The Effect Of Punicalagin In Murine Fungal Keratitis

Purpose: Fungal keratitis is an infectious corneal disease caused by pathogenic fungi,with a very high blinding rate.Our study confirmed the role of punicalagin(PUN)in fungal keratitis caused by Aspergillus fumigatus(A.fumigatus)in mice and its mechanism.Methods: 1.Disk diffusion and absorbance measurement were used to detect the antifangal effect of PUN and PUN combined with amphotericin B on Aspergillus fumigatus at different concentrations.Results of fluorescence staining indicated the germination status of Aspergillus fumigatus at different concentrations of punicalagin.Crystal violet staining statistics of the inhibitory effect of punicalagin on the biofilm of Aspergillus fumigatus.2.Standard Aspergillus fumigatus strain was used to infect the cornea of C57 BL / 6 mice.An animal model of fungal keratitis in mice was established.The number of viable bacteria on the cornea of the PBS control group and the PUN treatment group was counted by plate counting.3.The mRNA and protein expressions of corneal inflammatory factors were detected by RT-PCR and Western blotting in 5 days after infection.4.Flow cytometry was used to detect the number and ratio of corneal macrophages at 5 days.The number of macrophages in the cornea and the depth of infiltration were indicated by immunofluorescence staining.5.RAW264.7 cell line was pretreated with different concentrations of PUN for 2 hours.After 8 hours of inactivation of inactivated Aspergillus fumigatus mycelia,RT-PCR was used to detect the mRNA expression of inflammatory factors including IL-1?,IL-6,IL-10,TNF-?,MIP2,iNOS,CXCL1.6.After RAW264.7 cell line was pretreated with different concentrations of PUN for 2 hours,DCFA-DA fluorescent probes detected the ROS levels of cells at 0,1,2,4,8,12,and 24 hours.Results: 1.PUN increased the diameter of the bacteriostatic ring of amphotericin B against Aspergillus fumigatus.PUN reduced the degree of turbidity of Aspergillus fumigatus spore culture fluid and inhibited the formation of fungal biofilms.2.5 days after Aspergillus fumigatus infection in mice,compared with the PBS control group,the corneal inflammation response in the PUN treatment group was significantly reduced,and the clinical score was significantly reduced;the number of colonies cultured in the corneal abrasive solution was significantly reduced,and the difference was statistically significant(P <0.001).3.Compared with the PBS control group,the expression of mRNA and protein of corneal inflammatory cytokines such as IL-1?,TNF-?,and IL-6 in the PUN-treated mice was significantly reduced(P <0.001).4.2 days after Aspergillus fumigatus infection in mice,the number of macrophages and their proportion in immune cells in the cornea of the PUN treatment group was reduced compared with the PBS control group,and the difference was statistically significant(P <0.001).5.After PUN pretreated RAW264.7 cells for 2 hours,the increase of inflammatory factors induced by inactivation of inactivated Aspergillus fumigatus mycelium was suppressed,and the difference was statistically significant(P <0.001).6.After pretreatment with different concentrations of PUN for 2 hours,the ROS produced in the RAW264.7 cell line was reduced at 1,2,4,8,12,and 24 hours.The difference was statistically significant(P <0.001).Conclusion: These data provide evidence that punicalagin can reduce the number of fungi,inhibit the number of macrophages and secrete inflammatory factors in Aspergillus fumigatus keratitis in C57 BL / 6 mice,thereby inhibiting the inflammation of fungal keratitis.