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类胶原蛋白酶诱导人中性粒细胞产生活性氧依赖的 NLRP3 炎症小体。

Reactive oxygen species-dependent-NLRP3 inflammasome activation in human neutrophils induced by l-amino acid oxidase derived from Calloselasma rhodostoma venom.

机构信息

Laboratório de Imunologia Celular Aplicada à Saúde, Fundação Oswaldo Cruz, FIOCRUZ Rondônia, Porto Velho, RO, Brazil.

Centro de Estudos de Biomoléculas Aplicadas à Saúde (CEBio), Fundação Oswaldo Cruz, FIOCRUZ Rondônia e Departamento de Medicina, Universidade Federal de Rondônia, UNIR, Porto Velho, RO, Brazil.

出版信息

Life Sci. 2022 Nov 1;308:120962. doi: 10.1016/j.lfs.2022.120962. Epub 2022 Sep 13.

Abstract

l-Amino acid oxidase isolated from Calloselasma rhodostoma (Cr-LAAO) snake venom is a potent stimulus for neutrophil activation and production of inflammatory mediators, contributing to local inflammatory effects in victims of envenoming. Cr-LAAO triggered the activation of nicotinamide adenine dinucleotide phosphatase (NADPH) oxidase complex and protein kinase C (PKC)-α signaling protein for reactive oxygen species (ROS) production. This study aims to evaluate the ROS participation in the NLRP3 inflammasome complex activation in human neutrophil. Human neutrophils were isolated and stimulated for 1 or 2 h with RPMI (negative control), LPS (1 μg/mL, positive control) or Cr-LAAO (50 μg/mL). The neutrophil transcriptome was examined using the microarray technique, and RT-qPCR for confirmation of gene expression. Immunofluorescence assays for NLRP3, caspase-1, IL-1β and GSDMD proteins was performed by Western blot in the presence and/or absence of Apocynin, an inhibitor of NADPH oxidase. IL-1β release was also detected in the presence and/or absence of NLRP3, caspase-1 and NADPH oxidase inhibitors. Results showed that Cr-LAAO upregulated the expression of genes that participate in the NADPH oxidase complex formation and inflammasome assembly. NLRP3 was activated and accumulated in the cytosol forming punctas, indicating its activation. Gasdermin D was not cleaved but lactate dehydrogenase was released. Furthermore, ROS inhibition decreased the expression of NLRP3 inflammasome complex proteins, as observed by protein expression in the presence and/or absence of apocynin, an NADPH oxidase inhibitor. IL-1β was also released, and pharmacological inhibition of NLRP3, caspase-1, and ROS reduced the amount of released cytokine. This is the first report demonstrating the activation of the NLRP3 inflammasome complex via ROS generation by Cr-LAAO, which may lead to the development of local inflammatory effects observed in snakebite victims.

摘要

从尖吻蝮蛇(Calloselasma rhodostoma)蛇毒中分离出的 l-氨基酸氧化酶(Cr-LAAO)是一种强烈的中性粒细胞激活剂和炎症介质产生剂,导致中毒者局部炎症反应。Cr-LAAO 触发烟酰胺腺嘌呤二核苷酸磷酸酶(NADPH)氧化酶复合物和蛋白激酶 C(PKC)-α信号蛋白的激活,从而产生活性氧(ROS)。本研究旨在评估 ROS 在人中性粒细胞中 NOD、LRR 和富含亮氨酸重复蛋白 3(NLRP3)炎症小体复合物激活中的作用。分离出人中性粒细胞,并用 RPMI(阴性对照)、LPS(1μg/mL,阳性对照)或 Cr-LAAO(50μg/mL)刺激 1 或 2 小时。使用微阵列技术检测中性粒细胞转录组,并通过 RT-qPCR 对基因表达进行确认。通过 Western blot 在存在和/或不存在 NADPH 氧化酶抑制剂 Apocynin 的情况下进行 NLRP3、半胱天冬酶-1、IL-1β 和 GSDMD 蛋白的免疫荧光测定。在存在和/或不存在 NLRP3、半胱天冬酶-1 和 NADPH 氧化酶抑制剂的情况下,还检测了 IL-1β 的释放。结果表明,Cr-LAAO 上调了参与 NADPH 氧化酶复合物形成和炎症小体组装的基因表达。NLRP3 被激活并在细胞质中积累形成点状,表明其被激活。Gasdermin D 未被切割,但乳酸脱氢酶被释放。此外,ROS 抑制减少了 NLRP3 炎症小体复合物蛋白的表达,如在存在和/或不存在 NADPH 氧化酶抑制剂 Apocynin 的情况下通过蛋白表达观察到的。IL-1β 也被释放,并且 NLRP3、半胱天冬酶-1 和 ROS 的药理学抑制减少了释放的细胞因子的量。这是第一项表明 Cr-LAAO 通过 ROS 生成激活 NLRP3 炎症小体复合物的报告,这可能导致在蛇咬伤受害者中观察到的局部炎症反应的发展。

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