Effects Of Calcium Homeostasis Of Osteoblast In The Development Of Skeletal Fluorosis

Endemic fluorosis is an emphasis prevention and cure endemic in our country.The skeletal injury of it includes osteosclerosis、osteomalacia、osteoporosis and softtissue ossification. The pathogenesis of skeletal fluorosis is unclear. That the activityof osteoblast function was an early link which played a leading role was confirmed byobserving a lot of animal and in vitro cell culture. In the last few years, our researchgroup and domestic and overseas related research has proved that the genesis anddevelopment of skeletal fluorosis may be related to the changes of OB calciumhomeostasis. The maintenance of calcium homeostasis involved a series ofcomplicated pathogenesis. Ca²⁺increased in cell （Essentially in the cytosol）, includingthe increase of Ca²⁺internal flow outside the cell; Ca²⁺reverse concentration in cellwas stuck when it moved to outside plasma membrane; The release of Ca²⁺inintracellular calcium store increased and the mechanism of calcium store reverseconcentration’s uptaking Ca²⁺was stuck. Proposed from the aspects of the second,this study tried to prove that fluorine lead to the increase of OB Ca²⁺concentration byinfluencing the Ca²⁺transport from intracellular to extracellular. Thus the functions ofosteoblasts of stimulating OB increased significantly.Methods: Mice osteoblast cell lines（MC3T3-E1）were used in this study asresearch objects. Experiment was divided into the control group、exposed to fluoridegroup1、 exposed to fluoride group2and exposed to fluoride group3, and theconcentration of fluoride respectively were0、1、5、10mg/L F. The times of fluorideexposure were4h and48h。The methods of fluorescence quantitative、real-time quantitative PCR andELISA were used to detect OB calmodulin（CaM）、plasma membrane Ca²⁺-ATPase（PMCA）、Ca²⁺-Na⁺exchange carrier（NCS）and the expression of cbfa-1inmRNA and protein levels. Explore whether elevated dye fluorine OB Ca²⁺ concentration by influencing the bone-forming cell membrane Ca²⁺pump, andanalyzes the change and the relationship between the bone growth factor expression.Findings:1. When cells exposed to fluoride4h, OB Ca²⁺concentration increased obviously inevery fluoride exposure groups comparing with the control group.2. Compared with the control group, expression of CaM decreased greatly in the4-hour fluorine exposed5mg/L group and10mg/L group but increased greatly in the48-hour fluorine exposed5mg/L group and10mg/L group, while CaM protein in the48-hour fluorine exposed5mg/L group increased greatly.3. PMCA4B-1in fluoride exposure4h, every fluoride exposure group was less thancontrol group; But in fluoride exposure48h, the expression of PMCA4B-1mRNA andprotein in every fluoride exposure group were higher than control group.4. The expression of NCS3was was higher in every fluoride exposure group;5. The expressions of RUNX2in all fluoride exposure5mg/L group and10mg/Lgroup significantly increased.ï¼›6. Exposure fluoride48h, the expression of OCN was increased significantly ingroup of5mg/L.Conclusions：1. Under this experimental condition, Ca²⁺concentration increased significantly whenOB was excited by fluoride, and that further proved the stimulating effect of thefluoride on osteoblastï¼›2. The effect of short fluoride exposure time（4h） for CaM in OB mainly wasinhibition, and extending fluoride exposure time to48h, the effect mainly wasstimulation. At the same time, PMCA4B-1as a calmodulin-dependent enzymes alsochanged with the changes of CaM. That is to say, fluoride exposure4h, the expressionof PMCA4B-1reduced, and fluoride exposure48h, the expression of PMCA4B-1 increased. The results further demonstrated the regulatory role of the CaM toPMCA4B-1;3. The increase of RUNX2and OCN expression levels represented the enhancementof osteoblast function.Summing up the results, we can draw a clear route map for the role of fluoride:Fluoride could lead to the descent of PMCA4B-1expression levels by inhibitingthe expression of CaM, therefore it affected Ca²⁺running to the extracellular, and thefinal result was the increase of OB Ca²⁺concentration. The increase of OB Ca²⁺concentration could stimulate OB function enhancement. It acted as the expressionenhancement of RUNX2and OCN. The final result was the enhancement ofosteoblast function.The above study will suggest that calcium homeostasis imbalance is in a pivotalposition in the molecular mechanisms of fluorosis bone damage, and it will providenew arguments for the study of the occurrence mechanism of skeletal fluorosis.