Anti-inflammatory Effects Of Glabridin On Aspergillus Fumigatus Keratitis

PURPOSETo explore the anti-inflammatory effect of Glabridin(GLD)on fungal keratitis and its mechanism.METHODSThis study investigated the effects of GLD on the expression of pro-inflammatory cytokines and pattern recognition receptors(PRRs)in Aspergillus fumigatus-infected cells,as well as the effect on macrophage polarization in mouse corneal tissue and mouse peritoneal macrophages infected by A.fumigatus.1.In vitro cell experiments,to explore whether GLD has an influence on m RNA and protein expression of inflammatory factors(IL-1β,TNF-α),PRRs(TLR2,TLR4,Dectin-1)in fungal infected cells,human corneal epithelial cells(HCECs),mouse peritoneal macrophages and mouse peritoneal neutrophils were co-cultured with GLD(16μg/m L)and inactivated A.fumigatus hyphae(1×10~8cfu/m L).After 8 hours cultivation,m RNA was extracted from 12-well plate cells for Real-Time PCR experiment.After 24 hours cultivation,enzyme-linked immunosorbent assay(ELISA)and Western Blot were used to detect protein expression of inflammatory factors and PRRs in 6-well plate cells.2.The mouse models of A.fumigatus keratitis were randomly divided into GLD treatment group and PBS treatment group.Flow cytometry was used to detect the content of CD206+and CD86+macrophages in the corneas of each group to uncover the difference in proportion of M2/M1 macrophages between the two groups.The mouse peritoneal macrophages were cultivated and divided into blank control group,A.fumigatus treated group and A.fumigatus+GLD treated group.Flow cytometry was used to detect the content of CD206+and CD86+macrophages in each group to uncover the difference in proportion of M2/M1 macrophages between these groups.3.The mouse models of A.fumigatus keratitis were randomly divided into GLD treatment group and PBS treatment group.The eyeballs of mice infected for three days were collected and corneal sections were prepared.Periodic acid Schiff staining(PAS)was used to show the degree of corneal edema and mycelial load of the two groups and then we compared these results.RESULTS1.According to the results,in the A.fumigatus stimulation group the expressions of inflammatory factors(IL-1β,TNF-α),PRRs(TLR2,TLR4,Dectin-1)m RNA and protein of HCECs,mouse peritoneal macrophages and mouse peritoneal neutrophils were significantly up-regulated compared with the control group and GLD group.The m RNA and protein expressions of the above-mentioned inflammatory cytokines and PRRs in the A.fumigatus+GLD group were significantly lower than those of the A.fumigatus stimulation group.2.The results of flow cytometry showed that the ratio of M2/M1macrophages in the mouse corneas and mouse peritoneal macrophages stimulated with A.fumigatus mycelia and treated by GLD was higher than the other groups.3.PAS staining showed that the corneal stromal edema of the mice in the GLD treatment group was significantly reduced,the corneal epithelium was arranged more neatly and smoothly,and the fungal hyphae were significantly reduced compared with the PBS treatment group.CONCLUSIONGLD has anti-inflammatory effects on A.fumigatus keratitis.GLD inhibits the expression of PRRs(TLR2,TLR4,Dectin-1),pro-inflammatory cytokines(TNF-α,IL-1β)and mediates macrophages polarization to M2 type to inhibit inflammation.And it can reduce the degree of edema of A.fumigatus keratitis,promote corneal epithelial repair and reduce mycelial load.