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

<b>Background:</b> 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. <b>Methods:</b> 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.). <b>Results:</b> 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 <i>pMitoTimer</i> imaging. Furthermore, rutin reinstated the rhythmic expression of oxidative phosphorylation proteins and <i>Pgc1α</i>. 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. <b>Conclusions:</b> 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.