Isoorientin ameliorates DNCB-induced atopic dermatitis via regulating macrophage polarization and the AMPK/NLRP3 signaling axis

Atopic dermatitis (AD) is a chronic inflammatory skin disorder characterized by skin barrier dysfunction and immune dysregulation. Current treatments are often limited by side effects and relapse upon withdrawal, highlighting the need for safer, natural alternatives. Isoorientin, a natural flavonoid, exhibits diverse pharmacological activities, including anti-inflammatory and immunomodulatory properties, yet its role in AD remains unexplored. This study explored the therapeutic effects of isoorientin on 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) in male C57BL/6 mice and clarified the underlying mechanism involving macrophage polarization and the adenosine monophosphate-activated protein kinase (AMPK)/NOD-like receptor family pyrin domain containing 3 (NLRP3) axis. A DNCB-induced AD mouse model was established. In vitro, human THP-1 monocytes were differentiated into macrophages and polarized into M1 phenotypes using lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Counting Kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), and western blot were used to determine safe drug concentrations and polarization marker expression. Rescue experiments with the AMPK inhibitor Compound C and the NLRP3 inhibitor MCC950 were performed by co-administering these inhibitors with LPS/IFN-γ and isoorientin to verify the role of the AMPK/NLRP3 signaling axis. Isoorientin dose-dependently relieved DNCB-induced skin lesions, lowered clinical scores, epidermal thickness, serum levels of pro-inflammatory cytokines (tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interferon-γ (IFN-γ)), and total immunoglobulin E (IgE), and increased the anti-inflammatory cytokine interleukin-10 (IL-10). Concurrently, it inhibited aberrant infiltration of F4/80⁺ macrophages in skin tissue, attenuated excessive M1 activation as evidenced by reduced cluster of differentiation 86 (CD86) expression, and promoted M2 polarization as shown by elevated cluster of differentiation 206 (CD206) and arginase 1 (Arg1) levels, thereby restoring macrophage immune homeostasis. Both in vivo and in vitro experiments confirmed that isoorientin markedly enhanced AMPK phosphorylation and inhibited NLRP3 inflammasome activation along with downstream cleaved caspase-1. Mechanistic rescue experiments suggested that isoorientin negatively regulated NLRP3 in an AMPK-dependent manner, consistent with NLRP3 functioning downstream of AMPK in this context to modulate the inflammatory cytokine network. Notably, combined treatment with MCC950 (NLRP3 inflammasome-specific inhibitor) and isoorientin produced no significant additive effects, further corroborating the AMPK/NLRP3 signaling axis as a critical pathway underlying the dermoprotective and immunomodulatory actions of isoorientin. Isoorientin relieved DNCB-induced AD in a manner correlating with AMPK activation, NLRP3 inflammasome inhibition, and M1/M2 macrophage polarization balance. However, alternative mechanisms—including direct antioxidant effects independent of AMPK and the possibility of reverse causality—cannot be excluded. These findings suggest that isoorientin may represent a promising therapeutic candidate for AD management, though further mechanistic validation was warranted. Not applicable.