Integrating Metagenomics and Network Pharmacology Reveals That Hypericum perforatum L. Alleviates Depressive-Like Behaviors via Gut Microbiota-Associated Inflammatory and PI3K-Akt Signaling.

INTRODUCTION: Hypericum perforatum L. (HP) is a well-known herbal antidepressant with reliable antidepressant effects. However, its underlying mechanism, particularly the interplay with the gut-brain axis, remains poorly elucidated. In this study, aimed to explore the potential mechanism by which HP relieves depressive-like behaviors by integrating metagenomic sequencing and network pharmacology, with a focus on gut microbiota and host signaling pathways. METHODS: A rat model of depressive-like behaviors was established using Chronic Restraint Stress (CRS). Sucrose Preference Test (SPT), Open Field Test (OFT), and Forced Swimming Test (FST) were applied to evaluate behavioral performance. Histopathological changes in the hippocampus and colon were assessed by Hematoxylin and Eosin (HE) staining. Serum levels of inflammatory cytokines were measured by ELISA. Gut microbiota composition was profiled by metagenomic sequencing; intestinal barrier integrity was evaluated by assessing the expression of tight junction proteins Zona Occludens 1 (ZO-1) and occludin. Network pharmacology was used to predict active components, targets, and pathways of HP. Key hippocampal pathway proteins were validated by western blot analysis. RESULTS: HP intervention ameliorated CRS-induced depressive-like behaviors, alleviated hippocampal neuronal damage, and restored intestinal barrier integrity. Serum levels of proinflammatory cytokines were also reduced. Metagenomic analysis revealed that HP reversed CRS-induced gut dysbiosis, notably by increasing the relative abundance of beneficial bacteria (e.g., Prevotella) and decreasing pro-inflammatory taxa. Redundancy Analysis (RDA) revealed close correlations between microbial alteration and inflammatory cytokine levels. Network pharmacology identified six active components and 42 potential therapeutic targets; the phosphoinositide 3- Kinase-Protein Kinase B (PI3K-Akt) signaling pathway was determined as the core pathway. Experimental verification confirmed that HP could regulate the PI3K-Akt signaling pathway and modulate the expression of its downstream protein Nuclear Factor Kappa B (NF-κB) in the hippocampus. DISCUSSION: Our work provides integrative insight into the pharmacological characteristics of HP and supports that gut microbiota may be potentially involved in its antidepressant-like effects. CONCLUSION: This study provides evidence that HP ameliorates CRS-induced depressive-like behaviors, which is closely associated with the restoration of gut microbial homeostasis, suppression of systemic inflammation, and regulation of the brain PI3K-Akt signaling pathway.