Rutin as a Circadian Modulator Preserves Skeletal Muscle Mitochondrial Function and Reduces Oxidative Stress to Protect Against D-Galactose-Induced Aging In Vitro and In Vivo

Background: Skeletal muscle aging is characterized by impaired myogenic differentiation, disrupted circadian rhythms, elevated oxidative stress, and mitochondrial dysfunction. Rutin, a natural flavonoid with antioxidant properties, has been suggested to mitigate aging processes; however, its effects on circadian regulation and muscle homeostasis remain unclear. Methods: In vitro, differentiated C2C12 myotubes were treated with D-galactose (D-gal, 20 g/L) with or without rutin (20 μM). In vivo, C57BL/6 mice were supplemented with rutin (100 mg/kg b.w.) via oral gavage in a D-gal-induced aging mouse model (150 mg/kg b.w., i.p.). Results: D-gal induced cellular senescence, impaired myogenic differentiation, disrupted circadian oscillations, increased oxidative stress, and compromised mitochondrial function. Rutin treatment restored myotube formation, enhanced circadian rhythmicity of differentiation-related genes, and corrected the antiphase patterns of Per2 and Rorc. It also reduced reactive oxygen species and malondialdehyde levels; increased superoxide dismutase, catalase, and glutathione peroxidase activity; improved ATP production and membrane potential; and decreased mitochondrial oxidative aging, as confirmed by pMitoTimer imaging. Furthermore, rutin reinstated the rhythmic expression of oxidative phosphorylation proteins and Pgc1α. In vivo, rutin supplementation enhanced muscle performance (prolonged hanging time) and oxidative capacity, particularly at night (ZT14–ZT16), without altering muscle fiber-type distribution, and normalized circadian rhythmicity of core clock genes. Conclusions: Rutin attenuates D-gal-induced cellular senescence by modulating circadian rhythms, reducing oxidative stress, and improving mitochondrial function. Importantly, its in vivo effects on muscle performance and circadian regulation suggest that rutin is a promising therapeutic strategy to counteract skeletal muscle aging and sarcopenia.