| IntroductionEndemic fluorosis is a whole - body chronic poisoning disease caused by long - term excessive consumption of fluoride. Dental fluorosis and skeletal fluorosis are the main symptoms of chronic fluorosis. Fluorosis is a most severe and widespread emdemic disease in the world. Fluoride level is not the only cause of fluorosis, other factors, such as lack of calcium in foods are also involved in development of fluorosis. Osteoprotegerin ligand( OPGL) is a new -founded cyto-kine involved in bone turnover. In the animal experimental study we copied the drinking - water - type chronic fluorosis animal models under the conditions of normal calcium and low calcium and observed the changes of fluoride levels in bone, serum and urine, bone morphology and the expression of OPGL in bone to explore the relationship between OPGL expression and chronic fluorosis under two calcium nutritional conditions.Materials and Methods1. Animals and their groupingSixty 80g ± 10g Wistar rats weaned one week later were randomly classified into normal calcium group and low calcium group. Then the two groups were divided into control, middle F, and high F subgroups with 0, 50 and 100 mg/L F~- in distilled water, respectively. After 3 months the rats were killed for various samples.2. Fluoride ion selective electrode method was used for measurements of rats' fluoride concentrations in bone, serum and urine.3. The tibias of rats were embedded in paraffin for HE staining and immu-nohistochemical staining. Sections were stained by rabbit anti - rats OPGL poly-clonal antibody and goat anti - rabbits IgG. After immunohistochemical staining , sections were analyzed in a random order by computer - assisted image a-nalysis, analyzing 5 high power fields for each section. The grey value and the mean optical density were measured by computer - assisted image analysis system.4. SPSS 12. 0 software package for Windows was used to gather and analyze the experimental data.Results1. During the experiment, the rats' general status in normal calcium group was better than the rats' in low calcium group. Most of the rats in low calcium group had dental fluorosis before the rats in normal calcium group. Until the end of the experiment, all rats in the fluoride - treated groups had dental fluorosis in different degrees.2. While fed with the same fluoride concentration, the rats' bone, serum and urine fluoride levels in fluoride - treated groups in the status of low calcium were significantly higher than those in the status of normal calcium .3. Morphology and immunohistochemistry3. 1 HE stainingThe bone tissue structure in fluoride - treated rats had changed in the states of both normal calcium and low calcium. With the increasing fluoride concentrations in rats' drinking water, the bone cells quantities became more and more. The cortex bone cells were in disorder in low calcium group.3.2 ImmunohistochemistryOPGL was mainly expressed in some osteoblasts and bone matrix cells in marrow cavity. The cell membranes were stained but the nuclei not. The expression of OPGL in the fluoride - treated rats was enhanced in bone cortex. The difference of the expression of OPGL between the rats in normal calcium group and the rats in the low calcium group was not very clear under the microscopy.4. Semi - quantitative analysis of OPGL expressionThe expression of OPGL in bone tissue was significantly enhanced in fluoride — treated rats than in controls in both normal calcium group and the low calcium group( P <0.05). At the same fluoride concentration in fluoride - treated group, the OPGL expression was different in the different states of calcium. 5. Correlation analysis of bone OPGL with bone fluoride There was significant relationship of the expression of OPGL in bone with bone fluoride level (P <0. 05) in the states of both normal calcium and low calcium.DiscussionThe drinking - water - type chronic fluorosis rat models in the different states of calcium was copied in this study. And this study aimed to explore the expression of OPGL and relationship between OPGL and bone changes in chronic fluorosis rats in the different states of calcium.Levels of fluoride in bone, serum and urine were significantly increased compared with their respective control in both normal calcium group and low calcium group. In low calcium group, the rats' state was bad, and most of the rats had dental fluorosis before the rats in normal calcium group. With the same fluoride concentration in fluoride -treated rats, fluoride levels of bone, serum and urine were higher in low calcium group than those in the normal calcium group. All this suggested that low calcium increased the absorption of fluoride and the cumulation of bone fluoride.Osteoprotegerin ligand(OPGL) is a cytokine that regulates osteoclasts differentiation and activation. And OPGL is necessary and sufficient for bone re-sorption in osteoclasts. Mice with a knocked - off OPGL gene showed severe os-teopetrosis and a defect in tooth eruption, and completely lack osteoclasts as a result of an inability of osteoblasts to support osteoclastogenesis. In other animal studies, results implied that fluoride maybe enhance the activity of osteoclasts by up - regulating the expression of osteoblastic OPGL mRNA.In this study, the expression of OPGL in bone tissue in fluoride - treated rats was significantly more than that in control rats in the states of both normalcalcium and low calcium. And OPGL expression had significant relationship with bone fluoride. That suggested fluoride could enhance the activity and proliferation by increasing the expression of osteoblastic OPGL. In addition, there was significant difference of the expression of OPGL in fluoride - treated rats between normal calcium group and low calcium group. That indicated the state of calcium might have some effect on the expression of OPGL.This study was based on the study of relationship between bone osteoprote-gerin (OPG) and osteosclerosis in chronic fluorosis rats by my previous postgraduate. OPGL levels might associate with OPG levels to function. The balance of the two would determine whether osteoclastic or osteoblastic activity dominates.ConclusionsA drinking - water - type chronic fluorosis rat model in the different states of calcium was successfully copied in the animal experimental study. The expression of OPGL in bone tissue was markedly increased in fluorosis in the states of both normal calcium and low calcium. There were also significant relationships of OPGL with bone fluoride in the different states of calcium. We concluded that OPGL might be play an important role with OPG together in the process of bone changes in chronic fluorosis. And the states of calcium might have some effect on the expression of OPGL.
|
PDF Full Text Request