Gut microbiota-metabolite crosstalk mediates icariin's protection against obesity-induced spermatogenic dysfunction through NF-κB/AMH axis in Sertoli cells.

BACKGROUND: Chronic inflammation caused by obesity is a major contributor to male infertility, yet effective therapeutic options remain limited. Icariin (ICA), a flavonoid from Epimedium, shows anti-inflammatory and reproductive-protective properties. This study aims to investigate the mechanisms by which ICA ameliorates obesity-associated spermatogenic impairment. METHODS: A high-fat diet (HFD) mouse model was developed and treated with ICA. Multi-omics approaches, including serum metabolomics, gut microbiota profiling, and testicular transcriptomics, were combined with bioinformatics tools (WGCNA, GSEA, KEGG, and network pharmacology). Key targets and pathways were validated by qRT-PCR, Western blotting, immunohistochemistry, immunofluorescence, and CETSA assays. RESULTS: ICA treatment improved testicular morphology, enhanced spermatogenesis, and restored serum AMH levels. Metabolomic analysis revealed that ICA reduced the levels of pro-inflammatory metabolites (7-ketocholesterol, 8(S),15(S)-DiHETE, aldosterone) and increased the levels of anti-inflammatory metabolites (resveratrol, folic acid, eicosapentaenoic acid). Microbiota-metabolite correlations indicated that ICA enriched Akkermansia and suppressed pro-inflammatory Helicobacteraceae, which reshaped the gut-metabolite-inflammation axis. Transcriptomic analysis revealed that ICA suppressed HFD-induced activation of IL-1β, TNF-α, and NF-κB signaling, accompanied by recovery of AMH-related regulators (SF-1, SOX9, GATA4, WT-1). Network pharmacology further identified NF-κB as a core target of ICA. CETSA and co-localization assays confirmed the direct interaction between ICA and NF-κB. CONCLUSION: ICA alleviates obesity-induced spermatogenic impairment by inhibiting NF-κB-mediated inflammation and restoring AMH synthesis, partially through microbiota-metabolite modulation. These findings highlight that ICA serves as a promising therapeutic candidate for obesity-related male infertility.