The Mechanism Of Autophagy On Senescence Induced By D-galactose In C2C12 Myoblasts

Objective:Myoblast is a myogenic cell derived from muscle satellite cells,which has the ability of differentiation,proliferation,and etc.Myoblast senescence was the causes of skeletal muscle atrophy and sarcopenia.As a mechanism of cell survival,autophagy played a pivotal role in maintaining the homeostasis of the cell environment.Some studies have shown that autophagy activity is gradually inhibited as aging occurs.However,the regulatory effect and mechanism of autophagy on myoblast senescence has not been clear.In this study,C2C12 myoblasts induced by d-galactose were used to establish a skeletal muscle cell aging model in order to study the effect of autophagy on C2C12 myoblasts aging and to provide new reference for diagnosis and treatment of skeletal muscle reduction-related diseases.Methods:1.The effect of d-galactose on C2C12 myoblasts was studied.?-galactosidase staining,western blot,superoxide dismutase assay and mitochondrial membrane potential assay was used to detect the expression of aging-related ?-galactosidase,P53 and P16,the antioxidant stress activity and mitochondrial function.2.After D-galactose intervened C2C12 myoblasts,the expression of autophagy-related protein P62 and LC3 was tested by western-blot,and the changes of P62 were detected by immunofluorescence assay.3.The experiment was divided into four groups: control group(MOCK group),Dgalactose group(D-GAL group),rapamycin pretreatment d-galactose group(D-GAL+RAPA group)and 3-methylpurine pretreatment d-galactose group(D-GAL+3-MA group).?-galactosidase staining,mitochondrial membrane potential,superoxide dismutase and Western-blot assay were used to detect the changes of C2C12 myoblasts in aging level.EdU-488 was devoted to analyze cell proliferation and flow cytometry was used to test the distribution of cell cycle.4.The experiment was divided into four groups: control group(MOCK group),Dgalactose group(D-GAL group),rapamycin pretreatment d-galactose group(D-GAL+RAPA group)and 3-methylpurine pretreatment d-galactose group(D-GAL+3-MA group).Westernblot was used to detect the expressions of Hippo signaling pathway related proteins(pLATs1/2,Mst1/2,YAP,p-YAP1).Results:1.D-galactose induced C2C12 myoblast senescence: After d-galactose treatment,the number of C2C12 myoblast aging-related ?-galactosidase staining positive cells increased,The aging-related proteins P53 and P16 increased.Superoxide dismutase activity was degraded.Mitochondrial membrane potential staining showed increasing green-fluorescence.2.D-galactose inhibited autophagy activity: Western blotting results showed that after 9 hours of d-galactose treatment,the conversion of LC3-I to LC3-II is reduced,and degradation of autophagy associated protein P62(The 62 kDa sequestosome 1 protein,P62 protein)is inhibited;P62 granular fluorescence aggregation was observed under fluorescence microscope.3.Autophagy inhibited C2C12 myoblasts senescence induced by d-galactose: Compared with D-GAL group,in D-GAL+RAPA group,the activity of ?-galactosidase and the expression of aging-related proteins P53 and P16 were decreased,concomitant with the increases red-fluorescence of mitochondrial membrane potential and superoxide dismutase.4.Autophagy inhibited the effects of d-galactose on proliferation and cycle of C2C12 myoblasts: Compared with D-GAL group,in D-GAL+RAPA group,the number of EdU positive cells increased and G0/G1 phase cells decreased.5.Autophagy inhibited C2C12 myoblast senescence by blocking Hippo signaling pathway: Western blot results show that D-galactose promotes phosphorylation of Hippo signaling pathway related proteins LATS1/2 and YAP1,and phosphorylated proteins LATS1/2 and YAP1 decrease after rapamycin pretreatment.Conclusion:D-galactose induced C2C12 myoblast senescence,inhibited autophagy activity and proliferation,and arrest cell cycle.Enhancing autophagy activity can inhibit C2C12 myoblast senescence by regulating Hippo signaling pathway.These results may provide a theoretical basis for us to understand the occurrence and development of myogenic cell senescence,and provide new ideas for the prevention and cure of skeletal muscle reductionrelated diseases.