Hydroxytyrosol downregulates inflammatory responses via Nrf2/HO-1 axis during fungal keratitis and exerts antifungal effects.

PURPOSE: This study aims to explore the protective effect and underlying mechanism of hydroxytyrosol (HT) in fungal keratitis.

METHODS

Mouse models with Aspergillus fumigatus (A. fumigatus) keratitis, human corneal epithelial cells (HCECs) and RAW 264.7 cells were used in this study. Methods employed included MIC assay, biofilm formation test, hyphal immunofluorescence staining and adhesion test to assess the antifungal activity of HT. The severity of keratitis was evaluated using slit-lamp examination and HE staining. Draize eye test was used to measure corneal tolerance to HT. Corneal macrophages were detected by immunofluorescence staining. Reactive oxygen species (ROS) production in cytoplasm was quantified using DCFH-DA. Mitochondrial membrane potential was detected by JC-1. RT-PCR, ELISA and western blot were used to measure the expression of cytokines, as well as Nrf2 and HO-1 levels.

RESULTS

HT inhibited A. fumigatus growth, biofilm formation and conidial adhesion, and downregulated the expression of genes related to cell-wall assembly and morphogenesis. In fungal keratitis mouse models, HT significantly alleviated corneal inflammation, decreased the expression of cytokines and the accumulation of macrophages. In vitro, HT attenuated A. fumigatus-induced cytokine overexpression in HCECs or RAW 264.7 cells, and this effect was counteracted by an Nrf2 inhibitor. In RAW 264.7 cells stimulated with A. fumigatus, HT downregulated M1 markers expression, upregulated M2 markers expression, reduced ROS production, and restored mitochondrial membrane potential. Notably, these effects of HT were negated by pretreatment with an Nrf2 inhibitor.

CONCLUSIONS

This study underscores HT's efficacy against A. fumigatus growth and corneal invasion, its ability to mitigate fungi-induced inflammation, and its capacity to eliminate ROS via activation of the Nrf2/HO-1 signaling pathway. These findings suggest that HT holds therapeutic promise for fungal keratitis.