**Background:** Major depressive disorder (MDD) is a leading global psychiatric problem affecting over 350 million people, with high comorbidity of gastrointestinal abnormalities, particularly constipation (approximately 29.8% in epidemiological surveys). Current antidepressants often worsen constipation due to reduced serotonin transporter activity, and long-term laxative use causes dependence and intestinal damage. The gut microbiome-brain axis offers a potential therapeutic target, and previous studies showed individual probiotic strains (Bifidobacterium breve CCFM1025, Bifidobacterium longum CCFM687, Pediococcus acidilactici CCFM6432) had antidepressant and anxiolytic effects in mice via serotonergic system modulation. This study aimed to evaluate the combined multi-probiotic formula on both mental and gastrointestinal symptoms in MDD patients and validate mechanisms in an animal model.
**Methods:** The clinical trial was a two-arm, placebo-controlled, double-blinded RCT conducted at Tinghu People’s Hospital (Yancheng, China). Inclusion criteria: mild to moderate MDD patients aged >18 years, without restrictions on antidepressant drugs. Exclusion criteria: other mental disorders, refractory depression, severe suicide tendencies, schizophrenia, bipolar disorder, neurodegenerative diseases, severe physical diseases, pregnancy/breastfeeding. Power analysis (G*Power 3.1) required at least 20 participants per group, but due to COVID-19 lockdowns, only 28 completed (13 placebo, 15 multi-probiotics). The multi-probiotic formula (Shisheng Yisheng Co. Ltd.) contained freeze-dried B. breve CCFM1025, B. longum CCFM687, and P. acidilactici CCFM6432 (1:1:1, each 4×10^9 CFU/g) in maltodextrin; placebo was matched maltodextrin. Intervention lasted 4 weeks. Psychometric evaluations used HAMDS, MADRS, BPRS (doctor-administered) and GSRS (self-completed). Faecal 16S rRNA sequencing assessed gut microbiome. Animal experiments used male C57BL/6 mice (6 weeks old) with chronic unpredictable mild stress (CUMS) model. Probiotics (10^9 CFU/day) were given for 4 weeks. Behavioral tests: forced swim test, tail suspension test. Gut motility: gastrointestinal transit, first defecation time. 5-HT and 5-HIAA in prefrontal cortex, brainstem, and colon were quantified by HPLC. Hypothalamus CRF and serum corticosterone by ELISA. Hippocampal proBDNF and BDNF by immunohistochemistry/immunofluorescence. Colonic Tph1 and Slc6a4 expression by qRT-PCR. Caecal SCFAs by GC-MS. Statistical analysis: two-way ANOVA with Sidak's multiple comparisons, unpaired t-tests, Cohen's d effect size.
**Key Results:** In the clinical trial, HAMDS score significantly decreased in both placebo (P=.001) and multi-probiotics (P<.001) groups, but score reduction was greater in probiotics (d=0.553, P<.001). MADRS score decreased significantly only in probiotics (P<.001) vs placebo (P=.679), with greater reduction (d=0.319, P=.003). BPRS score decreased significantly only in probiotics (P<.001) vs placebo (P=.679), with greater reduction (d=0.473, P<.001). GSRS score decreased significantly in probiotics (P<.001) vs placebo, with greater reduction (d=0.198, P=.049); factor analysis showed 'bowel dysfunction syndrome' primarily contributed (d=0.184, P=.006). Gut microbiome: Shannon diversity increased only in probiotics (P=.038); observed OTUs increased in both groups (placebo P=.002, probiotics P<.001), but no significant difference in pre-post changes between groups (Shannon P=.843, OTUs P=.629). Beta diversity (Aitchison distance volatility) showed no significant difference (P=.568). LEfSe identified 4 taxa changed in placebo, 9 in probiotics, including increased Ruminococcaceae. In animal experiments, multi-probiotics significantly reduced depressive-like behavior in forced swim test and tail suspension test. Serum corticosterone and hypothalamus CRF were normalized. 5-HT turnover (5-HIAA/5-HT ratio) was reversed in prefrontal cortex and brainstem. Hippocampal proBDNF overexpression and BDNF insufficiency were balanced. Gut motility: gastrointestinal transit and first defecation time improved. Colonic 5-HT level increased, associated with reduced Slc6a4 expression (serotonin transporter) without affecting Tph1 (biosynthesis). Stool moisture recovered, and caecal SCFA levels increased.
**Clinical Implications:** This study provides evidence that a multi-strain probiotic formula can simultaneously ameliorate depressive symptoms and comorbid gastrointestinal dysfunction in MDD patients, with effect sizes ranging from small to moderate (Cohen's d 0.198–0.553). The mechanisms involve modulation of the HPA axis, brain serotonergic system, hippocampal neuroplasticity, and colonic serotonin transporter expression, along with increased SCFA production. These findings suggest probiotics as a promising adjunctive therapy for MDD, particularly for patients with constipation, potentially reducing reliance on laxatives. Limitations include small sample size (n=28), lack of serological or neuroimaging biomarkers, and need for dose-response and population-specific studies. Future research should confirm efficacy in larger cohorts and explore personalized probiotic therapies.