Experimental Study On Anti-inflammatory Mechanism Of LXA4 In Aspergillus Fumigatus Keratitis

Purpose: This study aims to investigate the anti-inflammatory effects of lipoxin A4(LXA4)in Aspergillus fumigatus(A.fumigatus)keratitis.Methods: 1.The model of A.fumigatus keratitis was established via A.fumigatus infection.ELISA was performed to detect the cornea level of LXA4 in normal group and infected group.2.5 μL 6μmol/L LXA4 was administrated to the infected corneas,while the control group was treated with the same dose of PBS.Slit lamp photographs were taken at 1,3,and 5 days post infection,and clinical scores were utilized to evaluate the severity of the disease.A plate count test was performed to assess the effect of LXA4 on corneal fungal load.3.Immunofluorescence staining,MPO test and HE staining were carried out to investigate the effect of LXA4 on neutrophil infiltration in A.fumigatus keratitis.4.The production of related inflammatory factors at 3 and 5 days post infection were evaluated by real-time fluorescent quantitative PCR(q RT-PCR)and ELISA.5.RAW264.7 macrophages were pretreated with LXA4 2 hours before A.fumigatus stimulation.The m RNA and protein levels of related inflammatory factors were detected via q RT-PCR and ELISA at 8 hours and 20 hours post A.fumigatus stimulation respectively.6.After pretreatment with the ALX/FPR2 receptor antagonist Boc-2,reactive oxygen species(ROS)detection,q RT-PCR and ELISA were performed at 2 hours,8 hours and 20 hours post A.fumigatus stimulation in RAW264.7macrophages respectively.Results: 1.Compared with the uninfected group,the level of LXA4 in mice with A.fumigatus keratitis increased and peaked at 3 days post infection.2.6 μmol/L LXA4 treatment significantly reduced the disease severity and clinical score at the 3 days and5 days post infection compared with the PBS control group.The results of immunofluorescence staining,MPO test,and HE staining demonstrated that LXA4 administration alleviated cornea edema and neutrophil infiltration at 3 days post infection;the plate count results showed the corneal fungal load were mitigated by LXA4 treatment.3.As displayed by q RT-PCR and ELISA,pattern recognition receptors(PRR)TLR-2,TLR-4,dectin-1 and inflammation-related mediators IL-1β,TNF-α,IL-6,i NOS,COX-2 expression were downregulated by LXA4 administration in A.fumigatus infected mice cornea.4.10 nmol/L LXA4 pretreatment for 2 hours decreased the generation of inflammation-related mediators IL-1β,TNF-α,IL-6,i NOS and COX-2,inhibited ROS production and promoted the expression of IL-10 and Arg-1 in RAW264.7 macrophages stimulated by A.fumigatus.5.The expression of IL-1β,TNF-α,IL-6,COX-2 and the production of ROS suppressed by LXA4 treatment were elevated by the ALX/FPR2 receptor antagonist Boc-2 in A.fumigatus stimulated RAW264.7 macrophages.Conclusion: The content of LXA4 in the cornea of mice infected with A.fumigatus increased in comparison with the uninfected group;LXA4 inhibited the infiltration of neutrophils,reduced corneal fungal burden,suppressed the expression of PRRs and inflammation-related mediators and facilitated the anti-inflammatory mediators generation in mice A.fumigatus keratitis.The production of ROS was also decreased by LXA4 in A.fumigatus stimulated-RAW264.7 macrophages.ALX / FPR2 receptor involved in its anti-inflammatory effect.