Exploring the Effects of Changes in Dietary Protein Content on Naturally Aging Mice Based on Comprehensive Quantitative Scoring and Metabolomic Analysis

Background: During aging, protein nutrition has a bidirectional role in regulating healthy lifespan by modulating body metabolism and neurological function. However, the current &ldquo:low-high&rdquo: hypothesis on the dynamics of protein requirements is mainly based on male animal models, and its applicability to female physiology (e.g., estrogen fluctuations) is unclear. The present study aims to fill the gap in the study of protein demand dynamics in female naturally aging mice and to investigate the effects of different protein levels on the health status of female C57BL/6J mice at different stages of aging. Methods: In this study, four dietary interventions (high protein, HP: low protein, LP: model test, MT: and control, C) were evaluated by constructing a C57BL/6J female mouse model at three ages, 9 M (9 months), 16 M (16 months), and 20 M (20 months), which are approximately equivalent to 34, 65, and 78 years of age in humans, respectively, to determine the effects on naturally aging mice. The effects of the interventions were quantitatively described by behavioral, neuropathological, oxidative, and inflammatory indices and NMR metabolomics using Principal Component Analysis to construct a comprehensive quantitative scoring method. Results: The comprehensive quantitative scores Fsum was highest in the HP group, lowest in the LP group, and in between in the MT group. The HP intervention showed the most significant improvement in the aged group (20 M) mice, with a 35.2% reduction in avoidance latency (p &lt: 0.01) and a 32.9% increase in pyramidal cell density in the hippocampal CA1 region (p &lt: 0.05), while the LP intervention led to a cognitive decline in the mice, with an avoidance latency that was prolonged by 15.2% (p &lt: 0.05). Metabolomics analysis revealed that mouse samples of all ages showed age-dependent metabolic re-adaptation: the 9 M group may reflect gut microbial metabolism rather than direct host TCA cycle activity, suggesting an indirect association with energy metabolism: an enhanced degradation of branched-chain amino acids (BCAAs) was seen in the middle-aged group (16 M): and amino acid biosynthesis was predominant in the old group (20 M). Conclusions: Female mice have sustained neuromotor benefits to high-protein diets at different aging stages, and the dynamics of their protein requirements differ significantly from those of males. The study reveals the critical role of gender factors in protein nutritional strategies and provides an experimental basis for precise protein supplementation in older women.