Study On The Effect And Molecular Mechanism Of Weight-bearing Treadmill Combined With LIPUS On The Improvement Of Osteoporosis And Bone Injury Healing In Ovariectomized Rats

Objective:With the increasing aging population,the incidence of chronic diseases such as osteoporosis is getting higher and higher,and the proportion of males and females in the elderly is unbalanced.The annual osteoporosis caused by menopause is more prominent,and the postmenopausal bones are more prominent.The incidence of fractures caused by looseness is also high,which has become a major health problem in medical and social fields.Exercise is a good doctor.Appropriate exercise has certain positive benefits for human body function and metabolism.As a resistance exercise,weight-bearing training can not only positively regulate the quality of skeletal muscle,but also regulate bone metabolism and improve bone growth.Ultrasound as a physical sound wave,in clinical and basic experimental research,the intervention of bone tissue is mainly reflected in the low-intensity pulsed ultrasound(LIPUS)intervention in the physical physiotherapy of fractures.However,the effects and potential mechanisms of exercise combined with LIPUS intervention on osteoporosis and bone injury have rarely been studied.In this study,rat femur and bone cells were used as research objects.By constructing osteoporosis and bone injury models,the effects of weight-bearing training combined with LIPUS on osteoporosis and bone injury healing in ovariectomized rats and potential musculoskeletal bone were studied.Provide a practical theoretical basis and innovative approach for the prevention and treatment of clinical osteoporosis,especially postmenopausal osteoporotic and bone injury.Methods:Animal experiments in this study:70 healthy female rats of 3 months old Sprague-Dawley were used as experimental subjects,and they were adaptively fed for one week and randomized into groups:Sham Operation(Sham),Ovariectomized(OVX),The OVX group was randomly divided into two groups after ovariectomy:Ovariectomized(OVX),Ovariectomized and Weight-bearing training(OWT),Ovariectomized and Ovariectomized Drilling and Ultrasound,(ODU),Ovariectomized and Weight-bearing training and Drilling and Ultrasound(OWTDU).The OWT group was operated at a speed of 20 min/m,6 d/w,20 min,and continuous exercise for 10 w.After the end of the exercise intervention,8 rats in the Sham group,OVX group and OWT group were sacrificed,and serum and related tissues were collected for subsequent testing.In the remaining group of rats,the middle part of the right femur was subjected to bone injury surgery to construct a bone injury model.After successful modeling,the ODU group and the OWTDU group were treated with LIPUS,the sound intensity was 30mW/cm2,the center frequency was 1.5MHz,the time was 20min/d,and the intervention was continued for 21d.In this experiment,the body weight was comearsurement and recording on Sunday mornings,and the force test and the bone density test were performed before the samples were taken.The activity and content of serum ALP,StrACP,E2 and MSTN were detected by biochemical kit.Micro-CT was used to detect the bone microstructure of the cancellous region of the distal femur and the middle part of the femur.The three-point 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 and RT-qPCR technique was used to analyze the musculoskeletal coupling mechanism of each group,namely the skeletal muscle Myostatin mediated signaling pathway and the gene and protein expression of Wnt/?-catenin signaling pathway in bone tissue.The cell experiments in this study were based on osteoblast MC3T3-E1 and osteoclast Raw264.7.Under low-intensity pulsed ultrasound,MC3T3-E1 cells were randomly divided into control group(Control)and ultrasound intervention group(LIPUS).MC3T3-E1 cells were randomly divided into control group(normal control,NC)and traction stimulation group(strain,ST)under mechanical traction.After each intervention,CCK-8 was used to detect cell proliferation,ALP activity assay was used to evaluate cell differentiation,and alizarin red staining was used to test cell mineralization results;LIPUS was used to interfere with the proliferation of osteoclast Raw264.7.Results:1.Body weight,grip strength,quadriceps wet weight,femur wet weight,uterine wet weight and living bone mineral density in routine indexes of rats:body weight increased significantly after 12 weeks of ovariectomy(P<0.01),The lateral femur BMD,uterus and quadriceps wet weight and grip strength were significantly decreased(P<0.01).After weight-bearing training,the body weight was significantly lower than that of OVX group(P<0.05),BMD(P<0.05)and holding power.(P<0.01)and wet weight of quadriceps muscle(P<0.05)were significantly increased(P<0.05).After the 21 d LIPUS intervention,the wet weight of the quadriceps muscle in the exercise group and the ultrasound group increased significantly(P<0.01),but the combined effect of the two groups was not significantly different from the single intervention group.2.Serum index results of rats:ALP activity and StrACP content in OVX group were significantly increased after ovarian surgery for 12 weeks(P<0.01),E2 content was significantly decreased(P<0.01),and training with 10w.After the 21d LIPUS intervention,ALP activity and E2 content increased significantly(P<0.01),and StrACP content decreased significantly(P<0.01).After 21d LIPUS intervention,ALP activity(P<0.05),StrACP content(P<0.01)and E2 content(P<0.05)were significantly decreased in the early exercise group,and the ALP activity in the ultrasound group was significantly increased(P<0.01).The content of StrACP and E2 was significantly decreased(/P<0.01),and the effect of exercise combined with ultrasound was better than that of single intervention group.3.BMD and bone microstructural results of cancellous bone in the distal femur of rats:BMD,Tb.N,Tb.Th and BV/TV in the distal femur of rats were significantly decreased(P<0.01)after 12 weeks of ovariectomy,while the Tb.Sp index increased significantly(P<0.01).After weight-bearing training,BMD and BV/TV increased significantly(P<0.05)and Tb.N significantly increased(P<0.01),Tb.Sp was significantly decreased(P<0.01).After 21 days of LIPUS intervention,the Tb.N of the early exercise group increased significantly(P<0.05),BMD and Tb.Th increased significantly(P<0.01),and the Tb.Sp index decreased significantly.(P<0.05);BMD and Tb.Th in the ultrasound group were significantly increased(P<0.01),Tb.Sp was significantly lower(P<0.05),and the exercise combined ultrasound group was only superior than a single intervention group in terms of BMD,Tb.N and Tb.Sp.4.Biomechanical properties of the middle femur of rats:The maximum load,stiffness,energy absorption and elastic modulus of the middle femur of rats were significantly decreased after 12 weeks of ovariectomy(P<0.01).The maximum load,stiffness,energy absorption and elastic modulus of the OVX group were significantly improved(P<0.01).After 21d LIPUS intervention,the maximum load(P<0.01),energy absorption(P<0.01)and elastic modulus(P<0.05)of the early exercise group increased significantly;the maximum load(P<0.01)and stiffness of the ultrasound group(P<0.05),energy absorption(P<0.05)and elastic modulus(P<0.05)were all significantly increased;but,there was no significant difference between the two groups and the single intervention group.5.Healing results of rat bone injury:In the case of LIPUS intervention for 21 days,the bone healing degree of the OD group was significantly slower(P<0.01).Compared with the OD group,the exercise group and the ultrasound group and the combination group of the two groups healed were significantly increased(P<0.01);but,there was no significant difference between the two groups and the single intervention group6.Myostatin expression:After 12 weeks of ovariectomy,the expression of Myostatin showed that the serum Myostatin content and the expression of Myotatin protein in the quadriceps muscle were significantly increased(P<0.01).The serum Myostatin content and the expression of Myotatin protein in the quadriceps muscle of the rats were significantly decreased in weight-bearing training group than the OVX group(P<0.01).After 21 days of LIPUS intervention,the serum levels of Myostatin and Myotatin in the quadriceps muscle were significantly decreased in the early exercise group and the ultrasound intervention group(P<0.01).However,only in the protein expression of Myotatin in the quadriceps muscle,the combination group was significantly lower than the single intervention group(P<0.05).7.Gene and protein expression in the Myostatin-mediated signaling pathway and Wnt/?-catenin signaling pathway in rats:and the gene and protein expression in the Myostatin receptor ActRIIB and downstream key molecules Smad2/3,GSK-3P of rats after 12 weeks of ovariectomy were significantly increased(P<0.01 or P<0.05),and the protein and gene expression of Wntl and ?-catenin in Wnt/?-catenin signaling pathway were significantly decreased(P<0.01 or P<0.05).The Myostatin signaling pathway was promoted in ovariectomized rats,while the Wnt/?-catenin signaling pathway was inhibited.After the weight-bearing training,it could effectively inhibit Myostatin signaling pathway and activate Wnt/(?-catenin signaling pathway;LIPUS intervention at 21d,in the ultrasound group,the Myostatin signaling pathway was activated and the Wnt/p-catenin signaling pathway was activated.The combination of ActRIIB,Wntl,GSK-3? and ?-catenin in gene and protein expression levels were significantly better than the single intervention group.8.Proliferation,differentiation and mineralization results of osteoblast MC3T3-E1:The tensile stress parameters showed a significant increase in the proliferation activity of MC3T3-E1 cells at a strain force of 2000ustrain,frequency 0.5Hz,time 120min(P<0.01);The proliferation activity of MC3T3-E1 cells was significantly increased at 3d,5d and 7d(P<0.01)by LIPUS intervention.The tensile stress stimulation and LIPUS intervention significantly increased the ALP activity of MC3T3-E1 cells(P<0.01).The tensile stress stimulation and LIPUS intervention in MC3T3-E1 cells for 14d,21d and 28d accelerated the formation of mineralized nodules.9.Proliferation results of osteoclast Raw264.7:LIPUS intervention in osteoclast Raw264.7 proliferation activity had significant inhibition at 5d(P<0.05),and significantly inhibited proliferation at 7d after LIPUS intervention(P<0.01).Conclusion:1.After 12 weeks of ovariectomy,the osteoporosis model was established successfully;weight-bearing training can delay the decline of bone mass caused by ovariectomy;2.LIPUS in the treatment of bone injury in ovariectomized rats can significantly promote the healing of bone pores,improve its structural and mechanical properties,and significantly accelerate the healing rate of bone injury within 21 days after the termination of bone injury.The combination of the two interventions strengthens the repair of bone injury.Process,there is a trend that is better than a single intervention,but there is no significant difference;3.The molecular mechanism of potential musculoskeletal coupling between weight-bearing training and LIPUS to improve bone mass and accelerate bone healing may be:by inhibiting the expression of Myostatin in skeletal muscle and thereby reducing its binding to receptors in bone tissue,while activating the Wnt/?-catenin signaling pathway signaling pathway works together to achieve;4.Pulling stress stimulation and LIPUS intervention osteoblast MC3T3-E1 can significantly promote proliferation,differentiation and mineralization;and LIPUS intervention on osteoclast Raw264.7 can significantly inhibit its proliferative activity at 5d,and further enhance the inhibitory effect at 7d and have extreme significantly difference.