Chelidonic acid ameliorates Aspergillus fumigatus keratitis via antifungal and anti-inflammatory activities.

PURPOSE: This study aimed to evaluate the therapeutic efficacy of chelidonic acid (CA) in Aspergillus fumigatus (AF)-induced keratitis and to elucidate the underlying molecular mechanisms.

METHODS

The safety of CA was assessed using a Cell Counting Kit-8 (CCK-8) assay, live-dead cell staining, cell scratch assay, corneal fluorescein sodium staining, and hematoxylin-eosin (HE) staining. The antifungal activity of CA was determined using minimum inhibitory concentration (MIC) assays, calcofluor white staining, propidium iodide (PI) staining, quantification of corneal fungal burden, periodic acid-Schiff (PAS) staining and quantitative real-time polymerase chain reaction (qRT-PCR). The therapeutic efficacy of CA was evaluated by slit-lamp biomicroscopy, corneal clinical scoring, HE staining, immunofluorescence staining, myeloperoxidase (MPO) measurement, and Transwell migration assay. The anti-inflammatory activity of CA was determined by qRT-PCR, western blotting, and immunofluorescence staining. The therapeutic efficacy of combined CA and natamycin treatment was assessed by slit-lamp examination, corneal clinical scoring, PAS staining, and HE staining.

RESULTS

5 mM CA exhibited no detectable cytotoxicity in RAW264.7 cells, human corneal epithelial cells (HCECs) or mouse corneas. CA exerted antifungal effects in AF keratitis. CA significantly ameliorated AF keratitis by suppressing neutrophil and macrophage infiltration. CA markedly downregulated the expression of pro-inflammatory cytokines, including interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α), and upregulated the expression of anti-inflammatory cytokine IL-10, through inhibition of the myeloid differentiation factor 88 (MyD88)/nuclear factor-kappa B (NF-κB) signaling pathway. Combined treatment with CA and natamycin further reduced clinical scores, fungal load, and inflammatory cell infiltration compared with monotherapy.

CONCLUSIONS

CA exhibits both antifungal and anti-inflammatory activities in AF keratitis and represents a promising candidate for the treatment of fungal keratitis.