| Purpose:To investigate the effect of Glabridin（GLD）on fungal keratitis and the underlying mechanisms.Methods:1.Aspergillus fumigatus（A.fumigatus）conidia was inoculated in 96-well plate,and minimal inhibitory concentration（MIC）and biofilm formation ability were evaluated after GLD treatment.2.Human corneal epithelial cells（HCECs）were treated with GLD at different concentrations for 24 hours,and the toxicity of GLD to HCECs was detected using the CCK-8 kit.Spore adhesion ability was evaluated in conidia infected HCECs.3.C57BL/6（B6）mouse keratitis model was created by corneal intrastromal injection with A.fumigatus conidia,and GLD（therapy group）treatment or DMSO（control group）treatment started after infection.The degree of corneal inflammation was assessed by clinical score at 1,3 and 5 days after infection,and the number of fungal colonies was calculated by plate count at 5 days post infection.4.Mice corneas were removed at 3 days post-infection,myeloperoxidase（MPO）,flow cytometry and immunofluorescence staining（IFS）experiments were used to evaluate neutrophil infiltration.5.The cornea of mice was collected at 1,3,5 days after infection,and the expression of TLR4 and Dectin-1 mRNA was detected by PCR.Western blot was used to detect the expression of TLR4 and Dectin-1 protein at 3 and 5 days after infection.6.The expressions of inflammatory factors HMGB1,IL-1β,and TNF-αmRNA were detected by PCR,the expressions of HMGB1,IL-1β,and TNF-αprotein levels were detected by enzyme-linked immunosorbent assay（ELISA）.Result:1.GLD inhibited the growth of A.fumigatus in vitro at 8μg/mL,the conidia density was reduced to 90%(MIC90)at 12μg/mL,and 99%(MIC99)at 16μg/mL.The biofilm formation was significantly inhibited by GLD at 12μg/mL.2.CCK-8 assay showed the concentration no more than 16μg/mL can be applied as the effective dose.Therefore,16μg/m L GLD was used in the following experiments.There are much less spores adhering to HCECs in GLD treatment group compared with DMSO group.3.The clinical score of GLD group was significantly lower than that DMSO group after infection.Plate count assay demonstrated that the number of fungal colonies at 5 d post-infection corneas was significantly reduced after GLD treatment compared with DMSO treated group.4.The flow cytometry result showed that compared with DMSO group,neutrophil infiltration in GLD treated corneas was significantly attenuated.IFS showed that the GLD-treated group showed weaker staining intensity than the DMSO-treated one.And GLD significantly reduced MPO level.5.Treatment with GLD significantly inhibited A.fumigatus induced augment of mRNA levels of TLR-4 and Dectin-1.6.GLD significantly reduced mRNA levels of IL-1β,TNF-a and HMGB1 in C57BL/6 mice corneas.Conclusion:1.GLD treatment inhibited the growth,biofilm formation ability and adhesion capability to HCECs of A.fumigatus.2.Upon A.fumigatus infection,treatment of GLD after infection in mice showed significantly decreased severity of corneal inflammation,reduced number of A.fumigatus in cornea,and suppressed neutrophil infiltration in cornea.3.GLD exerts anti-inflammatory effect in A.fumigatus keratitis via inhibiting the expression of TLR4,Dectin-1 and pro-inflammatory mediators such as HMGB1,IL-1βand TNF-α. |
