| Objective:To investigate the effect and mechanism of wedelolatone on the inflammatory response of Aspergillus fumigatus(A.fumigatus)keratitis in mice.Methods:1.Effect of wedelolatone pretreatment on fungal keratitis in mice:Mice were randomly divided into drug group and infection group.Fungal keratitis models were established by injecting A.fumigatus spores(2ul at a concentration of 0.5×10~5/?L)into the corneal stroma for both groups.5ul wedelolatone with a concentration of 30?M was injected into subconjunctiva in drug group 1 day before modeling and just before modeling.The same pretreatment with DMSO was performed to the infection group.Corneal infection pictures of mice were taken and observed under a slit lamp on the first and third day after modeling,then the clinical scores were recorded.Neutrophils were specifically labeled with immunofluorescence.Meanwhile,Myeloperoxidase(MPO)was used to evaluate the function of neutrophils and analyze the effect of wedelolatone on the recruitment of neutrophils in the corneal stroma of mice.2.Mechanism of wedelolatone effect on fungal keratitis in mice:Mice were randomly divided into infection control group,infection group,drug control group and drug group.5ul wedelolatone at a concentration of 30?M was injected into subconjunctiva for drug control group and drug group 1 day before modeling and just before modeling.The same pretreatment with DMSO was performed to infection control group and infection group.Pretreatment was only conducted to infection control group and infection group.Fungal keratitis models were established by injecting A.fumigatus spores into the corneal stroma after the pretreatment.1 day after modeling,reverse transcription-PCR(RT-PCR)was applied to detect the expression of proinflammatory cytokine IL-1?mRNA in the corneas of mice in each group.Western Blot(WB)was conducted to detect the expression of proteins of the antifungal pattern recognition receptors Dectin-1,LOX-1 and IL-1?protein(precursor and maturation)in the corneas of mice in each group.3.Mechanism of wedelolatone on fungal infection of human THP-1 macrophages:Human THP-1 cells were induced to become macrophages by PMA.Cells were divided into four groups:infection control group,infection group,drug control group and drug group.Both the drug control group and the drug group were pretreated with wedelolatone(final concentration 10?M)2hours before modeling.The same pretreatment with DMSO was performed to infection control group and infection group.Pretreatment was only conducted to infection control group and infection group.After pretreatment,both the infection group and the drug group were stimulated with the conidia of A.fumigatus(spores:cells=1:1)to establish a cell infection model.The expression of IL-1?mRNA in cornea of mice in each group was detected by PCR 4 hours after the establishment of the model,and the expression of anti-fungal pattern recognition receptor Dectin-1,LOX-1 and IL-1?(precursor and mature)protein in cornea of mice in each group was detected by WB 16 hours later.4.Mechanism of wedelolatone effect on IL-1?:Fungal keratitis model in mice was established by injecting A.fumigatus spores into the corneal stroma.Corneas of mice were collected on day 1/2,1,2,3 and 5 after modeling.Caspase-1 activation in the cornea of mice was quantitatively detected with the application of WB at various stages.Also tested in Experiment 2 with WB.5.Effect of wedelolatone on the treatment of fungal keratitis in mice:Mice were randomly divided into treatment group and combination treatment group.A.fumigatus spores were injected into corneal stroma to establish the fungal keratitis modles.5ul wedelolatone at a concentration of 30?M was injected in subconjunctival for combination treatment group 1 day after modeling.The same way with DMSO was performed to the treatment group.Both groups were then given Natacyn eye drops(Alcon Laboratories)4 times a day for 2 days.Corneal infection of mice was observed under a slit lamp and the clinical scores were recorded carefully.Results:1.Effect of wedelolatone treatment on fungal keratitis in mice:Compared with the infection group,the degree of inflammation of the corneas in drug group by wedelolatone intervention was lighter than that in the infected group on the first day after A.fumigatus infection,and the clinical scores were lower.However,on the third day,the inflammatory response was more severe than that of the infection group,and the clinical scores were higher.On the first day after infection,neutrophilic staining revealed that the number of neutrophils in the corneal stromal of mice was decreased in the group by wedelolatone intervention whilethe number of MPO in the cornea of mice in drug group by wedelolatone intervention was dropped,and the difference was statistically significant.2.Mechanism of wedelolatone effect on fungal keratitis in mice:The study found that the level of IL-1?mRNA and the expression of mature IL-1?proteins in the cornea of mice with A.fumigatus keratitis was inhibited by wedelolatone.However,it did not affect the expression of proteins of the antifungal pattern recognition receptors Dectin-1,LOX-1and IL-1?precursor protein.3.Mechanism of wedelolatone on fungal infection of human THP-1 macrophages:Wedelitrilactone could down regulate IL-1?mRNA level and inhibit the expression of mature IL-1?protein in THP-1 cells infected by A.fumigatus.The pretreatment of wedelolatone did not affect the expression of proteins of the antifungal pattern recognition receptors such as Dectin-1,LOX-1 and IL-1?precursor protein.4.Mechanism of wedelolatone effect on IL-1?:The expression level of Caspase-1 in A.fumigatus keratitis was increased gradually from the beginning to the first day and then began to decline.In the drug group with pretreatment of wedelolatone,the expression of Caspase-1 in the cornea of mice was significantly lower than that of the infection group.5.Effect of wedelolatone on the treatment of fungal keratitis in mice:A.fumigatus keratitis treated with wedelolatone had better corneal transparency on the third day after infection than the control group,and the clinical scores were lower than that of the control group.Conclusions:1.The pretreatment of wedelolatone reduced the recruitment of neutrophils in the corneal region during A.fumigatus keratitis in mice.2.Wedelolatone inhibited the maturation of IL-1?by down regulating the activity of caspase-1.3.Wedelolatone in combination with antifungal drugs helped maintain the transparency of cornea with A.fumigatus keratitis in mice. |
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