Free Radicals and Redox Signaling Triggered by Lipopolysccharide in Macrophages

在与环境和新陈代谢相关的人类疾病中，氧化应激诱导炎症的机制只有部分了解。这项建议的长期目标是应用在本项目中发现/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。具体的假设是：在巨噬细胞激活过程中，以生物大分子为中心的自由基是脂多糖(LPS)触发的氧化还原信号的一部分。这一假说基于以下观察：1)内毒素诱导细胞内的活性氧物种(ROS)；2)ROS可从溶酶体中动员氧化还原活性铁；3)铁与大分子结合并与ROS原位反应产生羟基自由基；4)羟基自由基从产生自由基的大分子中提取电子(S)。具体目的是使用免疫自旋捕获(1)与硝酮自旋陷阱5，5，-二甲基吡咯烷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。具体目的1：确定铁催化的氧化在触发氧化还原信号中的作用，氧化还原信号产生内毒素诱导的巨噬细胞激活。为了达到这一目的，我们将：a)确定触发氧化还原信号的ROS的来源和类型，b)确定内毒素诱导的ROS是否介导铁从溶酶体释放到细胞质，以及c)研究在巨噬细胞激活过程中内毒素诱导的铁催化的生物大分子氧化的标志物。具体目的#2：优化、验证和应用DMPO来表征在内毒素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)免疫(细胞)化学分析DMPO-蛋白质硝酮加合物，b)制备包被抗DMPO抗体的珠子以免疫纯化硝酮加合物，以及c)通过质谱分析对纯化的DMPO硝酮加合物进行表征。具体目的#3：优化、验证和应用DMPO检测脂多糖诱导的巨噬细胞激活过程中的总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)建立并验证First用于检测mRNA自由基，以及c)将First应用于检测内毒素激活的巨噬细胞和暴露于内毒素的小鼠肺组织中诱导的总mRNA自由基。相关性(见说明)：旨在确定暴露的新靶点和早期标记的新研究将帮助我们开发更好的诊断和治疗方法，用于治疗氧化应激诱导的炎症介导的人类疾病。

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.