Baicalin ameliorates Aspergillus fumigatus keratitis by its antifungal activity and suppression of pyroptosis.

PURPOSE: To investigate the therapeutic effect of Baicalin (BA) and its underlying mechanism in Aspergillus fumigatus (A. fumigatus) keratitis.

METHODS

First, the safety of BA was evaluated. In vitro cytotoxicity was tested with a CCK-8 assay on human corneal epithelial cells (HCECs) and peritoneal macrophages, while in vivo ocular toxicity was assessed in mice using corneal fluorescein sodium staining (CFS) and Draize scoring. Next, the direct antifungal effects of BA were evaluated through Minimum Inhibitory Concentration (MIC) tests, fluorescence staining, and biofilm formation and adhesion assays, supported by electron microscopy. Then, using a murine keratitis model, we assessed corneal pathology, fungal burden, and inflammatory responses via hematoxylin and eosin (H&E) staining, colony-forming unit (CFU) counting, quantitative PCR, and enzyme-linked immunosorbent assay (ELISA). Finally, activation of the NLRP3-caspase-1-GSDMD pathway was examined in peritoneal macrophages by Western blotting, immunofluorescence, RT-qPCR, and ELISA.

RESULTS

BA up to 75 μg/mL showed no cytotoxicity in vitro and no ocular toxicity in vivo. BA inhibited A. fumigatus growth, disrupted hyphal ultrastructure, and suppressed adhesion and biofilm formation. In keratitis models, BA treatment significantly decreased clinical scores, reduced corneal edema and inflammatory cell infiltration, and lowered fungal burden. Corneal samples from fungal keratitis mice exhibited enhanced expression of pro-inflammatory cytokines, as determined by quantitative reverse transcription PCR (RT-qPCR) and ELISA. This effect was markedly reversed by BA. In peritoneal macrophages, BA markedly downregulated the NLRP3-caspase-1-GSDMD signaling pathway.

CONCLUSION

BA demonstrates antifungal effects by disrupting the cell wall, cell membrane, and intracellular redox homeostasis of A. fumigatus. Additionally, it alleviates inflammation by suppressing the NLRP3 inflammasome-mediated pyroptosis.