| Objective:(1)Explore how Sirtl in the bone of T2DM mice regulates the NF-κB pathway,which in turn affects bone resorption and metabolism.(2)Explore how the Sirtl/NF-κB pathway affects the bone resorption metabolism of T2DM mice through different exercise methods.Methods:Fifty-five 4-week-old C57BL/6 male mice were purchased from Yangzhou University,and they were randomly divided into control group(NC,12)and experimental group(T2DM model,43),and they were adaptively fed with ordinary feed for 1 week.The mice in the NC group continued to be fed with normal diet,while the mice in the experimental group were fed with a high-fat diet.After 8 weeks,mice in the experimental group were modeled with T2DM.After 24 hours of fasting,STZ(110mg/kg)(purchased from Sigma)was injected at a time.The NC group was injected with citric acid/sodium citrate solution(110mg/kg)after 24 hours of fasting.Three days later,fasting for 12 hours,tail docking to measure blood glucose concentration,≥16.7 mmol/L were successfully modeled T2DM mice,a total of 40 mice.T2DM mice were randomly divided into T2DM control group(TDC,13),T2DM high-intensity intermittent exercise group(TDG,13)and T2DM downhill running exercise group(TDP,14).The NC group was fed with normal diet,and T2DM mice were fed with high-fat diet.Carry out breeding and training according to the established plan.After 8 weeks,the mice in each group were weighed,and the femur bone length and bone wet weight of each group were measured with vernier calipers;Micro-CT scans the cancellous and cortical bone BMD of the distal femur of the mice in each group;the tibia of the mice Perform HE staining to observe the bone tissue morphology and structure of each group of mice;perform primary culture of mouse bone marrow BMM cells to observe the differentiation of OC in each group of mice;blood was taken by eyeball extraction to detect the activity of StrACP in the serum;RT-PCR detection was small The mRNA expression of Sirtl,NF-κB,IL-6,c-fos and NFATcl in mouse bones;Western Blot detected the protein expression of Sirtl,IκBα,NF-κB,c-fos and NFATc1 in mouse bones of each group.Results:(1)Compared with the TDC group,the weight of the mice in the TDC group increased,and the difference was significant(P<0.01);the wet weight,length,distal sagittal axis width and coronal axis width of the mouse femur in the TDC group.The width of the median sagittal axis and the width of the coronal axis,the width of the proximal sagittal axis and the width of the coronal axis were significantly decreased(P<0.01 or P<0.05);the cortical bone BMD and the cancellous bone BMD of the TDC group decreased,and the difference was significant The results of HE staining showed that the number of distal tibia trabeculae in the TDC group decreased significantly;the results of TRAP staining showed that the number of multinucleated OC in the TDC group increased significantly.(2)Compared with the TDC group,the StrACP activity of the TDC group was enhanced,and the difference was significant(P<0.01);the mRNA expression of Sirt1 in the bone was down-regulated,and the difference was significant(P<0.01),NF-κB The mRNA expression of,IL-6,c-fos and NFATcl were all significantly up-regulated(P<0.01 or P<0.05);the protein expression of IκBα,NF-κB,IL-6,c-fos and NFATcl in bone increased significantly(P<0.01),Sirt1 protein expression was significantly down-regulated(P<0.01).(3)Compared with the TDG group and TDP group,the body weight of the mice in the TDC group was significantly reduced(P<0.05);the length of the femur,the width of the distal sagittal axis and the width of the coronal axis,and the width of the middle coronal axis of the mice in the TDG group The width of the proximal sagittal axis and the proximal sagittal axis were significantly increased(P<0.05).The cortical bone BMD and the cancellous bone BMD of the TDG group increased,and the difference was significant(P<0.05);the HE staining results showed that the distal tibia of the TDG group The number of end bone trabeculae was significantly increased;TRAP staining results showed that the number of multinuclear OC in the TDG group was significantly reduced.The body weight of the mice in the TDP group decreased significantly(P<0.01);the wet weight of the femur,the width of the distal sagittal axis and the width of the coronal axis,the width of the median sagittal axis and the width of the coronal axis,the width of the proximal sagittal axis and the coronal axis of the mice in the TDP group Axis width increased significantly(P<0.05);Cortical bone BMD and cancellous bone BMD increased,and the difference was significant(P<0.05);HE staining results showed that the number of distal tibia trabeculae in the TDP group increased significantly;TRAP staining results showed that the number of multi-core OC in the TDP group was significantly reduced.Compared with the TDP group,the femur length,distal sagittal axis width and coronal axis width,median sagittal axis width and proximal coronal axis width of mice in the TDP group increased,but the difference was not significant(P>0.05)),while the wet weight of femur,the width of the median coronal axis and the width of the proximal sagittal axis all increased,and the difference was significant(P<0.05);the BMD of cancellous bone increased,and the difference was significant(P<0.05);HE The staining results showed that the number of trabecular bones in the distal tibia of the TDP group was significantly increased;the TRAP staining results showed that the number of multinucleated OC in the TDP group was significantly reduced.(4)Compared with the TDG group and TDP group,the TDC group has a decreased StrACP activity,and the difference is significant(P<0.05);the TDG group Sirt1 mRNA expression is up-regulated,and the difference is significant(P<0.05),NF-κB and NFATcl mRNA expression were significantly down-regulated(P<0.05),while the mRNA expression of c-fos and IL-6 showed a downward-regulated trend,but the difference was not significant(P>0.05);TDG group mice Sirt1 protein expression in bone was significantly up-regulated(P<0.05),protein expression of IκBα,NF-κB and IL-6 was down-regulated,but the difference was not significant(P>0.05),c-fos and NFATcl protein expression was down-regulated,and there was a difference It is significant(P<0.05).The activity of StrACP in the TDP group decreased,and the difference was significant(P<0.01);the mRNA expression of Sirt1 in the TDP group was up-regulated,and the difference was significant(P<0.01),and the differences in NF-κB,IL-6,c-fos and NFATc1 were significant.mRNA expression was all down-regulated,and the difference was significant(P<0.01 or P<0.05):IκBα,NF-κB,IL-6,c-fos and NFATc1 protein expression in the bones of mice in the TDP group were significantly down-regulated(P<0.01 or P<0.05),Sirt1 protein expression increased significantly(P<0.05).Compared with the TDG group,the StrACP activity of the TDP group was significantly decreased(P<0.05);the mRNA expression of Sirt1 in the TDP group was up-regulated,and the difference was significant(P<0.01),and the mRNA expression of IL-6 and NFATc1 was significantly down-regulated(P<0.01 or P<0.05),while the mRNA expression of NF-κB and c-fos decreased,but the difference was not significant(P>0.05);Sirt1 protein expression in the bones of mice in the TDP group was significantly up-regulated(P<0.05),IκBα,NF-κB,IL-6,c-fos and NFATcl protein expression were significantly down-regulated(P<0.01 or P<0.05).Conclusion:(1)T2DM caused the down-regulation of Sirt1 in mouse bones and then activated the NF-κB pathway to promote OC differentiation and bone resorption.(2)The direct effect of downhill running on the bones of T2DM mice inhibits OC differentiation,nucleation,and bone resorption through the Sirt1/NF-κB pathway,thereby improving bone health,and its effect is better than high-intensity intermittent movement. |
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