Nitric oxide and mitochondrial biogenesis in sepsis

这是一种研究线粒体中一氧化氮(NO)基本作用机制的改进应用 脓毒症时肝脏的生物发生。NO合酶诱导是脓毒症诱导免疫反应的基础 但产生的活性氮和氧物种(RNS，ROS)是驱动线粒体的一种压力 功能障碍，在多器官衰竭(MOF)的发病机制中起重要作用。我们发现， 实验性脓毒症时RNS和ROS的产生损伤线粒体DNA(MtDNA)，从而损害 线粒体转录、蛋白质组保真度和线粒体功能。这种线粒体损伤 刺激涉及线粒体生物发生的细胞补偿，这需要激活线粒体 转录因子A(MtTFA)和两个核转录因子-1和-2 (NRF-1和-2)和共激活剂PGC-1。这些独特的细胞反应，在核控制下，意味着 线粒体生物学中受RNS和ROS调控的关键方面，也是支持生存的方面。虽然我们 我们的数据显示，我们对炎症中生物发生的调节或其在脓毒症中的破坏知之甚少 显然，线粒体病理学并不局限于破坏线粒体和 杀死细胞。因此，我们建议检验iNOS刺激线粒体生物发生的假设 反对脓毒症中的细胞坏死，因此，生物发生的调节是 细胞存活。为了检验这一假设，我们提出了三个具体目标：目标1：衡量 诱导型一氧化氮合酶在脓毒症肝线粒体DNA和蛋白质损伤发病机制中的作用 和iNOS基因敲除小鼠；目标2：确定iNOS在线粒体转录因子A中的重要性 (TFAM)激活及肝线粒体DNA拷贝数和转录的恢复 脓毒症中iNOS基因敲除小鼠；目的3：确定iNOS在核转录中的作用 通过野生型和iNOS基因敲除小鼠NRF-1和NRF-2的表达激活脓毒症的生物发生。 这项工作将提供一个更好的机制理解核-线粒体通讯在 宿主炎症反应，这应该有助于培养新的分子策略来评估 线粒体反应标记物预测多器官功能衰竭并指导干预措施预防并最终治疗 脓毒症诱导的多器官功能衰竭。

This is an amended application to study the basic mechanisms of action of nitric oxide (NO) in mitochondrial biogenesis in the liver in sepsis. NO synthase induction is fundamental to the sepsis-induced immune response but the production reactive nitrogen and oxygen species (RNS, ROS) is a stress that drives mitochondrial dysfunction and is important in the pathogenesis of multiple organ failure (MOF). We have discovered that RNS and ROS production in experimental sepsis damage mitochondrial DNA (mtDNA), thereby impairing mitochondrial transcription, proteome fidelity, and mitochondrial function. This mitochondrial damage stimulates cellular compensation involvingmitochondrialbiogenesis, which requires activation ofmitochondrial transcription factor A (mtTFA) and two nuclear transcription factors, nuclear respiratory factor-1 and -2 (NRF-1 and -2),and a co-activator, PGC-1. These unique cell responses, under nuclear control, signify a crucial side of mitochondrial biology modulated by RNS and ROS, and one that is pro-survival. Though we still know very little about regulation of biogenesis in inflammation or its disruption in sepsis, our data show clearly that mitochondrial pathology is not limited simply to the NO-chemistry that damages mitochondria and kills cells. Therefore, we propose to test the hypothesis that iNOS-stimulated mitochondrial biogenesis opposes cell necrosis in sepsis, and thereby, regulation of biogenesis is an important determinant of cell survival. To test this hypothesis we propose three Specific Aims: Aim 1: Measure the contribution of iNOS to the pathogenesis of damage to hepatic mitochondrial DNA and proteins in sepsis using wild type and iNOS knockout mice; Aim 2: Define the importance of iNOS in mitochondrial transcription factor A (Tfam) activation and the restoration of hepatic mtDNA copy number and transcription in wild type and iNOS knockout mice in sepsis; Aim 3: Determine the contribution of iNOS to nuclear transcriptional activation of biogenesis in sepsis via NRF-1 and NRF-2 expression in wild type and iNOS knockout mice. This work will provide a better mechanistic understanding of the nuclear-mitochondrial communication during the host inflammatory response, which should help foster new molecular strategies to assess the ability of mitochondrial response markers to predict MOF and to guide interventions to prevent and eventually to treat sepsis-induced MOF.