Objective:

2

To investigate the antidepressant effects of Xiaoyao Pill (XYP) by exploring its interactions with gut microbiota and tryptophan metabolism.

Methods:

2

Utilizing network pharmacology

the functional substance groups

key targets

and pathways of XYP in the treatment of depression were identified. The chronic unpredictable mild stress (CUMS) protocol was implemented in male Sprague-Dawley rats to establish depression model. Thirty rats were randomly divided into 3 groups according to their body weight (10 for each): control

CUMS and XYP groups (1.8 g/kg). After 28-day interventions

behavioral phenotyping including sucrose preference test (SPT) and open field test (OFT) were performed. Biochemical validation encompassed enzyme-linked immunosorbent assay for serum cortisol

hematoxylin-eosin histopathology

and immunohistochemistry. Liquid chromatography-mass spectrometry was utilized to profile serum metabolites

while fecal samples underwent metagenomic sequencing for gut microbiota characterization.

Results:

2

Network pharmacology studies predicted that key components can protect the nervous system by regulating inflammatory pathways through the blood-brain barrier. SPT and OFT showed that XYP treatment significantly ameliorated depressive-like behaviors (all

P

<

0.05). XYP treatment also restored hippocampal neuronal density

increased serum neurotransmitter levels of neurotransmitters such as 5-hydroxytryptamine and vasoactive intestinal peptide

and while suppressing inflammatory markers such as tumor necrosis factor-alpha

interleukin-1 beta (IL-1β)

and IL-6 (all

P

<

0.05). Metagenomics revealed significant restructuring of gut microbiota

notably the regulation of

Parabacteroides distasonis

(

P

<

0.05). Non-targeted metabolomics analysis showed that the level of metabolites in the tryptophan and kynurenine pathway significantly changed (variable importance in the projection

>

1

P

<

0.05)

and the change of metabolic flux was significantly correlated with behavioral improvement (

P

<

0.05).

Conclusions:

2

XYP exerts antidepressant effects by increasing neurotransmitter levels

reducing inflammatory makers and modulating

Parabacteroides distasonis

. Through further exploration of metabolomics

we found that XYP may play a protective role in depression by regulating tryptophan metabolism.