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.

中文翻译：

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安石榴苷对鼠烟曲霉角膜炎的抗真菌和抗炎作用

摘要

目的

本研究旨在探讨安石榴苷在小鼠真菌性角膜炎中的抗炎作用和抗真菌作用。

方法

我们使用体外和体内方案来评估安石榴苷的抗炎作用和抗真菌作用。在体外，进行时间杀伤和菌丝体染色。在体内，建立了鼠真菌性角膜炎并用 PBS 或 PUN 治疗。在感染后第 1、3 和 5 天进行临床评分。RT-PCR和Western blot检测炎症因子mRNA和蛋白水平，流式细胞仪和免疫荧光分析巨噬细胞数量和位置。此外，真菌平板计数用于评估抗真菌效果。DCFH-DA荧光探针检测ROS水平。

结果

在体外，PUN 显示出对烟曲霉的活性。( AF .)，MIC90 值为 250 μg/ml，并显着降低AF。生物膜形成 (p<0.001)。在体内，punicalagin 治疗后的小鼠真菌性角膜炎模型表现出比用 PBS 治疗的小鼠更少的疾病、更低的临床评分 (p<0.05)、更低的巨噬细胞浸润减少 (p<0.001) 和真菌负荷 (p<0.001)。punicalagin 治疗也降低了促炎因子的 mRNA 表达和蛋白质水平。在细胞水平上，PUN 显着降低了 RAW264.7 中由菌丝体刺激引起的炎症因子的 mRNA 表达和 ROS 的产生（p<0.001）。

结论

结果表明，punicalagin对治疗鼠真菌性角膜炎有益。其抗炎作用的机制是综合性的，包括抗真菌活性、促炎因子和巨噬细胞的抑制作用以及抗氧化作用。