Effects Of Aerobic Exercise Combined With LIPUS On Gastrocnemius Muscle In Simulated Weightlessness Rats And Its Molecular Metabolic Mechanism

Purpose:In space,astronauts undergo mid-to-long-term flight,which will cause changes in their physiological systems,mainly manifested by reduced athletic ability,causing muscle atrophy and bone loss.Among them,muscle atrophy is particularly obvious,and such changes will seriously affect the physical health and working ability of astronauts in flight and the ability to readjust after returning.Scientific sports can prevent muscle atrophy,and have a better effect on muscle mass,area,and muscle strength.Targeted exercise methods have a more positive effect on skeletal muscle.The mechanism of action has also been the focus of research.Low-intensity pulse ultrasound(LIPUS)is a safe,effective,and convenient treatment.Studies have shown that LIPUS has biological effects on tissues,promotes muscle damage repair,cell proliferation,and protein synthesis.Explore space and value.However,there are few researches on combating and treating muscle atrophy in the aviation field using dual interventions of exercise combined with low-intensity pulsed ultrasound.Therefore,in this experiment,a rat tail suspension weightless muscular atrophy model was established.Endurance exercise,low-intensity pulsed ultrasound,and a combination of the two were used to study the effects of different intervention methods on the gastrocnemius muscle of muscular atrophy rats from the perspective of protein and histology.Related mechanism research.In addition,metabolomics analysis analysis of the changes of endogenous market by means of technical detection.metabolites and the differential metabolic pathways related to them after exercise,ultrasound and the combination of the three intervention methods.Explore effective methods to combat and treat muscle atrophy caused by weightlessness and its mechanism of action,and provide a reliable basis and guidance for the recovery of muscle atrophy caused by weightlessness in space.Methods:The subjects of this study were 72 healthy pure male Sprague-Dawley rats weighing 180-200 g.Randomly divided into normal control group(NC,n=8),model control group(MC,n=8),high sound intensity ultrasound group(HLS+80mW/cm~2,n=8),high and low sound intensity combined ultrasound group(HLS+30/80mW/cm~2,n=8),low sound intensity ultrasound group(HLS+30mW/cm~2,n=8),high sound intensity combined exercise group(HLS-E+80mW/cm~2,n=8),height Sound intensity ultrasound combined exercise group(HLS-E+30/80mW/cm~2,n=8),low sound intensity ultrasound combined exercise group(HLS-E+30mW/cm~2,n=8),and exercise group(EC,n=8)Nine groups,after one week of adaptive feeding,except for the normal control group,were tail-suspended for 28 days.The ultrasound group and the combined exercise ultrasound group used the LIPUS apparatus for ultrasound diagnosis and treatment.The intensity of the ultrasound therapy apparatus was 30 mW/cm~2 and 80 mW/cm~2,the center frequency was 1.5 MHz,the duty cycle was 20%,and the treatment time was 20 minutes.The treatment time was weekly.7 days,4 weeks of ultrasound treatment until the end of the tail suspension.The rats in the exercise group and the ultrasound combined exercise group were subjected to adaptive exercise for 1 week,followed by a formal treadmill exercise.The treadmill speed was set to 25 m/min,each group time was 40 minutes,the total exercise period was 4 w,and the weekly exercise was 7 d.On the 28th day,the remaining rats were collected for the second time.Finally,the serum and tissues of each group were examined for biochemical indicators such as CK and LDH.H E staining was used to analyze the morphology of the quadriceps of the rats.Using Western Blot to analyze key signal molecules on the MSTN/Akt pathway from the protein level,the effects of aerobic endurance exercise and low-intensity pulsed ultrasound on gastrocnemius atrophy caused by weightlessness and the biological mechanism were explored.Exercise gas chromatography-mass spectrometry(GC-MC)technology is used to detect and analyze rat urine after exercise,ultrasound and exercise combined with ultrasound intervention.A variety of data statistical methods are used,It mainly includes principal component analysis and orthogonal partial least squares method.Analyze to find potential differential metabolites,identify the biomarkers selected through the Kyoto Gene and Genomic Encyclopedia(KEGG)database,and enter them into the MetPA database to analyze the metabolic pathways corresponding to these biomarkers.Results:1.According to the results of gastrocnemius muscle wet weight,at 28 days,the gastrocnemius muscles of other groups were significantly reduced compared with the NC group(P<0.01),and the HLS-E+30mW/cm~2 group was significantly different from the MC group(P<0.05).2.Gastrocnemius muscle tension showed that the muscle tension in the MC group was significantly reduced compared with the NC group at 28 days(P<0.01).The muscle tension in the EC group,HLS+80mW/cm~2 group and HLS-E+3 0mW/cm~2 group was significantly higher than MC Increase(P<0.05).3.The test results of serum biochemical indexes of rats showed that after 28 days of exercise and ultrasound intervention,the serum CK activity of HLS+30mW/cm~2 group was significantly increased compared with HLS+30mW/cm~2 group and MC group(P<0.01),HLS+30 Serum CK in the/80mW/cm~2 group was significantly lower than that in the MC group,NC group,and HLS+30mW/cm~2 group;The serum LDH activity of HLS+30/80mw/cm~2 group was significantly lower than that of MC group,NC group,HLS-E+30mw/cm~2 group and EC group(P<0.01).4.The results of H&E staining on the gastrocnemius morphology of rats showed that,compared with the MC group,NC,HLS+30/80mW/cm~2,HLS+80mW/cm~2,EC,HLS-E+30mW/cm~2,HLS-E The average cross-sectional area of muscle fibers in the+30/80mW/cm~2 and HLS-E+80mW/cm~2 groups increased,and there was a very significant difference(P<0.01).5.WB results showed that MSTN in gastrocnemius muscles of tail-suspended rats showed that compared with the NC group,the gastrocnemius MSTN protein expression levels of the remaining groups were significantly increased(P<0.01);compared with the MC group,HLS+30/The 80mW/cm~2,HLS+30mW/cm~2,HLS-E+80mW/cm~2 groups all increased significantly while the HLS+80mW/cm~2,HLS-E+30/80mW/cm~2 and exercise groups were compared with the HLS+30mW/cm~2 group.Significant differences and a decreasing trend.The MSTN receptor Act ? B protein level test results showed that compared with the NC group and the MC group,HLS+80,W/cm~2,HLS+30/80mW/cm~2,HLS+80mW/cm~2,HLS-E+30mW/cm~2,and Compared with the HLS-E+30mW/cm~2 group,the protein expression in the HLS-E+80mW/cm~2 and HLS-E+30/80mW/cm~2 groups increased significantly in the EC group(P<0.01).Akt protein expression in gastrocnemius muscle of tail-suspended rats:Compared with MC group,except for HLS+80mW/cm~2 group,the other groups were significantly reduced;HLS+30/80mW/cm~2 and HLS-E+30mW/cm~2 group It is significantly higher than the protein expression of HLS+80mW/cm~2 group,and the trend of ATOR downstream molecule mTOR is completely consistent with Akt.It is suggested that the intervention methods have effectively increased the protein expression of Akt and mTOR except for ultrasound with a sound intensity of 80 mW/cm~2.6.The NC group and the MC group rats,the EC group and the MC group rats have two common metabolic differences in the same pathway:linoleic acid metabolism and phenylalanine,tyrosine,tryptophan biosynthesis.Muscle atrophy and exercise intervention are directly related to the changes in the levels of metabolic intermediates linoleic acid and tyrosine;there are 5 potential metabolic targets for the same intensity of 30/80mW/cm~2 and 80mW/cm~2.Pathways:(1)Alanine,glutamate(glu)and aspartic acid(Asp)metabolism;(2)Linoleic acid metabolism;(3)biosynthesis of Phe,Tyr and Try;Glycerine metabolism;(5)the metabolism of glycine(Gly),serine(Ser)and threonine(Thr),And the metabolic target pathways under the intervention of 30mW/cm~2 sound intensity are alanine,aspartic acid and glutamate metabolism;compared with the HLS-E+30mW/cm~2 group in the NC group,three metabolic pathways were analyzed:linoleic Acid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis and glycerol lipid metabolism.Compared with the HLS-E+30/80mW/cm~2 group,the NC group obtained 5 differential metabolic pathways:(1)Biosynthesis of Phe,Tyr and Try;(2)Glycerol metabolism;(3)metabolism of Gly,Ser and Thr;(4)metabolism of glyoxylic acid and dicarboxylic acid;Metabolism of alanine,Asp and glu.Compared with the HLS-E+80mW/cm~2 group,the NC group obtained three metabolic pathways:linoleic acid metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,and glycine,serine and threonine metabolism.Conclusion:1.Aerobic treadmill exercise and three different sound intensity ultrasound can effectively fight the rat gastrocnemius atrophy caused by tail suspension.2.Among the three kinds of ultrasound with different sound intensity,80mW/cm~2 sound intensity ultrasound is better than 30mW/cm~2 sound intensity and 30/80mW/cm~2 sound intensity in improving muscle tone;exercise and ultrasound can improve muscle atrophy because of hindlimb unloading.3.The molecular mechanism of exercise and ultrasound to improve gastrocnemius muscle atrophy in weightless rats may be related to the increased expression of intracellular signaling molecule Akt and downstream signaling protein mTOR.4.According to metabolomics,gastrocnemius muscle atrophy and exercise against muscle atrophy mainly depend on the regulation of linoleic acid metabolism and the biosynthetic pathways of phenylalanine,tyrosine,and tryptophan,mainly related to linoleic acid and tyrosine.The changes are closely related,and these two metabolites can be used as targeted indicators for the diagnosis and treatment of weightless muscular atrophy.