The Effect Of Ladder Exercise Combined With Low-intensity Pulsed Ultrasound On The Improvement Of Quadriceps Atrophy In Ovariectomized Rats And Its Molecular Mechanism

Objective:As the elderly grow older,their physical functions continue to decline,especially in postmenopausal women.because of the decline of ovarian function,the level of estrogen in the body is low,which causes a series of metabolic diseases.Skeletal muscle atrophy is a common disease,clinically characterized by recession and decline in the quality of the skeletal muscle function.This increases the risk of falls and fractures,greatly increases morbidity and mortality,and the quality of life of postmenopausal women continues to decline.How to prevent estrogen-induced skeletal muscle atrophy is an urgent clinical problem.Resistance exercise is a more effective way to improve skeletal muscle atrophy,which can effectively stimulate skeletal muscle,improve skeletal muscle metabolism,and make skeletal muscle hypertrophy,but the mechanism of resistance exercise to improve skeletal muscle atrophy is still unclear.Low-intensity Pulsed ultrasound(LIPUS)is a convenient,safe and non-invasive approach treatment,which has received much attention due to its many advantages.Studies have shown that LIPUS has a good effect on the treatment of sports injuries,but its treatment of skeletal muscle atrophy has not been widely investigated.Myostatin(MSTN)is a growth differentiation factor 8,and exercise can inhibit the expression of MSTN,activate the downstream key molecule Akt,inhibit FoxO1,activate mTOR,promote protein synthesis,and promote skeletal muscle hypertrophy.However,studies on the effects of resistance exercise and LIPUS on postmenopausal skeletal muscle atrophy have been limited.In this study,we investigated the effect and molecular mechanism of weight-bearing climbing exercise combined with LIPUS on skeletal muscle atrophy in castrated rats,providing a theoretical basis for the prevention of skeletal muscle atrophy induced by estrogen reduction.Methods:68 healthy female Sprague-Dawley(SD)rats with body weight of 220-240 g and age of 3 months were randomly divided into two groups:Ovariectomized group(OVX)and Sham Operation group(Sham Operation,Sham).The ovaries of Sham group were not resected,but the same volume of fat beside the ovaries was removed,and the ovaries of OVX group were all removed.After Ovariectomized surgery,rats were kept in silence for 2 w,and after the wound recovery,the OVX group was randomly divided into:castrated control group(Ovariectomized,OVX),castrated Ultrasound intervention group(Ovariectomized+Ultrasound,OUS),castrated weight-climbing group(Ovariectomized+Exercise,OEX),castrated weight-climbing group and LIPUS combined intervention group(Ovariectomized+Exercise+Ultrasound,OEU).Independent patent products using laboratory gym,height is 1 m,slope 85°,2 cm per step span,the OEX,OEU group rats implement 6 d/w,continuous 10 w increasing load gym exercise intervention,after 10 w to OUS group and OEU LIPUS treatment intervention,20 min/d,6 d/w,3 w in a row,the intensity of low intensity pulsed ultrasound instrument for 30 mW/cm2,the frequency of 1.5 MHz,duty ratio was 20%,the pulse frequency of 100 Hz.After 10w,7 rats were randomly selected from Sham group and OVX group,and 7 rats were randomly selected from OEX group.Serum and tissue were stored for detection after sampling,and all remaining rats were sampled after 13 w.Record and detect each group rats physiological and biochemical indexes,including:weight,holding power,skeletal muscle weight,creatine kinase(CK),lactate dehydrogenase(LDH),skeletal muscle glycogen,use the RT-qPCR(policy Real-time PCR)and Western Blot from gene level,protein level to detect MSTN/Akt signaling pathway related signaling molecules expression,to analyze the weight gym movement and LIPUS quadriceps atrophy of castrated rats to improve the effect and molecular mechanism.To investigate the effects of LIPUS and mechanical traction on myoblasts at the cellular level.Myoblasts were treated with mechanical traction and LIPUS.The mechanical traction intervention was divided into four groups:normal group,1000?strain group,2000 ?strain group and 4000 ?strain group.The intervention frequency was 0.5 Hz and the time was 1 h.Intervention by LIPUS was divided into four groups:the normal group,10 mW/cm2 group,30 mW/cm2 group and 75 mW/cm2 group.The duration of intervention was 20 min/d for 3 days.This experiment will focus on the use of flow cytometry,in order to detect the proliferation of cells,this instrument will be used to get the cell cycle,according to the corresponding formula to calculate the cell proliferation index.Results:1.Results of rats' physiological indexes:after 10 w of ladder exercise,compared with Sham group rats,OVX group rats had significantly increased body weight(P<0.01),decreased grip(P<0.05),and reduced quadriceps muscle weight(P<0.05);OEX group rats had significantly increased body weight(P<0.01),and significantly increased grip(P<0.01).Compared with OVX group,OEX group rats had significantly reduced body weight(P<0.05),significantly increased grip(P<0.01),and significantly increased weight of quadriceps femoris(P<0.05).After 3 w LIPUS irradiation,compared with Sham group,the weight of OVX,OUS,OEX and OEU group rats was significantly increased(P<0.01)and the grasping power was significantly reduced(P<0.01),and the weight of quadriceps femoris was significantly reduced(P<0.01).Compared with OVX group,the weight of quadriceps femoris in OUS,OEX and OEU groups was significantly increased(P<0.01).2.Biochemical indexes of rats:after 10 w of ladder exercise,compared with Sham group rats,serum LDH content in OVX group significantly increased(P<0.05)and quadriceps femoris glycogen content significantly decreased(P<0.05).Compared with the OVX group,the serum LDH content in OEX group decreased significantly(P<0.05),the serum CK content decreased significantly(P<0.05),and the quadriceps femoris glycogen content increased significantly(P<0.05).After 3 w LIPUS of irradiation,compared with the Sham group rats,the OVX group rats serum LDH increased significantly(P<0.01),the content of LDH content OUS,the OEX group rats increased significantly(P<0.05),group OVX rats serum CK increased significantly(P<0.01),the content of OUS,the OEX group rats CK levels increased significantly(P<0.05),the OVX group,OUS glycogen content in the quadriceps muscle of rats significantly reduced(P<0.01),the OEX group rats quadriceps glycogen content decreased significantly(P<0.05);Compared with the OVX group,the serum LDH and CK contents in OEU group were significantly decreased(P<0.05),the glycogen content in the quadriceps femoris of OEX group was significantly increased(P<0.05),and the glycogen content in the quadriceps femoris of OEU group was significantly increased(P<0.01).Compared with the OUS group,the glycogen content of the quadriceps femoris in OEX group was significantly increased(P<0.05),and the glycogen content of the quadriceps femoris in OEU group was significantly increased(P<0.01).3.Expression of intracellular Akt and downstream signaling molecules mediated by MSTN and its receptor:after 10 w of ladder exercise,mRNA and protein expressions of MSTN,ActRIIB and FoxO1 in the quadriceps femoris of OVX group were significantly increased,Akt,p-Akt and mTOR protein expressions were significantly decreased,and mTOR mRNA expression was significantly decreased compared with that of Sham group.Compared with OVX group,protein expressions of MSTN,ActRIIB and FoxO1 in rat quadriceps femoris in OEX group were significantly decreased,mrna expressions of MSTN and FoxO1 were significantly decreased,and protein expressions of Akt and mTOR were significantly increased.mRNA and protein expressions of MSTN,ActRIIB and FoxO1 in the quadriceps femoris of OVX group were significantly increased after 3 w LIPUS irradiation compared with Sham group,while mRNA and protein expressions of Akt,p-Akt and mTOR were significantly decreased.Compared with OVX group,OUS,the OEX,OEU group rats quadriceps MSTN,ActRIIB,FoxO1 protein expression was significantly reduced,OUS,the OEX group rats quadriceps MSTN,ActRIIB,OEU group rats quadriceps FoxO1 mRNA expression decreased significantly,the OEX,OEU group rats quadriceps Akt,p-Akt,mTOR protein expression increased significantly,OUS group rats quadriceps Akt,mTOR protein expression increased significantly.The combined effect of the two intervention methods on the expression of ActRIIB,p-Akt and mTOR protein was better than that of the single intervention method.4.Results of myoblast proliferation:the proliferation of myoblasts was significantly increased in the 10 mW/cm2 and 30 mW/cm2 groups after the intervention of low-intensity pulsed ultrasound,compared with the NC group(P<0.05,P<0.01).Compared with the 10 mW/cm2 group,the proliferation level of the 30 mW/cm2 group was significantly increased(P<0.05).Compared with the 30 mW/cm2 group,the proliferation level of 75 mW/cm2 group decreased significantly(p<0.01).The proliferation of myoblast cells was significantly increased in the 1000 ?strain and 2000?strain groups compared with the NC group(P<0.01).Compared with the 1000 ?strain group,the proliferation level of the 2000 ?strain group was significantly increased(P<0.01),and that of the 4000 ?strain group was significantly decreased(P<0.01).Compared with the 2000 ?strain group,the proliferation level of the 4000 ?strain group decreased significantly(P<0.01).Conclusion:1.Ladder climbing exercise can effectively inhibit the weight gain,reduced grip and weight loss of quadriceps femoris caused by castlessness in rats.The effect of weight gain on the quadriceps still persists after the exercise stopped for 3 w.LIPUS intervention can significantly reduce the weight loss of the quadriceps femoris caused by castration.2.Climbing exercise,low-intensity pulsed ultrasound intervention and the joint interventions have an improved effect on quad femoris atrophy caused by castration.The mechanism alleviates the atrophy of quad femoris in castrated rats by regulating MSTN/Akt signaling pathway,inhibiting MSTN expression,and mediating the downstream FoxO1 and mTOR pathways of Akt to promote protein synthesis.According to the analysis of several different intervention methods,the therapeutic effect of climbing exercise is better than that of LIPUS intervention,and the combined intervention of the two methods is better than that of the single method for the treatment of quadriceps femoris atrophy caused by castration.3.2000 ?strain mechanical strain and 30 mW/cm2 LIPUS can effectively promote the proliferation of myoblasts,further explanation of the cellular mechanism of traction and LIPUS improving skeletal muscle function from in vitro experiments.