Isorhamnetin Ameliorates Aspergillus Fumigatus Keratitis By Reducing Fungal Load And Inhibiting Inflammatory Reaction

Objective:The current study aims to investigate the antifungal and the anti-inflammatory effects of isorhamnetin against mouse Aspergillus fumigatus（A.fumigatus）keratitis.Methods:1.In vitro,the lowest effective concentration of isorhamnetin was assessed by MIC and cytotoxicity tests（CCK-8）in human corneal epithelial cells（HCECs）and RAW264.7cells.2.The effects of isorhamnetin on A.fumigatus morphology and membrane integrity were observed by SEM and PI uptake test.3.The anti-inflammatory effect of isorhamnetin in HCECs and RAW264.7 cells was observed by q RT-PCR.4.In the eyes of mice with A.fumigatus keratitis,FK severity was evaluated using slit lamp photographs and clinical score;the fungal load in mice corneas was observed by plate counting and PAS staining.5.Immunofluorescence staining（IFS）,myeloperoxidase（MPO）assay and histological staining were performed to detect the level of neutrophil infiltration in mice with A.fumigatus keratitis 6.In vivo,the anti-inflammatory effect of isorhamnetin was examined by q RT-PCR,ELISA,and Western Blot.Results:1.The MIC₉₀of isorhamnetin to Aspergillus fumigatus was 80μg/mL,and inhibited A.fumigatus hyphae growth at 80μg/mL.Cell viability of HCECs and RAW264.7 cells was not affected when isorhamnetin concentration was lower than 160μg/mL.2.In vitro,80μg/mL isorhamnetin altered A.fumigatus hyphal morphology and membrane integrity.3.After stimulating HCECs and RAW264.7 cells with A.fumigatus,isorhamnetin（80μg/mL）significantly inhibited the m RNA expression of proinflammatory factors including TLR-2,TLR-4,Dectin-1,IL-1β,TNF-α.4.In A.fumigatus keratitis mouse model,isorhamnetin treatment alleviated the severity of FK,compared to the control group,the corneas of the isorhamnetin group were more transparent,had smaller ulcers,and did not perforate.The clinical score of isorhamnetin treated group was significantly lower than that of DMSO group at 3 and 5 days p.i.5.PAS staining showed that isorhamnetin treatment can inhibit hyphal growth and deep invasion into corneal tissue on the third day after infection.Plate counting experiment showed that the number of viable fungus in corneas of isorhamnetin group was significantly less than control group on the5th day after infection.6.Isorhamnetin significantly reduced inflammatory cell infiltration and alleviated corneal edema at 3 days p.i.Isorhamnetin attenuated inflammatory response by inhibiting neutrophil recruitment in A.fumigatus infected corneas,which is supported by MPO activity assay that neutrophil activity was significantly reduced by isorhamnetin treatment.7.The m RNA and protein expression levels of pattern recognition receptors TLR-2,TLR-4,Dectin-1 and proinflammatory mediators L-1β,TNF-αwere significantly decreased in isorhamnetin treated groups in vivo.Conclusion:Isorhamnetin improves the prognosis of A.fumigatus keratitis in mice by inhibiting the growth of A.fumigatus,reducing the recruitment of neutrophils and downregulating inflammatory factors.