Astragalus polysaccharide enhances OGT-mediated O-GlcNAcylation to stabilize PINK1 to induce mitophagy in D-galactose treated C2C12 myoblasts.

BACKGROUND

Astragalus polysaccharide (APS) has been shown to alleviate muscle atrophy. This study investigated the effects and underlying mechanisms of APS on D-galactose (D-gal)-induced mitochondrial autophagy in C2C12 myoblasts.

METHODS

Cell viability in C2C12 myoblasts was assessed using the CCK-8 assay. To further elucidate the role of APS, we evaluated skeletal muscle cell diameter and mitochondrial autophagy in C2C12 myoblasts, with and without O-GlcNAc transferase (OGT). Immunofluorescence staining for myosin heavy chain (MyHC) and western blot analysis were employed. Co-immunoprecipitation (Co-IP) experiments and immunofluorescence staining were conducted to examine the interaction between OGT and PTEN-induced putative kinase 1 (PINK1). In vivo, male C57BL/6 J mice were treated with D-gal to induce sarcopenia, and APS was administered to assess its effects on muscle function and mitochondrial health.

RESULTS

APS promoted mitophagy in vitro by inducing O-GlcNAcylation through OGT. Knockdown of OGT significantly weakened the protective effects of APS. OGT modifies PINK1 with O-GlcNAcylation through the S425 site. In vivo, APS treatment significantly improved grip strength and muscle mass in D-gal-induced sarcopenia mice. Histological analysis showed increased cross-sectional area of gastrocnemius muscle fibers, and Western blot analysis revealed enhanced expression of LC3II, PINK1, and Parkin in muscle tissues.

CONCLUSION

Collectively, APS promotes OGT-mediated O-GlcNAcylation to stabilize PINK1, thereby facilitating mitophagy in D-gal-treated C2C12 myoblasts in vitro. In vivo, APS improves muscle function and mitochondrial health in a mouse model of sarcopenia. These findings suggest that APS could serve as a potential therapeutic agent for muscle atrophy and related conditions.