Nerolidol Inhibits The LOX-1/IL-1? Signaling To Protect Against The Aspergillus Fumigatus Keratitis Inflammation Damage To The Cornea

Purpose: This study investigated the protective effect of Nerolidol on Aspergillus fumigatus(AF)in mouse keratitis,and explored its feasibility and molecular mechanism for treatment.Methods: 1.The MIC and cytotoxicity experiments were used to determine the inhibitory concentration range of Nerolidol on Aspergillus fumigatus and its toxic effect on cells,so as to select the optimal drug concentration.2.C57 BL / 6 mice were randomly divided into 4 groups: control group,fungal keratitis group,drug group,and drug-treatment fungal keratitis group.A mouse fungal keratitis model was established by intrastromal injection of Aspergillus fumigatus spores.200 ?M / 5ul of Nerolidol was injected into the drug group and drug-treatment fungal keratitis group at 2 hours,1 day,3 days,and 5 days after infection.The control group and the fungal keratitis group were injected with an equal volume of PBS under the conjunctiva.Use a slit lamp microscope to observe the corneal infection in mice every day and take pictures.3.Immunofluorescence was used to specifically mark the recruitment of neutrophils and macrophages in the corneal stroma of each group of mice.The myeloperoxidase activity in the corneal stroma of fungal-infected mice was determined by the MPO method.4.RT-PCR and Western Bolt were used to detect the mRNA and protein levels of pattern recognition receptor LOX-1 and inflammatory factor IL-1? in the cornea of mice.5.HCECs were used to establish a cell model of Aspergillus fumigatus infection,which was divided into control group,fungal stimulation group,drug group and drug-treatment fungal stimulation group.Nerolidol was added to the cell culture solution of the drug group and the drug-treatment fungal stimulation group to a final concentration of 200 ?M.An equal volume of PBS was added to the cell culture solution of the control group and the fungal stimulation group.The cells were collected at 8 and 16 hours after fungal stimulation and used for RT-PCR and Western Bolt experiments,respectively,to detect the mRNA and protein expression of pattern recognition receptor LOX-1 and inflammatory factor IL-1?.Results: 1.Nerolidol began to inhibit fungal growth at a concentration of 100 ?M,and the inhibitory effect was concentration-dependent.At a concentration of 400 ?M,cell viability of HCECs began to be damaged.The maximum concentration that does not damage cell viability within the effective antifungal concentration range is selected for subsequent experiments,that is,200 ?M.2.On the 3rd and 5th day after infection,compared with the fungal keratitis group,the corneal inflammation response and the clinical score was significantly reduced in the drug-treatment fungal keratitis group.3.Compared with the fungal keratitis group,the specific staining of neutrophils and macrophages and the MPO activity of corneal stroma was significantly reduced in the drug-treatment fungal keratitis group.4.The mRNA and protein levels of pattern recognition receptor LOX-1 and inflammatory factor IL-1? in the mouse cornea of the drug-treatment fungal keratitis group was significantly reduced compared with the fungal keratitis group.5.In the cell experiment,compared with the fungal stimulating group,the tendency of the increased expression of the LOX-1 and the IL-1? in the drug-treatment fungal stimulating group was significantly inhibited.Conclusions: Nerolidol has an inhibitory effect on the growth of Aspergillus fumigatus.In Aspergillus fumigatus keratitis,Nerolidol can play a protective role by inhibiting the recruitment of neutrophils and macrophages and inhibiting the LOX-1 / IL-1? signaling pathway to reduce the inflammatory response.