| Objective:To investigate the effect of Gliotoxin(GT)on the progress of A.fumigatus keratitis by affecting macrophage polarization and inducing apoptosis in mouse.Methods:1.Corneas of C57BL/6 mice were infected with A.fumigatus wild-type strain B5233,its mutant strain gli PΔ strain(deletion of the peptide synthetase gli P)and the gli P reconstituted strain gli PR.The progressions of corneal inflammation on day 1,3,and 5 p.i.were observed under the slit lamp,and the severity of corneal disease of mice was determined by slit lamp photography and clinical inflammation score.Macrophages recruitment in corneas were evaluated using immunofluorescence staining.Apoptosis in cornea was detected by TUNEL.2.RT-PCR was used to detect m RNA expression levels of macrophage-related cytokines(M1: IL-12,i NOS;M2: Arg-1、IL-10),inflammation-related factors(TNF-α,IL-1β)and apoptosis-related factors(Caspase-3,Caspase-9).RT-PCR was also used to detect the m RNA expression levels of macrophage-related cytokines(M1: IL-12,i NOS;M2: Arg-1、IL-10),inflammation-related factors(TNF-α,IL-1β)in peritoneal macrophages stimulated by GT(125ng/ml)for 24 h.3.Peritoneal macrophages and human corneal epithelial cells(HCECs)treated with GT at concentrations of 0,62.5ng/ml,125ng/ml,250ng/ml,500ng/ml and1000ng/ml,cell activity were observed using cell counting kit-8(CCK-8),and the expression level of Caspase-3 m RNA in each group was detected by RT-PCR.4.Flow cytometry was used to detect the change of the proportion of corneal CD206+/CD86+macrophages in each group,and the changes of CD206+/CD86+ ratio in peritoneal macrophages were detected after 24 hours of GT(125ng/ml)stimulation.The influence of GT on the change of the proportion of corneal or peritoneal macrophages M1 and M2 was analyzed.Results:1.Clinical score showed that inflammation in the gli PΔ group was significantly severer than that in the other two groups on 3 days p.i.Expression of IL-1β and TNF-α in group gli PΔ were significantly higher than those in groups B-5233 and gli PR on 3days p.i.2.Compared with groups gli PΔ,the m RNA levels of Caspase-3 were significantly increased on3 days and 5 days p.i.in groups B-5233 and gli PR.Tunel staining showed that the number of apoptotic cells in gli PΔ group was significantly lower than the other two groups.3.There were significantly more macrophages in group gli PΔ on 3 days p.i.than the other two groups.4.A significant decline in macrophage activity occurred at 250ng/ml after 8h stimulation of GT.The activity of HCECs begin to decline from a concentration of 1000ng/ml at 8h.5.M2 macrophage inflammatory factors Arg-1,IL-10 were significantly higher in groups B-5233 and gli PR,especially on 3 days p.i.M1 macrophage factor i NOS was higher in the gli PΔgroups of corneas on 3 days p.i.The expression of polarization-related factors in vitro macrophages after 125ng/ml GT treatment for 24 h was M1-related factors i NOS,IL-12 in AF+GT group decreased,M2-related factors Arg-1,IL-10 increased.Flow cytometry were used to detect the difference of GT influence in M1 and M2 ratios among the three groups,we found that M1 macrophage ratio in group gli PΔ was significantly higher than that in the other two groups.Vitro study showed that,treatment by GT and hyphae together significantly increase the proportion of CD206+/CD86+ compared with the only hyphae treatment group.Conclusion:GT prolongs the disease process of A.fumigatus keratitis in mice.GT delays onset up and cause long course of A.fumigatus keratitis by interfering macrophage recruitment,promoting apoptosis and increasing the M2/M1 differentiation ratio of macrophages.In addition,compared with HCECs,GT inhibited the activity of macrophages more obviously. |
PDF Full Text Request