Nitric oxide and mitochondrial biogenesis in sepsis

描述（由申请人提供）：这是一份修改后的申请，用于研究脓毒症中肝脏线粒体生物发生中一氧化氮（NO）作用的基本机制。一氧化氮合酶的诱导是脓毒症诱导的免疫应答的基础，但活性氮和活性氧（RNS， ROS）的产生是一种驱动线粒体功能障碍的应激，在多器官功能衰竭（MOF）的发病机制中起重要作用。我们发现实验性败血症中RNS和ROS的产生会损害线粒体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了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，核呼吸因子-1和-2 (NRF-1和-2)，以及一个共激活因子PGC-1。这些独特的细胞反应，在核控制下，表明由RNS和ROS调节的线粒体生物学的一个重要方面，一个有利于生存。尽管我们对炎症或败血症中生物发生的调节知之甚少，但我们的数据清楚地表明，线粒体病理不仅仅局限于破坏线粒体和杀死细胞的no化学。因此，我们提出验证inos刺激的线粒体生物发生反对脓毒症中细胞坏死的假设，因此，生物发生的调节是细胞存活的重要决定因素。为了验证这一假设，我们提出了三个具体目的：目的1：利用野生型和iNOS敲除小鼠，测量iNOS在败血症中肝脏线粒体DNA和蛋白质损伤的发病机制中的作用；目的2：明确iNOS在脓毒症中野生型和iNOS敲除小鼠线粒体转录因子A （Tfam）激活和肝脏mtDNA拷贝数和转录恢复中的重要性；目的3：通过野生型和iNOS敲除小鼠NRF-1和NRF-2的表达，确定iNOS对脓毒症生物发生的核转录激活的贡献。这项工作将为宿主炎症反应过程中核-线粒体通讯的机制提供更好的理解，这将有助于培养新的分子策略来评估线粒体反应标志物预测MOF的能力，并指导干预措施预防和最终治疗败血症诱导的MOF。

DESCRIPTION (provided by applicant): 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 involving mitochondrial biogenesis, which requires activation of mitochondrial 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.