| Aging-related changes in skeletal muscle atrophy occur during aging.In the past studies,whether studies on degenerative changes of aging skeletal muscle,or studies on exercise and caloric restriction as interventions to slow down aging skeletal muscle degeneration,mostly focused on muscle synthesis.In recent years,people have gradually realized the importance of degradation on skeletal muscle function and quality,becoming the research direction gradually shifting to skeletal muscle degradation.80% of proteins in vivo are degraded by the ubiquitin-proteasome system,so the level of ubiquitination modification of proteins in vivo is essential for the quality control of proteins.?Objective: This study was conducted from the perspective of ubiquitination modification level.The relationship between changes in ubiquitination modification levels and aging process was explored through quantitative proteomic research strategies of non-standard quantification and ubiquitination modification enrichment techniques,as well as high resolution liquid chromatography-mass spectrometry.In addition,caloric restriction and exercise were used as intervention means to explore the effect of exercise and caloric restriction on the level of ubiquitination modification of proteins in aging organisms.Selection of experimental animals: In this experiment,40 male SD rats(purchased from Tianjin Aoyi Laboratory Animal Breeding Co.,Ltd.,license number: SCXK(Beijing)2007-0003,grade VAF)with no significant difference in body weight at 9months of age were selected.Firstly,8 rats were randomly selected as young control group(n=8)for sampling.The remaining rats(n=32)were kept in the animal room of our laboratory until the age of 24 months,when our experimental intervention was started.Animal model construction: The purchased rats were raised in the animal room of our laboratory,which was a clean-grade experimental animal room.Two animals per cage,natural lighting,natural circadian rhythm,free to eat,indoor temperature20-25?,humidity 40%-60%.Training program: This study is based on ZsoltRadak reported the method to carry out the experiment: model animal grouping: when SD rats were reared until 24 months of age,aging rats(n=32)were randomly divided into four groups: normal diet quiet control group(S,herein referred to as the aging group),caloric intake restriction group(X,herein referred to as the restriction group),exercise intervention group(Y,herein referred to as the exercise group),caloric restriction plus exercise group(YX,This article is referred to as the exercise restriction group).Exercise intervention: This experiment was conducted after a week of adaptive exercise.The same intensity of treadmill training was conducted on six days a week,strength 15 m/min,45 min/time for three months.Caloric restriction intervention: single cage feeding,free drinking water,The food intake of rats in the caloric restriction group,exercise and caloric restriction group was 60% of that in the free-feeding group,and the non-caloric restriction group was fed with sufficient feed.Methods:After the successful construction of animal models,sampling,protein extraction,SDS-PAGE,trypsin digestion,affinity enrichment,LC-MS/MS analysis,database search,KEGG pathway annotation,subcellular localization.Statistical analysis: Two-sample two-tailed T-test was used to calculate the p-value between groups.Let p value < 0.05 be the significant difference,and the change of differential modifier more than 1.5 as the change threshold of significant upregulation,and less than 1/1.5 as the change threshold of significant downregulation.Experimental result:1.A total of 255754 secondary spectra were obtained by mass spectrometry.After searching the library of protein theory,the available spectrum number is 70009,the spectrum utilization ratio is 27.4%,and 6316 peptide segments and 2646 ubiquitinated modified peptide segments are identified.2.In the comparison group of aging and young people,there were 33up-regulated proteins with 65 sites.There are 114 down regulated proteins and 233 down regulated sites.In the control group,103 proteins and 229 loci were up-regulated.There are 15 down regulated proteins and 21 down regulated sites.In the exercise group and the aging group,there were 43 up-regulated proteins with 82 sites.There are 31 down regulated proteins and 55 down regulated sites.In the exercise restriction group and the aging group,there were 138 up-regulated proteins with 344 sites.There are 8 down regulated proteins and 10 down regulated sites.3.Access obtained by KEGG the database.Pathways obtained through KEGG database.Aging group and young group comparison group: metabolic pathways:Glycolysis/Gluconeogenesis,Citrate cycle(TCA cycle),Pentose phosphate pathway,Fructose and mannose metabolism,Oxidative phosphorylation,etc.Signaling pathways: Glucagon signaling pathway;pathways highly related to metabolic pathways and signaling pathways in this comparison group are:Thyroid hormone synthesis,Alzheimer's disease,Parkinson's disease,etc.Caloric restriction group and aging group: metabolic pathway:Glycolysis/Gluconeogenesis,Citrate cycle(TCA cycle),Fructose and mannose metabolism,Oxidative phosphorylation,Metabolic pathway;signaling pathway: Calcium signaling pathway,cGMP-PKG signaling pathway,Apelin signaling pathway;pathways highly related to metabolic pathways and signaling pathways in this comparison group: Huntington disease,Legionellosis,Circadian entrainment,etc.The comparison group of exercise group and aging group: metabolic pathways: Arginine biosynthesis,Tyrosine metabolism,Phenylalanine metabolism,etc;signaling pathways: Calcium signaling pathway,cGMP-PKG signaling pathway;the pathways highly related to the signaling pathways in this comparison group are: Estrogen signaling pathway,Thyroid hormone signaling pathway,Huntington disease,etc.The exercise caloric restriction group and the aging group comparison group: metabolic pathways:Glycolysis/Gluconeogenesis,Citrate cycle(TCA cycle),Purine metabolism,etc.Signaling pathways: Calcium signaling pathway,cGMP-PKG signaling pathway,cAMP signaling pathway;The pathways highly associated with the metabolic pathways in this comparison group are: Adrenergic signaling in cardiomyocytes,circadian rhythm,Insulin secretion,Glucagon signaling pathway,etc.Exercise caloric restriction group and caloric restriction group comp-arison group: metabolic pathways: Glycolysis/Gluconeogenesis,Glutathione metabo-lism,Carbon metabolism;signaling pathways: PPAR signaling pathway,Calcium signaling pathway,cGMP-PKG signaling pathway;pathways highlyassociated with the metabolic pathways in this comparison group are: Alzheimer's disease.Exercise caloric restriction group and exercise group comparison group:metabolic pathways: Glycolysis/Gluconeogenesis,Citrate cycle(TCA cycle),Oxidative phosphorylation,Pyruvate metabolism,Carbon metabolism,etc;signaling pathways: Calcium signaling pathway,cGMP-PKG signaling pathway,cAMP signaling pathway;pathways highly related to the metabolic pathways in this comparison group are Thyroid Hormone signaling pathway,salivary secretion,bile secretion,etc.4.The KEGG pathway database was used to annotate the protein pathways of proteins that showed significant differences in ubiquitination modification levels among the aging group and young group comparison group,caloric restriction group and aging group,exercise group and aging group comparison group,exercise caloric restriction group and aging group comparison group,so as to obtain multiple protein pathways,among which TCA cycle pathway had significant change consistency in ubiquitin modification level.5.relative expression and significance of the Malate dehydrogenase,cytoplasmic: compared with the aging group,the expression of the protein in the young group was significantly reduced(p < 0.01),the relative expression of the protein in the caloric restriction group was not significantly different,and the relative expression of the protein in the exercise caloric restriction group was significantly reduced(p < 0.05).Relative expression and significance of the Malate dehydrogenase,mitochondrial: compared with the aging group,there was no significant change in the expression of the protein in the young group,the relative expression of the protein in the caloric restriction group was significantly reduced(p < 0.05),and the relative expression of the protein in the exercise caloric restriction group was not significantly changed.Relative expression and significance of the Isocitrate dehydrogenase [NADP],mitochondrial: compared with the aging group,the protein expression in the young group decreased significantly(p < 0.01),The relative expression of this protein wassignificantly increased in the caloric restriction group(p < 0.01),there was no significant change in the relative expression of this protein in the exercise caloric restriction group.Relative expression and significance of the Isocitrate dehydrogenase [NAD]subunit,mitochondrial: compared with the aging group,the protein expression in the young group increased significantly(p < 0.05),there was no significant difference in the relative expression of this protein between the caloric restriction group and the exercise caloric restriction group.Relative expression and significance of the Isocitrate dehydrogenase [NAD]subunit beta,mitochondrial: compared with the aging group,the protein expression in the young group increased significantly(p < 0.01),the relative expression of this protein increased significantly in the caloric restriction group(p < 0.01),the relative expression of this protein increased significantly in the exercise caloric restriction group(p < 0.01).Relative expression and significance of the Dihydrolipoamide S-succinyltransferase(E2 component of 2-oxo-glutarate complex): compared with the aging group,there was no significant difference in the expression of the protein in the young group,and the relative expression of the protein increased significantly in the caloric restriction group(p < 0.01),the relative expression of this protein increased significantly in the exercise caloric restriction group(p < 0.05).Relative expression and significance of the Aconitate hydratase,mitochondrial:compared with the aging group,the protein expression in the young group was significantly decreased(p < 0.05),the relative expression of this protein in the caloric restriction group was not significantly different from that in the exercise caloric restriction group.6.Among the proteins with significantly different levels of ubiquitination modification,there are 40 proteins that target the mitochondria(Appendix 2),among them,ADP / ATP translocase 1 is the protein with significant difference in the level of ubiquitination modification exercise calorie restriction group and the aging group,inthe aging group and the youth group comparison group,calorie restriction group and aging group comparison group,comparison between exercise group and aging group,Conclusion: 1.In aging organisms,the level of ubiquitination modification of skeletal muscle proteins decreases as a whole,which may suggest that the aging performance of skeletal muscle is closely related to protein ubiquitination degradation.Calorie restriction and exercise can restore or increase the level of ubiquitination modification of skeletal muscle proteins.2.To further verify the possible mechanism of the effect of the level of ubiquitination modification on skeletal muscle aging and the possible mechanism of exercise and calorie restriction to increase the level of ubiquitination modification of skeletal muscle proteins,we annotate the protein pathway of ubiquitination modification differential proteins.Among the obtained multiple protein pathways,TCA cycle changes were significantly consistent,so it was speculated that TCA cycle might be the key mechanism contributing to aging manifestations,or that exercise and calorie restriction intervention could be the key mechanism resisting aging manifestations of skeletal muscle.Exercise and calorie restriction can increase the level of ubiquitination modification of proteins in the TCA cycle pathway,presumably improving the rate of protein turnover in the pathway.If the renewal rate of protein in the pathway is increased,it can enhance the pathway activity and function level of TCA circulatory pathway in aging skeletal muscle,restore the aerobic oxidative function of aging skeletal muscle to a certain extent,and even resist the damage caused by aging skeletal muscle.3.According to the key protein pathway(TCA cycle)identified in Result 2,and in order to further study the effect of changes in the level of ubiquitination modification of mitochondrial-related proteins on skeletal muscle aging,subcellular structural localization of all ubiquitination-differentiated proteins was performed.It was found that ADP/ATP translocase 1 changed significantly in the exercise group,the calorie restriction group,the exercise calorie restriction group and the aging group compared with the aging group.It is speculated that ADP/ATP translocase 1 may be akey protein in the mitochondrial mechanism that contributes to the manifestation of skeletal muscle aging during aging,as well as a target of exercise and calorie restriction intervention to delay skeletal muscle aging.It is speculated that exercise and calorie restriction interventions can improve the rate of ADP/ATP translocase 1renewal. |
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