The Metabolic Signatures Associated with the Effects of Aerobic Exercise on Depressive-like Behaviors in CUMS Rats

<b>Objectives</b>: This study explored the antidepressant mechanisms of aerobic exercise in CUMS rats by analyzing urinary metabolomics (LC-MS and NMR), with the aim of providing both theoretical and practical support for exercise-based depression interventions. <b>Methods</b>: (1) Thirty-two Sprague-Dawley rats were acclimatized for one week and then randomly assigned to four groups (n = 8 per group): control (C), control + aerobic exercise group (E), CUMS model (D), and CUMS + exercise (DE). Groups D and DE were subjected to nine types of CUMS stimuli. Behavioral indicators were assessed weekly, and the successful establishment of the CUMS model was confirmed at week 3. Following successful modeling, rats in groups E and DE underwent four weeks of aerobic exercise training. Throughout this period, groups D and DE continued to receive CUMS exposure, while groups C and E were maintained under standard control conditions. (2) At the end of week 7, behavioral tests were repeated. Twelve-hour urine samples were collected for metabolomic analysis using liquid chromatography-mass spectrometry (LC-MS) and ¹H-NMR spectroscopy. The following morning, rats were euthanized under anesthesia. Whole blood was collected from the abdominal aorta, and serum was separated for subsequent biochemical assays. Bioinformatics approaches were employed to identify potential targets and signaling pathways associated with the antidepressant effects of aerobic exercise. (3) For statistical analysis, one-way or two-way analysis of variance (ANOVA) was applied to behavioral, physiological, and biochemical data, whereas multivariate statistical analysis was used for metabolomic data. <b>Results</b>: (1) By week 3, body mass, sucrose preference, rearing frequency, and the number of grid crossings were significantly lower in groups D and DE than in groups C and E (<i>p</i> < 0.05 or <i>p</i> < 0.01). These findings confirmed the successful establishment of the depression model. At week 7, all behavioral indicators in group DE showed significant recovery relative to group D (<i>p</i> < 0.05 or <i>p</i> < 0.01). (2) Compared with group C, corticosterone and blood ammonia levels were significantly elevated in group D (<i>p</i> < 0.01). In contrast, these levels were markedly reduced in group DE compared with group D (<i>p</i> < 0.01). (3) LC-MS analysis identified 25 urinary metabolites associated with depression in group D relative to group C. Among these, 21 were significantly downregulated and 4 were upregulated (<i>p</i> < 0.05 or <i>p</i> < 0.01), involving seven metabolic pathways. Following aerobic exercise intervention, six of these depression-related metabolites in group DE showed significant recovery (<i>p</i> < 0.05 or <i>p</i> < 0.01), which were associated with two metabolic pathways. (4) Integrated analysis of LC-MS and ¹H-NMR data revealed glutamine as a common differential metabolite, linked to three metabolic pathways. All metabolic pathways modulated by aerobic exercise were related to amino acid metabolism. (5) Bioinformatics analysis indicated that AKT1, MTOR, IL6, RAF1, and TNF were core targets through which aerobic exercise regulated urinary metabolism in CUMS rats. <b>Conclusions</b>: A four-week regimen of aerobic exercise significantly improved depressive-like behaviors and enhanced anti-fatigue capacity in CUMS rats. This exercise regimen promoted urinary metabolic remodeling, primarily through the modulation of amino acid metabolism. Furthermore, its antidepressant effect is likely mediated through the regulation of core tissue targets-including AKT1, mTOR, IL-6, RAF1, and TNF-thereby influencing key pathways such as PI3K-AKT, MAPK/ERK, and neuroinflammatory signaling.