Kaempferol Protects Intestinal Health in Chinese Forest Musk Deer Possibly by Regulating Intestinal Microbiota and Inhibiting the NF‐κB/NLRP3 Signaling Pathway

Captive Chinese forest musk deer (FMD; Moschus berezovskii) frequently experience diarrhea linked to gut microbiota dysbiosis. This study uses network pharmacology analysis to clarify kaempferol's therapeutic potential as a dietary supplement for FMD intestinal health, especially under antibiotic restrictions. To explore its mechanisms, we employed 16S rRNA gene sequencing to profile gut microbiota composition, untargeted metabolomics to identify key metabolites, biochemical assays (ELISA) to measure inflammatory and oxidative stress markers, and RT-qPCR to quantify gene expression related to inflammatory pathways. Network pharmacology analysis suggested that kaempferol mitigates intestinal inflammation by targeting critical molecules, such as TNF. Kaempferol significantly reduced fecal scores and diarrhea frequencies compared to controls (p < 0.05). ELISA analyses of fecal samples demonstrated that kaempferol suppressed IL-6, TNF-α, and MDA, while enhancing sIgA, IgG, GSH-Px, and CAT. 16S rRNA gene sequencing indicated that 400 mg/kg kaempferol treatment led to a 64.98% increase in the Firmicutes/Bacteroidetes ratio, accompanied by significant enrichment of beneficial microbiota identified via linear discriminant analysis (LDA > 3.0), including the Christensenellaceae R7 group and Blautia. Metabolomic analysis showed significantly increased intestinal levels of indoleacetic acid and indolelactic acid. RT-qPCR confirmed kaempferol's suppression of NF-κB/NLRP3 inflammasome pathway genes. This study establishes an integrated "active ingredient-host target-intestinal microbiota-metabolic regulation-pathway network" framework through multi-omics analysis and highlights that kaempferol promotes intestinal health in FMD by modulating the gut microbiota-metabolite axis to suppress NF-κB/NLRP3 inflammasome activation. This work deepens research on natural plant extracts for FMD intestinal health and provides new perspectives to advance sustainable FMD breeding.