Antifungal And Anti-inflammatory Effects Of Sanguinarine In Aspergillus Fumigatus Keratitis In Mice

Purpose: To investigate the anti-inflammatory effect of Sanguinarine(SAN)in Aspergillus fumigatus keratitis and the potential mechanism of antifungal effect.Method:1.Determination of the effect of various concentrations of SAN on the impact of human corneal epithelial cells(HCECs)viability by CCK-8 assay.The effect of SAN on the proliferation and migration ability of HCECs was detected by cell scratch experiment.The toxicity of SAN to the cornea of mice was detected by the Draize ocular surface toxicity experiment.2.The minimum inhibitory concentration(MIC)of Aspergillus fumigatus conidia and hyphae was determined by microdilution method,and the time killing curve of Aspergillus fumigatus conidia was recorded.Propidium iodide uptake experiment,fluorescent white staining,biofilm formation experiment,fungal adhesion experiment,scanning electron microscopy and transmission electron microscopy to detect the antifungal effect of SAN in vitro.3.The animal model of Aspergillus fumigatus keratitis in C57BL/6mice was established,and the treatment was carried out by using 2 mg/L SAN or 0.1%DMSO solution,and the therapeutic effect of SAN was observed by clinical scoring and slit lamp photography on 1,3 and 5 days post infection(p.i.).The antifungal effect of SAN in vivo was assessed by plate colony counting experiment and periodic acid-Schiff staining(PAS).4.Immunofluorescence staining and myeloperoxidase assay(MPO)were performed on mouse corneal tissues to detect the effect of SAN on neutrophil infiltration and recruitment after Aspergillus fumigatus infection.5.Detect the gene and protein expression of inflammatory mediators(IL-1β,IL-6 and CCL2)in the model of Aspergillus fumigatus keratitis in mice and HCECs by RT-PCR and ELISA,and evaluate the antiinflammatory effect of SAN in vitro and in vivo.Results:1.The results of CCK-8 experiments showed that when the concentration of SAN was 0.5mg/L,there was no cytotoxicity to HCECs and did not affect the proliferation of cells.Scratch experiments showed that the concentration of SAN at 0.5 mg/L did not affect the migration ability of HCECs.In vivo,the results of Draize experiment showed that 2 mg/L of SAN was not toxic to the cornea of mice,so this concentration was used for subsequent experiments in vitro and in vivo,respectively.2.In vitro,the growth of Aspergillus fumigatus conidia was significantly inhibited by 0.5mg/L SAN,and the growth of Aspergillus fumigatus hyphae was significantly inhibited by 2 mg/L SAN.0.5 mg/L SAN could significantly inhibit the formation of Aspergillus fumigatus biofilm,the adhesion of conidia and increase the permeability of Aspergillus fumigatus cell membrane.In scanning electron microscopy,compared with the 0.1% DMSO control group,after 2 mg/L SAN treatment,the surface of Aspergillus fumigatus mycelium was bent and deformed,the outer wall was wrinkled and the surface of the conidia was visible pitted.In transmission electron microscopy,compared with the control group,2 mg/L SAN treatment showed that the cell wall lysis of hyphae became thinner,the continuity of the cell membrane was interrupted,the intracellular mitochondrial crest was widened,and even the organelles were missing.3.In the mouse Aspergillus fumigatus keratitis model,compared with the DMSO control group,2 mg/L SAN treatment can significantly reduce the severity of keratitis in mice.PAS staining showed significant reduction in fungal load in the corneal stroma of mice after SAN treatment.4.Immunofluorescence staining and MPO of mouse cornea showed that 2mg/L SAN could inhibit the mobilization and infiltration of neutrophils in Aspergillus fumigatus keratitis.5.In the infectious response to Aspergillus fumigatus keratitis,In,SAN exerts a protective role by downregulating the expression levels of genes and proteins of inflammatory mediators IL-1β,IL-6 and CCL2 in vitro and in vivo.Conclusions:In Aspergillus fumigatus keratitis,SAN exerts antifungal effect by inhibiting the growth and adhesion of Aspergillus fumigatus,inhibiting the formation of mycelial biofilms,and destroying the ultrastructure of Aspergillus fumigatus.By reducing the recruitment and infiltration of neutrophils and downregulating the expression of inflammatory mediators,it plays a protective role in Aspergillus fumigatus keratitis.