Therapeutic Effect Of Gallic Acid-natamycin On Aspergillus Fumigatus Keratitis

Purpose.In order to improve the therapeutic effect of natamycin on fungal keratitis,a new compound gallic acid-natamycin(GA-NAT)was obtained by grafting natamycin and gallic acid.To study the therapeutic effect of this compound on Aspergillus fumigatus keratitis.Methods.1.Structural characterization by Fourier transform infrared(FTIR)and ultravioletvisible spectroscopy(UV-vis).2.Use CCK-8 test and mouse corneal Draize test to determine drug toxicity.3.The antifungal activity of the compounds against Aspergillus fumigatus in vitro was determined by MIC experiment,adhesion experiment,biofilm formation and fluorescent white staining of hyphae.4.The fungal keratitis mouse model was established by C57BL/6 mice,and the mouse model was treated with DMSO,natamycin and gallic acid-natamycin with a frequency of 4 times a day.On days 3 and 5after drug treatment,the corneal condition of the mice was scored and recorded by slit lamp microscopy.5.The infiltration of neutrophils in the corneal stroma of the mouse model was assessed by immunofluorescence staining and myeloperoxidase assay.6.q RTPCR was used to detect the m RNA expression of pattern recognition receptor LOX-1 and downstream pro-inflammatory factors IL-1β and TNF-α in corneal mice on day 3 and 5after fungal infection.The mouse corneal tissue on the 3rd day after Aspergillus fumigatus infection was collected to detect the protein expression levels of proinflammatory factors in mouse corneal tissue by western blot and ELISA experiments.7.RAW264.7 cells were infected with Aspergillus fumigatus,and they were divided into DMSO control group,fungal infection group,natamycin treatment group and GA-NAT treatment group according to the treatment methods.The study used q RT-PCR,Western blot and ELISA to detect the m RNA and protein expression levels of pattern recognition receptor LOX-1 and downstream inflammatory factors IL-1β and TNF-α in cell samples.Results.1.It was proved by FT-IR and UV-vis experiments that natamycin and gallic acid were successfully grafted to generate gallic acid-natamycin.2.It was found that GA-NAT was not toxic to HCEC and RAW264.7 cells at 8μg/m L by CCK-8 experiment.And the results of the mouse cornea Draize test showed that the concentration of 8μg/m L GANAT did not cause damage to the mouse cornea.3.GA-NAT significantly inhibited the growth of Aspergillus fumigatus at 8μg/m L,and significantly inhibited biofilm formation and adhesion to corneal epithelial cells.4.Corneal disease records and clinical scoring experiments found that after different treatments of corneal tissue in mice with Aspergillus fumigatitis,the corneal tissue of the GA-NAT treatment group was significantly better than the DMSO control group and the natamycin treatment group.The degree of corneal inflammation was significantly attenuated,and the clinical score of the corneal tissue of the mice in the GA-NAT treatment group was significantly lower than that in the DMSO control group and the NAT treatment group.5.The plate count experiment showed that GA-NAT significantly reduced the residual amount of fungi in the infected corneal tissue on the 5th day after corneal infection in the mouse model.6.Compared with the natamycin-treated group,GA-NAT treatment can significantly reduce the inflammatory response of mouse cornea,inhibit the recruitment of neutrophils in the inflammatory site and down-regulate the expression of myeloperoxidase.7.The m RNA and protein expressions of LOX-1,IL-1β and TNF-α in the fungal keratitis model of mice treated with GA-NAT were significantly lower than those in the DMSO control group.8.After RAW264.7 was stimulated by Aspergillus fumigatus,the trend of increased expression of pattern recognition receptor LOX-1 and pro-inflammatory factors IL-1β and TNF-α in cells of GA-NAT drug treatment group was significantly inhibited.Conclusions.GA-NAT is a novel compound with low cytotoxicity and obvious antibacterial effect on Aspergillus fumigatus.In Aspergillus fumigatus keratitis,GA-NAT exerts good antiinflammatory activity by inhibiting fungal load,reducing neutrophil infiltration,and downregulating the expression of inflammatory factors,providing a new option for the treatment of fungal keratitis.