Nitric oxide and mitochondrial biogenesis in sepsis

描述（由申请人提供）：这是一份研究一氧化氮（NO）在脓毒症肝脏线粒体生物合成中的基本作用机制的修订申请。NO合酶诱导是脓毒症诱导的免疫应答的基础，但活性氮和氧物质（RNS，ROS）的产生是驱动线粒体功能障碍的应激，并且在多器官衰竭（MOF）的发病机制中是重要的。我们已经发现实验性脓毒症中RNS和ROS的产生损害线粒体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）和两种核转录因子，核呼吸因子-1和-2（NRF-1和-2），以及共激活因子PGC-1。这些独特的细胞反应，在核控制下，意味着由RNS和ROS调节的线粒体生物学的关键方面，并且是促生存的一个方面。虽然我们对炎症中生物发生的调节或脓毒症中生物发生的破坏仍然知之甚少，但我们的数据清楚地表明，线粒体病理学不仅限于破坏线粒体和杀死细胞的NO化学。因此，我们建议测试的假设，iNOS刺激的线粒体生物合成反对细胞坏死脓毒症，从而，调节生物合成是一个重要的决定因素细胞存活。为了验证这一假设，我们提出了三个具体的目的：目的1：利用野生型和iNOS基因敲除小鼠，测量iNOS在脓毒症中对肝线粒体DNA和蛋白质损伤的发病机制中的贡献;目的2：确定iNOS在脓毒症中在线粒体转录因子A（Tfam）激活和野生型和iNOS基因敲除小鼠中肝线粒体DNA拷贝数和转录恢复中的重要性;目标3：通过野生型和iNOS敲除小鼠中NRF-1和NRF-2的表达，确定iNOS对脓毒症中生物发生的核转录激活的贡献。这项工作将提供一个更好的机制理解的核线粒体通信在主机炎症反应，这应该有助于促进新的分子策略，以评估线粒体反应标志物预测MOF的能力，并指导干预措施，以预防和最终治疗脓毒症诱导的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.