| Purpose: To investigate the anti-inflammatory and antifungal effects of kaempferol(KAE)on Aspergillus fumigatus(A.fumigatus)keratitis in C57BL/6 mice.Method: 1.CCK-8 test was used to test whether KAE has a toxic effect on the RAW264.7cells and human corneal epithelial cells(HCECs),and then to select a suitable concentration of KAE for subsequent experiments.2.KAE or DMSO were administrated to the corneas after the corneas of C57BL/6 mice were infected with A.fumigatus.Then the degree of keratitis was observed by slit lamp at 1,3 days post infection(p.i.),and the severity of the keratitis was evaluated by clinical scores.HE staining and plate count were performed to test the effects of KAE on infiltration of inflammatory cell and fungal load respectively in corneas at 3 days p.i.3.Immunofluorescence staining and MPO test were utilized to evaluate the effects of KAE on neutrophil and macrophages infiltration in A.fumigatus infected corneas.4.At 3 days p.i.,Real-time fluorescent quantitative PCR(q RT-PCR)was used to detect the m RNA expression of IL-1β,TNF-α,MIP-2,TLR4,Dectin-1;ELISA was used to detect the protein expression of IL-1β,TNF-α,MIP-2;and Western Blot was used to detect the protein expression of TLR4 and Dectin-1 as well as the phosphorylated-p38(p-p38)/p38 MAPK ratio in corneas.5.Macrophages were pretreated with KAE for 2h,followed by 8hs’ and 24hs’ stimulation of A.fumigatus for testing the m RNA and protein expression of IL-1β,TNF-α and MIP-2 respectively,and 1h’s stimulation of A.fumigatus for testing the ratio of p-p38/p38 MAPK.6.RAW264.7 cells were stimulated by Curdlan for 8,24 and 1h after 2hs’ pretreatment with KAE.And the m RNA and protein expression of TNF-α and MIP-2 as well as the p-p38/p38 MAPK ratio were evaluated respectively.Results: 1.Cell viability of HCECs was not affected when KAE concentration was lower than 60μg/m L,and Cell viability of RAW264.7 cells was not affected when KAE concentration was lower than 30μg/m L.2.Compared with the DMSO treated group,60μg/m L KAE treatment significantly slowed down the course of fungal keratitis,reduced the clinical score and fungal load of corneas at the 3 days p.i.3.KAE administration alleviated cornea edema and infiltration of inflammatory cells such as neutrophil and macrophages at 3 days p.i.4.Compared with the DMSO treated group,KAE treatment significantly reduced the m RNA and protein expression of IL-1β,TNF-α,MIP-2,TLR4 and Dectin-1,as well as the phosphorylation levels of p38 MAPK in A.fumigatus infected corneas.5.Compared with the DMSO treated group,30μg/m L KAE pretreatment significantly reduced the m RNA and protein expression of IL-1β,TNF-α,MIP-2 and the phosphorylation levels of p38 MAPK in A.fumigatus stimulated macrophages.6.Compared with the DMSO treated group,30μg/m L KAE pretreatment significantly reduced the m RNA and protein expression of TNF-α,MIP-2 and the phosphorylation levels of p38 MAPK in Curdlan stimulated RAW264.7 cells.Conclusions: KAE protects against fungal keratitis by reducing corneal fungal load,depressing the inflammatory cells recruitment,and downregulating the expression of inflammatory factors such as IL-1β,TNF-α and MIP-2,and those effects depended on the inhibition of Dectin-1 and p38 MAPK pathway. |
