The Effect Of Aerobic Exercise On Doxorubicin-induced Skeletal Muscle Atrophy And GCN2 Expression In Mice

ObjectiveDoxorubicin(DOX)is a highly effective antitumor agent widely used in cancer treatment.However,it is well established that doxorubicin induces muscular atrophy and impairs force production.Although no therapeutic interventions exist to combat doxorubicin-induced muscle weakness,aerobic exercise training has been shown to reduce skeletal muscle damage caused by doxorubicin administration.Studies have attempted to determine the molecular mechanism of exercise against the myotoxicity induced by doxorubicin administration.Nevertheless,the mechanisms by which aerobic exercise protects against doxorubicin-induced muscle weakness remain elusive.Studies indicate that General control nonderepressible 2 kinase(GCN2)as an amino acid sensor can regulate skeletal muscle wasting,Therefore,this study aims to explore the mechanism of aerobic exercise training performed during and after doxorubicin administration to reduce doxorubicin-induced skeletal muscle atrophy,to investigate whether the GCN2 signaling pathway plays a role in the process of aerobic exercise training in regulating doxorubicin-induced skeletal muscle atrophy in mice.MethodsEight-week-old Male C57 mice(n=36)were randomized to the quiet saline group(SED-SAL,n=6),the quiet doxorubicin group(SED-DOX,n=12),the exercise saline group(EX-SAL,n=6),and the exercise doxorubicin group(EX-DOX,n=12).The doxorubicicin injection group was injected for 4 consecutive weeks,Injection once a week,5mg/kg each time,the total dose is 20mg/kg,and mice were injected with the same amount of saline in the saline group.After 8 weeks of intervention,the mice were anesthetized to obtain the gastrocnemius muscle and tibia.The gastrocnemius muscle morphology was analyzed by HE staining and WGA staining,and the gastrocnemius muscle related protein expression was detected by Western blot.Results(1)Changes of mice body weight and the ratio of gastrocnemius weight to the length of tibia(GAS/TL)after 8 weeks of intervention:In the quiet group,compared with SED-SAL,the weight of mice(P<0.01)and the GAS/TL ratio(P<0.05)was reduced significantly in the SED-DOX group.In the exercise group,compared with EX-SAL,the weight of mice(P<0.01)and the GAS/TL ratio(P<0.05)was reduced significantly in the EX-DOX group.Compared with the EX-DOX group and the SAL-DOX group,there was no significant difference in the weight of the mice,but the GAS/TL ratio was significantly increased in the EX-DOX group(P<0.05).(2)Morphological changes of mice gastrocnemius muscle:Doxorubicin administration can cause muscle fibers vary in size,increase polygonal atrophy muscle fibers,and the cell nucleus shifting inward.In the quiet group,compared with SED-SAL,the cross-sectional area of gastrocnemius muscle cells in SED-DOX group reduced significantly(P<0.01).In the exercise group,compared with EX-SAL,the cross-section of gastrocnemius muscle cells in the EX-DOX group was reduced significantly(P<0.01).Compared with the SED-DOX group,the cross-sectional area of gastrocnemius muscle fibers in the EX-DOX group was increased significantly(P<0.05).(3)GCN2/eIF2α/ATF4 protein expression changes in gastrocnemius muscle:In the quiet group,compared with SED-SAL group,the protein expression of GCN2,P-eIF2α and ATF4 was increased significantly in the SED-DOX group(P<0.01).In the exercise group,compared with EX-SED group,the protein expression of GCN2,P-eIF2α and ATF4 was increased in the EX-DOX group,but there is no significant difference(P>0.05).Compared with the SED-DOX group,the protein expression of GCN2,P-eIF2α and ATF4 was significantly reduced in the EX-DOX group(P<0.05).(4)Ubiquitin proteasome system and Caspase-3 expression in mice gastrocnemius muscle:In the quiet group,compared with SED-SAL,P-Fox03a(P<0.05),MuRF1(P<0.01),Fbx32(P<0.01),Caspase-3(P<0.01)protein expression was significantly increased in the SED-DOX group.In the exercise group,compared with EX-SAL,P-FoxO3α,MuRF1,Fbx32 protein expression was increased in the EX-DOX group,but there was no significant difference(P>0.05),Caspase-3 protein expression was increased significantl(P<0.05).Compared with SED-DOX group,P-FoxO3α,MuRF1,Fbx32 and Caspase-3 protein expression levels was significantly decreased in the EX-DOX group(P<0.01).(5)Changes in protein expression of AKt/mTOR/P70S6 in the protein synthesis pathway of gastrocnemius muscle:the expression of P-AKt in the SED-DOX group and EX-SAL group was significantly higher than that in the SED-SAL group.Compared with the SED-DOX group,P-AKt expression significantly decreased in the EX-DOX group.Compared with SED-SAL,the expression of P-mTOR and P-P70S6 was decreased in the SED-DOX group,but there was no significant difference(P>0.05).Compared with the SED-DOX group,the expression of P-mTOR(P<0.01)and P-P70S6(P<0.05)in the EX-DOX group were significantly increased.Conclusions(1)Doxorubicin injection can significantly reduce the body weight and wet weight of skeletal muscle in mice,induce skeletal muscle cell structure damage in mice and skeletal muscle atrophy.Aerobic exercise can improve doxorubicin-induced skeletal muscle cell structure damage and muscle fiber atrophy.(2)Aerobic exercise during and after doxorubicin injection can alleviate doxorubicin-induced skeletal muscle atrophy in mice by inhibiting protein degradation signal pathways,such as the ubiquitin proteasome system and Caspase-3,and improving protein synthesis signal pathway mTOR/P70S6.(3)Aerobic exercise reduces mouse skeletal muscle atrophy induced by doxorubicin,which may be related to the GCN2/eIF2α/ATF4 signaling pathway.