Unlocking Puerarin's Antidepressant Mechanism: TLR4/MYD88/NF-kB Signaling Pathway and Metabolomics-Macrogenomics associated Gut Microbiome Signatures

Objective Depression is a highly prevalent mental disorder in which dysfunction of the gut microbiota is implicated as a significant factor in its pathogenesis. Puerarin has been suggested to alleviate depression via the microbe-gut-brain (BGM) axis, although the precise mechanisms remain elusive. This study aimed to elucidate the association between the antidepressant effects of Puerarin and its role in regulating intestinal flora imbalance and inhibiting subsequent activation of the LPS/TLR4 inflammatory pathway from metabolomics and macrogenomics perspectives. Methods A rat model of depression was established using a 6-week chronic unpredictable mild stress (CUMS) protocol. Depressive-like behaviors were assessed through the sucrose preference test (SPT), forced swim test (FST), and open field test (OFT). Inflammatory cytokines (TNF-α, IL−1β, IL−6), LPS, corticosterone, and 5-HT were measured via ELISA. Hippocampal and colonic protein expression of TLR4, MyD88, IκBα, and NF-κB was analyzed by western blot. Colon tissue integrity was evaluated using H&E staining, PAS staining, and transmission electron microscopy. Immunofluorescence was employed to detect Iba−1  ⁺  microglia, TLR4  ⁺  cells, and ZO−1 expression. Fecal metabolomics and metagenomics were conducted to identify differential metabolites and microbial composition, followed by KEGG and KO enrichment analyses to predict relevant pathways. Spearman correlation analysis was used to explore relationships among gut microbiota, metabolites, and behavioral indices. Results Puerarin markedly ameliorated depression-like behaviors in chronic unpredictable mild stress (CUMS) rats. Concurrently, Puerarin inhibited the LPS/TLR 4 signaling pathway and its downstream pro-inflammatory mediators in both the hippocampus and colon, resulting in a significant reduction in inflammatory responses across these regions, as well as in the serum. Macrogenomic sequencing revealed that Puerarin suppressed inflammation-associated bacteria, enhanced the abundance of Firmicutes, and induced alterations in the microbial community structure and composition. Metabolomic analysis demonstrated that Puerarin could counteract dysregulated fecal metabolism, identifying 17 metabolites as potential key mediators in restoring metabolic homeostasis in CUMS rats. These biomarkers were implicated in several metabolic pathways, including aminoacyl-tRNA biosynthesis, alanine, aspartate, and glutamate metabolism, and pyrimidine metabolism. Conclusion Puerarin may exert its antidepressant effects by modulating the gut microbial structure and metabolite profiles, thereby alleviating inflammatory stress in the colon, bloodstream, and hippocampus, potentially through inhibition of the LPS/TLR4 signaling pathway.