Berberine from Coptidis Rhizoma enhances host antifungal immunity against dermatophytosis and cutaneous candidiasis via JNK signaling and autophagy

<title>Abstract</title> <p> <bold>Background:</bold> Dermatophytosis and cutaneous candidiasis are prevalent fungal skin diseases worldwide, with rising antifungal resistance rendering conventional therapies increasingly inadequate. <italic>Coptidis Rhizoma</italic> (Huanglian) is a traditional Chinese medicinal herb historically used for treating skin infections and inflammatory conditions. Berberine, its major active alkaloid, exhibits broad-spectrum antimicrobial activity, yet its host-directed immunomodulatory mechanisms against fungal infections remain poorly understood. <bold>Methods:</bold> We employed network pharmacology and molecular docking to identify core targets of berberine against dermatophytosis and cutaneous candidiasis. In vitro antifungal activity was assessed by broth microdilution, checkerboard synergy assays, scanning electron microscopy, crystal violet biofilm staining, and confocal laser scanning microscopy with live/dead staining. In vivo efficacy was evaluated using <italic>Galleria mellonella</italic> survival assays, time-kill curves, and mouse models of ordinary and itraconazole-refractory <italic>Trichophyton rubrum</italic> and <italic>Candida albicans</italic> skin infections. Host immune responses, JNK pathway activation, and autophagic flux were examined by qPCR, histopathology, and immunohistochemistry. <bold>Results:</bold> Network pharmacology identified 19 core targets, with JNK signaling prominently enriched. Molecular docking showed stable berberine binding to MAPK8 (JNK) and other targets (≤ −7.0 kcal/mol). Berberine MICs ranged 16–128 μg/mL and synergized with itraconazole (FICI 0.125–0.375). Berberine induced severe ultrastructural damage, significantly inhibited biofilm formation (P &lt; 0.0001), and increased dead cell proportions to 40.4% ( <italic>T. rubrum</italic> ) and 54.9% ( <italic>C. albicans</italic> ). In vivo, berberine activated JNK (increased p-JNK), enhanced autophagic flux (elevated LC3-II/I, reduced p62), and suppressed excessive inflammation (downregulated <italic>TNF</italic> , <italic>PTGS2</italic> ). In refractory models, berberine alone or combined with itraconazole reduced fungal burden by ~2.5–3.4 log₁₀ CFU/g (P &lt; 0.01) and ameliorated histopathological damage. <bold>Conclusions:</bold> Berberine from <italic>Coptidis Rhizoma</italic> enhances host antifungal immunity by activating the JNK-autophagy axis, while exerting direct fungicidal and antibiofilm effects. Its synergy with itraconazole against refractory infections provides a mechanistic rationale for the ethnopharmacological use of <italic>Coptidis Rhizoma</italic> and supports its development as a promising antifungal sensitizer. </p>