Antifungal and anti-inflammatory effect of punicalagin on murine Aspergillus fumigatus keratitis

Abstract

Purpose This study aimed to investigate the anti-inflammatory effect and antifungal effect of punicalagin in murine fungal keratitis. Methods We used in vitro and in vivo protocols to assess the anti-inflammatory effect and antifungal effect of punicalagin. In vitro, time kill and mycelial stain were done. In vivo, murine fungal keratitis was established and treated with PBS or PUN. clinical scores were taken on days 1, 3, and 5 post infection. The mRNA and protein levels of inflammatory factors were detected by RT-PCR and Western blot, and the number and location of macrophages were analyzed by flow cytometry and immunofluorescence. Also, fungal plate counting was used to assess the antifungal effect. DCFH-DA fluorescence Probe detected the ROS level. Results In vitro, PUN showed activity against A.fumigatus. (A.F.), with MIC90 values of 250 μg/ml, and significantly reduced A.F. biofilm formation (p<0.001). In vivo, mouse fungal keratitis model after punicalagin treatment exhibited less disease, lower clinical scores (p<0.05), lower reduced macrophage infiltrate (p<0.001), and fungal load (p<0.001) than those treated with PBS. Treatment with punicalagin also reduced the mRNA expression and protein level of pro-inflammatory factors. At the cellular level, PUN significantly reduced the mRNA expression of inflammatory factors and ROS production caused by stimulation of mycelia in RAW264.7 (p<0.001). Conclusions The results show that punicalagin is beneficial in the treatment of murine fungal keratitis. The mechanism of its anti-inflammatory effect was synthetical, including antifungal activity, an inhibitory effect of proinflammatory factor and macrophages, and anti-oxidation.