The Effects Of Aerobic Exercise On Mitochondrial Quality Control In Skeletal Muscle Of Aging Mice

Background:With age,the balance between skeletal muscle protein synthesis and degradation is disrupted,resulting in loss of skeletal muscle mass and function,resulting in a net loss of myofibrillar protein,resulting in sarcopenia and an increased risk of other diseases.The high incidence of muscular dystrophy in the elderly also increases the risk of falls and fractures,which in turn reduces the quality of life and self-care ability,seriously affecting the health and longevity of older persons.Therefore,inhibiting skeletal muscle loss,improving muscle mass and function,and promoting protein synthesis are extremely important for protecting the quality and function of aging skeletal muscle.Skeletal muscle mass and functional integrity can be maintained through mitochondrial quality control and oxidative stress.Although aerobic exercise can improve or delay muscle loss caused by aging by regulating mitochondrial quality control and inhibiting oxidative stress,the specific molecular mechanism is still not fully understood.Studies show that aerobic exercise intervention can effectively promote the up-regulation of β-hydroxybutyrate(β-HB)expression,and exogenous supplementation of β-HB can also improve exercise ability.However,the mechanism of whether the up-regulation of β-HB can mediate the protective effect of aerobic exercise on aging skeletal muscle is still not completely clear.Therefore,it is still worth paying attention to explore the mechanism of delaying and improving the mass and function decline of aging skeletal muscle.Object:In this study,the effects of aerobic exercise on mitochondrial quality control and oxidative stress of skeletal muscle in aging mice were studied to explore the improvement effect of aerobic exercise on the quality and function of skeletal muscle in aging mice,and the possible mechanisms were analyzed,providing important theoretical basis for aerobic exercise to improve the quality and function of skeletal muscle in aging mice,promote protein synthesis and prevent muscle atrophy.Method:(1)Experimental animals and their grouping: 4-month-old male C57BL/6J wild-type mice were randomly divided into young control group(YC)and young exercise group(YE),with 10 mice in each group;The 15-month-old male C57BL/6J wild-type mice were randomly divided into senile control group(OC)and senile exercise group(OE)with 10 mice in each group.(2)Aerobic exercise intervention scheme: The experiment adopted moderate intensity training on the treadmill as the aerobic exercise intervention way.The mice in aerobic exercise group received adaptive training for 1 week before formal intervention to adapt to treadmill exercise.The speed was 15m/min,the slope was 0°,60minutes/time,5 days of exercise and 2 days of rest,and the intervention period was 16 weeks.(3)Motor ability test: Mouse muscle strength was assessed using a grip test.After fixing the grip tester,place the mouse in the center of the instrument,hold the middle and rear of the tail of the mouse and gently pull back.The reading recorded on the instrument is the grip of the mouse.Each mouse was tested for 5 times,and the average value was taken(each interval was 10min).Fatigue resistance and motor coordination of mice were evaluated by rotating-rod fatigue meter.In the adaptation stage,the rotary rod fatigue instrument was used for adaptive training of mice for 2days,and the rotational speed was set at 5-25 rpm for adaptive training,and the time was not more than 5min.In the formal test,the speed was set to accelerate from 4RPM to 40 RPM within 5 minutes,and three experiments were carried out with a5 min rest interval at each interval to record the falling time and speed of mice.The final result was the best time in the three experiments.If no falling time was recorded within 5 minutes,5min was recorded.The timing was stopped when the mouse fell from the rotating rod.If there was a holding rod,the falling time was recorded when the mouse turned from the holding rod to the second circle).(4)Index detection and methods: Blood β-HB level of mice was detected by ketograp--h;mouse gastrocnemius wet-weight ratio was used to evaluate the muscle mass of mice;The morphological changes of mouse skeletal muscle fibers were observed by hematoxylin-eosin staining.CAT,GSH and MDA kits were used to detect antioxidant capacity.The changes of β-HB/HCAR2/PPARG pathway,oxidative stress related markers,mitochondrial biogenesis and mitochondrial dynamics related genes were detected by RT-PCR.Western Blotting detected the protein expressions of BDH1,HCAR2 PPARG,P70S6 K,P-P70S6 K,4EBP-1,P-4EBP-1,PGC-1α,TFAM,MFN2,OPA1,DRP1 and FIS1.Result:(1)Effects of aerobic exercise on skeletal muscle exercise ability in aging mice The grip test results showed that the grip of aging mice decreased significantly compared with that of young mice(P<0.01).Aerobic exercise enhanced the grasping power of aging mice and young mice(P<0.05,P<0.01).The rotating rod fatigue test results showed that the rotating rod time of aging mice was significantly shorter than that of young mice(P<0.01).Aerobic exercise can significantly increase the stick retention time of old mice and young mice(P<0.05,P<0.05).The results showed that the muscle strength,fatigue resistance and motor coordination of mice were weakened by aging,but aerobic exercise could improve the muscle strength,fatigue resistance and motor coordination.(2)Effects of aerobic exercise on muscle mass in aging mice Compared with young group,the wet to weight ratio of gastrocnemius muscle in old group was significantly decreased(P<0.01);However,compared with the aged and young control mice,aerobic exercise significantly increased the wet to weight ratio of gastrocnemius muscle in both aged and young mice(P<0.05,P<0.01),indicating that aerobic exercise can effectively slow down the decline of skeletal muscle mass.(3)Effect of aerobic exercise on fiber morphology of gastrocnemius muscle in aging mice HE staining results showed that the influence of fiber,compared with young mice,mice aging makes the gastrocnemius muscle fiber cross-sectional area decreased significantly(P<0.01);Compared with the control group,aerobic exercise significantly increased the cross-sectional area of muscle fibers in both aging and young mice(P<0.05,P<0.05).(4)Effects of aerobic exercise on signal molecules related to protein synthesis signali--ng pathway in skeletal muscle of aging mice Western Blot results showed that compared with young mice,the protein expressions of protein synthesis-related factors 4EBP-1 and P70S6 K in skeletal muscle of aging mice were significantly decreased(P<0.01,P<0.01).However,aerobic exercise could increase the protein expression of the protein synthesis-related factors(P<0.05,P<0.05).Phosphorylation of 4EBP-1 and P70S6 K was activated without significant differences.(5)Effects of aerobic exercise on m RNA expression of molecules related to mitochondrial quality control in skeletal muscle of aging mice RT-PCR results showed that compared with young mice,m RNA levels of Sirt1,Pgc-1α and Nrf2 related factors related to mitochondrial biogenesis in skeletal muscle decreased with aging(P<0.05,P<0.01,P<0.01),and Tfam tended to decrease,but there was no significant difference.Aerobic exercise could increase the m RNA expressions of Pgc-1α and Nrf2 in skeletal muscle of mice(P<0.05,P<0.05),but the m RNA expressions of Sirt1 and Tfam were increased,but the difference was not obvious.Compared with young mice,m RNA expressions of mitochondrial fusion-related factors Mfn1 and Mfn2 in skeletal muscle of aging mice tended to decrease,but the difference was not obvious.Aerobic exercise could significantly increase m RNA expressions of mitochondrial fusion-related factors Mfn1 and Mfn2 in skeletal muscle of aging mice(P<0.05,P<0.05).The m RNA expression of mitochondrial fission-related factor Fis1 in skeletal muscle decreased due to aging(P<0.05),and the m RNA expression of Drp1 showed a downward trend without significant difference.However,aerobic exercise could increase m RNA expressions of mitochondrial fission-related factors Drp1 and Fis1 in skeletal muscle of aging mice(P<0.05,P<0.05).(6)Effects of aerobic exercise on expression of molecular proteins related to mitocho--ndrial quality control in skeletal muscle of aging mice Western Blot results showed that compared with the young group,the protein expressions of mitochondrial biogenesis related factors PGC-1α and TFAM in skeletal muscle of aging group were decreased(P<0.05,P<0.05).Aerobic exercise increased the protein expressions of PGC-1α and TFAM in skeletal muscle(P<0.05,P<0.05).In addition,protein expressions of mitochondrial fusion and fission-related factors MFN2,OPA1,DRP1 and FIS1 in skeletal muscle of aging mice were decreased(P<0.05,P<0.05,P<0.05,P<0.05).In addition,aerobic exercise increased the protein expressions of mitochondrial fusion and fission-related factors MFN2,OPA1,DRP1 and FIS1 in skeletal muscle of aging mice(P<0.05,P<0.05,P<0.05,P<0.05).(7)Effects of aerobic exercise on antioxidant capacity of skeletal muscle in aging mice Compared with the young group,CAT activity in the old group showed a decreasing trend,but the difference was not obvious.And the aerobic exercise only increased,but there was no significant difference.Compared with the young group,the activity of GSH in the aging group decreased(P<0.01),while the content of MDA increased significantly(P<0.01).However,aerobic exercise could significantly increase the GSH activity of aging mice(P<0.05),decrease MDA content(P<0.01),and also significantly decrease the MDA content of young mice(P<0.01).RT-PCR results showed that the m RNA expression of oxidative stress-related factor NOX2 in skeletal muscle of aged mice increased(P<0.01),but aerobic exercise could reduce the m RNA expression of NOX2(P<0.05).In addition,the m RNA level of anti-oxidative stress-related factor SOD1 in skeletal muscle of mice was decreased by aging(P<0.05),and was significantly increased by aerobic exercise(P<0.05).(8)Effects of aerobic exercise on blood β-HB levels in mice The results showed that the blood levels of β-HB in YE and OE groups immediately after exercise were significantly higher than those before exercise(P<0.05,P<0.05).(9)Effects of aerobic exercise on m RNA and protein expression of β-HB/HCAR2/PPARG signaling pathway in skeletal muscle of aging mice RT-PCR results showed that compared with aging mice,the m RNA expressions of Bdh1,Hcar2 and Pparg in skeletal muscle of aging mice were significantly increased by aerobic exercise(P<0.05,P<0.05,P<0.01).The same results were found in young mice.Western Blot analysis of skeletal muscle BDH1,HCAR2,PPARG protein expression results showed that compared with aging mice,aerobic exercise can significantly increase the skeletal muscle BDH1,HCAR2 and PPARG protein expression(P<0.05,P<0.05,P<0.05).The results were consistent with those of young mice.Conclusion:Aging can lead to skeletal muscle mitochondrial quality control disorders,increased oxidative stress,and decreased protein expression of signaling molecules related to protein synthesis,thus resulting in decreased muscle mass and function.Aerobic exercise may regulate mitochondrial quality control,improve protein synthesis,muscle mass and function,and the β-HB/HCAR2/PPARG signaling pathway may play an important role.