Dimethyl Itaconate Protects Against Fungal Keratitis By Activating The Nrf2/HO-1 Signaling Pathway

Purpose: To test the protective effect of DI against fungal keratitis and investigate the specific mechanism in this process.Methods: 1.The cornea of C57BL/6 mice was infected with Aspergillus fumigatus(A.fumigatus).The cornea of mice was treated with 2 m M DI or PBS,and the clinical score of corneal infection in mice was observed.Fungal load in corneas of infected mice was detected by colony count method.Immunofluorescence,MPO and flow cytometry were used to detect the number of neutrophils in infected corneas treated with PBS and DI.The m RNA and protein expressions of IL-1?,CXCL1,nuclear factor E2 related factor 2(Nrf2)and Heme oxygenase 1(HO-1)in the corneas of each group were detected by PCR and Western blot.2.After being pretreated with 0.25 m M DI or PBS for 2 hours,human corneal epithelial cells were stimulated by A.fumigatus for 8 hours or 24 hours.The m RNA and protein expressions of IL-1?,IL-8 and IL-6 in each group were detected by PCR and ELISA.The expression and localization of Nrf2 in human corneal epithelial cells were detected by cellular immunofluorescence.3.Human corneal epithelial cells were pretreated with Nrf2 inhibitor Brusatol(BT)or HO-1 inhibitor Zinc protoporphyrin(Znpp)for 2 hours,and then treated with DI for 2 hours.The expression of IL-1?,IL-8 and IL-6 m RNA and protein in the cells were detected by PCR and ELISA.The expression of HO-1 protein was detected by Western blot.4.Different concentrations of DI(2,5,10 and 20 m M)were cultured with A.fumigatus for 48 hours,and the changes in the number of fungi were detected by absorbance and fluorescence staining.Results: 1.Compared with the PBS control group,after 2 m M DI was given to the cornea of mice,the inflammatory response of the cornea of mice infected by aspergillus fumigatus for 3 days and 5 days was significantly reduced,and the clinical score was significantly reduced.The count of corneal colony was significantly decreased at 3 days after infection.The number of neutrophils in the cornea after 3 days of infection decreased significantly.2.Compared with PBS control group,m RNA and protein expressions of IL-1?,CXCL1 in corneas infected for 3 days after 2 m M DI treatment were significantly decreased.3.0.25 m M DI pretreatment of human corneal epithelial cells stimulated by A.fumigatus significantly reduced the expression of IL-1?,IL-8 and IL-6 m RNA and protein in the cells.4.2 m M DI treatment of the cornea of infected mice significantly increased the m RNA and protein expression of Nrf2 and HO-1 in the cornea.Pretreatment with 0.25 m M DI in infected human corneal epithelial cells significantly increased the expression of Nrf2 in the cells,and Nrf2 expression was mainly concentrated in the nucleus.5.Compared with the treatment of human corneal epithelial cells with 0.25 m M DI alone,pretreatment of human corneal epithelial cells with 15 n M BT before DI significantly reduced the expression of HO-1 m RNA and protein in infected corneal epithelial cells.Compared with human corneal epithelial cells treated with 0.25 m M DI alone,the m RNA and protein expression of IL-1?,IL-8 and IL-6 in human corneal epithelial cells increased after pretreatment with 15 n M BT or 10 ?M Znpp and treatment with 0.25 m M DI.The difference was statistically significant.6.With the increase of cocultured DI concentration,the number of A.fumigatus in vitro gradually decreased.Conclusion: These data indicate that DI protects against fungal keratitis by limiting inflammation via the Nrf2/HO-1 signaling pathway and that DI inhibits the growth of A.fumigatus.