| Background: With the increase in elderly population and the prolongation of life expectancy,sarcopenia as one of hot issues has gradually drawn attraction from public health.Preventing and delaying the onset of sarcopenia and improving the quality of self-care life of the elderly have been the focus of aging research.Recent studies have shown that iron accumulation and ferroptosis are related to the aging process.Iron accumulation may induce Fenton reaction,an increase of reactive oxygen species（ROS）,and then lead to oxidative stress,resulting in growing sensitivity to ferroptosis.Different exercises have important effects on oxidative stress and antioxidant balance of the body.Whether exercise can improve antioxidant capacity to relieve oxidative stress caused by iron overload,so as to improve skeletal muscle mass and delay aging?And Whether exercise can affect iron metabolism deserves further study.Objectives:（1）To investigate the effects of aging on ferroptosis and antioxidant capacity of skeletal muscle in mice;（2）To study the relationship between ferroptosis and sarcopenia;（3）To explore the effect of resistance or aerobic exercise intervention on iron metabolism and antioxidant capacity.It provides theoretical basis for exercise to intervene iron metabolism and reduce sensitivity to ferroptosis.Methods: Thirty-two 2-month-old C57BL/6J mice were purchased and reared to 3and 18 months of age in an IVC independent air supply system to establish young mice and natural aging mouse models,a total of 4 experimental groups were set up（including: young quiet control group,elderly quiet control group,elderly aerobic exercise group,elderly resistance exercise group）.The 3-month-old mice were included in the young control group（YC group: n=8）,and the 18-month-old mice were randomly divided into three groups: The elderly quiet control group（OC group:n=8）,who ate and drank freely and did not participate in exercise training.The Elderly resistance exercise group（OR group: n=8）,carried out resistance exercise with tail load climbing ladder for 8 weeks on Mondays,Wednesdays and Fridays,and measured the maximum weight bearing capacity（Max）of Mice in OR group on Mondays.Taking Max value as training standard,the "Pyramid training model" was adopted,and the ladder climbing training was conducted for 3 times,2 times,1 time,2 times and 3 times respectively in the sequence of 50%,75%,100%,75% and 50%of the maximum load of mice.Rest between groups for 1min and rest between groups for 30 s are needed in the process.The elderly aerobic exercise group（OA group: n=8）underwent aerobic exercise at a speed of 6m/min on the running platform for 30 minutes for 8 weeks on Mondays,Wednesdays and Fridays.The body weight of OC,OA and OR groups was measured every week,and the grip strength was measured at week 1,4 and 8.The maximum loading capacity of OC and OA groups was measured at week 1 and 4.At the age of 3 months,mice in YC group were anesthetized by intraperitoneal injection of 10% chloral hydrate（3.5ml/kg body weight）12 hours after the last training,and the mice were killed by neck removal and samples were taken.The quadriceps femoris muscle was fixed in 4% paraformaldehyde solution,followed by histochemistry experiments and HE staining.After electron microscopy,Image Pro Plus 6.0 software was used to calculate the cross-sectional area of quadriceps muscle fiber in aging mice.ROS was detected by fluorescence staining,and protein changes of TF,TFR1,DMT1,FPN1,Ferritin,NCOA4,NRF2,x CT,GPX4,SOD1,SOD2,CAT and HMOX1 in mice quadriceps femoris were detected by western blot.The contents of total iron,MDA and GSH,T-AOC and T-SOD activities,GPX and CAT enzyme activities of mice quadriceps femoris were detected by kit.Image Pro Plus 6.0 and Case Viewer were used for statistical processing of the experimental data,IBM SPSS Statistics 26 was used for statistical analysis,and Graphpad Prism8 was used to plot the data analysis results.Results:（1）Body weight and skeletal muscle fiber cross-sectional area,weight,strength and function indexes: Compared with the changes in weight of mice in each group,the body weight of aging mice was significantly increased compared with that of young mice（P < 0.001）.After 8 weeks of experiment,the body weight of OC mice was significantly increased（P < 0.01）,while there were no significant changes in the body weight of OR and OA mice.The body weight of mice in OR group was significantly lower than that in OC group（P < 0.05）.Compared with YC group,the skeletal muscle fiber cross-sectional area,weight,strength and function indexes of skeletal muscle fiber in OC group were significantly decreased,including skeletal muscle fiber cross-sectional area（P < 0.001）,quadriceps mass index（P < 0.001）,relative grip strength（P < 0.01）and relative maximum load-bearing capacity（Max）（P < 0.001）.Compared with OC group,muscle fiber cross-sectional area,quadriceps femoris mass index and relative Max of mice in OA group showed an increasing trend but no significant difference,while relative grip strength was extremely significantly increased（P < 0.01）.Compared with OC group,the morphology,mass,strength and function indexes of skeletal muscle in OR group were significantly improved,including muscle fiber cross-sectional area（P < 0.01）,quadriceps muscle mass index（P < 0.01）,relative grip strength（P < 0.001）and relative Max（P < 0.001）.（2）Total iron and related indexes of iron metabolism in skeletal muscle: Compared with YC group,iron content in quadriceps femoris in OC group was significantly increased（P < 0.001）;The protein expression of TFR1,HMOX1,FPN1,Ferritin and NCOA4 was significantly down-regulated（P < 0.001）.Compared with OC group,HMOX1 protein expression in quadriceps femoris of mice in OA group was significantly up-regulated（P < 0.05）;Compared with OC group,the iron content in OA and OR groups was significantly decreased（P < 0.001）.The expression of FPN1 protein was significantly up-regulated（P < 0.05）,ferritin protein was extremely significantly down-regulated（P < 0.001）,and NCOA4 protein was extremely significantly down-regulated（P < 0.01）.（3）Oxidative stress and antioxidant related indexes:Oxidative stress index: Compared with YC group,ROS content of quadriceps femoris were extremely significantly increased（P < 0.001）and MDA content of quadriceps femoris in OC group were extremely significantly increased（P < 0.01）;Compared with OC group,ROS content in OA and OR groups was significantly decreased（P <0.001、P < 0.001）,MDA content in was remarkably decreased（P < 0.05、P < 0.001）.Indexes related to NRF2-x CT regulatory mechanism: Compared with YC group,NRF2 protein expression in OC group was extremely significantly down-regulated（P< 0.01）,x CT protein expression was extremely significantly down-regulated（P <0.05）;Compared with OC group,NRF2 protein expression in OA and OR groups was significantly up-regulated（P < 0.05 、 P < 0.001）,x CT protein expression was significantly up-regulated（P < 0.05、P < 0.01）,GPX4 protein expression in OR group was significantly up-regulated（P < 0.001）.The GPX enzyme activity of mice quadriceps femoris in OA group was significantly increased（P < 0.05）.Indexes related to NRF2-ARE regulatory mechanism: Compared with YC group,the protein of CAT and SOD1 in OC group were significantly down-regulated（P < 0.05）,and the activities of T-AOC,CAT,T-SOD and GST were significantly decreased（P<0.001、P <0.05、P <0.05、P <0.001）.Compared with OC group,SOD2 protein was increased（P <0.05、P <0.01）,T-AOC activity was significantly increased（P <0.05、P <0.01）,GST enzyme activity was significantly increased（P <0.01、P <0.05）in OA and OR groups.CAT protein was significantly up-regulated（P< 0.001）and CAT enzyme activity was significantly increased（P < 0.05）in OR group.Conclusion:（1）Aging makes the body weight of mice increase significantly,and the cross-sectional area,mass,strength and function indexes of skeletal muscle fibers decrease significantly;Both resistance exercise and aerobic exercise can improve skeletal muscle mass,strength and function in aging mice,and resistance exercise intervention has better effect.Aging results in iron metabolism imbalance in mice,and an increase in the total iron content in skeletal muscle and a rise in sensitivity to ferroptosis;Both aerobic and resistance exercise can improve the iron metabolism imbalance,reduce the excessive accumulation of iron content in tissues,and reduce the sensitivity of ferroptosis in aging mice.（3）Aging leads to the imbalance of oxidation/antioxidant balance in mice and an increase in oxidative stress;Both of aerobic exercise and resistance exercise can improve the antioxidant capacity and resist oxidative stress of aging mice through the regulation mechanism of NRF2-XCT and NRF2-ARE.（4）Ferroptosis may be one of important targets associated with oxidation and antioxidant activity of skeletal muscle attenuation,which affects skeletal muscle mass in multiple ways.As a negative regulator of ferroptosis,NRF2 regulates the sensitivity of ferroptosis at different levels and is one of the core factors of exercise intervention on ferroptosis in aging mice. |
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