The Role And Mechanism Of Resveratrol In Aspergillus Fumigatus Keratitis

Purpose: To determine the antifungal,anti-inflammatory and neuroprotective effects of resveratrol(RES)on Aspergillus fumigatus(A.fumigatus)keratitis.Methods:1.To assess the toxicity of different concentrations of RES on human corneal epithelial cells(HCECs),cytotoxicity assay and Draize eye test were performed;2.The antifungal effect of RES was assessed by minimum inhibitory concentration,scanning or transmission electron microscopy,propidium iodide test and Calcofluor white staining;3.In the fungal keratitis model in mice,0.8 m M of RES,12 μM natamycin(NATA)and0.1% DMSO were dripped into the corneas of mice.The degree of corneal inflammation was observed by slit lamp at days 1,3 and 5 post infection(p.i.),and clinical scores was performed.Mice were executed on day 3 and HE staining,plate colony counting and MPO were performed to detect the effect of RES on inflammatory cell infiltration and fungal load in corneal tissue;4.p-p38/p38 MAPK ratio as well as mRNA and protein expression of inflammatory factors such as IL-1β,IL-6 and pattern recognition receptor such as Dectin-1 in the cornea at 3 and 5 days p.i.were detected by qRT-PCR,Western blot and ELISA;5.HCECs were pretreated with RES for 2 hours and cells were collected after stimulation of A.fumigatus,and the ratio of p-p38/p38 MAPK as well as the mRNA and protein levels of IL-1β,IL-6 and Dectin-1 were measured by qRT-PCR,Western blot and ELISA;6.HCECs were pretreated with RES for 2 hours,and cells were collected after stimulation of Curdlan,and qRT-PCR,Western blot and ELISA was used to detect the p-p38/p38 MAPK ratio as well as mRNA and protein expression of inflammatory factors such as IL-1β and IL-6;7.Immunofluorescence staining,qRT-PCR and Von Frey assay were used to assess the neuroprotective effect of RES.Results: 1.cytotoxicity assay showed no toxic effect of RES on HCECs cells when concentration was below or equal to 100μM,the corneas of mice exposed to DMSO,0.2m M RES and 0.8 m M RES at 3 and 5 days showed no sodium fluorescein staining;2.RES inhibited the growth of A.fumigatus hyphae and altered the morphology of hyphae in vitro;3.0.8 m M RES treatment reduced the clinical scores and reduced the severity of keratitis,and RES also decreased inflammatory cell infiltration,neutrophil activity and fungal load;4.RES down-regulated Dectin-1,IL-1β,and IL-6 levels in the corneas of A.fumigatus-infected mice,and also reduced the phosphorylation level of p38 MAPK.RES pretreatment down-regulated the expression of IL-1β,IL-6,and Dectin-1 as well as the phosphorylation level of p38 MAPK in HCECs stimulated by A.fumigatus.RES pretreatment decreased the expression of IL-1β and IL-6 as well as the level of p38 MAPK phosphorylation in HCECs stimulated by Curdlan;5.RES protected corneal basal nerve fibers,down-regulated mechanosensitivity threshold,and increased CGRP and TRPV-1 expression.Conclusions: RES can inhibit the growth of A.fumigatus and destroy the morphology of hyphae,and also attenuates neutrophil infiltration in the corneal tissue of mice with A.fumigatus keratitis and reduces fungal load,thus exerting a protective effect in keratitis.Meanwhile,RES down-regulates the expression of inflammatory factors as well as pattern recognition receptors and inhibits p38 MAPK phosphorylation,and RES exerts its anti-inflammatory effect by inhibiting the Dectin-1/p38 pathway,and RES also has a neuroprotective effect on the corneas of mice in A.fumigatus keratitis.