Intervention Mechanism Of Aerobic And Resistance Exercise On P53-Mediated Ferroptosis In Skeletal Muscle Of Aging Mice

Background:China has gradually entered an aging society,with the emergence of aging,the skeletal muscle in the elderly in quality,function in different degrees of decline,called sarcopenia.The causes of sarcopenia are not fully understood,and there are still no definitive treatments or interventions for this disease.Ferroptosis is an iron-dependent form of cell death in which a"Fenton reaction"occurs in cells due to a large accumulation of iron,producing toxic lipid peroxides and extremely active reactive oxygen species（ROS）.It has been shown that ferroptosis may be a cause of muscle atrophy and that p53 production is increased in aging human myocytes,inhibiting the transport of cysteine and cysteine by light chain subunit solute carrier family 7 member11（SLC7A11）and regulating glutathione（GSH）levels,thereby affecting lipid peroxidation and ROS levels.Similarly,it has been shown that spermidine/spermine N1-acetyltransferase 1（SAT1）is regulated by p53,while arachidonic acid 15-lipoxygenase（ALOX15）is a target protein downstream of SAT1 that affects lipid peroxidation and plays an important regulatory role in the process of Ferroptosis.whether different forms of motility have an effect on the p53/SLC7A11 signaling pathway and whether different forms of movement have a role for p53/SLC7A11signaling pathway and for downstream related molecules SAT1 and ALOX15 in the regulation of ferroptosis still need further investigation.Objective:In order to investigate the relevant mechanisms and molecular roles of the p53/SLC7A11 signaling pathway and the expression of p53 on SAT1 and ALOX15 in the regulation of ferroptosis,C57BL/6J naturally aged mice were used for 8 weeks of aerobic,resistance training to investigate the effects of exercise on iron content,the p53/SLC7A11 signaling pathway and downstream SAT1 and ALOX15 to provide a theoretical basis for improving ferroptosis and muscle atrophy.Methods:After the mice were raised to 18 months of age,they were randomly divided into 3 groups:the quiet control group,the aerobic training group,the resistance training group,and the young control group,with 6 mice in each group.Afterwards,an 8-week training plan was carried out.The training group started sports training at8:00 am on January,3,and 5 every week.The aerobic training group performed treadmill training.The resistance training group used pyramid training for ladder training.Weekly measurement The mouse grip strength,body weight,maximum weight and other indicators were recorded.After the last training,the mice were sacrificed by neck dislocation,and the quadriceps femoris muscle was harvested.HE staining was used to observe the changes of muscle cross-sectional area,and ROS immunofluorescence was used to observe the area of ROS;tissue iron,malondialdehyde（MDA）,and glutathione assay kits were used to measure various contents;Wes^TM automatic protein expression analysis system was used to measure p53,Protein expression levels of SLC7A11,GPX4,ALOX15,SAT1,ZIP14,and TFr1.Image J was used to analyze muscle cross-sectional area and number of ROS,SPSS25.0 was used for data statistics and analysis,and Graph Pad Prism 8.0 was used for drawing.Results:（1）Body weight,muscle mass index,grip strength,and muscle cross-sectional area changes:（a）The body weight of mice in group C was markedly larger than that of mice in group RT（p<0.05）;there was a highly substantial change in body weight in group C before and after the experiment（p<0.01）,and there was no substantial variation between groups AT and RT（p>0.05）.（b）The muscle mass index of mice in group C was drastically lower than that of mice in group YC（p<0.01）;group RT was vastly larger than that of group C（p<0.05）;and there was no big variation between mice in groups AT and C（p>0.05）.（c）The grip strength of group C mice declined substantially（P<0.001）when compared to the YC group;the grip strength of the RT group was markedly larger than that of the C group（P<0.01）;the grip strength was consistently greater in group C at 4 weeks compared to 0 weeks（p<0.05）,but there was no substantial variation in grip strength in group C at 8 weeks compared to 0 weeks（p>0.05）;the grip strength was extremely significantly greater AT at 4 weeks compared to（d）Group C mice had a considerably smaller muscle cross-sectional area than group YC mice（p<0.05）;group RT mice had a substantially larger muscle cross-sectional area than group C mice（p<0.05）.The muscle cross-sectional area of mice in groups AT and C did not differ significantly（p>0.05）.（2）Protein expression levels associated to the p53/SLC7A11 signalling pathway:（a）p53 protein levels are elevated in group C mice than in group YC mice（p<0.01）;p53protein levels were considerably lower in training group mice compared to group C mice（p<0.05）;there was no substantial variation between AT and RT groups（p>0.05）;（b）group C mice SLC7A11 protein levels were remarkably lower in group C mice than in group YC mice（p<0.05）;there was no statistical difference between AT and RT groups（p>0.05）;（c）There was no statistical difference between group C mice and group YC mice（p>0.05）,and the GPX4 protein level was markedly lower in group RT mice compared to group C mice（p<0.05）,and there was no statistical difference between group AT mice and group C mice（p>0.05）;（d）there was no statistical difference between group C mice and group YC mice（p>0.05）.The mice were not statistically significant from group C（p>0.05）.（3）Comparison of SAT1 and ALOX15 protein expression levels:（a）SAT1 protein expression was statistically highly significant C mice than in group YC mice（p<0.05）;SAT1 protein expression was markedly lower in group AT mice（p<0.05）,and no change was observed in group RT mice（p>0.05）;（b）ALOX15 protein expression was statistically higher C mice than in group YC mice（p<0.01）.ALOX15 protein expression was substantially greater in group C mice than in group YC mice（p<0.01）;ALOX15 protein expression was dramatically lower in group AT mice（p<0.05）than in group C mice,and there was no notable change in group RT mice（p>0.05）.（4）Comparison of ZIP14 and TFr1 protein expression levels:（a）ZIP14 protein expression level was considerably greater in mice from group C（p<0.01）;in mice from AT and RT groups,ZIP14 protein expression level was markedly smaller（p<0.05）.There was no statistical difference between the AT and RT groups;（b）TFr1 protein expression was considerably higher in group C mice.（b）TFr1 protein expression in mice in group C was substantially lower than in mice in group YC（p<0.05）;there was no statistical difference in TFr1 protein expression levels in mice in the training group compared to mice in group C（p>0.05）.（5）Comparing tissue iron,GSH,and MDA levels,as well as the region of ROS fluorescence:（a）Compared to mice in the YC group,the quadriceps iron content was extremely significant higher in the C group（p<0.01）;compared to mice in the C group,the iron content was extremely substantial lower in the AT group（p<0.01）and dramatically decrease in the RT group（p<0.05）,with no statistical difference between the AT and RT groups(p>（p0.05）;（b）The GSH content of the quadriceps muscle of mice in group C decreased over time and was markedly smaller than that of mice in group YC（p<0.05）;the GSH content of the quadriceps muscle of mice in group RT was dramatically higher（p<0.05）than that of mice in group C;and there was no statistical difference between mice in group AT and mice in group C（p>0.05）.（c）The quadriceps MDA content was remarkably higher in group C mice（p<0.05）;compared with group C mice,the quadriceps MDA content was statistically lower in group AT and group RT（p<0.05）,and there was no statistical difference in MDA content between group AT and group RT（p>0.05）;（d）The ROS fluorescence area was statistically higher in group C mice than in group YC mice（p<0.001）;the ROS fluorescence area was dramatically lower in mice in the AT group（p<0.01）and remarkably lower in mice in the RT group（p<0.001）compared with mice in the C group.the ROS fluorescence area was statistically higher in mice in the AT group compared with the RT group（p<0.05）.Conclusion:（1）Resistance training can effectively increase the wet weight/body weight ratio of quadriceps muscle,which can improve the muscle mass and relative grip strength of skeletal muscle of the limbs in mice.（2）Resistance training may improve the disordered iron metabolism in mice by down-regulating p53 negative regulation of the SLC39A11 signaling pathway and its downstream related molecules,and regulating GSH content to inhibit the antioxidant imbalance occurring in muscle.（3）Aerobic training may regulate lipid peroxide production by down-regulating p53-activated SAT1,ALOX15 protein expression,thereby ameliorating the iron overload that occurs in mice muscle.