| Objective:To investigate the antifungal activity of chelerythrine(CHE)against Aspergullus fumigatus(A.fumigatus),and its anti-inflammatory effect in A.fumigatus keratitis and related mechanisms.Method:1.The cytotoxic effects of different concentrations of CHE on immortalized human corneal epithelial cells(HCECs)and mice corneas were evaluated by CCK-8 assay and the Draize test,respectively.2.The in vitro antifungal activity of CHE was assessed by minimum inhibitory concentration test,scanning electron microscopy,transmission electron microscopy,propidium iodide uptake test,calcofluor white test,and crystal violet staining.3.C57BL/6 mouse model of A.fumigatus keratitis was established,2μg/mL CHE solution was treated with experimental mice and 0.1%DMSO solution or 8μg/mL natamycin solution was treated with control mice at 1 to 5 days post infection.The therapeutic effects of CHE were estimated by slit lamp photographs and clinical score.Plate count was used to measure the fungal load in the mice corneas;Hematoxylin-eosin staining was used to detect inflammatory cells infiltration in the corneas of mice.4.Immunofluorescence staining was utilized to evaluate the effects of CHE on neutrophil recuitment and infiltration;Myeloperoxidase(MPO)test was used to measure neutrophil activity.5.In animal and cellular A.fumigatus inflammation models,the m RNA expression of pro-inflammatory cytokines(IL-1β and IL-6)and pathway factors(Nrf2,HO-1 and LOX-1)were detected by RT-PCR;the protein expression of IL-1β and IL-6were detected by ELISA;Western Blot was used to detect the protein expression of Nrf2,HO-1 and LOX-1,and p38 MAPK phosphorylation.6.HCECs were transfected with LOX-1 plasmid and blank plasmid.The effect of CHE on the LOX-1/p38 MAPK signaling pathway was assessed by detecting the protein expression of LOX-1 and phosphorylation of p38 MAPK,and the protein expression of downstream inflammatory factors(IL-1β and IL-6).Results:1.CHE at 2μg/mL concentration showed no cytotoxic effects on HCECs and mice corneas.2.In in vitro tests,CHE inhibited A.fumigatus conidia growth,decreased fungal hyphae survival,damaged the fungal morphological structure,and prevented fungal biofilm formation.3.In the A.fumigatus keratitis mice model,2μg/mL CHE significantly improved the severity of A.fumigatus keratitis,reduced corneal fungal load,and inhibited inflammatory cell infiltration,exhibiting antifungal and anti-inflammatory effects in vivo.4.Immunofluorescence staining and myeloperoxidase test showed that CHE inhibited neutrophil recruitment and infiltration,and reduced MPO activity.5.In the in vitro and in vivo inflammatory response assays,RT-PCR results suggested that 2μg/mL CHE decreased the m RNA expression of IL-1β,IL-6 and LOX-1,and increased the m RNA expression of Nrf2 and HO-1;ELISA results showed that 2μg/mL CHE inhibited the protein expression of IL-1β,IL-6;Western Blot results showed that 2μg/mL CHE decreased the protein expression of LOX-1,inhibited p38 MAPK phosphorylation and increased the protein expression of Nrf2/HO-1.6.LOX-1 plasmid treatment significantly increased LOX-1 protein expression levels,the p-p38/p38 MAPK ratio,and protein expression levels of IL-1β and IL-6.2μg/mL CHE pretreatment down-regulated plasmid stimulation-induced LOX-1 protein expression,phosphorylation of p38 MAPK and expression of IL-1β and IL-6.Conclusions:In A.fumigatus keratitis,2μg/mL CHE had no significant toxic effects on HCECs and mouse corneas,and could exert antifungal and anti-inflammatory effects.The antifungal effect of CHE was achieved by inhibiting the growth of A.fumigatus conidia,disrupting the morphological structure of fungal hyphae,reducing the survival of fungal hyphae and preventing the formation of fungal biofilms.CHE reduced neutrophil recruitment and infiltration,down-regulated the expression of pro-inflammatory cytokines,increased the expression of Nrf2/HO-1 and inhibited the LOX-1/p38 MAPK signaling pathway to exert anti-inflammatory effects. |
