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, MIC 90 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.

Repurposing of auranofin and honokiol as antifungals against Scedosporium species and the related fungus Lomentospora prolificans

“…neoformans, and fluconazole-resistant C. albicans with relatively high MIC values [47,49,63]. HOK underwent successful in vivo trials for several fungal species exhibiting very high MIC values in vitro [48,49,64]. Therefore, considering the very low MIC values found in the present study, in vivo investigations should be conducted on murine models of Scedosporium and Lomentospora infections.…”

“…Antifungal activity of HOK has been investigated in many species, including dermatophytes, phytopathogenic fungi, A. fumigatus, Cr. neoformans, and fluconazole-resistant C. albicans with relatively high MIC values [47,49,63]. HOK underwent successful in vivo trials for several fungal species exhibiting very high MIC values in vitro [48,49,64].…”

“…Consistently, the HOG pathway is considered a common virulence trait since it is the leading contributor to stress-related responses in fungi [46]. Taking advantage of its biphenolic nature and its safety properties, HOK has been extensively investigated in many clinical fields, and its antifungal activity against a wide range of pathogenic fungi is now ascertained [47][48][49].…”

Isorhamnetin Ameliorates Aspergillus fumigatus Keratitis by Reducing Fungal Load, Inhibiting Pattern-Recognition Receptors and Inflammatory Cytokines

“…The isorhamnetin MIC 90 was determined spectrophotometrically and was recognized as the lowest concentration that could inhibit 90% growth of A. fumigatus. 29…”

“…The viable fungi on the treated cornea (n = 3/group/time) were reflected by taking pictures and counting the colonies. 29…”