Exercise Intervention Alleviates Sarcopenia By Activating Autophagy Via The MiR-34a-5p/Sirt1/FoxO3a Signaling Pathway

Objective:The rapid advancement of high-throughput sequencing technology has enabled the use of miRNA sequencing in sports science for identifying and quantitatively analyzing miRNAs in the context of preventing and treating chronic diseases through exercise.Physical activity can stimulate the release of specific miRNAs and modulate their expression patterns,thereby influencing the impact of exercise on skeletal muscle myopathy.This research utilized miRNA sequencing to identify and evaluate differentially expressed miRNAs associated with age-related muscle atrophy（sarcopenia）and exercise interventions.The results indicated a significant increase in the level of miR-34a-5p in age-related muscle wasting,showing a negative correlation with skeletal muscle mass and exercise capacity decline.This finding suggests that further exploration of the regulatory molecular mechanisms of miR-34a-5p could enhance the use of miRNA as a research tool for exercise interventions in age-related muscle wasting.Additionally,these findings may support the potential of miR-34a-5p as a biomarker candidate for targeted prevention and treatment strategies under these conditions.Methods:1.To investigate the correlation between miR-34a-5p and age-related skeletal muscle atrophy,61 patients with sarcopenia were divided into two groups:sarcopenia quiet control group（OC）and sarcopenia Biduanjin exercise intervention group（OB）,and 18 young people were included as control group（YC）.Changes in muscle strength,muscle mass,and physical activity were assessed following a 12-week Baduanjin exercise.Peripheral blood samples were collected for miRNA sequencing,target gene GO and KEGG enrichment analysis,protein interaction network construction,and identification of Hub genes.RT-q PCR was utilized to validate differential miRNAs and Hub gene expression,while receiver operating characteristic（ROC）analysis was used to determine the clinical diagnostic specificity of the miRNAs.Additionally,a dual-luciferase reporter gene system was used to examine the interaction between miR-34a-5p and the Sirt1 3’UTR sequence.2.To investigate the impact of miR-34a-5p on natural aging skeletal muscle atrophy under various exercise regimens,SPF C57BL/6 male mice aged 9 months were allowed to age naturally until 21 months.The participants were then randomly assigned to the elderly control group（OC）,aerobic exercise group（OE）,resistance exercise group（OR）,and alternating between aerobic and resistance exercise group（OM）.Young control（YC）mice were 4months old and of the same strain.The aerobic exercise involved treadmill training at a speed of 12 m/min and a 10°slope,which was conducted 3 times a week.Resistance exercise consisted of weight-bearing climbing ladder exercises,starting at 10%of the body weight of mice and increasing by 10%weekly until reaching 60%,which were also performed 3 times a week.In the mixed exercise group,both aerobic and resistance exercises were alternated and were performed 3 times a week over a 12-week period.3.To investigate the key role of miR-34a-5p in skeletal muscle maintenance,a mouse model with skeletal muscle-specific miR-34a-5p knockout was generated using CRISPR/Cas9technology.This model aimed to elucidate the molecular mechanism of miR-34a-5p in regulating skeletal muscle aging.The mice were fed until 18 months of age to establish a natural aging model and then categorized into three groups:the miR-34a homozygous wild-type sedentary control group(miR-34a^f/f-SE),the miR-34a homozygous resistance exercise group(miR-34a^f/f-RE),and the miR-34a skeletal muscle-specific miR-34a^-knockout sedentary control group(miR-34a^-/--SE).The exercise intervention protocol remained consistent with the procedures outlined in experiment 2.Skeletal muscle atrophy and exercise capacity were assessed in all animal models following the exercise intervention.Pathological changes in skeletal muscle were evaluated using HE staining and immunofluorescence staining.Ultrastructural changes in skeletal muscle fibers were observed through transmission electron microscopy（TEM）.RT-q PCR and Western blot analyses were used to detect molecular changes in the ubiquitin?proteasome system,autophagy,and apoptosis mediated by the miR-34a-5p/Sirt1/Fox O3a signaling pathway.Results:1.Intragroup comparisons showed a significant reduction in walking speed in the OC group after the intervention period（p<0.05）.Female sarcopenia patients in the OB group exhibited a significant increase in relative muscle mass（RASM;p<0.01）.Grip strength in males significantly improved（p<0.05）.Both men and women demonstrated significant improvements in gait speed and SPPB scores（p<0.05,p<0.05）.Compared with those in the YC group,body weight significantly increased in the OC group（p<0.001）,while other muscle mass,muscle strength,and physical function indicators decreased significantly.Compared with those in the OC group,the total muscle volume and RASM of the female limbs in the OB group significantly increased following the Baduanjin exercise intervention（p<0.001,p<0.001）.Gait speed and the SPPB score also showed significant improvements（p<0.001,p<0.05）.Bioinformatics analysis revealed that miR-144-5p and miR-34a-5p were intersecting genes.Subsequent RT-q PCR verification confirmed the expression of miR-144-5p and miR-34a-5p,along with the expression of the hub genes SIRT1,SRC,and AR,which was consistent with the initial bioinformatics findings.ROC curve analysis demonstrated the specificity of miR-144-5p and miR-34a-5p as potential molecular diagnostic markers for sarcopenia.Furthermore,dual luciferase reporter gene assay confirmed the direct targeting binding of miR-34a-5p to Sirt1.Pearson correlation analysis indicated a negative correlation between high miR-34a-5p expression and the RASM index,grip strength,and step speed in patients with sarcopenia.2.Compared to the YC group,the OC group exhibited a significant decrease in the wet weight ratio of various muscles and the weight ratio of gastrocnemius to tibia length.However,exercise intervention led to improvements in these indices to varying degrees.Muscle function and exercise ability tests revealed significant improvements in mice grip strength,performance on the Kondziela inverted screen test,and weight test scores postexercise intervention,with the resistance exercise group showing greater improvement.Histological analyses revealed that muscle fibers in the OC group displayed characteristics such as decreased circularization,centralization of nuclei,reduced fiber cross-sectional area（CSA）,and increased fibrosis.Conversely,However,a significant increase in CSA,a decrease in the number of atrophic fibers,a decrease in the number of central nuclear fibers,and a decrease in the degree of muscle fibrosis were observed in the exercise intervention group.TEM revealed myotome disorder,abnormal myofilament arrangement,mitochondrial swelling,and blurred ridges in the OC group,exercise intervention effectively improved the ultrastructure of skeletal muscle fibers and the morphology and number of mitochondria,and the formation of autophagosomes was observed.Moreover,exercise intervention reduced the overexpression of miR-34a-5p in aging skeletal muscle and increased the m RNA level of the target gene Sirt1.Western blot analysis revealed that exercise activated the Sirt1/Fox O3a signaling pathway,further inhibiting the expression of the downstream E3 ubiquitin ligases Atrogin-1 and Mu RF1,reducing skeletal muscle protein degradation,activating autophagy,and reducing cell apoptosis,thereby slowing the process of skeletal muscle atrophy in aging mice.3.Compared to those in the normal aging miR-34a^f/f-SEgroup,the wet-to-weight ratio and tibial length ratio were greater in both the miR-34a^f/f-REand miR-34a^-/--SEgroups.Improvements in forelimb grip strength,flip screen performance,and weight test scores were also noted（p<0.001,p<0.05;p<0.01,p<0.05;p<0.001,p<0.05）.Muscle fibers appeared more densely packed,central aggregation of nuclear fibers decreased,the CSA increased significantly,and fibrosis levels decreased.The skeletal muscle ultrastructure exhibited orderly and compact features,with an increased number of mitochondria.Western blot analysis revealed a significant increase in the protein expression of Sirt1 in both the miR-34a^f/f-REand miR-34a^-/--SEgroups compared to that in the miR-34a^f/f-SEgroup（p<0.001,p<0.05）.Furthermore,the protein expression of Fox O3a in the cell nucleus significantly decreased（p<0.001,p<0.05）,while the protein expression of Atrogin-1 and Myostatin significantly decreased（p<0.01,p<0.05;p<0.01,p<0.05）.The levels of the autophagy-related proteins Beclin-1 and Atg7 and the LC3-II/LC3-I ratio were significantly increased（p<0.001,p<0.05;p<0.001,p<0.001;p<0.001,p<0.001）,with a simultaneous decrease in p62 protein expression（p<0.01,p<0.05）.Notably,the expression of the antiapoptotic protein Bcl-2 significantly increased（p<0.01,p<0.01）,while the expression of the proapoptotic protein Bax decreased significantly（p<0.01,p<0.01）,leading to a notable decrease in the percentage of TUNEL-positive cells（p<0.01,p<0.05）.Conclusion:Twelve weeks of Baduanjin exercise intervention has been shown to enhance muscle mass,muscle strength,and physical activity in individuals with sarcopenia.miRNA sequencing analysis revealed a negative correlation between high levels of miR-34a-5p in the peripheral blood of sarcopenic patients and indicators of muscular atrophy.A 12-week exercise program effectively improved skeletal muscle atrophy,muscle mass,and exercise capacity in naturally aged mice at 24 months.This improvement may be attributed to the inhibition of elevated miR-34a-5p expression in the skeletal muscle of aged mice,leading to the activation of the Sirt1/Fox O3a signalingpathway,enhanced autophagy activity,reduced apoptosis,and decreased ubiquitin?proteasome system activity.Compared with the control group of aging mice,the specific knockout of miR-34a-5p in skeletal muscle at 21 months of age can activate Sirt1/Fox O3a signaling pathway,improve autophagy,reduce apoptosis,inhibit the activity of ubiquitin-proteasomal system,reduce the aging process of skeletal muscle,improve exercise ability,and delay the process of skeletal muscle atrophy.