Synergistic antidepressant effects of gastrodin combined with repetitive transcranial magnetic stimulation: Mediation by the gut-brain axis and endocannabinoid system

Gastrodin combined with r-TMS treats depression via brain-gut axis by targeting endocannabinoid components: NAPE-PLD, DAGL, CB1R. • Identify key gut microbiota and serum metabolites as potential antidepressant biomarkers. • Multi-omics analysis of synergistic antidepressant pathway via "microbiota-metabolite-behavior axis". This study investigates the synergistic antidepressant mechanisms of gastrodin (GAS) combined with repetitive transcranial magnetic stimulation (r-TMS) through the gut-brain axis and the endocannabinoid system. A depression model was established in male C57BL/6J mice using Chronic Unpredictable Mild Stress (CUMS). Following four weeks of modeling, animals were divided into five groups and received respective treatments for two weeks. A multi-modal approach was employed, encompassing behavioral tests, hematoxylin and eosin (HE) staining, 16S rRNA sequencing, liquid chromatography-mass spectrometry (LC-MS) metabolomics, Western blot analysis, and mediation analysis. Results demonstrated that the combined therapy significantly ameliorated depressive-like behaviors and cognitive deficits in CUMS mice, exhibiting superior efficacy compared to monotherapies. The treatment repaired hippocampal neuronal architecture and colonic mucosal integrity, upregulated the tight junction protein ZO-1, and reshaped the serum metabolic profile. Western blot analysis confirmed the activation of key proteins within the target pathway. Mediation analysis established a credible microbiota-metabolite-behavioral association, indicating that specific gut microbiota influence behavioral outcomes by modulating differential metabolites, with this relationship mediated through the endocannabinoid system. This study confirms that the combination of GAS and r-TMS exerts synergistic antidepressant effects by regulating gut-brain axis homeostasis and activating the target signaling pathway, providing a novel mechanistic foundation for combined depression treatment strategies. Gut Microbiota-Metabolite Mediation of the Antidepressant Effects of r-TMS Combined with GAS.r-TMS acts on the prefrontal cortex, improving hippocampal DG morphology and neuronal survival. GAS modulates gut microbiota composition, restores intestinal barrier integrity, and ameliorates colonic histology. Both interventions activate the ECS. Western blotting confirms altered expression of key ECS proteins (CB1R, DA-L, NAPE-PLD), and untargeted serum metabolomics identifies the endocannabinoid pathway and related metabolites. These central and peripheral changes collectively produce antidepressant-like behavioral effects. A mediation model (Microbiota-Metabolite-Behavior axis) validates that gut microbiota shifts mediate behavioral effects via metabolite alterations. This graphical summary highlights the gut-brain axis as a critical hub for the multi-target antidepressant actions of r-TMS and GAS. Download: Download high-res image (453KB) Download: Download full-size image