Effects Of Different Hypoxic Training Modes On Skeletal Muscle Autophagy And MTOR Signaling Pathway In Mice

PurposesAltitude training is the main training method to improve aerobic exercise ability.In order to avoid the disadvantages of altitude training(such as environment,small exercise load,etc.),hypoxic training was proposed.Living High-Training low and Living Low-Training High have also become the main methods of hypoxic training.Skeletal muscle adaptation to exercise is highly correlated with autophagy activation.m TOR is a negative regulator of autophagy,and the effect of different hypoxic training modes on m TOR is still controversial.This study aims to explore the effects of two hypoxic training methods on the autophagy and m TOR signaling pathway of skeletal muscle in mice,so as to provide theoretical basis for scientific training in the future.MethodsC57BL/6J healthy male mice were randomly divided into 4 groups with 8 mice in each group,which were Control group(C),Exercise group(E),Living low-training low(Hi Lo)group,and Living low-training High(Lo Hi)group.Mice in group E were given treadmill training at 25 m/min for 1 h,5 d/ week for 6 weeks.The oxygen concentration of hypoxia training was 13.6%(equivalent to 3500 m),and mice in the Hi Lo group were exposed to hypoxia at 13.6% O2 from 8:00 to 18:00,and then the exercise intervention of the normal oxygen training group was carried out again.Mice in the Lo Hi group were trained at 13.6% O2,and the exercise regimen was the same as that in the normoxia group.At the end of 6 weeks of training,the exercise ability of increasing load was tested.After 48 hours,the patients were anesthetized and sacrificed.Bilateral gastrocnemius muscles were taken.LC3 and LAMP2 immunofluorescence were used to observe the level of autophagosome clearance in skeletal muscle cells,m RNA levels of m TOR and P70S6 K were detected by RT-PCR,and expression levels of autophagy and m TOR signaling pathway and other related proteins were detected by Western-blot.Results(1)After 6 weeks of hypoxic training in different modes,the exhaustive running distance of mice in groups E,Hi Lo and Lo Hi were significantly increased compared with group C(P < 0.01);The running distance of mice in Hi Lo group was significantly increased compared with that in E group(P < 0.01),but there was no significant change in Lo Hi group compared with that in E group.(2)Effects of 6 weeks of hypoxia training in different modes on the fluorescence colocalization coefficient of LC3 and LAMP2 in skeletal muscle of mice.Compared with group C,the fluorescence co-localization levels of LC3 and LAMP2 in the three training modes were significantly increased(P < 0.01).Compared with E group,the fluorescence co-localization of skeletal muscle was significantly increased after Hi Lo mode hypoxia training(P < 0.01).(3)Effects of 6 weeks of hypoxic training in different modes on the level of skeletal muscle autophagy in mice.Compared with C group,AMPK protein in skeletal muscle in E group,Hi Lo group and Lo Hi group increased,but there was no significant difference(P > 0.05).LC3II/I ratio was increased(P <0.05),and there was no significant difference among the three training methods.Beclin1 protein expressions were significantly increased in all groups,and there was a significant difference in group E(P < 0.05),and there was a very significant difference in group Hi Lo and Lo Hi(P < 0.01).The expressions of P62 protein in skeletal muscle of mice in all three groups were decreased,but there was no significant change in group E,and it was significantly decreased in group Hi Lo and Lo Hi(P < 0.01).Compared with group E,the expression of P62 protein in group Hi Lo was significantly decreased(P < 0.05).The expression of BNIP3 protein in skeletal muscle of mice in all three groups were increased,but there were no significant change in group E and Hi Lo,while the expressions of BNIP3 protein in Lohi group was significantly increased(P < 0.01).The expressions of LAMP2 protein in skeletal muscle of mice in all three groups were increased,and the change of LAMP2 protein in group E and Lo Hi was significant(P < 0.05).(4)Effects of 6 weeks of different hypoxic training modes on m TOR signaling pathway in skeletal muscle of mice.Compared with group C,m TOR m RNA and protein expression in group E were increased,and m TOR protein expression was increased significantly(P < 0.05),m TOR m RNA and protein expressions in group Hi Lo and Lo Hi were decreased,m TOR m RNA and m TOR protein in group Lo Hi were decreased significantly(P < 0.05).Compared with group E,m TOR protein expression level of the two hypoxic training modes were significantly decreased(P < 0.01),and there was no significant difference between the two hypoxic training modes(P > 0.05).Akt protein expressions were significantly increased among the three groups(P < 0.05),but there were no significant difference among the three groups(P > 0.05).The m RNA level of P70S6 K in group E was significantly increased(P < 0.05),and it was significantly decreased in group Hi Lo compared with group E(P < 0.05),but there was no significant change in group Lo Hi(P > 0.05).Conclusion(1)Both HiLo and Lo Hi can improve the aerobic exercise capacity and the level of skeletal muscle autophagy in mice.The up-regulation of autophagy level may accelerate the clearance of damaged organelle in skeletal muscle,so that the body can produce good adaptive changes.(2)Exercise can activate m TOR signaling pathway in the skeletal muscle of mice.Hi Lo and Lo Hi can weaken the activation of m TOR signaling pathway by exercise.Hi Lo can weaken m TOR signaling pathway more obviously,is beneficial to the activation of autophagy,which may be Hi Lo makes mice aerobic exercise capacity is superior to the Lo Hi mode of mechanism.