| Purpose: To investigate the antifungal mechanism of Benzyl isothiocyanate(BITC)against Aspergillus fumigatus(A.fumigatus)and its therapeutic effects on A.fumigatus keratitis.Methods: The suitable concentrations of BITC for in vivo and in vitro experiments were obtained by cell counting kit(CCK-8)for immortalized human corneal epithelial cells(HCECs),mouse macrophage(RAW 264.7)cell activity and Draize assay.In vitro,the minimum inhibitory concentration(MIC)assay,calcium fluorescence white staining and time-kill assay were used to evaluate the antifungal activity of BITC against A.fumigatus.The disruption of BITC on A.fumigatus cell membrane integrity was detected by propidium iodide(PI)uptake assay.The intracellular ROS accumulation in A.fumigatus was detected by reactive oxygen species(ROS)kit.Adhesion assay was used to verify the inhibitory effect of BITC on the adhesion capacity of conidia.Corneas of A.fumigatus keratitis were treated with BITC or DMSO by subconjunctival injection.Corneal conditions were observed,photographed under slit lamp and scored at 3 and 5 days post infection(p.i.).The efficacy of BITC on A.fumigatus keratitis was evaluated by clinical score and plate counting.The infiltration of inflammatory cell was assessed by HE staining at 3 and 5 days p.i.Corneal neutrophils were quantified by myeloperoxidase(MPO)kit.The m RNA and protein levels of IL-1β,TNF-α,and IL-6 in mice cornea were assessed by qRT-PCR and ELISA at 3 and 5 days p.i.The m RNA and protein levels of Mincle were detected by qRT-PCR and western blot at 3 and 5 days p.i.After BITC pretreatment for 2 h,RAW264.7 cells were stimulated by inactivated A.fumigatus hyphae.The m RNA and protein of IL-1β,TNF-α,IL-6 and Mincle were detected by qRT-PCR,ELISA and Western Blot.After pretreatment with BITC for 2 h,Mincle-specific agonist TDB was used to stimulate RAW264.7 cells,and the expression of m RNA and protein of IL-1β、TNF-α,and IL-6 were detected by qRT-PCR and ELISA.The protein expression levels of Mincle were detected by Western Blot and immunofluorescence.Results:1.Cell viability of HCECs was unaffected by BITC at concentrations of 0-100μg/m L,and cell viability of RAW264.7 was unaffected by BITC at concentrations of 0-6μg/m L.The Draize eye test revealed that BITC was not toxic to mouse cornea at a concentration of 0-400 μg/m L.2.In vitro experiments,BITC excerted antifungal effects against A.fumigatus at the concentration of 25 μg/m L in a time-dependent and concentration-dependent manner.BITC killed A.fumigatus by damaging cell membranes integrity,mitochondria morphology and function,inhibiting its adhesion to HCECs and biofilms.3.In the mouse keratitis model,compared with the the DMSO-treated group,clinical score was significantly lower in the 200 μg/m L BITC-treated group,and the fungal load of infected corneas was reduced in BITC-treated group.HE staining revealed that the inflammatory cell infiltration in the corneas of mice was significantly reduced in the BITC-treated group compared to the DMSO-treated group at 3 and 5 days p.i.RT-PCR,Western Blot and ELISA experiments showed that the m RNA and protein expression of IL-1β,TNF-α and IL-6 and pattern recognition receptor Mincle were significantly reduced in the infected corneas of the BITC-treated group compared to the DMSO-treated group.4.The results of qRT-PCR,Western Blot,ELISA,and immunofluorescence experiments showed that 3 μg/m L BITC significantly downregulated the m RNA and protein expression of Mincle,IL-1β,TNF-α and IL-6 compared with DMSO in RAW264.7 cells stimulated by hyphae of A.fumigatus or TDB.Conclusions: BITC possessed fungicidal activities against A.fumigatus through disrupting cell membranes,mitochondria,fungal adhesion,and biofilms.BITC exerted protective effects in fungal keratitis.BITC improved clinical scores,reduced fungal load,depressed the infiltration of inflammatory cells and levels of pro-inflammatory cytokines such as IL-1β,TNF-α and IL-6 in the infected cornea.BITC mediated anti-inflammatory effects by downregulating Mincle. |
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