This study aimed to investigate the protective effects of gut-derived urolithin D (Uro D) against neurodegenerative lesions in a D-galactose (D-gal)-induced chemical aging mouse model and its underlying molecular mechanisms. In vivo, mice received 8-week daily intraperitoneal injection of 150 mg/kg D-gal plus oral gavage of 50-150 mg/kg/day Uro D. The regulatory protein networks in related pathways and structural features of mitophagy-associated proteins were further explored. A D-gal-induced cellular stress model in HT22 neuronal cells was established to evaluate mitochondrial parameters, protein expression levels, and alterations in mitophagy-related molecular machinery. In vivo, Uro D treatment improved cognitive performance and behavioral impairment, reduced hippocampal pathology, preserved synaptic plasticity, and alleviated aging symptoms. Mechanistically, Uro D upregulated AMPK, PGC-1α, and mitochondrial SIRT3 expression to modulate mitochondrial functional activity. Uro D modulates mitophagy-associated signaling, accompanied by altered expression of Beclin1, LC3, P62, Parkin, and PINK1. Further in vitro experiments suggested that Uro D enhances mitochondrial integrity in cellular stress models. Progressive normalization of mitochondrial shape, oxidative stress, and calcium ion homeostasis contribute to the alleviation of pathological symptoms associated with aging. SIRT3 knockdown in HT22 cells increased mitochondrial damage and suppressed the activation of mitophagy-related molecular machinery. Further results from AMPK/PGC-1α inhibition and SIRT3 rescue experiments revealed that inhibition of AMPK or PGC-1α abolished the protective effects of Uro D, whereas SIRT3 overexpression alleviated D-gal-induced cellular abnormalities. Uro D partially triggers molecular changes consistent with mitophagy activation through the AMPK/PGC-1α/SIRT3 cascade, contributing to the mitigation of hippocampal neuronal injury and the improvement of learning and memory deficits in D-gal-induced aging mice.
