Free Radicals and Redox Signaling Triggered by Lipopolysccharide in Macrophages

巨噬细胞中脂多糖触发的自由基和氧化还原信号传导

• 批准号: 7567807
• 负责人: DARIO C RAMIREZ
• 金额: $ 24.4万
• 依托单位: OKLAHOMA MEDICAL RESEARCH FOUNDATION
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7567807
• 关键词: Antibodies; Asthma; Binding; Cells; Cellular biology; Chronic; Cytoplasm; Detection; Diagnostic; Disease; Electrons; Free Radicals; Freezing; Goals; Hydroxyl Radical; In Situ; Inflammation; Inflammatory; Instruction; Iron; Lipopolysaccharides; Lung; Lysosomes; Macrophage Activation; Mammalian Cell; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolic; Metabolism; Methods; Modeling; Molecular; Mus; Numbers; Obesity; Oxidation-Reduction; Oxidative Stress; Oxides; Pathogenesis; Proteins; Reactive Oxygen Species; Research Design; Role; Signal Transduction; Source; Spin Trapping; Techniques; Testing; Therapeutic; abstracting; adduct; base; concept; design; human disease; improved; insight; macromolecule; macrophage; nitrone; novel; oxidation; pyrroline; response; stressor

The mechanism of oxidative stress-induced inflammation in environmental- and metabolism-related human diseases is only partially understood. The long-term goal of this proposal is to apply insights and techniques discovered/developed in the present project to understand the molecular basis of environmental-lung-chronic inflammatory diseases. The specific hypothesis is: biomacromolecule-centered free radicals are part of the redox signaling triggered by lipopolysaccharide (LPS) during macrophage activation. This hypothesis is based on the following observations: 1) LPS induces reactive oxygen species (ROS) in cells; 2) ROS can mobilize redox-active iron from lysosomes; 3) iron binds to macromolecules and reacts in situ with ROS producing hydroxyl radicals; 4) hydroxyl radicals abstract electron(s) from macromolecules producing free radicals. The specific aims are designed to test our hypothesis using immuno-spin trapping (1ST) with the nitrone spin trap 5,5,-dimethyM-pyrroline A/-oxide (DMPO) and to improve our methods of detecting and characterizing those free radicals as their DMPO nitrone adduct-derivatives. SPECIFIC AIM#1: To determine the role of iron-catalyzed oxidations in triggering the redox signaling that produces LPS-induced activation of macrophages. To achieve this aim we will: a) determine the source and the type of ROS that triggers the redox signaling, b) determine whether the ROS induced by LPS mediates the release of iron from lysosomes to the cytoplasm, and c) study LPS-induced markers of iron-catalyzed oxidation of biomacromolecules during macrophage activation. SPECIFIC AIM # 2: To optimize, validate, and apply 1ST with DMPO to characterize protein radicals produced during LPS-induced activation of macrophages. To achieve this aim we will: a) immuno(cyto)chemically analyze DMPO-protein nitrone adducts, b) prepare beads coated with anti-DMPO antibodies to immunopurify nitrone adducts, and c) characterize purified DMPO nitrone adducts by mass spectrometric analyses. SPECIFIC AIM #3: To optimize, validate, and apply 1ST with DMPO to.detect total mRNA radicals in LPS- induced activation of macrophages. To achieve this aim we will: a) detect DMPO-RNA nitrone adducts, b) develop and validate 1ST for detecting mRNA radicals, and c) apply 1ST to the detection of total mRNA radicals induced in LPS-activated macrophages and in the lungs of mice exposed to LPS. RELEVANCE (See instructions): New studies aimed at identifying novel targets and early markers of exposure will help us develop better diagnostic and therapeutic approaches for oxidative stress-induced inflammation-mediated human diseases.

在与环境和代谢相关的人类疾病中氧化应激诱导炎症的机制仅部分了解。该提案的长期目标是应用本项目中发现/开发的见解和技术来了解环境肺慢性炎症性疾病的分子基础。具体假设是：以生物大分子为中心的自由基是巨噬细胞激活过程中脂多糖（LPS）触发的氧化还原信号的一部分。该假设基于以下观察：1）LPS 诱导细胞内活性氧（ROS）； 2）ROS可以动员溶酶体中具有氧化还原活性的铁； 3）铁与大分子结合并与ROS原位反应产生羟基自由基； 4) 羟基自由基从大分子中夺取电子，产生自由基。具体目标是使用免疫自旋捕获 (1ST) 和硝酮自旋捕获 5,5,-二甲基吡咯啉 A/-氧化物 (DMPO) 来检验我们的假设，并改进我们检测和表征这些自由基作为其 DMPO 硝酮加合物衍生物的方法。具体目标#1：确定铁催化氧化在触发氧化还原信号传导（产生 LPS 诱导的巨噬细胞激活）中的作用。为了实现这一目标，我们将：a）确定触发氧化还原信号传导的ROS的来源和类型，b）确定LPS诱导的ROS是否介导铁从溶酶体释放到细胞质，以及c）研究LPS诱导的巨噬细胞活化过程中铁催化生物大分子氧化的标记。具体目标#2：优化、验证并应用 1ST 与 DMPO 来表征 LPS 诱导的巨噬细胞激活过程中产生的蛋白质自由基。为了实现这一目标，我们将：a) 对 DMPO-蛋白质硝酮加合物进行免疫（细胞）化学分析，b) 制备涂有抗 DMPO 抗体的珠子以免疫纯化硝酮加合物，以及 c) 通过质谱分析表征纯化的 DMPO 硝酮加合物。具体目标#3：优化、验证并应用 1ST 与 DMPO 来检测 LPS 诱导的巨噬细胞激活中的总 mRNA 自由基。为了实现这一目标，我们将：a) 检测 DMPO-RNA 硝酮加合物，b) 开发并验证用于检测 mRNA 自由基的 1ST，以及 c) 将 1ST 应用于检测 LPS 激活的巨噬细胞和暴露于 LPS 的小鼠肺部诱导的总 mRNA 自由基。相关性（参见说明）：旨在确定新靶点和早期暴露标记的新研究将帮助我们开发更好的诊断和治疗方法，以治疗氧化应激诱导的炎症介导的人类疾病。