Jiu-Wei-Yong-An decoction alleviates atopic dermatitis by suppressing TLR4/MyD88/NF-κB signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Jiu-Wei-Yong-An (JWYA) decoction is a clinical traditional Chinese medicine prescription for atopic dermatitis (AD). Its anti-inflammatory and anti-pruritic activities have been previously confirmed, yet the underlying molecular mechanism remains unclear. AIM OF STUDY: This work aimed to identify JWYA's bioactive compounds and clarify its therapeutic mechanisms against AD. MATERIALS AND METHODS: We established MC903-induced AD mouse models and TNF-α/IFN-γ-stimulated HaCaT keratinocytes to assess JWYA's efficacy. LC-MS/MS combined with bioinformatics, public microarray datasets, and compound-target prediction was used to screen active components and core pathways. In vivo dermatitis severity and epidermal thickness were quantified after 14-day JWYA oral administration. Western blotting detected pathway protein expression in vitro and in vivo. TLR4 inhibitor TAK-242 rescue assays verified TLR4-dependent regulation. Molecular docking, molecular dynamics (MD) simulations, DARTS and CETSA assays further validated the direct binding of candidate compounds to TLR4/MyD88/NF-κB. RESULTS: Thirteen absorbable ingredients of JWYA were identified, with the TLR4/MyD88/NF-κB cascade screened as the core anti-AD pathway. JWYA alleviated AD-like lesions, reduced IgE and proinflammatory cytokines, and suppressed TLR4/MyD88/NF-κB overactivation in mice and keratinocytes; TAK-242 assays confirmed JWYA's effects rely on intact TLR4 signaling. Phylliroside and Suspenoidside B exhibited high affinity toward core targets, with direct physical binding validated by MD, DARTS and CETSA. CONCLUSIONS: JWYA ameliorates AD through multi-component, multi-target modulation of the TLR4/MyD88/NF-κB inflammatory axis. Phylliroside and Suspenoidside B are key bioactive substances that directly bind TLR4/MyD88/NF-κB to inhibit NF-κB hyperactivation, relieving AD-related inflammation. This study provides experimental basis for JWYA's clinical application and subsequent monomer drug development.