MANF inhibits NLRP3 inflammasome activation by competitively binding to DDX3X in paraquat-stimulated alveolar macrophages

IF 6.2 2区环境科学与生态学 Q1 ENVIRONMENTAL SCIENCES

Ecotoxicology and Environmental Safety Pub Date : 2024-11-15 DOI:10.1016/j.ecoenv.2024.117331

Yi Pu , Siying Han , Jie Chen , Zhenning Liu

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引用次数: 0

Abstract

NLRP3 inflammasome activation in macrophages is involved in paraquat-induced acute lung injury (ALI). MANF exerts an inhibitory effect against inflammation and cell death. The aim of this study was to investigate the role of MANF in paraquat-stimulated alveolar macrophages and the potential mechanism. Paraquat-induced ALI mouse model was established by intraperitoneally injection of 30 mg/kg of paraquat. The lung pathological changes were observed by hematoxylin and eosin staining. The expression of MANF/DDX3X/NLRP3/Caspase-1 in mice lung macrophages was evaluated by double immunofluorescence staining and western blot. NLRP3 inflammasome activation and pro-inflammatory cytokines (IL-1β and IL-18) in paraquat-stimulated macrophage transfected with MANF overexpression plasmid (pcDNA3.1-MANF) or siRNA-MANF were measured by Western blot. The protein–protein interaction of MANF/DDX3X/NLRP3 was verified by Co-immunoprecipitation. As a result, MANF/DDX3X/NLRP3/Caspase-1 were upregulated in alveolar macrophages of paraquat-induced ALI in mice. In paraquat-stimulated alveolar macrophages, upregulation of MANF and DDX3X were also observed, accompanied by NLRP3 inflammasome activation. In addition, overexpression of MANF inhibited NLRP3 inflammasome activation in paraquat-stimulated alveolar macrophages. In contrast, knockdown of MANF aggravated NLRP3 inflammasome activation. Co-immunoprecipitation results revealed that DDX3X could bind to MANF and NLRP3, but MANF could not bind to NLRP3 in paraquat-stimulated alveolar macrophages. Furthermore, Co-immunoprecipitation of truncated three fragments of DDX3X confirmed MANF can interact with the helicase core of DDX3X which is the binding site for NLRP3. Taken together, MANF exerted a protective effect against paraquat-induced cytotoxicity by inhibiting the NLRP3 inflammasome activation in macrophages via competitive binding to the helicase core of DDX3X.

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MANF 通过与百草枯刺激的肺泡巨噬细胞中的 DDX3X 竞争性结合，抑制 NLRP3 炎症小体的激活。

巨噬细胞中NLRP3炎性体的激活参与了百草枯诱导的急性肺损伤（ALI）。MANF具有抑制炎症和细胞死亡的作用。本研究旨在探讨MANF在百草枯刺激的肺泡巨噬细胞中的作用及其潜在机制。通过腹腔注射30毫克/千克百草枯，建立了百草枯诱导的ALI小鼠模型。苏木精和伊红染色观察肺部病理变化。小鼠肺巨噬细胞中 MANF/DDX3X/NLRP3/Caspase-1 的表达通过双重免疫荧光染色和 Western 印迹进行评估。Western印迹法测定了转染MANF过表达质粒（pcDNA3.1-MANF）或siRNA-MANF的百草枯刺激巨噬细胞的NLRP3炎性体活化和促炎细胞因子（IL-1β和IL-18）。共免疫沉淀法验证了MANF/DDX3X/NLRP3的蛋白相互作用。结果表明，MANF/DDX3X/NLRP3/Caspase-1在百草枯诱导的小鼠ALI的肺泡巨噬细胞中上调。在百草枯刺激的肺泡巨噬细胞中，也观察到 MANF 和 DDX3X 的上调，并伴随着 NLRP3 炎性体的激活。此外，在百草枯刺激的肺泡巨噬细胞中，过表达 MANF 可抑制 NLRP3 炎性体的激活。相反，敲除MANF会加剧NLRP3炎症小体的激活。共免疫沉淀结果显示，在百草枯刺激的肺泡巨噬细胞中，DDX3X能与MANF和NLRP3结合，但MANF不能与NLRP3结合。此外，DDX3X三个截短片段的共免疫沉淀证实，MANF能与DDX3X的螺旋酶核心相互作用，而该核心正是NLRP3的结合位点。综上所述，MANF通过与DDX3X的螺旋酶核心竞争性结合，抑制巨噬细胞中NLRP3炎性体的激活，从而对百草枯诱导的细胞毒性起到保护作用。

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来源期刊

Ecotoxicology and Environmental Safety 环境科学-毒理学

CiteScore

12.10

自引率

5.90%

发文量

1234

审稿时长

88 days

期刊介绍： Ecotoxicology and Environmental Safety is a multi-disciplinary journal that focuses on understanding the exposure and effects of environmental contamination on organisms including human health. The scope of the journal covers three main themes. The topics within these themes, indicated below, include (but are not limited to) the following: Ecotoxicology、Environmental Chemistry、Environmental Safety etc.