Gallic Acid Ameliorates Aspergillus Fumigatus Keratitis Through Reducing Fungal Load And Suppressing The Inflammatory Response

Objective:To explore the antifungal and anti-inflammatory effects of Gallic Acid(GA)on Aspergillus fumigatus(A.fumigatus)keratitis and its potential mechanism.Methods:1.CCK-8 assay applied to human corneal epithelial cells(HCECs)and Draize eye test applied to mouse cornea were used to determine the non-cytotoxic concentration of GA in subsequent experiments.2.In vitro,the minimal inhibitory concentration(MIC)test,Biofilm formation test,fungal adherence assay,calcofluor white staining,and propidium iodide uptake test were taken to evaluate the antifungal effect of GA.3.Establish A.fumigatus keratitis mice model,separately treated with GA and phosphate buffer(PBS)for 5 days.The therapeutic effects of GA were estimated by slit lamp photographs and clinical score.Periodic acid-Schiff staining and HE staining was taken to evaluate antifungal and anti-inflammatory effects of GA in vivo.4.Immunofluorescence staining and myeloperoxidase assay were conducted to identify neutrophil infiltration and activity.5.RT-PCR,ELISA,and Western blot were used to detect the expression of pro-inflammatory cytokines such as IL-1β、TNF-α、COX-2、LOX-1,and Nrf2/HO-1 that may releated to anti-inflammatory mechanism of GA.Results:1.In HCECs cells and healthy mouse corneal epithelium,GA at 100 μg/m L did not affect cell viability and was non toxic to cornea,thus this concentration could applied to subsequent experiments.2.In vitro,100 μg/m L GA significantly inhibited A.fumigatus growth,biofilm formation,and spore adhesion.Alos it altered fungus membrane integrity.3.In vivo,100 μg/m L GA reduced A.fumigatus infected mouse cornea inflammation and clinical score.PAS staining revealed GA could repress fungal load in mouse cornea slice,HE staining suggested GA could reduce inflammatory cell infiltration.4.Immunofluorescence staining and myeloperoxidase assay showed GA inhibited neutrophil recruitment and reduced MPO activity.5.The expression of IL-1β,TNF-α,LOX-1,and COX-2 was inhibited in m RNA and protein level,while Nrf2 and HO-1 expression was enhanced both in m RNA and protein levels in 100 μg/m L GA treated group in comparison to PBS control.Conclusion:GA ameliorates fungal keratitis severity through inhibiting A.fumigatus load,reducing neutrophils infiltration,downregulating the expression of pro-inflammatory cytokines,and increasing the Nrf2/HO-1 pathway.