Timosaponin AIII from Anemarrhena asphodeloides binds and inhibits S100A8-mediated neutrophil infiltration and NET formation ameliorates MASH

This study aimed to identify the active compound from Anemarrhena asphodeloides Bge. (AA) that ameliorates metabolic dysfunction-associated steatohepatitis (MASH) and to elucidate its mechanism of action. A screening was conducted using a high-cholesterol diet-induced zebrafish MASH model. The active ingredients and compounds were subsequently evaluated in a feed-induced mice model of methionine-choline deficiency (MCD) and a primary neutrophil model induced by PMA. Immune cell depletion, network pharmacology, transcriptomics, molecular docking, and surface plasmon resonance (SPR) were utilized to identify targets and pathways. Timosaponin AIII (TA3) from AA n-butanol extract could significantly improve the pathological characteristics of MASH. In mice, TA3 reduced lipid accumulation, liver injury, inflammation, and neutrophil infiltration. Neutrophil depletion studies confirmed that TA3 primarily acts by targeting neutrophils. It inhibited neutrophil extracellular trap (NET) formation, an effect validated in PAD4-/- mice. Integrated analyses identified S100A8 as a key target; TA3 bound directly to S100A8, disrupting the S100A8/A9 heterodimer and subsequently inhibiting the TLR4/NF-κB pathway and reducing ROS production. These effects were abolished upon S100A8 knockdown. TA3's ability to ameliorate MASH may be associated with the binding of S100A8, which attenuates NETosis and neutrophil infiltration, and inhibits the activation of the TLR4/NF-κB pathway and ROS production.