Schaftoside reduces inflammation in Aspergillus fumigatus keratitis through the inhibition of the TLR4/MyD88 pathway

Purpose: The purpose of this research study was to investigate the impact of schaftoside on Aspergillus fumigatus (A. fumigatus) keratitis and elucidate its underlying mechanisms. Methods: In order to establish safe experimental concentrations of schaftoside in human corneal epithelial cells (HCECs), RAW264.7 cells, and mouse models, various techniques were employed including cytotoxicity assay (CCK-8) assay, cell scratch assay, and Draize test. The therapeutic effect of schaftoside was assessed using slit -lamp biomicroscopy, clinical scores, as well as determination of neutrophil infiltration through hematoxylin and eosin (HE) staining, immunofluorescence (IF) staining, and myeloperoxidase (MPO) assay. The levels of Toll -like receptor 4 (TLR4), myeloid differentiation primary response 88 (MyD88), pro-inflammatory mediators interleukin (IL)-1 beta, tumor necrosis factor (TNF)-alpha, and IL-6 were determined using quantitative real-time poly-merase chain reaction (qRT-PCR), western blotting, and IF techniques. Results: Schaftoside at a concentration of 160 mu M displayed no harmful side effects on HCECs, RAW cells, and mouse corneas, rendering it suitable for further experiments. In a murine fungal keratitis model, schaftoside mitigated the severity of fungal keratitis by inhibiting neutrophil infiltration and reducing MPO activity. Both in vitro and in vivo experiments demonstrated that schaftoside treatment suppressed the upregulation of IL-1 beta, TNF-alpha, and IL-6 expression, while also downregulating the expressions of TLR4 as well as MyD88 at both mRNA and protein levels. Conclusions: Schaftoside demonstrated a protective effect against A. fumigatus keratitis by reducing corneal damage through inhibition of neutrophil recruitment and downstream inflammatory cytokines. The anti-inflammatory properties of schaftoside in A. fumigatus keratitis may involve modulation of the TLR4/MyD88 pathway.