The Effect Of Weight-bearing Exercise And LIPUS Dual Intervention On The Quadriceps Femoris Of Ovariectomized Rats And Its Biological Mechanism

Purpose:Muscle atrophy becomes a common clinical disease.Especially in the middle-aged and elderly women after 60 years old,menopause occurs frequently.After menopause,the level of estrogen in the human body drops sharply.The decline of muscle mass and strength produces a series of physiological functions and the negative effects increase the risk of multiple diseases.Skeletal muscle is the dynamic part of the motor system.Under the nerve drive,normal contraction occurs,and the joint acts as a hub to affect bone activity.Muscle atrophy seriously affects the daily life of menopausal women,while scientific sports can prevent muscle atrophy,and has a good effect on muscle mass,area,and muscle strength.Targeted exercise is more active on skeletal muscle.The effect,such as one of the resistance movements,is weight-bearing exercise.Low-intensity pulsed ultrasound(LIPUS)has been widely used in the treatment of injury sites and fracture healing in recent years as a recognized non-invasive,safe and reliable treatment tool.As a kind of mechanical stimulation,it improves the cell proliferation and survival,and has a great concern on the soft tissue and skeletal muscle.Its exploration space and value are very large.The use of exercise and low-intensity pulsed ultrasound in the field of prevention and treatment of muscle atrophy is very scarce,and the impact of the combined intervention on muscle is rare.This study first established a bilateral ovarian model in rats,using weight-bearing exercise and low-intensity pulsed ultrasound and a combination of the two interventions to study different interventions for ovariectomized rats from the perspectives of genes,proteins,histology and pathology,and find the effects of quadriceps and related mechanisms.At the cellular level,the effects of four-point bending mechanics loader simulated motion and low-intensity pulsed ultrasound on myoblasts and related mechanisms were studied.Exploring effective methods for preventing and treating muscle wasting diseases caused by postmenopausal estrogen deficiency provides a theoretical basis for clinical medicine.Methods:The subjects of this study was 70 healthy pure female SD(Sprague-Dawley)rats that weighted 220-240 g.These rats were randomly divided into normal control group(Sham group,Sham)and the ovaries removed group(OVX group,OVX).Ovaries removed group bilateral ovarian surgery is adopted to establish the model of the Sham group no ovaries removed.After 2 weeks,the healing rats were randomly divided into four groups,ovaries removed groups(Ovariectomized group,the OVX).Weight-bearing exercise group(O+W),Ovariectomized ultrasound treatment group(O+U)and group of combining weight-bearing exercise and ultrasound combined treatment group(O+W+U).The female rats in the exercise group could bear about 35%of their own body weight.The rats were subjected to adaptive exercise for 1 w,and then the official treadmill exercise was carried out.The speed of the treadmill was set to 20 m/min,each group was 30 min,the total weight exercise period was 10 w,and the exercise was performed for 6 d per week.After 10 weeks of weight-bearing exercise,3 weeks of ultrasound diagnosis was performed.The ultrasound treatment group and the weight-bearing ultrasound group were treated with ultrasound using the LIPUS instrument.The intensity of the ultrasound therapy device was 30 mW/cm2,the center frequency was 1.5 MHz,the duty cycle was 20%and treatment time was 20 min.Meanwhile,6 days a week and 3 w altoghter.After 10 w weight-bearing exercise,8 rats were randomly selected from Sham group and OVX group,and 4 rats from O+W and O+W+U groups were randomly selected,and the serum and tissue were retained for test.The serum and tissue CK and LDH biochemical indexes of each group were detected,and the morphological analysis of rat quadriceps muscle was performed by H&E staining.Western Blot and real-time PCR were used to analyze the key signal molecules in the MSTN/Akt pathway from protein and gene levels,and to investigate the effects of weight-bearing exercise and low-intensity pulsed ultrasound on the quadriceps of ovariectomized rats and its biological mechanism.In order to further explore the effects of exercise and low-intensity pulsed ultrasound on skeletal muscle,this experiment was further studied in the cell aspect.Four-point bending mechanics loader was used to intervene into myoblast proliferation group:cells were randomly grouped and divided into control group(Control)without force and the pull group(Strain,ST).The pull group parameters were set to:frequency of 0.5 Hz,afterburning for 60 min,respectively,mechanical stress stimulation of tensile stress 1000 ?strain,2000 ?strain,4000 ?strain.After the completion of the traction intervention,the shape and distribution of the cells were observed.The next day,the CCK-8 kit was used to detect cell viability and protein extraction.Low-intensity pulsed ultrasound instruments intervened into myoblast proliferation group:cells were randomly grouped:control group and LIPUS group,LI PUS parameters were set to:center frequency was 1.5 MHz,stimulation intensity was 30 mW/cm2,duty cycle was 20%,time is 20 min/d,continuous intervention for 24 1h 72 h,120 h.The next day,the CCK-8 kit was used to detect cell viability and protein extraction.The specific steps were performed according to the kit instructions,and finally the required data were obtained and analyzed.Western Blot method was used to detect the expression of MSTN and upstream key signaling molecules.Results:1.According to the results of body weight,grasping power,wet weight of quadriceps femoris and wet weight of uterus,compared with Sham group,weight of OVX group rats increased with a very significant difference(P<0.01),grasping power of OVX group rats decreased significantly(P<0.01),wet weight of quadriceps femoris and uterus of rats also decreased significantly with a significant difference(P<0.01,P<0.01).After 10 w weight-bearing exercise,the body weight of the O+W group decreased(P<0.05),while the grasping power and the wet weight of the quadriceps femoris increased(P<0.01,P<0.01).After 3 w LIPUS treatment,compared with OVX group,the weight of O+W,O+W+U group rats decreased significantly(P<0.01,P<0.01),the grasping power of O+W,O+U,O+W+U group rats increased significantly(P<0.05,P<0.01,P<0.05),the wet weight of the quadriceps femoris increased significantly(P<0.01),and the wet weight of the uterus did not change significantly.2.According to the detection results of serum and tissue biochemical indexes in rats,through 10 w weight-bearing exercise,compared with Sham group,CK and LDH activities in OVX group were significantly increased(P<0.01,P<0,01);Compared with OVX group,CK and LDH activities of O+W group were significantly different(P<0.01,P<0.01).After 3 w low-intensity pulsed ultrasound treatment,CK activity and LDH activity in serum of OVX group,O+W group,O+U group and O+W+U group showed significant changes compared with Sham group(P<0.01).CK and LDH activity of O+W,O+U,O+W+U group decreased significantly compared with OVX group,and CK and LDH activity of O+W+U group decreased most significantly.3.H&E staining method was used to detect the morphological indexes of the quadriceps femoris in rats.It was found that in the prevention period of 10 w weight-bearing exercise,compared with the Sham group,the average cross-sectional area of muscle fibers in OVX and O+W groups decreased and showed significant difference(P<0.01,P<0.01).Compared with OVX group,the muscle fiber cross-sectional area of the quadriceps femoris in the O+W group was increased,and there was a significant difference(P<0.01).During the diagnosis and treatment of LIPUS,compared with the Sham group,the OVX,O+W,O+U and O+W+U groups all showed significant decreases in the average muscle fiber cross-sectional area(P<0.01,P<0.01,P<0.05,P<0.01).Compared with the OVX group,the average cross-sectional area of the muscle fibers of the quadriceps femoris in the O+W,O+U and O+W+U groups was increased,with significant differences(P<0.01,P<0.01,P<0.05).The average cross-sectional area of quadriceps femoris muscle fiber in the O+U group was higher than that in the O+W group.4.Protein and gene expression result can be concluded that the weight-bearing exercise and LIPUS significantly inhibited the MSTN mRNA expression level and its receptor ActRIIB mRNA and ActRIIB protein expression levels,obviously raised important molecules in the muscles of the Akt mRNA,mTOR mRNA and mTOR and Akt phosphorylation p-Akt protein expression levels and downstream signaling molecules FoxO1 mRNA expression and FoxOl protein expression levels.5.The experimental results from cell can be concluded that the four-point bending mechanical force and low intensity pulsed ultrasound intervention into muscle cells,cell proliferation by CCK 8 detection,compared with the Control group,four-point bending mechanical force group and the low intensity pulsed ultrasound of muscle cells have obvious proliferation,and there are significant differences,of which 2000 ?strain and LIPUS proliferation effect was most pronounced-72 h group,using Western Blot method,extraction of myoblast do protein detection,four-point bending mechanical force and low intensity pulsed ultrasound after intervention into muscle cells,MSTN protein expression was significantly inhibited and the expression levels of ERK1/2 and Integrin proteins were up-regulated with significant differences.It was also shown that the two best parameters for the intervention of myoblasts were 2000 ?strain and LIPUS-72 h.Conclusion:1.Weight-bearing exercise can effectively prevent the skeletal muscle atrophy caused by ovulation removal in rats,and effectively increase the cross-sectional area of quadriceps femoris muscle cells.This good effect was not significantly changed during the 3w period of ultrasonic treatment without exercise training.2.Weight-bearing exercise and low-intensity pulsed ultrasound intervention,as well as the combined intervention,have a good effect on improving the symptoms of quadriceps femoris atrophy in ovariectomized rats.3.The molecular mechanism of the combined effect of weight-bearing exercise and low-intensity pulsed ultrasound on the quadriceps femoris of ovariectomated rats may be that by inhibiting the expression of MSTN/ActRIIBand activating downstream Akt,mTOR and FoxO1.protein synthesis is accelerated and degradation is inhibited,thereby improving muscle atrophy.4.Cell experiments showed that four-point bending and low-intensity pulsed ultrasound both promoted proliferation of myoblasts,and Integrin ??ERK1/2 may be an upstream signaling pathway regulating MSNT expression.