CT decreased but proteinexpression was increased (P<0.01); OPG, RANKL mRNA and protein expression,RANKL/OPG mRNA and protein ratios were increased in bone tissue.2) Compared to the eclipse low calcium group (G2group), serum free calciumconcentration of eclipse low calcium and fluoride group (G3group) was significantlyreduced, mRNA and protein expression of PTH and PTH-rp were significantlyincreased (P<0.01, P<0.05), CaSR mRNA and protein expression was significantlyincreased (P<0.01, P<0.05), CT mRNA and protein expression was significantlyreduce (P<0.01), OPG mRNA expression was significantly increased (P<0.01);RANKL mRNA obvious decline, RANKL/OPG mRNA ratio was significantlydecreased. OPG and RANKL protein expression of G3group was increased, RANKL/OPG protein ratio was decreased significantly compared with G2group.2. In vitro experiments: 1) compared to the control group (C group), supernatant free calciumconcentration of F2group was significantly higher (P<0.01), while that of F5, F10group were decreased (P<0.05, P<0.01).2) In this experiment, a lower fluorine concentration (2mg/L F-,5mg/L F-)stimulates cell activity, whereas high doses fluoride (10mg/L F-) lead to thecytostatic activity.3) compared with C group, PTH, PTH-rp mRNA and protein expression of F2,F5group were significantly lower, but that of F10group was significantly higher(P<0.05);4) compared with C group, CT mRNA and protein reduced in each group, whileCaSR mRNA and protein increased (P<0.05, P<0.01).Conclusion:1. Calcium ion concentration outside the OBs is closely associated with fluorideconcentration.2. fluoride effects on osteoblasts in a dose dependent manner.3. Under fluorine effect, either in vivo or in vitro, PTH, PTH-rp gene and proteinexpression were significant fluctuated, and PTH protein was enhanced in the richcalcium balanced diet group, but it was inhibited in other groups. In the high dose offluoride (10mg/LF-), PTH, PTH-rp gene and protein levels of osteoblasts showedhigher expression (P<0.01);4. CaSR gene and protein expression in vitro and in vivo were significantlyincreased.5. CT gene expression in vivo and in vitro were inhibited by fluoride,but theexpression of CT protein exists differences in vivo and vitro, the reasons are worthy tobe explored.6. OPGL/OPG mRNA expression and protein expression, and the ratio of themwere different in the calcium-rich balance group and eclipse hypocalcemia group,which were significantly increased in the former group, while they were opposited inthe latter one.These results suggest a dose of fluoride, whether in vivo or in vitro, can lead tosignificant fluctuations in PTH and PTH-rp. This change in the body is affected byboth dietary calcium content, and regulated by complex metabolic networks in vivo.However, the general trend is fluoride can lead to significantly enhanced expression of PTH and PTH-rp, it suggests that overall low calcium stimulates PTH secretion anda series of subsequent changes has an important position in the pathogenesis offluorosis. Stimulating effect of fluoride on CaSR in vivo and in vitro suggested thatCaSR may holds an important position in the pathogenesis of skeletalfluorosis——overall hypocalcemia-increased secretion of PTH-osteoblasts Ca2+increases. Inhibition of fluoride on CT and stimulation or inhibition of that on theRANKL/OPG mRNA ratio supplied further evidence to the important role offluoride in bone turnover and synergy of dietary hypocalcemia in the occurrence offluorosis. |
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