| Glucocorticoid-induced osteoporosis is the most frequent secondary osteoporosis, Osteoporotic fractures often result in delayed union and nounion. The ratio of disability is very high, which is a serious problem in the orthopaedic domain of the today' s world. The pathogenesis of glucocorticoid-induced osteoporosis is not clear now, and there is not an effective therapy. Obviousely, to study how to prevent glucocorticoid-induced osteoporosis is very necessary. In vitro and in rodents, this experiment has studied the effectiveness of nitric oxide (NO) which may be regulated by simvastatin on glucocorticoid-induced osteoporosis, and simvastatin can prevent the osteoporosis induced by glucocorticoid.Material and method1. Experiment in vitro: The newborn SD rats' skull was taken out and osteoblast had be separated from it, osteoblast was randomly divided into 3 groups (8 samples for each group): Group A was fed with culture medium RPMI 1640 of simvastatin of 10~-7mol/L, dexamethasone of 10~-7mol/L and DMSO of 0.1%; Group B was fed with culture medium RPMI 1640 of dexamethasone of 10~-7mol/L and DMSO of 0.1%; Group C was cultured to add only DMSO of 0.1%. After 96 hours, all the groups was observed the expression of eNOS mRNA and osteocalcin mRNA by ReverseTranscription-Polymerase Chain Reaction, the ratio of proliferation of osteoblast was measured using MTT reduction assay, the amount of NO in culture medium was detected by the method of nitric acid reduction enzyme.2. Experiment in rodents: Thirty-six male SD rats, weighted 0.45±0.02kg each, were randomly divided into 3 groups (12 rats for each group): Experiment group, given simvastatin 10mg/(kg-d) through gastric tube and dexamethasone 2.5mg/kg twice a week through intramuscular injection; Model group, given 0.9% natrium chloride 2ml/(kg-d) through gastric tube and dexamethasone 2.5mg/kg twice a week through intramuscular injection; Control group, given 0.9% natrium chloride 2ml/(kg-d) through gastric tube and 0.5ml/kg twice a week through intramuscular injection. After the experiment lasted for 8 weeks, using Norland Dual Energy X-ray Absorptiometry, the bone mineral density of the third lumbar vertebral bodies of all the rats was measured, the structure of bone trabeculae of the third lumbar vertebral bodies were observed, the bone histomorphometric study of left femur was analyzed, immunohistochemistry was used to study the expression of iNOS and eNOS that the image analysis was done to measure the grayscale and average integral optical density, and the serum levels of Ca, P, ALP and NO were measured.Result1. Compard with group B, the expression of eNOS mRNA in group A increasedsignificantly, there was significant defference between group A and group B (PO.01), and no significant defference was observed in group A and group C (p>0.05). The expression of eNOS mRNA of group B is lower than that of group C, there was significant defference between the two groups (PO.01).2. The expression of osteocalcin mRNA of group A was much higher than that ofgroup B, there was significant defference between group A and group B (PO.01), and there was no significant defference between group A and group C (p>0.05). The expression quantity of osteocalcin mRNA in group B was decreased remarkably than that of group C, there was significant defference between thetwo groups (PO.01).3. The ratio of osteoblast proliferation in group A was significantly higher than that of group B, there was significant defference between group A and group B (PO.Ol), and no significant defference was observed in group A and group C (p>0.05). The ratio of proliferation of osteoblast in group B is lower than that of group C, there was significant defference between the two groups (P<0.01).4. In the culture medium, the amount of NO of group A is higher than that of group B, there was significant defference between group A and group B (PO.05), and no significant defference was observed in group A and group C (p>0.05). The amount of NO in group B was declined sharply which was compared with that of group C, there was significant defference between the two groups (PO.05).5. Compard with the control group, the values of bone mineral density of the model group decreased significantly, there was significant defference between the two groups (PO.01), which proved that the glucocorticoid-induced osteoporosis rat model was developed successfully by intramuscular dexamethasone. The values of bone mineral density in the experiment group were significantly higher than that of the model group, there was significant defference between the two groups (pO.Ol). No significant defference was observed in bone mineral density between the experiment group and control group (p>0.05).6. The structure of bone trabeculae of the experiment group was similar to that of the control group, and the model group presented the feature of osteoporosis.7. ALP levels of the experiment group was significantly decreased compared to the corresponding value of the model group, there was significant defference between the two groups (pO.Ol), and no significant defference was observed in the experiment group and control group (p>0.05). ALP levels of the model group was significantly increased compared to the corresponding value of the control group, there was significant defference between the two groups (pO.Ol).8. The serum levels of Ca, P, ALP and NO had no marked defference among theexperiment group, model group and control group (p>0.05).9. In the three groups, the expression of iNOS was negative which was observed by immunohistochemistry method.10. Using the immunohistochemistry mothod to detect the influence of simvastatin and dexamethasone on the expression of eNOS, the experiment group and control group were strong positive, whereas, the model group was weak positive. Compard with the model group, the grayscale and average integral optical density of the experiment group increased 5.7% and 13.5%, there was significant defference between the two above groups(P<0.01). The grayscale and average integral optical density of the model group which compard with the control group decreased 6.4% and 13.4%, there was also significant defference between them(P<0.01), however, the grayscale and average integral optical density had no defference between the experiment group and control group(p>0.05).11. In the bone histomorphometrical study, the average of bone volume, osteoid surface, osteoid thickness, and osteoblast surface of the experiment group were significantly higher than that of the model group, there was significant defference between the two groups (p<0.01), however, no defference was observed in the bone absorption surface, mineral apposition rate, adjusted apposition rate and mineralization lag time among the three groups (p>0.05).Conclusion1. Dexamethasone restrains significantly the expression of eNOS mRNA and osteocalcin mRNA in osteoblast, decreases the proliferation of osteoblast and the amount of NO in the culture medium. Dexamethasone declines significantly the expression of eNOS and the amount of bone volume, osteoid surface, osteoid thickness, osteoblast surface of rats, it results in the lower amount of bone formation of rats. It proves that the glucocorticoid-induced osteoporosis rat model had been developed successfully by dexamethasone.2. It demonstrates that the lower expression of nitric oxide depended on eNOS is animportant pathogenesis of glucocorticoid-induced osteoporosis. 3. Simvastatin is resistant to the restraint of dexamethasone, enhances the expression of eNOS mRNA and osteocaicin mRNA in osteoblast. Simvastatin inreases the expression of eNOS and the amount of bone volume, osteoid surface, osteoid thickness and osteoblast surface of rats, it results in the higher amount of bone formation of rats. Simvastatin can prevent the osteoporosis induced by glucocorticoid. |
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