Dimethyl itaconate (DI) is a membrane-permeable itaconate derivative with anti-inflammatory functions. However, the anti-inflammatory effect of DI has never been studied in fungal keratitis. In this study, we tested the protective effect of DI against fungal keratitis and assessed the role of NF-E2-related factor-2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in this process. Eyes of C57BL/6 (B6) mice were treated with 2 mm DI after infection with Aspergillus fumigatus. Human corneal epithelial cells (HCECs) were pretreated with 0.25 mm DI and then incubated with A. fumigatus. Clinical scoring, slit-lamp photography, myeloperoxidase determination, flow cytometry and immunostaining were used to assess the disease response and treatment efficacy. PCR, Western blot and ELISA were used to assess the expression of interleukin-1β (IL-1β), chemokine (C-X-C motif) ligand 1, IL-6, IL-8, Nrf2 and HO-1. In addition, quantification of viable fungi, absorbance assays and fluorimetry were used to measure DI fungistatic activity. We observed that DI-treated eyes showed decreased clinical scores, fungal loads, polymorphonuclear neutrophil (PMN) infiltration and cytokine expression, compared with phosphate-buffered saline-treated infected eyes. DI treatment decreased the cytokine levels in infected corneas and in HCECs stimulated with A. fumigatus. Moreover, DI treatment increased Nrf2 and HO-1 expression in corneas and nuclear Nrf2 accumulation in HCECs. DI-induced cytokine downregulation was inhibited by pretreatment with an Nrf2 or HO-1 inhibitor. Finally, DI treatment reduced the A. fumigatus absorbance and fungal mass. These data indicate that DI protects against fungal keratitis by limiting inflammation via the Nrf2/HO-1 signaling pathway and that DI inhibits the growth of A. fumigatus.

中文翻译：

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衣康酸二甲酯通过激活Nrf2 / HO-1信号通路来预防真菌性角膜炎。

衣康酸二甲酯（DI）是具有抗炎功能的可透膜衣康酸酯衍生物。然而，从未在真菌性角膜炎中研究DI的抗炎作用。在这项研究中，我们测试了DI对真菌性角膜炎的保护作用，并评估了NF-E2相关因子2（Nrf2）/血红素加氧酶1（HO-1）信号在此过程中的作用。烟曲霉感染后，用2 mm DI处理C57BL / 6（B6）小鼠的眼睛。用0.25mm DI预处理人角膜上皮细胞（HCEC），然后与烟曲霉一起孵育。临床评分，裂隙灯照相，髓过氧化物酶测定，流式细胞术和免疫染色被用于评估疾病反应和治疗效果。PCR，Western blot和ELISA检测白细胞介素1β（IL-1β）的表达，趋化因子（CXC基序）配体1，IL-6，IL-8，Nrf2和HO-1。另外，活菌的定量，吸光度测定法和荧光法用于测量DI抑菌活性。我们观察到，与磷酸盐缓冲盐水治疗感染的眼睛相比，经DI处理的眼睛显示出临床评分，真菌负荷，多形核中性粒细胞（PMN）浸润和细胞因子表达降低。DI处理可降低感染的角膜和烟曲霉刺激的HCEC中的细胞因子水平。此外，DI处理可增加HCEC中角膜中Nrf2和HO-1的表达以及核Nrf2的积累。用Nrf2或HO-1抑制剂预处理可抑制DI诱导的细胞因子下调。最后，去离子处理降低了烟曲霉的吸光度和真菌量。