| Objective:This study aimed to investigate the relationship between the oxidative stress(OS)level of bone microenvironment of the type 2 diabetes mellitus(DM)rats and the Runx2 factor of the osteoblast differentiation.Meanwhile,we observed that whether adiponectin(APN)could affect the differentiation and function of osteoblast(OB)by inhibiting the level of bone marrow microenvironment(OS).Further more,we explored the pathogenesis and early intervention of diabetes osteoporosis(DOP),and to provide new ideas for clinical prevention and treatment of DOP.Methods:4 week old male SD rats were randomly divided into normal control group(n=18)and diabetic model group(n = 42).The rats in the control group were fed with normal diet.The rats in the model group were fed with high cholesterol and high glucose for 2 months,and then were injected a single intraperitoneal injection of streptozotocin of 30mg/kg to prepare type 2 diabetic model.The successfully-induced diabetic rats were divided into diabetic group(DM=18)and adiponectin intervention group(APN=18),then the rats in APN group were injected with APN 10 g/kg*d by intraperitoneal injection.The rats were sacrificed at 4,8 and 12 weeks after the intervention,removed the bilateral femur and tibia,extracted the bone marrow from one side of femur and tibia,and then obtained bone marrow cells and bone marrow supernatant by centrifugation.After bone density measurement and decalcification in EDTA,the other side of the femur and lumbar spine was embedded in paraffin and 4-7?m sections was cut.ELISA and RT-PCR were used to detect the expression of Runx-2,BALP and AOPPs in the bone marrow cells and bone marrow supernatant.Immunohistochemical staining was used to observe the expression of AGE of the bone tissue.HE staining was used to observe the bone microstructure(trabecular bone density and cortical thickness)and the amount of fat cells.Results: 1.Comparison of the blood glucose level in rats in each group:With the extension of feeding time,the level of blood glucose of DM group was significantly increased.After APN intervention,the level of blood glucose was improved.2.Comparison of the AOPP level in three groups of rat bone marrow:With the extension of feeding time,there was no significant change of AOPP in normal group.However,the AOPP level in the DM group and APN intervention group increased(P<0.05),but the increase of APN intervention group was smaller than that of DM group.At 4 weeks,the level of AOPP in the DM group was higher than that in the control group(P<0.05);there was no significant difference between APN group and DM group(P>0.05).At 8 and 12 weeks,the AOPP level were significantly higher in the DM group(P<0.05).After the intervention of APN,the level was lower than that of DM group(P<0.05),and the level decreased significantly in 12 weeks than in the 8 week.3.Comparison of the expression level of AGEs in bone tissue of three groups of rats:With the change of time,the expression of AGEs in bone tissue of normal rats was not significantly different(P>0.05);the expression of AGEs was increased in DM group and APN intervention group(P<0.05),but the increase amplitude of APN intervention group was smaller than that of DM group.At 4 weeks,the expression level of AGEs in the DM group was higher than that in the control group(P<0.05);compared with DM group,the expression of AGEs decreased after APN intervention(P<0.05).At 8 and 12 weeks,the expression of AGEs in DM group was significantly higher(P<0.05);compared with DM group,the expression of AGEs decreased after APN intervention(P<0.05),and the AGEs decreased significantly in 12 weeks than in the 8 week.4.Comparison of the expression levels of Runx2 in the bone marrow cells and the BALP level in bone marrow supernatant at three groups of rats:With the increase of feeding time,the Runx2 and BALP levels in normal control group,DM group and APN intervention group were gradually decreased,and the DM group decreased more significantly(P<0.05).At 4 weeks,there was no significant difference between any two groups;at 8 weeks,the Runx2 and BALP levels in the DM group were lower than those in the normal control group.After the APN intervention,compared with DM group,the descent amplitude of above markers decreased,and the levels increased(P<0.05).At 12 weeks,the Runx2 and BALP levels in DM group were significantly lower than those in control group,after the APN intervention,which increased than those in DM group.The difference between the APN group and the DM group was significantly higher than that in the 8 weeks.5.Comparion of the femoral bone mineral density(BMD)in three groups:The BMD values of the three group were decreased compared with the 4 week and the 12 week.At 4 weeks,the BMD difference was not statistically significant between any two groups(P>0.05).But at 12 weeks,the BMD in the DM group was lower than that in the control group,and it was significantly higher than that in the DM group after the APN intervention(P<0.05).6.Observe the structure of trabecular bone and the content of fat cells by HE staining of bone tissue sections :After 4 weeks of culture,the trabecular bone structure of the three groups was fair,and the amount of fat cells was less.However,at 8 and 12 weeks,the trabecular structure of the three groups showed different degrees of damage,especially in the DM group.Trabecular bone was thinning,fracture,partial trabecular bone structure disappeared and the medullary cavity enlarged,and the fat cells increased.After the APN intervention,the trabecular bone structure was restored,and the content of fat cells decreased,and these changes were more obvious at 12 weeks.Conclusion1.The level of OS in bone micro-environment of diabetic rats was significantly increased,which may have a direct inhibitory effect on the regulation of osteogenic differentiation factor Runx2,may cause the decrease of osteoblast differentiation and bone formation,and combined with the high level of AGEs,non enzymatic glycation of bone collagen affects bone quality and bone mineral density,which may be involved in the occurrence of diabetic osteoporosis,all of which may be involved in the occurrence of diabetic osteoporosis.2.Adiponectin can improve glucose metabolism,reduce AGEs production,and inhibit the level of oxidative stress in bone microenvironment of diabetic rats,which may play a protective role in bone.But specific functions still need further study. |
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