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，旨在研究一氧化氮(NO)在脓毒症肝脏线粒体生物发生中的基本作用机制。NO合酶诱导是脓毒症诱导的免疫反应的基础，但其产生的活性氮和氧物种(RNS，ROS)是一种导致线粒体功能障碍的应激反应，在多器官衰竭(MOF)的发病机制中起重要作用。我们发现，在实验性脓毒症中，RNS和ROS的产生会损伤线粒体DNA(MtDNA)，从而损害线粒体的转录、蛋白质组的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-1和-2(NRF-1和-2)，以及辅助激活剂PGC-1。这些独特的细胞反应，在核控制下，标志着线粒体生物学中受RNS和ROS调控的关键方面，也是有利于生存的一面。尽管我们对炎症中生物发生的调节或对脓毒症中生物发生的破坏知之甚少，但我们的数据清楚地表明，线粒体病理不仅仅限于破坏线粒体和杀死细胞的非化学物质。因此，我们建议检验iNOS刺激的线粒体生物发生与脓毒症细胞坏死相反的假说，因此，生物发生的调节是细胞存活的重要决定因素。为了验证这一假说，我们提出了三个特定的目标：目的1：利用野生型和iNOS基因敲除小鼠，测量iNOS在脓毒症肝脏线粒体DNA和蛋白质损伤的发病机制中的作用；目的2：确定iNOS在脓毒症中线粒体转录因子A(Tfam)激活以及肝脏mtDNA拷贝数和转录恢复中的重要性；目的3：通过野生型和iNOS基因敲除小鼠NRF-1和NRF-2的表达，确定iNOS在脓毒症生物发生核转录激活中的作用。这项工作将提供对宿主炎症反应过程中核-线粒体通讯的更好的机制理解，这将有助于开发新的分子策略来评估线粒体反应标记物预测MOF的能力，并指导预防和最终治疗脓毒症诱导的MOF的干预措施。

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.