Honokiol reduces fungal load, toll-like receptor-2, and inflammatory cytokines in aspergillus fumigatus keratitis

Abstract

PURPOSE. We characterized the effects of Honokiol (HNK) on Aspergillus fumigatus-caused keratomycosis and the underlying mechanisms. HNK is known to have anti-inflammatory and antifungal properties, but the influence on fungal keratitis (FK) remains unknown. METHODS. In ex vivo, minimum inhibitory concentration and Cell Count Kit-8 assay were carried out spectrophotometrically to provide preferred concentration applied in vivo. Time kill assay pointed that HNK was fungicidal and fungistatic chronologically. Adherence assay, crystal violet staining, and membrane permeability assay tested HNK effects on different fungal stages. In vivo, clinical scores reflected the improvement degree of keratitis outcome. Myeloperoxidase (MPO) assay, flow cytometry (FCM), and immunohistofluorescence staining (IFS) were done to evaluate neutrophil infiltration. Plate count detected HNK fungicidal potentiality. RT-PCR, Western blot, and enzyme-linked immunosorbent assay (ELISA) verified the anti-inflammatory activity of HNK collaboratively. RESULTS. In vitro, MIC90 HNK was 8 μg/mL (no cytotoxicity), and Minimal Fungicidal Concentration (MFC) was 12 μg/mL for A. fumigatus. HNK played the fungistatic and fungicidal roles at 6 and 24 hours, respectively, inhibiting adherence at the beginning, diminishing biofilms formation, and increasing membrane permeability all the time. In vivo, HNK improved C57BL/6 mice outcome by reducing disease severity (clinical scores), neutrophil infiltration (MPO, FCM, and IFS), and fungal loading (plate count). RT-PCR, Western blot, and ELISA revealed that HNK downregulated mRNA and protein expression levels of Toll-like receptor-2 (TLR-2), high mobility group box 1 (HMGB1), IL-1β, and TNF-α. CONCLUSIONS. Our study suggested HNK played antifungal and anti-inflammatory roles on keratomycosis by reducing survival of fungi, infiltration of leucocytes, and expression of HMGB1, TLR-2, and proinflammatory cytokines, providing a potential treatment for FK.