Alnustone ameliorates atopic dermatitis by rebalancing TAp63/ΔNp63 to upregulate filaggrin and restore skin barrier...

Atopic dermatitis (AD) is a chronic, pruritic, erythematous skin condition with skin barrier dysfunction, driven largely by filaggrin (FLG) deficiency; however, direct FLG-targeting therapeutics remains lacking. To identify novel natural products capable of enhancing FLG expression, we screened an in-house natural product library using a dual-luciferase reporter assay in HaCaT keratinocytes, which revealed that alnustone dose-dependently activated the human FLG promoter. RT-qPCR and Western blot analysis confirmed that alnustone upregulated FLG mRNA and protein levels in keratinocytes, independently of the aryl hydrocarbon receptor (AhR). The therapeutic efficacy of alnustone was evaluated in oxazolone-induced AD mouse model, where topical application significantly alleviated clinical severity, inflammation, and skin barrier defects. The molecular mechanism was explored using ChIP-seq data mining, isoform-specific modulation, promoter truncation, and DNA pull-down assays. The results demostrated that alnustone rebalanced p63 isoforms by downregulating the transcriptional repressor TAp63 and upregulating the transcriptional activator ΔNp63. Both isoforms directly bound to a core response element (-390/-381) within the FLG promoter to mediate its transcriptional activation. To our knowledge, alnustone represents the first barrier-repair agent that ameliorates AD by transcriptionally upregulating FLG via restoring TAp63/ΔNp63 homeostasis, offering a novel therapeutic strategy targeting skin barrier regeneration in AD.