**Background:** Stress activates the hypothalamic–pituitary–adrenal (HPA) axis, increasing circulating glucocorticoids. However, whether plasma corticosteroid levels accurately reflect local tissue levels is unclear, as tissues can locally synthesize, metabolize, and sequester steroids via enzymes such as 11β-hydroxysteroid dehydrogenases (11HSD1 and 11HSD2). Additionally, stress alters gut microbiota, which may modulate host stress responses. This study aimed to profile multiple corticosteroids across blood, brain, and peripheral tissues in chronically stressed mice and to assess concurrent changes in gut microbiota.
**Methods:** Nine-week-old male BALB/c mice were randomly assigned to stressed (n=5) or control (n=5) groups. Stressed mice underwent a repeated social defeat paradigm (resident-intruder) 3 times per week for a total of 16 sessions. Each session involved 10 min of direct physical contact followed by 50 min of sensory contact via a steel mesh. Control mice remained undisturbed. Mice were sacrificed 80 min after the final stressor initiation (20 min after the last session). Blood, brain (cerebral cortex, hippocampus), liver, kidney, colon, thymus, and mesenteric lymph node (MLN) samples were collected. Steroids (PREG, PROG, 11DOC, CORT, 11DHC) were quantified using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Fecal samples were collected before and after the stress period for 16S rRNA gene sequencing (V4V5 region) on the Ion Torrent platform. Statistical analyses used one-way/two-way ANOVA, Tukey's post hoc test, unpaired Student's t-test, and for microbiome: Kruskal–Wallis, PERMANOVA (Adonis), and LEfSe (LDA score >2).
**Key Results:** In the adrenal glands, stress significantly upregulated PREG and CORT but not PROG, 11DOC, or 11DHC. In plasma, stress significantly increased CORT (×26), PROG (×45), 11DOC (×27), and 11DHC (×6). Two-way ANOVA showed significant effects of stress (F₁,₃₀=76.03, P<0.001), steroid type (F₃,₃₀=68.52, P<0.001), and stress×steroid interaction (F₃,₃₀=59.10, P<0.01). In tissues, CORT increased ~20-fold in colon, ~40-fold in kidney, and >60-fold in liver and brain. 11DHC was highest in colon, kidney, and liver of stressed mice, and much lower in brain and lymphoid organs. The CORT/11DHC ratio in plasma was similar to brain but significantly lower in colon, liver, kidney, and MLN (P<0.05 or P<0.01). Stress increased PROG and 11DOC in most tissues except liver. The PROG/11DOC ratio was significantly higher in lymphoid organs (MLN, thymus) than in other tissues (P<0.001). For gut microbiota, α-diversity (Shannon index) did not differ significantly (P=0.51), but β-diversity (Bray–Curtis PCoA) showed a significant stress effect (R²=0.160, P=0.038). LEfSe analysis identified five genera associated with stress: Alistipes, Rikenella, Ruminiclostridium 5, Roseburia, and Helicobacter, while Ruminiclostridium 9 was associated with unstressed controls.
**Clinical Implications:** This study demonstrates that chronic stress induces tissue-dependent changes in local corticosteroid levels that do not always mirror plasma concentrations. This suggests that measuring only circulating glucocorticoids may inadequately reflect tissue-specific corticosteroid exposure, which has implications for understanding stress-related pathophysiology in organs such as the brain, liver, kidney, and immune tissues. The concurrent alterations in gut microbiota composition highlight a potential bidirectional interaction between the HPA axis and the microbiome. These findings underscore the need for tissue-level assessments in stress research and may inform future therapeutic strategies targeting local steroid metabolism or the gut–brain axis.