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Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

ABSTRACT: 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-1? (IL-1?), 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.

SUBMITTER: Gu L

PROVIDER: S-EPMC7065235 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

Gu Lingwen L Lin Jing J Wang Qian Q Li Cui C Peng Xudong X Fan Yiqun Y Lu Chunli C Lin Hao H Niu Yawen Y Zhu Guoqiang G Zhao Guiqiu G

Immunology and cell biology 20200211 3

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 cell ...[more]

PMID: 31943336

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Project description:PurposeThe purpose of this study was to investigate the therapeutic effect of perillaldehyde (PAE) on Aspergillus fumigatus (A. fumigatus) keratitis.MethodsHuman corneal epithelial cells (HCECs) were pretreated with PAE and stimulated with A. fumigatus mycelium. C57BL/6 mice were infected with A. fumigatus and treated with or without PAE 1 day after infection. Clinical scores, PCR, ELISA, and Western blotting were used to detect the expression of pro-inflammatory mediators, dendritic cell-associated c-type lectin-1 (Dectin-1), nuclear factor (erythroid-derived 2) like 2 (Nrf2), and heme oxygenase (HO-1). Nrf2 expression in HCECs pretreated with PAE was observed by immunofluorescence. NIMP-R14 protein expression and localization in mouse corneas were observed by immunofluorescence staining after treatment with PAE. Corneal colony counting, time-kill tests, and mycelial transformation inhibition tests were used to evaluate the antifungal effect of PAE.ResultsC57BL/6 mice treated with PAE at 1 day after infection had a lower clinical score and decreased IL-1β, TNF-α, IL-6, Dectin-1, and MPO levels. PAE treatment significantly reduced neutrophil recruitments to the corneal stroma. Compared with the DMSO-treated group, PAE treatment significantly decreased mRNA and protein levels of pro-inflammatory cytokines and Dectin-1 in HCECs. PAE pretreatment before A. fumigatus stimulation obviously elevated the mRNA and protein levels of components of the Nrf2/HO-1 axis. HCECs pretreated with PAE before infection showed a weakened ability to inhibit inflammation in the presence of brusatol (BT; an Nrf2 inhibitor) or ZnPP (an HO-1 inhibitor). PAE treatment significantly reduced the fungal load of C57BL/6 mouse corneas and inhibited fungal growth in vitro.ConclusionsThese data proved that PAE may ameliorate A. fumigatus keratitis by activating the Nrf2/HO-1 signaling pathway and inhibiting the Dectin-1 mediated inflammatory response and neutrophil recruitment. Furthermore, PAE exerts direct fungicidal activity on A. fumigatus.

Project description:BackgroundPaeonia lactiflora Pall. (PLP), a traditional Chinese herbal medicine, has been used for hepatic disease treatment over thousands of years. In our previous study, PLP was shown to demonstrate therapeutic effect on hepatitis with severe cholestasis. The aim of this study was to evaluate the antioxidative effect of PLP on alpha-naphthylisothiocyanate (ANIT)-induced cholestasis by activating NF-E2-related factor 2 (Nrf2) via phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway.Materials and methodsLiquid chromatography-mass spectrometry (LC-MS) was performed to identify the main compounds present in PLP. The mechanism of action of PLP and its therapeutic effect on cholestasis, induced by ANIT, were further investigated. Serum indices such as total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GT), and total bile acid (TBA) were measured, and histopathology of liver was also performed to determine the efficacy of treatment with PLP. Moreover, in order to illustrate the underlying signaling pathway, liver glutathione (GSH) content and mRNA or protein levels of glutamate-cysteine ligase catalytic subunit (GCLc), glutamate-cysteine ligase modulatory subunit (GCLm), Akt, heme oxygenase-1 (HO-1), NAD(P)H/quinone oxidoreductase 1 (Nqo1), and Nrf2 were further analyzed. In addition, validation of PLP putative target network was also performed in silico.ResultsFour major compounds including paeoniflorin, albiflorin, oxypaeoniflorin, and benzoylpaeoniflorin were identified by LC-MS analysis in water extract of PLP. Moreover, PLP could remarkably downregulate serum levels of TBIL, DBIL, AST, ALT, ALP, γ-GT, and TBA, and alleviate the histological damage of liver tissue caused by ANIT. It enhanced antioxidative system by activating PI3K/Akt/Nrf2 pathway through increasing Akt, Nrf2, HO-1, Nqo1, GCLc, and GCLm expression. The putative targets network validation also confirmed the important role of PLP in activating Akt expression.ConclusionThe potential mechanism of PLP in alleviating ANIT-induced cholestasis could to be related to the induction of GSH synthesis by activating Nrf2 through PI3K/Akt-dependent pathway. This indicates that PLP might be a potential therapeutic agent for cholestasis.

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Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

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Dimethyl itaconate protects against fungal keratitis by activating the Nrf2/HO-1 signaling pathway.

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