| Objective:To explore the effect of T2DM on bone formation and the mechanism of Sirtlmediated Wnt3a/β-catenin pathway in exercise-promoted bone formation in T2DM.Methods:After 1 week of adaptive feeding,70 4-week-old C57BL/6 male mice were randomly divided into normal control group（ZC,12 mice）and T2DM model group（58 mice）.The ZC group was fed with normal feed,and the T2DM group was fed with high-fat feed.After 8 weeks of feeding and fasting for 24 hours,the T2DM model group was injected with streptozotocin（STZ）（at 110 mg/kg）,and the ZC group was injected with citric acid/sodium citrate solution（at 110 mg/kg）.3 days after STZ injection,the mice were fasted for 12 hours,and the blood glucose concentration in the tail vein of the mice was detected by using the Abbott Antoine blood glucose meter,blood glucose concentration≥16.7 mmol/L is a T2DM mouse.53 T2DM mice were successfully established and randomly divided into T2DM control group（TC,13 mice）,T2DM+inhibitor group（TY,13 mice）,T2DM+downhill running group（TP,14 mice）,T2DM+inhibitor group+Downhill running group（TYP,13 mice）.The ZC group and T2DM mice were fed with normal chow and high-fat diet,respectively,with a day-to-night ratio of 1:1,with free access to water,and exercise intervention according to the training program.After 8 weeks,the body weight of the mice in each group and the change in body weight within 8 weeks were measured;the glucose tolerance level of the mice in each group was tested by gavage;the blood glucose and insulin levels of the mice in each group were detected,and the insulin resistance index and insulin sensitivity index were calculated;The wet weight of the tibia in each group was measured with a scale,and the length of the tibia,proximal sagittal width,proximal coronal width,mid-sagittal width,mid-coronal width,distal sagittal width,distal sagittal width and distal End-coronal axis width;paraffin tissue sections of mouse tibia and femur,HE staining and safranin fast green staining were performed to observe the changes in bone tissue morphology and structure of mice in each group;MicroCT scans the distal femur of mice in each group to observe Changes in bone histometry indexes of mice in each group;primary culture of mouse bone marrow BMSCs was performed,and ALP staining was used to observe the OB differentiation of mice in each group after differentiation;serum of mice in each group was collected to detect ALP activity and calcium ion in serum,phosphorus ion concentration;RT-PCR detection of Sirtl,Wnt3a,β-catenin,Runx2 mRNA expression in mouse bone;Western Blotting detection of Sirtl,Wnt3a,βcatenin,Runx2 protein expression in mouse bone in each group.Results:（1）Compared with the ZC group,the body weight,AUC,and insulin resistance index of the TC group were significantly increased（p<0.01）,while the fasting insulin and insulin sensitivity index were significantly decreased（p<0.01）,Tibial wet weight,Tibial length,distal sagittal width,distal coronal width,mid-sagittal width,proximal sagittal width,and proximal coronal width were significantly decreased（p<0.05 or p<0.01）,the number of trabecular bone was significantly decreased,the thickness of cortical bone was significantly decreased（p<0.01）,the BMD,BV/TV,Tb.Th of cortical bone were significantly decreased（p<0.05 or p<0.01）,cancellous bone BMD,BV/TV,Tb.Th were significantly decreased（p<0.01）,Tb.Sp was significantly increased（p<0.01）,AKP staining was decreased,Serum ALP activity was significantly increased（P<0.01）,calcium and phosphorus ion concentrations were significantly decreased（P<0.01）,the mRNA and protein expressions of Sirtl,Wnt3a,βcatenin,Runx2 in bone were significantly decreased（P<0.01）.（2）Compared with the TC group,the body weight,AUC and insulin resistance of the mice in the TY group were significantly increased（p<0.05）,and the insulin level and insulin sensitivity were significantly decreased（p<0.05）,Tibial wet weight,Tibial length,distal coronal width,and proximal coronal width were significantly decreased（p<0.05 or p<0.01）,the number of trabecular bone in femur and Tibia was further reduced,the thickness of cortical bone was significantly reduced（p<0.05）,the BMD,BV/TV and Tb.Th of cortical bone were significantly reduced（p<0.05）,and the BMD and BV/TV of cancellous bone were significantly reduced（p<0.05）,Tb.Sp was significantly increased（p<0.05）,AKP staining was further decreased,Serum ALP activity was significantly increased（P<0.01）,calcium and phosphorus ion concentrations were significantly decreased（P<0.01）,and the mRNA and protein expressions of Sirtl,Wnt3a,β-catenin,Runx2 in bone were significantly decreased（p<0.05 or p<0.01）;the body weight,AUC,and insulin resistance of mice in the TP group were significantly decreased（p<0.01）,and the insulin level and insulin sensitivity were significantly increased（p<0.05 or p<0.01）,Tibial wet weight,tibial length,distal sagittal width,distal coronal width,mid-sagittal width,and proximal coronal width were significantly increased（p<0.05 or p<0.01）,the BV/TV and Tb.Th of cancellous bone were significantly increased（p<0.05）,the number of trabecular bone of femur and tibia was significantly increased,the thickness of cortical bone was significantly increased（p<0.01）,the BMD,BV/TV,Tb.Th was significantly elevated（p<0.05 or p<0.01）,AKP staining increased,Serum ALP activity was significantly decreased（p<0.01）,and phosphorus and calcium ion concentrations were significantly increased（p<0.05 or p<0.01）,the mRNA and protein expressions of Sirtl,Wnt3a,β-catenin,Runx2 in bone were significantly increased（p<0.05 or p<0.01）.（3）Compared with the TP group,the body weight,AUC,and insulin resistance of the TYP group were significantly increased（p<0.05）,and the insulin level and insulin sensitivity were significantly decreased（p<0.05 or p<0.01）,Tibial wet weight,Tibial length,distal coronal width were significantly decreased（p<0.05 or p<0.01）,cortical bone BV/TV,Tb.Th were significantly decreased（p<0.05）,Femoral and Tibia bone trabecular numbers decreased,cortical bone thickness decreased significantly（p<0.05）,cancellous bone BMD（p<0.05）,BV/TV,Tb.Th significantly decreased（p<0.05）,AKP reduced staining,Serum ALP activity was significantly increased（p<0.05）,and phosphorus and calcium ion concentrations were significantly decreased（p<0.05 or p<0.01）,the mRNA and protein expressions of Sirtl,Wnt3a,β-catenin,Runx2 in bone were significantly decreased（p<0.05 or p<0.01）.Conclusion:（1）Inactivation of Sirtl/Wnt3a/β-catenin pathway in bone of T2DM mice inhibited OB differentiation and bone formation.（2）Exercise promotes OB differentiation and improves bone tissue microstructure by up-regulating Sirtl expression in T2DM mouse bone and activating the Wnt3a/β-catenin pathway,thereby promoting bone formation. |
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