The Mechanistic Study Of Sestrin2 In The Process Of Aerobic Exercise Ameliorating Sarcopenia

Objectives:Sarcopenia brings about many detrimental effects on the health of the elderly.Accumulation of abnormal mitochondria is an important reason why skeletal muscle cells age.Sestrins is stress induced protein with antiaging effect which is closely related to autophagy,so the antioxidative and autophagic function of Sestrins may play an important role in protecting the damaged mitochondria of aging skeletal muscle,and its specific regulatory mechanism needs to be studied in depth.Aerobic exercise effectively improves aging skeletal muscle atrophy,but its regulation mechanism is not yet clear.In order to provide a theoretical basis for revealing the mechanism of Sestrins in improving skeletal muscle atrophy in the aged,this topic focuses on the interaction of Sestrins and mitochondria in aging skeletal muscle.Methods:（1）Animal model:3 months old C57BL/6 young male mice（YS）were 8,and 12 months old C57BL/6 aged male mice were 16,divided into the elderly sedentary group（OS）and the elderly exercise group（OE）randomly.In addition,3months old male wild type and AMPKα2 gene knockout mice（n=8）were divided into sedentary group and the exercise group.After the exercise intervention,the muscle strength test,the body composition analysis,the weight of the body was weighed,and then the mice were sacrificed;（2）Exercise protocol:the intervention time in the elderly exercise group is 8 months.The AMPKα2 knockout mice exercise group for 6 weeks.The aerobic exercise intensity is 75%VO₂max for 1 hour,1 time per day,5 times per week;（3）The body composition testing:after exercise intervention the use of“ImpediVET analyzer”output body mass index,fat mass,fat free mass,followed by statistical analysis;（4）Electron microscope:after the mice were killed,the gastrocnemius muscle was rapidly separated,fixed by 2.5%glutaraldehyde and 1%osmium acid,dehydrated by alcohol and acetone,encapsulated by epoxy resin,and prepared ultrathin sections,and the mitochondria morphology was observed under 30000 times of vision;（5）Cell culture:C2C12 cells was induced to differentiate into myotubes by 2%equine serum,and using Sesn1-siRNA,Sesn2-siRNA to intervene cells,and ROS content in cell was detected by ROS detection kit,then extracted cell protein;（6）The gastrocnemius muscle of mice was fixed by 10%neutral formaldehyde,then paraffin embedded,sectioned and stained with hematoxylin-eosin.Finally,the cross-sectional area of gastrocnemius of each group was observed under microscope;（7）Western blot was used to detect Sesn1,Sesn2,Sesn3,pAMPK-Thr172,Drp1,Mfn2,Pink1,p62,PGC-1αCOX4,IL6and TNF-αprotein expression in skeletal muscle tissue or cells.Results:（1）Long-term aerobic exercise had no significant effect on increased body weight during aging of mice,but increased the ratio of gastrocnemius muscle mass to body weight,lean body weight,grip strength,the ratio of grip strength to weight,muscle fiber cross-sectional area,meanwhile reduced the fat content and the production of TNF-αin the aging skeletal muscle.（2）Long-term aerobic exercise effectively reduced the giant mitochondria in aging skeletal muscle,and increased mitochondrial function-related protein（Drp1,Mfn2,PGC-1α,COX4）and mitophagy-related protein（Pink1）,simultaneously reduced the expression of P62.（3）Expression of Sesn1 and Sesn2 decreased in skeletal muscle of aged mice.Inhibiting Sesn1 expression had no significant effect on mitochondrial regulatory proteins,while inhibiting the expression of Sesn2 significantly reduced the expression of Drp1,PGC-1 and COX4,and the ratio of Mfn2 and Drp1 was imbalanced（fusion index increased）.Pink1 expression decreased while P62 accumulation increased.In addition,the inhibition of Sesn2 expression also increased ROS levels in myotubes.（4）There was no significant change in the expression of Drp1 in the skeletal muscle of AMPKα2 knockout mice,but Mfn2 and Pink1 expression decreased significantly.Conclusion:（1）Long-term aerobic exercise effectively improves skeletal muscle atrophy during the aging of mice and reduces the low-grade inflammatory state in the aging skeletal muscles.（2）Long-term aerobic exercise may reduce the Occurrence rate of abnormal mitochondria by improving the ability of mitochondrial formation and repair.（3）Sesn2 may modulate the function of mitochondria and reduce the production of ROS in myotube,thereby improving mitochondrial dysfunction in the aging.（4）AMPK may mediate the course of Sesn2 to promote mitochondrial fusion and autophagy in the skeletal muscle.