| IntroductionEndemic fluorosis is a systemic chronic toxic poisoning by long - term over ingested fluoride with dental fluorosis and skeletal fluorosis as its two main symptoms. In China, endemic fluorosis is one of the most serious endemic diseases. There is no available therapy for endemic skeletal fluorosis due to its complex pathogenic mechanism. The main character of pathology in skeletal fluorosis is the increasing bone turnover. Osteoblasts, osteoclasts and many kinds of cytokines are involved in bone turnover and become the research hotspots related to skeletal fluorosis. Osteoprotegerin ( OPG) is a new cytokine involved in the bone turnover, which is secreted by osteoblasts and plays an important role in osteoclastogenesis. The biological functions of OPG in skeletal fluorosis onset are still unclear. This experimental study aimed to explore the relationship between OPG and osteosclerosis in chronic fluorosis rats by observing the changes of serum chemical biomarker, bone mineral density, bone morphology and the expression of OPG in bone after fluoride - treated for 3 months.Materials and Methods1. Animals and their groupingAfter weaned one week 32 female Wistar rats were randomly classified into 4 groups. Fluoride was administered to rats through drinking water with 4 dosesof 0,25,50 or 100 mg F-/ L for a period of 3 months.2. Fluoride ion selective electrode method was used for measurements of rats'serum, bone and urine fluoride concentrations.3. Serum alkaline phosphatase, calcium, phosphor were analyzed using an auto analyzer.4. Bone mineral density of rat thighbone was measured with dual energy x - ray absorptiometry.5. The tibias of rats were embedded in paraffin for HE staining and immu-nohistochemical staining. Sections were stained by rabbit anti - rats OPG polyclonal 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. All of the threshold area percent of staining, the integrated optical density, the mean optical density, and the grey were measured using computer - assisted image analysis system.6. Statistical analysis method: One - way ANOVA and Pearson correlation were performed with SPSS 11.5 software package.Results1. Until the end of the experiment, all rats in three fluoride - treated groups occurred in different degrees of dental fluorosis.2. The levels of fluoride in serum, bone, urine in fluoride - treated rats were significantly higher than controls (P <0.05).3. The levels of serum alkaline phosphatase and phosphorus in fluoride -treated rats and controls had no significant differences ( P > 0. 05). The serum calcium level of high F group was increased (P <0.05).4. The bone mineral density was increased in fluoride - treated rats, though the difference had no statistical difference(P >0.05).5. Morphology and immunohistochemistry 5.1 H -E stainingIn control group rats, the thickness of tibial cortex was equable, and there were many small stained blue bone cells in cortex. The ambit of the marrow cav-ity and cortex was clear. The structure of bone tissue in fluoride - treated rats had been changed. The ambit of cortex and marrow cavity was unclear. The trabecular bones were significantly increased.5.2 ImmunohistochemistryOPG was expressed in osteoblasts near the margin of bone cortex and bone matrix cells in marrow cavity. The cell membranes were stained but the nuclei not. The numbers of osteoblasts in bone cortex in the fluoride - treated rats were increased, and the hyperplasia of osteoblasts was remarkable. The expression of OPG in bone matrix cells was also enhanced. The stained cells were closely arrayed on the surface of hyperplastic trabecular bones.6. Semiquantitative analysis of OPG expressionThe expression of OPG in bone tissue was significantly enhanced in fluoride - treated rats than that in controls ( P <0.05).7. Correlation analysis of bone OPG to bone mineral density, bone fluoride and serum alkaline phosphataseThere were significant relationships of the expression of OPG in bone with both bone mineral density and the bone fluoride ( P <0.05). But there was no significant relationship between OPG and serum ALP ( P >0.05).DiscussionOsteoprotegerin (OPG) is a new soluble member of TNFR family and an important cytokine in bone turnover. It functions in inhibitions of osteoclastogen-esis and activation. In chronic fluorosis, the bioactivity of OPG and its role in the pathogenic mechanism of skeletal fluorosis is still unclear. This study aimed to explore the effect of OPG in skeletal fluorosis in rats.By the end of the experiment, all of the fluoride - treated rats occurred in dental fluorosis, and the levels of fluoride in serum, bone, and urine were significantly increased compared with controls. The rats presented chronic fluorosis. The pathological changes of fluorosis are complex, including osteosclerosis, osteolysis and osteoporosis. In this study, the rats were treated with fluoride and enough nutrition. The morphology of bone tissue showed the characters of osteo-sclerosis. The thickness of bone cortex and the volume of trabecular bone were markedly increased. These results were consistent with the report that the changes of bone morphology in the chronic fluorosis rabbits by Turner.In this study, the hyperplasia of osteoblasts in fluoride - treated rats was obvious. The activity of bone forming was increased and resulted in the increase of bone volume. At the same time, the bone mineral density in fluoride - treated rats seemed to be enhanced. Accompanying with the hyperplasia of osteoblasts, the expression of OPG in bone tissue was enhanced in fluoride - treated rats, and the intensity of expression was significantly correlated with both the bone mineral density and the bone fluoride level. In other animal studies, transgenic mice expressing OPG exhibited a general increase in bone density, associated with a decrease in bone resorption. Because exogenous OPG is specific to bone tissues, the increasing of bone mineral density and bone volume were exhibited, endogenous OPG may act only on bone tissues in the way of endocrine to inhibit the osteoclastogenesis and activation.The significant role of OPG on the regulation of osteoclastogenesis may function through the OPG/RANKL/RANK system. OPG is the competitive inhibitor of RANK, which binds to RANKL. RANKL is critical for differentiation, activation and survival of osteoclasts. OPG inhibits osteoclastogenesis by blocking the effect of RANKL. In this study, we found the expression of OPG in bone tissue had significant relationships with bone fluoride in fluoride - treated rats. Thus, it might be inferred that within the micro - circumstance of bone tissue in skeletal fluorosis, fluoride stimulated osteoblasts to over - express OPG, then triggered a series of reactions, and ultimately resulted in the inhibition of osteoclasts activities and the weakening of bone resorption.In conclusion, in bone tissue of chronic fluorosis rats, fluoride might increase bone formation by stimulating the osteoblasts directly, and weaken bone resorption by inhabiting the osteoclasts through OPG which were secreted by osteoblasts through the inducement of fluoride. The ultimate result was the bone tissue manifesting the pathological characters of osteosclerosis.
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