| Objective: We injected mice with activated enzyme inhibitors and subjected the mice to treadmill exercise intervention.BMD,biomechanical parameters,bone mass,the index of bone histomorphology,the index of serum and urine,the content of important genes and proteins in BMP-Smad signaling pathwaywere tested.We also tested the numbers of bone mesenchymal stem cells differentiating into osteoblasts and serum starvation in vitro.In this study,we aimed to investigate the influence of exercise on bone metabolism in mice with or without the inhibition of BMP-Smad signaling pathway.Materials and methods Part one Eighteen 5-week-old C57BL/6 male mice were divided into two groups in accordance with weight with nine mice in each group: control group(Control),and exercise group(Exercise).Exercise group was assigned treadmill running for 5 weeks.After a period of adaptation,from the second week to the end,the speed was 18 m/min,on a 5° slope,with the training duration of 50 min per day.Training was continued for 5 weeks,6 days per week.Mouse bones were labelled with subcutaneously injected calcein at day 1 and day 8 before the mice were killed at day 9.All the mice were weighted before killed.The left femurs of six mice were used for BMD,and the right femurs of six mice were used for bone histomorphometry analysis.The left tibias of three mice used for Real Time Polymerase Chain Reaction(Real-Time PCR),and other tibias of three mice used for Western blot.All the data were expressed as mean ± SEM.Statistical differences were calculated using independent sample t-test.P values < 0.05 were considered statistically significant.All statistical analysis was performed by statistical software SPSS 13.0 for Windows.Part two Animal experiments in vivo Seventy-two 6-week-old C57BL/6 male mice were divided into four groups in accordance with weight and BMD of mice with 18 mice in each group: control group(C),exercise group(E),LDN group(LDN+C),and exercise with LDN group(LDN+E).Mice in the exercise group and exercise with LDN group were assigned treadmill running exercise at intensities for 6 weeks.Briefly,the mice were trained on a treadmill.The starting speed was 12 m/min,for 20 min per day,with 0° slope.In the firstly two weeks,the speed was increased by 3 m/min every other day,and the duration was increased by 10 min every other day.After a period of adaptation,from week 3 to the end,the speed was 18 m/min,on a 5° slope,with the training duration of 50 min per day.Training was continued for 6 weeks,6 days per week.Mice in the LDN groups were injected with LDN(LDN-193189 HCL,3mg/kg)on Wednesday from the third week to sixth week.Each mouse was weighed before injecting and the doses of LDN were adjusted.Mouse bones were labelled with subcutaneously injected calcein at day 1 and day 8 before the mice were killed at day 9.Massage technique was used to obtain urine samples,which were stored at-80? until further analysis.All the mice were anesthetized with ether inhalation;after blood samples were collected,the animals were killed.The serum samples were collected from the blood.Then,the femurs of three mice were used for MSC extraction and culture.The left femurs of ten mice were used for bone mineral density,the left femurs of eight mice were used forbone biomechanical index,and the right femurs of six mice were used for bone histomorphometry analysis.The left tibias of four mice used for Real-Time PCR,and other left tibias of eight mice used for Western blot.The right tibias of three mice were used for the micro-computed tomography(?CT)test.Cell experiments in vitro The BMSCs were washed out with ?-MEM.Alkaline phosphatase(ALP)staining was performed 7 days after seeding.Colony-forming units(CFUs)were calculated from those images.Methods for extracting total RNA from the BMSC,reverse-transcription,and Real-Time PCR were similar to part one.OCN mRNA,Osterix mRNA,Runx2 mRNA,MSX2 mRNA,and DLX5 mRNA were measured by quantitative testing.Serum starvation experiment got protein,and proteins were used for Western blot assay.The blotting membranes incubated with phospho-Smad 1 antibody,Smad 1 antibody,phospho-p44/42 MAPK antibody,p44/42 MAPK antibody,phospho-p38 MAPK antibody,p38 MAPK antibody,and ?-Actin antibody.All statistical analysis was performed by statistical software SPSS 13.0 for Windows.All the data in tables were expressed as mean ± SD,and data in figures were expressed as mean ± SEM.Two-way analysis of variance was performed,and statistical differences were calculated using independent sample T-test.Values of p < 0.05 were considered statistically significant.Result Part one(1)Exercise increases BMD and bone volume.We found that BMD and BV/TV of mice were significantly higher in the exercise group than the control group.(2)Exercise activates BMP signaling pathway.The expression of OCN and OPN mRNA was higher in the exercise group than the control group.Furthermore,western blot analysis showed that exercise activated the Smad1 protein phosphorylation.Part two(1)LDN injection decreased the mouse body weight;however,the exercise did not show a significant effect on mouse body weight during LDN injection for 3 weeks.(2)BMD in the E group was significantly higherthan that in the C group(P<0.01),and BMD in the LDN+E group was higherthan that in the LDN+C group(P<0.05);four times LDN injection decreased the BMD that was significantly lowerin the LDN+E group than that in the E group(P<0.01).There were no significant changes in biomechanical parameters of left femur.(3)BV/TV and Tb.N in the LDN+E group were higher than those in the LDN+C group,Tb.Sp in the LDN+E group was lower than that in the LDN+C group,BFR/TV and BFR/BS in the E group were higher than those in the C group.Also,we found that BFR/BV,BFR/TV,and BFR/BS were lower in the LDN+E group than the E group(P<0.05).Mice that were exercised for 6 weeks or injected with LDN alone revealed that BV/TV,Tb.N,and BFR/TV in the LDN+C group were lower than those in the E group,while Tb.Sp in the LDN+C group was higher than that in the E group.BV/TV and Tb.N in the E group were higher than those in the C group,while Tb.Sp in the E group was lower than that in the C group.Moreover,BV/TV in the LDN+C group was lower than that in the E group,while Tb.Sp in the LDN+C group was higher than that in the E group when mice were exercised for 6 weeks or injected with LDN alone.(4)The exercise was found to increase the cortical bone BV/TV and Tb.Th of tibia: cortical bone BV/TV in the E group was higherthan that in the C group(P<0.05),cortical bone BV/TV in the LDN+E group was significantly higherthan that in the LDN+C group(P<0.01);whereas,the cortical bone Tb.Th in the E group was higherthan that in the C group(P<0.05).(5)The serum calcium level in the LDN+C group was significantly higher than that in the C group(P<0.01),while the serum calcium and phosphorus levels were significantly higher in the LDN+E group than that in the E group(P<0.01).The serum calcium level in the LDN+E group was significantly higher than that in the LDN+C group(P<0.05).DPD/Ucr in the LDN+C group was significantly higherthan that in the C group(P<0.01);DPD/Ucr and CTX-I levels were higher in the LDN+E group than the E group(P<0.05).(6)The mRNA expression of OCN was lower in the LDN+E group than the E group(P<0.05).(7)Western blot analysis with antibodies to p-Smad1 and Smad1 showed that p-Smad1 expression was higher in the E group than in the C group(P<0.05).Also,Smad1 expression was higher in the LDN+E group than the LDN+C group(P<0.05).(8)The medium level of exercise increased the number of osteogenic MSCs.LDN injection inhibited the osteoblast-related genes in BMSCs: Dlx5 mRNA expression in the LDN+E group was lower than that in the E group(P<0.05).(9)Smad1 protein phosphorylation in the C group,the LDN+C group,and the LDN+E group was lower than that in the E group(P<0.05).Conclusion(1)Medium-intensity treadmill exercise activates Smad1 protein phosphorylation,increases Smad1 protein content in vivo,and increases the number of osteogenic MSCs,promotes bone formation and inhibits bone resorption,increases BMD and bone mass in young mice.(2)LDN injection inhibits Smad1 protein phosphorylation in young mice,inhibits the expression of osteoblast-related genes in BMSCs and tibia,and LDN injection promotes bone resorption inhibits bone formation.LDN injection shows a significant effect on mouse body weight and BMD.(3)Medium-intensity treadmill exercise could partially antagonize the effects of enzyme inhibitor,possibly by affecting the phosphorylation and total amount of Smad1 protein,regulating the expression of osteoblasts-specific transcription factors and target genes in BMSCs and the expression of osteoblasts-related genes downstream of BMP-Smad signaling pathway in mice,thereby affecting the number of BMSCs differentiation into osteoblasts.In vivo bone mass,BMD and body weight of mice were affected by bone formation rate and bone resorption rate. |
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