Formononetin From Sophora flavescens Aiton Alleviates Atopic Dermatitis by Suppressing Neutrophil Extracellular Traps

ABSTRACT Atopic dermatitis (AD) is a chronic inflammatory skin disorder with limited effective treatment. Sophora flavescens Aiton (Kushen) exhibits anti‐inflammatory properties, but its key active components and mechanisms against AD remain unclear. This study aimed to identify its therapeutic compounds and the underlying molecular mechanisms. Network pharmacology and ultra‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometry were applied to screen for the potential bioactive compounds from Kushen, which were validated in AD mouse models. Formononetin (FMN) was topically administered to evaluate its curative efficacy through histopathological and immunofluorescent staining of skin lesions. Transcriptomic profiling explored the molecular mechanisms, with subsequent validation of neutrophil extracellular traps (NETs)‐associated markers by immunofluorescent staining and western blotting. Co‐treatment with PAD4 inhibitor GSK484 and molecular docking were used to study the underlying mechanisms. The study identified FMN as an important active component in Kushen, which showed therapeutic effects comparable to dexamethasone in AD models. FMN normalized cutaneous hyperplasia, reduced infiltration of CD3 + T cells, mast cells, and neutrophils, and suppressed inflammatory cytokine expression. Mechanistically, FMN inhibited NETs formation evidenced by decreased levels of citrullinated histone H3 (citH3), myeloperoxidase (MPO), and peptidylarginine deiminase 4 (PAD4). GSK484 co‐treatment showed non‐synergistic effects, suggesting that FMN suppresses NETs formation primarily through PAD4/MPO inhibition, which was confirmed by molecular docking. These findings highlight FMN as a primary anti‐AD constituent of Kushen, therapeutically suppressing NETs‐driven crosstalk of innate and adaptive immunity. FMN's multitarget mechanisms provide mechanistic insights and position it as a candidate for AD treatment.