Exploring the In Vitro Mechanism of Action of β-Acetoxyisovalerylalkannin on Inflammatory Skin Diseases Using Network-Based Pharmacology and Non-Targeted Metabolomics

<b>Objective:</b> Lithospermum erythrorhizon has been extensively used for the clinical treatment of skin diseases, but its material basis and mechanism of action remain unclear. This study integrates network pharmacology, untargeted metabolomics, and in vitro experimental validation to elucidate the anti-inflammatory effects and underlying mechanisms of β-acetoxyisovalerylalkannin, a bioactive naphthoquinone compound isolated from Arnebiae Radix, using inflammatory skin disease models. <b>Methods</b>: Core targets for β-Acetoxyisovalerylalkannin and skin inflammation were identified via network pharmacology and validated through molecular docking. In vitro assays assessed β-Acetoxyisovalerylalkannin's impact on keratinocyte proliferation, migration, apoptosis, and inflammatory factors (CXCL1, CXCL2, CXCL8, CCL20, IFN-γ, MCP-1, TNF-α, NF-κB). Non-targeted metabolomics identified differential metabolites and pathways. <b>Results</b>: Network pharmacology revealed 66 common targets significantly enriched in the MAPK/STAT3 signaling pathway. In vitro, β-Acetoxyisovalerylalkannin suppressed proliferative viability and hypermigration and induced apoptosis in HaCaTs. Moreover, it downregulated the mRNA levels of inflammatory markers (CXCL1, CXCL2, CXCL8, CCL20, IFN-γ, MCP-1, TNF-α, and NF-κB) by inhibiting the activation of the MAPK/STAT3 signaling pathway. Metabolomics identified 177 modified metabolites, associating them with the arginine/proline, glycine/serine/threonine, glutathione, and nitrogen metabolic pathways. <b>Conclusions</b>: β-Acetoxyisovalerylalkannin exerts protective effects against skin inflammation by reducing abnormal cell proliferation and inflammatory responses, promoting apoptosis, and effectively improving the metabolic abnormalities of HaCaTs. β-Acetoxyisovalerylalkannin is, therefore, a potential therapeutic option for mitigating skin inflammation-related damage.