Effects Of Exercise Training On Bone Formation And Bone Resorption In Obese Mice

Objective Exercise plays an important role in regulating bone growth and bone resorption.Obesity not only increases the risk of chronic metabolic diseases such as diabetes and hypertension,but also is closely related to the health status of bones.Previous clinical observations suggest that bone mass in obese people is higher than that in normal people,so obesity is regarded as a protective factor for adult bone health.However,more and more studies are challenging this view with more accurate clinical measurement methods and more detailed observation angles.At present,the effect of obesity on bone turnover and the effect of exercise on bone turnover under obesity are still unclear.In this study,obese mice were modeled by high-fat diet,and10-week treadmill training intervention was conducted to study the changes of bone mass in obese mice and explore the related bone turnover mechanism,trying to provide early research basis for the changes of bone mass in obese people during exercise and better serve the society.Methods Sixty male C57BL/6 mice were fed to 5 weeks of age and randomly divided into normal diet group(CTR group,n=30)and high fat diet group(HFD group,n=30)according to body weight for 12 weeks.CTR was fed with ordinary diet,and HFD was fed with special high-fat diet(D12492,Phan Biol).The body weight of mice in each group was recorded weekly.The difference in AUC of GTT and ITT in HFD group was used as the criterion of modeling success.Obese mice were divided into obese control group(HFD group,n=10)and obese exercise group(HFD+EX group,n=10).At the same time,20 ordinary diet mice were randomly divided into control group(CTR group,n=10)and exercise group(EX group,n=10)according to their body weight.Exercise intervention was conducted for 10 weeks,and the feeding of each group remained unchanged.BMSCs were extracted from the two sides of femurs of each of the CTR group and HFD group.The expression levels of osteogenic and osteoclastic genes in each group were detected by Q-PCR.End of exercise intervention,the mice were subcutaneously injected with Calcein(5μ L /g)8 days before sampling and 1 day before sampling for bone fluorescence labeling.Urine and blood supernatant were collected before sampling,and adipose tissue samples and bone tissue samples were extracted.micro-ct was used to scan bone microstructure.The maximum load of femur was detected by three-point bending method.Bone morphometric detection;Bone morphology was observed by HE staining.The expression levels of osteogenesis and osteoclast factor in each group were detected by Q-PCR.The protein expression level in bone was detected by WB.The expression of femur protein was detected by immunofluorescence.Biochemical markers of bone metabolism in blood and urine were detected by spectrophotometry and enzyme-linked immunosorbent assay.Results1.Changes of mice during the modeling period(1)Changes in body weight and blood glucose of mice during obesity modeling period: the body weight of mice in HFD group was always higher than that in CTR group(P<0.05),and the difference was significant at week 13(P<0.01).GTT and ITT AUC in HFD group were significantly higher than those in CTR group(P<0.01).(2)Changes in body weight and blood glucose of mice during exercise modeling period: from week 17 to 20,the body weight of HFD group was significantly higher than that of CTR group(P<0.001),and the body weight of HFD+EX group was significantly higher than that of EX group(P<0.001).From week 21 to 26,HFD+EX significantly reduced body weight compared to HFD,except that HFD had the same effect as HFD at week 17 to 20.The resting blood glucose in EX group was significantly higher than that in CTR group(P<0.01),and that in HFD+EX group was higher than that in HFD group(P<0.05).The GTT AUC in HFD+EX group was significantly higher than that in EX group(P<0.01).(3)Results of body weight,fat weight and blood lipid of mice after exercise intervention: the body weight of HFD group was significantly higher than that of CTR group(P<0.01),and the body weight of HFD+EX group was significantly higher than that of EX group(P<0.001).The body length of HFD group was longer than that of CTR group(P<0.05).EX group mice had the least and lightest fat content,while HFD group mice had the most and heaviest fat content.The CHOL content in HFD group was higher than that in CTR group(P<0.05),and the CHOL and TG contents in HFD+EX group were higher than that in EX group(P<0.05).There were no significant differences in NEFA and INS contents among the four groups.(4)The results of bone microstructure and biomechanics of mice: Tb.N in EX group was higher than that in CTR group(P<0.05),and TB.N in HFD+EX group was higher than that in HFD group(P<0.05);Tb.N in HFD group was lower than that in CTR group(P<0.05).There were no significant differences in BV/TV,Tb.Th,Tb.Sp and Ct.Th among all groups.Biomechanical properties of femur did not change significantly.2.Effect of exercise on bone formation in obese mice(1)HE staining results of mouse femur: There was no significant overall difference in the distal femur of mice in the CTR group and the EX group,but the bone mass of the distal femur of mice in the HFD group was lower than that in the HFD+EX group,and the overall trend of obesity was observed.(2)Results of fluorescence detection indexes of femur in mice: there were no significant differences in bone mineralization indexes among all groups.(3)Masson staining of bone formation indicators of mouse femur: The distal femur of HFD and HFD+EX groups showed a trend of obesity,but the distal femur bone mass of HFD group significantly decreased,while that of HFD+EX group significantly increased.BV/TV and TB.N in HFD group were lower than those in CTR group(P<0.05).There was no significant difference in other bone formation indexes.(4)Results of osteogenic genes and proteins in mice: the expression levels of ATF4 and Runx2 m RNA in BMSCs in Stretch group were significantly higher than those in CTR group(P<0.01,P<0.05),and the expression levels of Runx2 m RNA in BMSCs in HFD+Stretch group were significantly higher than those in HFD group(P<0.01).The expression of Runx2 protein in tibia of mice in EX group was significantly higher than that in CTR group,HFD+EX group was higher than that in HFD group.OCN protein expression in femur of mice in EX group was higher than that in CTR group,HFD+EX group was higher than that in HFD group(5)The content of biochemical markers of bone formation in serum of mice: the serum ALP content in HFD group was significantly lower than that in CTR group(P<0.01),and the serum ALP content in EX group was higher than that in HFD+EX group(P<0.05).the serum PINP content in EX group was higher than that in CTR group(P<0.05).3.Effect of exercise on bone resorption in obese mice(1)Results of bone resorption test indexes of mouse femur by Masson staining:The bone resorption indexes of mice in HFD groups tended to be higher than those in the other three groups,but there was no significant difference.(2)Results of osteoclast gene and protein in mice: the expression level of Ctsk m RNA in BMSCs in HFD group was significantly higher than that in CTR,Stretch and HFD+Stretch groups(P<0.001).the expression level of BMSCs MMP9 m RNA in Stretch group was significantly lower than that in CTR group(P<0.01),and the expression level of BMSCs MMP9 m RNA in HFD+Stretch group was significantly lower than that in HFD group(P<0.001).There was no significant difference in NFATc1 m RNA expression in the tibia of mice.The expression of Ctsk protein in the tibia of HFD group was significantly higher than that of the other three groups.The expression of Ctsk protein in femur of EX group was lower than that of CTR group,HFD group was higher than that of HFD+EX group.(3)Results of biochemical markers of serum bone resorption in mice: Serum calcium content in HFD+EX group was significantly higher than that in CTR,EX and HFD groups(P<0.01).There was no significant difference in serum phosphorus content among all groups.The urine CTx-1 content in HFD group was significantly higher than that in CTR group(P<0.01).4 Conclusion(1)High fat diet can inhibit bone formation and promote bone resorption in mice,resulting in the decrease of bone mass in obese mice.(2)Exercise training can promote bone formation in obese mice,inhibit bone resorption,and alleviate the decline in bone mass in obese mice.