The Effect And Molecular Mechanism Of Treadmill Exercise Combined With Low-intensity Pulsed Ultrasound In Improving Weightless Bone Loss

Purpose:In the microgravity environment or bedridden condition for a long time,the bone resorption and bone formation balance will be damaged,and the function of osteoclast will increase due to the continuous no load,no load,no stress stimulation[1].Development will lead to decreased bone density and increased bone fragility,causing osteoporosis and increasing the risk of fractures,which will increase the overweight burden of astronauts returning to the ground or affect the rehabilitation of bedridden patients,which will seriously endanger life.External forces stimulate the development and maintenance of bones and strength of life.The contraction of muscles generated by exercise is directly and closely related to the growth and development of bone tissue.Treadmill exercise,as a whole-body exercise method,has an effect on muscle mass and bone mass in a weightless environment.Maintenance has a positive effect.Low-intensity pulsed ultrasound(LIPUS)has a positive effect on fractures and bone healing in clinical and basic experimental research due to its non-invasiveness and convenience.However,the effect of LIPUS with different sound intensities and two sound intensity rotations on bone loss or healing of bone injury is still unclear,and the effect of exercise combined with LIPUS on weightless bone loss has not been studied.Myostatin(myostatin(MSTN),as an important factor that affects muscle and bone growth,has not been explored whether it participates in the bone protection process of exercise combined with LIPUS and its influence mechanism.Therefore,in this experiment,a weightless bone loss model and a midfemoral injury model were constructed by simulating weightlessness in tail-suspended rats,and different sound intensities of LIPUS and treadmill exercise were explored to improve bone loss or damage to bones The effect of healing is studied.The molecular mechanism of musculoskeletal coupling is studied in order to provide basic experimental evidence for the treatment and protection of weightless bone loss and provide innovative methods.Methods:Animal experiment:108 healthy 5-week-old SD male rats were used as experimental objects.After one week of adaptive breeding,the rats were drilled(diameter 1.00mm)in the middle of the left femur of each rat and randomly divided into groups after 3 days of recovery:Normal control group(NC),Hindlimb suspension(HLS),30mW/cm~2 sound intensity group(Hindlimb suspension with 30mW/cm~2,HLS-30mW/cm~2),80mW/cm~2 sound intensity group(Hindlimb suspension with 80mW/cm~2,HLS-80mW/cm~2),30mW/cm~2 and 80mW/cm~2 sound intensity rotation group(Hindlimb suspension with 30mW/cm~2 and 80mW/cm~2,HLS-30/80mW/cm~2),sports group(Hindlimb suspension with exercise(HLS+E),exercise combined with 30mW/cm~2 sound intensity group(Hindlimb suspension with exercise anu 30mW/cm~2,HLS-E-30mW/cm~2),exercise combined with 80mV/cm~2 sound intensity group(Hindlimb suspension with exercise and 80mW/cm~2,HLS-E+80mW/cm~2),exercise combined 30mW/cm~2+80mW/cm~2 sound intensity group(Hindlimb suspension with 30mW/cm~2-80mW/cm~2 and exercise,HLS-E+30/80mW/cm~2).The ultrasound group was irradiated with a center frequency of 1 MHz,a duty cycle of 20%,and different sound intensities for 20 min/d.The exercise group used speeds of 24m/min,40min/d,and 4w.On the 14th day of intervention,4 rats were sacrificed in each group,and on the 28th day,8 rats were sacrificed in each group.Serum and femur were collected for subsequent testing and use.At the end of the experiment,the body weight,bone weight,and quadriceps weight of the rats were weighed,and the serum ALP and TRACP activities were measured using a biochemical kit.Micro-CT was used to detect the bone microstructure of the cancellous bone region of the distal femur and the bone injury region of the middle femur.Threepoint bending mechanics test was used to detect the biomechanical properties of the middle femur.H&E staining was used to analyze the morphological changes of the distal femur.Western Blot technology was used to analyze each The skeletal muscle coupling mechanism,that is,the skeletal muscle MSTN-mediated signaling pathway and the intracellular Wnt/?-catenin signaling pathway in the expression of genes and proteins.Cell experiments:osteoblasts MC3T3-E1,bone marrow mesenchymal stem cells BMSC and osteoclast RAW264.7 were used as research objects.After the cells were resuscitated and cultured to the logarithmic phase,they were seeded in 96-well plates,and the control group(normal control,NC)and the ultrasound irradiation group(LIPUS)were set up,with 4 replicates in each group.During the intervention period,each group changed the culture medium every 3 days.The LIPUS group performed 20 minutes of intervention per day,with the intensity of the center frequency of 1 MHz,the duty cycle of 20%,and the sound intensity of 30 mW/cm~2 and 80 mW/cm~2.Cell proliferation was detected after 7 days.(CCK-8 kit),immunofluorescence staining(nucleus,mitochondria,microfilaments),RUNX-2 protein level detection;wild type mouse serum medium and Myostatin gene knockout mouse serum medium at 1%concentration,respectively Cells were cultured,and cell proliferation assay(CCK-8 kit)and cell cycle assay(flow cytometry)were performed after 7 days.Results:1.Rat weight,femur and quadriceps weight results:Rat weight,femur and quadriceps weight decreased significantly after 4 weeks of tail suspension(p<0.01,p<0.01,P<0.01);single treadmill Exercise or LIPUS and the combined intervention have no significant effect on improving weight loss in tail-suspended rats;femoral weight results show that exercise or LIPUS and the combined intervention can significantly improve femoral weight in tail-suspended rats,of which HLS-30mW The bone weight of the/cm~2 group was significantly lower than that of the HLS group(P<0.05).Compared with the HLS group,the bone weight of the other intervention groups was extremely significant(p<0.01).The quadriceps weight results showed that HLS+E and The quadriceps weight of the HLS-E+80mW/cm~2 group was significantly higher than that of the HLS group(p<0.05),and the quadriceps weight of the HLS-E+80mW/cm~2 group was extremely significant compared with the HLS group(p<0.01).2.Rat serum index results:After 4 weeks of tail suspension,the alkaline phosphatase and tartrate-resistant acid phosphatase activities in HLS group rats significantly increased(p<0.01);single treadmill exercise or LIPUS and the combined intervention The serum alkaline phosphatase increased in tail-suspended rats,but it was not significant;HLS+E group,HLS-80mW/cm~2 group and HLS-30mW/cm~2 group could significantly improve the serum resistance of tail-suspended rats.Tartrate acid phosphatase activity(p<0.01)was not significantly improved in the single LIPUS group.3.Results of BMD and bone microstructure of the cancellous bone of the distal femur in rats:BMD,BV/TV,and DA of the cancellous bone of the distal femur of the rats decreased significantly after 4 weeks of tail suspension(p<0.01);single treadmill exercise Compared with HLS group,BMD,Tb.N and DA were significantly increased(p<0.01),BV/TV was significantly increased(p<0.05);HLS-80mW/cm~2 group could significantly improve Tb.Th,BV/TV in tail-suspended rats And DA(p<0.05,p<0.01,p<0.05);the HLS-E+80mW/cm~2 group significantly increased BMD and BV/TV compared with the HLS group(p<0.05,p<0.05),and significantly reduced Tb.Sp(p<0.01);HLS-30mW/cm~2 group significantly increased BV/TV in tail-suspended rats(p<0.01);HLS-E+80mW/cm~2 group and HLS-30/80mW/cm~2 significantly increased BV/TV in tail-suspended rats/TV and DA(p<0.05,p<0.05;p<0.01,p<0.05);The HLSE+30/80mW/cm~2 group significantly increased BV/TV(p<0.01)and significantly reduced Tb.Sp compared with the HLS group(p<0.01).4.Results of the biomechanical performance of the middle femur of rats:The maximum load,energy absorption,stiffness,and elastic modulus of the middle femur of rats showed a significant decrease after 4 weeks of tail suspension(p<0.01);LIPUS and the combined intervention have significantly improved the elastic modulus of the midfemur of the tail-suspended rats(p<0.01);compared with the HLS group,the maximum femoral load,stiffness and energy of the rats after a single treadmill exercise Absorption index showed a very significant increase(p<0.01);HLS-80mW/cm~2 group can significantly increase the femoral stiffness of rats after tail suspension(p<0.05);HLS-E+80mW/cm~2 group can significantly increase tail-suspended rats Maximum load,energy absorption and stiffness(p<0.05,p<0.01,p<0.01);HLS-E+30mW/cm~2 group can significantly increase the stiffness of tail-suspended rats(p<0.01);single rotation with different sound intensity The irradiation or combined exercise group had no significant effect on improving the mechanical properties of the rat femur.5.Healing results of bone injury in rats:The two-dimensional and 3D reconstructions of the mid-femoral injury site in tail-suspended rats show that compared with the normal control group,the injury site in the tail-suspension group has obvious gaps and slow healing.At 14 days,the diameter of the damaged surface of the cortical bone is relatively complete,and at 28 days,the surface of the defect is still not smooth;compared with the single tail suspension group,the treadmill exercise group or the LIPUS group and the combination of the two can promote the healing of the injury site.Compared with the control group,the area of the surface defect of the middle femur of the rats in each intervention group was smaller at 14 days,and the surface of the defect was basically smooth at 28 days,and the two-dimensional reconstruction map showed that there was obvious new tissue connected at 14 days to the defect recovery at 28 days.intact.6.Myostatin protein expression results of quadriceps femoris:Myostatin protein expression in rat quadriceps femoris increased significantly after 4 weeks of tail suspension(p<0.01);single treadmill exercise or LIPUS and the combination of the two could significantly interfere Improve the expression level of Myostatin protein in the quadriceps femoris of tail-suspended rats(p<0.01).7.Myostatin receptor and downstream key molecules in femur and protein expression in Wnt/P-catenin signaling pathway:Compared with the normal control group,tail suspension for 4 weeks can significantly increase Myostatin receptor ActRIIB and downstream key in rat femur.The protein expression levels of the molecules Smad2/3 and GSK-3?(p<0.01 or p<0.05),while significantly reducing the protein expression levels of Wntl and ?-catenin in the Wnt/?-catenin signaling pathway(p<0.01 orp<0.05);single treadmill exercise or LIPUS and the combined intervention can effectively inhibit the Myostatin signaling pathway and activate the Wnt/?-catenin signaling pathway.8.RUNX-2 protein expression and proliferation of osteoblast MC3T3-E1 and immunofluorescence results:Compared with the normal control group,LIPUS with different sound intensity can significantly promote the expression level of RUNX-2 molecule in osteoblasts(p<0.01);CCK-8 test results showed that compared with the NC group,the LIPUS intervention with 30mW/cm~2 and 80mW/cm~2 sound intensity significantly promoted osteoblast proliferation(p<0.01,p<0.01),30+80mW/cm~2 The group significantly promoted the proliferation of osteoblasts(p<0.05).The immunofluorescence results showed that LIPUS with different sound intensities could increase the number of osteoblast nuclei,increase the number and density of mitochondria,and the microfilaments were compact and trended after LIPUS stimulation.9.Proliferation results of bone marrow mesenchymal stem cells BMSC and osteoclast Raw264.7:LIPUS has a tendency to promote BMSC proliferation,but it is not statistically significant;the effect of LIPUS on Raw264.7 cells is not obvious.10.The effect of Myostatin knockout mouse serum on the osteoblast MC3T3-E1 cycle results:Compared with the WT group,the proportion of cells in the MSTNKO group into the S phase increased and the cell proliferation rate in the MSTNKO group was significantly higher than that in the WT group(p<0.01).).Conclusion:1.Treadmill exercise and LIPUS can prevent bone loss in tail-suspended rats,improve bone microstructure damage,and improve femoral biomechanical performance.Among them,the single treadmill exercise group and the treadmill combined with high sound intensity group are better than low sound intensity Group or high and low sound.rotation group;2.Treadmill exercise and LIPUS can promote bone healing in tail-suspended rats,and the effect of the treadmill exercise group is better than the combined exercise and LIPUS group;3.Treadmill exercise and LIPUS can inhibit skeletal muscle MSTN expression and its osteocyte signaling pathway,and activate intracellular Wnt/?-catenin signaling molecule expression to promote osteogenesis;4.Different intensity of LIPUS can promote osteoblast proliferation and intracellular RUNX-2 expression and have regulatory effects on osteoblast nuclei,mitochondria,microfilaments and other cell microstructures.Among them,80mW/cm~2 is better than low sound intensity or high sound Strong rotation irradiation group.5.MSTN knockout mouse serum can significantly promote osteoblast proliferation and increase the proportion of S phase in its cycle.