AIM:To investigate the regulatory mechanism of silent information regulator 6(Sirt6)on nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway and ferroptosis in skeletal muscle aging in-duced by D-galactose(D-Gal)in mice.METHODS:A D-Gal-induced mouse aging model was established and randomly divided into control and D-Gal groups.In vitro,D-Gal-treated C2C12 mouse myoblasts were treated with ferroptosis ago-nist erastin(Era)and inhibitor ferrostatin-1(Fer-1),Sirt6 agonist MDL-800 and inhibitor OSS-128167,and Nrf2 siRNA.Mouse body weight and forelimb relative grip strength were monitored.RT-qPCR and Western blot were used to measure the expression of Sirt6,Nrf2,HO-1,P53,P21,P16,muscle ring finger protein 1,muscle atrophy F-box,solute carrier family 7 member 11,and glutathione peroxidase 4 in gastrocnemius muscle and myoblasts.Hematoxylin-eosin staining was performed to examine muscle fiber diameter.Levels of reactive oxygen species(ROS),mitochondrial ROS,mitochon-drial membrane potential,senescence-associated β-galactosidase activity,glutathione,lipid peroxidation,and Fe2? con-centration were measured in myoblasts and myotubes.Immunofluorescence staining was used to detect myosin heavy chain(MyHC)expression in myotubes.RESULTS:Mice in the D-Gal group exhibited significant reductions in body weight and forelimb grip strength(P<0.01),upregulation of aging and muscle atrophy markers,and decreased mRNA and pro-tein levels of ferroptosis markers and Sirt6(P<0.01).Additionally,gastrocnemius muscle fiber diameter significantly de-creased(P<0.01).In D-Gal-treated myoblasts and myotubes,aging and muscle atrophy markers were elevated(P<0.01),MyHC expression was reduced,and protein levels of ferroptosis-related markers,Sirt6,Nrf2,and HO-1 were de-creased(P<0.05 or P<0.01).Fer-1 pre-treatment alleviated these changes(P<0.05 or P<0.01).MDL-800 significantly improved D-Gal-induced aging and muscle atrophy in myoblasts and myotubes,while increasing the expression of ferropto-sis-related proteins(P<0.05 or P<0.01).However,the addition of Erastin abolished the beneficial effects of MDL-800(P<0.05 or P<0.01).Following Nrf2 siRNA transfection,the ability of MDL-800 to improve ferroptosis and the quality of myotube formation was significantly diminished(P<0.05 or P<0.01).CONCLUSION:Sirt6 inhibits ferroptosis in myo-blasts through the Nrf2/HO-1 signaling pathway,thereby alleviating age-related changes in myoblasts and the decline in myotube formation quality,which is beneficial for improving skeletal muscle aging.

AIM:To investigate the regulatory mechanism of silent information regulator 6(Sirt6)on nuclear factor E2-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway and ferroptosis in skeletal muscle aging in-duced by D-galactose(D-Gal)in mice.METHODS:A D-Gal-induced mouse aging model was established and randomly divided into control and D-Gal groups.In vitro,D-Gal-treated C2C12 mouse myoblasts were treated with ferroptosis ago-nist erastin(Era)and inhibitor ferrostatin-1(Fer-1),Sirt6 agonist MDL-800 and inhibitor OSS-128167,and Nrf2 siRNA.Mouse body weight and forelimb relative grip strength were monitored.RT-qPCR and Western blot were used to measure the expression of Sirt6,Nrf2,HO-1,P53,P21,P16,muscle ring finger protein 1,muscle atrophy F-box,solute carrier family 7 member 11,and glutathione peroxidase 4 in gastrocnemius muscle and myoblasts.Hematoxylin-eosin staining was performed to examine muscle fiber diameter.Levels of reactive oxygen species(ROS),mitochondrial ROS,mitochon-drial membrane potential,senescence-associated β-galactosidase activity,glutathione,lipid peroxidation,and Fe2? con-centration were measured in myoblasts and myotubes.Immunofluorescence staining was used to detect myosin heavy chain(MyHC)expression in myotubes.RESULTS:Mice in the D-Gal group exhibited significant reductions in body weight and forelimb grip strength(P<0.01),upregulation of aging and muscle atrophy markers,and decreased mRNA and pro-tein levels of ferroptosis markers and Sirt6(P<0.01).Additionally,gastrocnemius muscle fiber diameter significantly de-creased(P<0.01).In D-Gal-treated myoblasts and myotubes,aging and muscle atrophy markers were elevated(P<0.01),MyHC expression was reduced,and protein levels of ferroptosis-related markers,Sirt6,Nrf2,and HO-1 were de-creased(P<0.05 or P<0.01).Fer-1 pre-treatment alleviated these changes(P<0.05 or P<0.01).MDL-800 significantly improved D-Gal-induced aging and muscle atrophy in myoblasts and myotubes,while increasing the expression of ferropto-sis-related proteins(P<0.05 or P<0.01).However,the addition of Erastin abolished the beneficial effects of MDL-800(P<0.05 or P<0.01).Following Nrf2 siRNA transfection,the ability of MDL-800 to improve ferroptosis and the quality of myotube formation was significantly diminished(P<0.05 or P<0.01).CONCLUSION:Sirt6 inhibits ferroptosis in myo-blasts through the Nrf2/HO-1 signaling pathway,thereby alleviating age-related changes in myoblasts and the decline in myotube formation quality,which is beneficial for improving skeletal muscle aging.