Effect And Mechanism Of Disulfiram On Aspergillus Fumigatus Keratitis In Mice

Purpose: This study investigated the effect and molecular mechanism of Disulfiram on the immune reaction in mouse Aspergillus fumigatus keratitis.Methods: 1.Cell Count Kit-8(CCK-8)assay was performed to detect the effect of disulfiram on the proliferation and division capacity of human corneal epithelial cells(HCECs)to obtain available concentration without cytotoxicity.The effect of Disulfiram on the migration ability was observed through wound healing test on HCECs.2.Effect of disulfiram intervention on mouse fungal keratitis: Mice were divided into fungal keratitis group and fungal keratitis drug-intervention group randomly.The corneas of C57BL/6 mice were infected with Aspergillus fumigatus(A.fumigatus).In drug group,subconjunctival injection of 20 μM/5 μL disulfiram at 1 day before modeling and every day after modeling.In fungal keratitis group,DMSO was injected into subconjunctiva.Observed corneal opacity as well as ulcer morphology of mice and calculate clinical scores using a slit lamp on every other day after modeling.3.Fluorescent antibody technique was performed to label neutrophils and macrophages.Meanwhile,the quantity and activity of neutrophils and macrophages were monitored by myeloperoxidase(MPO)and nitric oxide synthase(NOS).4.Mechanism of disulfiram effect on fungal keratitis in mice: The cornea of C57BL/6 mice was infected with Aspergillus fumigatus(A.fumigatus).In drug group and drugintervention fungal keratitis group,subconjunctival injection of 20 μM/5 μL disulfiram at1 day before modeling,every day after modeling.And in the other two groups,subconjunctival injection of equivalent DMSO.Detect the expression of cytokines involving Gasdermin D,caspase-1 and IL-1β in mouse corneas.Techniques used including RT-PCR,and Western Blot.5.Mechanism of disulfiram effect on fungal infection in cells:Human peripheral blood neutrophils and mouse peritoneal primary macrophages were respectively divided into 4 groups according to experimental requirements.In drug group and drug-treatment fungal stimulation group,add experimental concentration of disulfiram into the cell culture medium.And in the other two groups,added an equivalent DMSO.After 8 or 16 hours of stimulation with inactivated mycelium,the cells were collected periodically for experimental samples,to detect the m RNA and protein expression of inflammatory factor IL-1β p31.And cell supernatant was collected at 16 hours for enzymelinked immunosorbent assay(ELISA)to detect the secretion of IL-1β p17.6.Therapeutic effect of disulfiram combined with natamycin in mouse fungal keratitis: Mice were randomly divided into natamycin group,drug and natamycin group.After fungal infection,in drug and natamycin group,mice regularly received both natamycin eye drops and disulfiram every day until 5 days after infection.In natamycin group,mice received natamycin eye drops and DMSO regularly.Using a slit lamp microscope and the clinical scores to assess responses to fungal keratitis.Results: 1.Disulfiram had no significant cytotoxic effect on proliferation at no more than 20 μM in CCK-8 test and does not affect cell migration compared with control group in scratch-wound assay.2.Compared with the fungal keratitis group,on the 1 d and 3 d day after infection,mice in drug-intervention fungal keratitis group had decreased corneal opacity and decreased clinical score.On the 5 d day after infection,the drug-treatment fungal keratitis group mice had severe inflammatory reaction and higher clinical scores.3.After one and two days of corneal infection in mice,neutrophils and macrophages staining and MPO and NOS activity were significantly reduced in drug-intervention fungal keratitis group compared with the fungal keratitis group.4.GSDMD,caspase-1 and IL-1β m RNA and protein in the cornea of fungal keratitis mice were not affected by disulfiram.5.In the cell experiment,disulfiram inhibited the release of IL-1β p17 by inhibiting the formation of GSDMD mediated plasma membrane pores in macrophages,but had no effect on the secretion of IL-1β p17 in neutrophils.6.Therapeutic effect of disulfiram combined with natamycin in mouse fungal keratitis: Mice in disulfiram combined with natamycin group had lighter corneal opacity and lower clinical scores compared with the control group.Conclusion: 1.Disulfiram intervention reduced corneal inflammation in the earlier stage,but enhanced inflammatory reaction in the later stage of fungal keratitis.2.Disulfiram had no significant effect on production of IL-1β,GSDMD and caspase-1.3.Disulfiram inhibits the release of IL-1β p17 from macrophages,but has no effect on neutrophils.4.Disulfiram combined with natamycin can reduce the damage to corneal transparency in mouse fungal keratitis.