Experimental Study on Anxiolytic and Antidepressant Effects of Zhi-Chi-Jie-Yu Granules in CUMS Mice and Prediction of Underlying Molecular Mechanisms.

Objective: This study investigated the in‑vivo pharmacological effects of Zhi-Chi-Jie-Yu Granules(ZCK) on CUMS‑induced anxiety‑ and depression‑like behaviors in mice. Post‑hoc exploratory analyses including network pharmacology and molecular docking were further performed to generate potential mechanistic hypotheses for its anti‑depressant action. Methods: An anxiety‑depression model was established in ICR mice via chronic unpredictable mild stress (CUMS) combined with solitary housing. Mice were divided into six groups (n=10): blank control, model, fluoxetine, and ZCK low‑, medium‑, and high‑dose groups. Behavioral changes were assessed by secondary‑auxiliary (SPT), together with FST, TST, OFT and EPM. Serum inflammatory cytokines and hippocampal neurotransmitters (5‑HT, NE) were measured by ELISA, and hippocampal pathological damage was observed by HE staining. UPLC‑Q‑TOF‑MS was used to characterize the chemical profile of ZCK. Non‑targeted serum metabolomics, network pharmacology, and molecular docking were integrated to predict targets and pathways. Results: ZCK significantly alleviated CUMS‑induced depressive‑ and anxiety‑like behaviors (P < 0.05 or P < 0.01). It downregulated serum pro‑inflammatory cytokines (IL‑6, TNF‑α), increased hippocampal 5‑HT and NE levels, and attenuated neuronal damage. UPLC‑Q‑TOF‑MS identified 39 core components. Metabolomics revealed 56 differential metabolites, mainly enriched in alanine‑aspartate‑glutamate and arachidonic acid metabolism. Network pharmacology predicted 410 overlapping targets, and the top‑10 core targets contained TP53, AKT1, STAT3, SRC, TNF and others. Considering our experimental findings on neuroinflammation, inflammatory‑related key targets (SRC, TNF, IL-6, NF‑κB and IL-1B) were selected for subsequent molecular‑docking verification. Molecular‑docking simulations suggested potential binding interactions between core components and key targets (binding energies <-5.0 kcal/mol). Conclusion: ZCK exerts anti‑anxiety and antidepressant effects in CUMS mice, accompanied by reduced peripheral neuroinflammation and improved hippocampal neurotransmitter levels. Multi‑omics and network‑pharmacology analyses further predict potential multi‑dimensional regulatory candidates, including neuroinflammation‑related signaling, neurotransmitter homeostasis and metabolic perturbation. These computational hypotheses require further protein‑level experimental validation. This study provides preliminary in‑vivo evidence for ZCK and an integrated methodological reference for TCM compound research.