Respiration in Sepsis

描述(由申请人提供)： 这项VA Merit Review更新申请建议研究实验性金黄色葡萄球菌败血症期间线粒体生物发生亲生存程序的转录调控。由于败血症引起的多器官功能障碍综合征(MODS)在老年退伍军人中的高患病率和高成本，这一领域的基础研究的需求很高。这种高死亡率综合症的部分原因是线粒体损伤，从机制上讲，人们对线粒体的损伤知之甚少。利用早期的资金，我们发现脓毒症的线粒体生物发生在能量危机之前就被激活了，而涉及一磷酸腺苷活化的蛋白激酶和pGC-1�共激活物的救援途径被推迟了。新的初步数据将AMPK激活与一氧化氮合酶-2(NOS2)诱导的脓毒症联系在一起，不需要高AMP/ATP，在一氧化氮合酶-2(NOS2)缺陷的小鼠中AMPK激活的丧失伴随着炎症的恶化。AMPK可能是脓毒症线粒体生物发生中三个关键转录因子CREB、NRF-1和NRF-2(GABPA)的激活剂，但NO调节也通过丧失NO信号特异性和NO物种(NOx)对线粒体的不分青红皂白的化学攻击而导致线粒体损伤。我们认为，通过即将发生的能量衰竭激活AMPK可以上调脓毒症时线粒体的损伤控制程序，如果是这样的话，非依赖于NO的药理学AMPK激活可以控制过量的NO产生，减轻脓毒症诱导的MODS。我们将通过药物激活AMPK来限制NO诱导的线粒体损伤，重点研究肝脏作为前哨器官的AMPK和NOS2对线粒体周转和细胞凋亡的定量影响。我们的具体目标是：目的1：了解脓毒症时肝脏AMPK的激活与NOS2诱导、线粒体生物发生程序以及细胞凋亡的预防之间的关系。1A.使用NOS2基因滴定，确定AMPK激活和NOx介导的线粒体DNA和蛋白质损伤、呼吸能力、高能代谢产物和脓毒症诱导的肝细胞死亡之间的关系。1B.确定通过NO依赖的CREB/NRF-1活性激活AMPK是否促进线粒体的生物发生和抑制促凋亡的Bad和BNIP3的磷酸化，从而防止脓毒症的细胞凋亡。目的：探讨不依赖于NOS2激活或抑制AMPK的特异性策略是否能调节脓毒症时肝线粒体的生物发生和细胞存活，减轻NO依赖的细胞损伤。2a.确定在脓毒症中，AMPK的药理激活是否独立于NOS2促进线粒体的生物发生和/或抑制细胞凋亡。我们还将使用高通量筛选来鉴定一个或多个新的AMPK选择性激动剂。2B。使用CREB和pGC-1�作为关键读数，比较NO依赖和NO非依赖AMPK激活对脓毒症线粒体生物发生转录调控的影响。这些研究将为AMPK在脓毒症中对线粒体生物发生的调控以及NO产生在多大程度上受到调控提供新的分子数据。概念验证将导致合理的药理学方法来激活和支持线粒体生物发生，同时将NO诱导的附带损害降至最低。了解这些基本的调控机制是设计前瞻性治疗方法以保护脓毒症期间线粒体质量控制所必需的。

DESCRIPTION (provided by applicant): This VA Merit Review renewal application proposes to study transcriptional regulation of the pro-survival program of mitochondrial biogenesis during experimental S. aureus sepsis. The need for basic research in this area is high due to the high prevalence and cost of sepsis-induced multiple organ dysfunction syndrome (MODS) in older Veterans. This high mortality syndrome is due in part to mitochondrial damage, which is mechanistically poorly understood. Using earlier funding, we discovered that mitochondrial biogenesis in sepsis is activated before energy crisis, while a rescue pathway is delayed involving the adenosine monophosphate (AMP)-activated protein kinase (AMPK) and PGC-1� co-activator. New preliminary data links AMPK activation to nitric oxide synthase-2 (NOS2) induction in sepsis, does not require high AMP/ATP, and loss of AMPK activation in nitric oxide synthase-2 (NOS2) deficient mice is accompanied by worsening inflammation. AMPK may serve as an activator of three crucial transcription factors- CREB, NRF-1, and NRF- 2 (GABPA) for mitochondrial biogenesis in sepsis, but NO regulation also induces mitochondrial damage by loss of NO signal specificity and indiscriminate chemical attack on mitochondria by NO species (NOx). We propose that AMPK activation by impending energy failure up-regulates the mitochondrial damage control program in sepsis and if so, NO-independent pharmacological AMPK activation could control excess NO production and mitigate sepsis-induced MODS. We will focus on AMPK in the liver, a sentinel organ, and NOS2 as a quantitative influence on mitochondrial turnover and apoptosis using pharmacological AMPK activation to limit NO-induced mitochondrial damage. Our Specific Aims are: Aim 1: To understand hepatic AMPK activation in sepsis in relation to NOS2 induction, the program of mitochondrial biogenesis, and the prevention of apoptosis. 1A. Define the relationships between AMPK activation and NOx-mediated mtDNA and protein damage, respiratory capacity, high-energy metabolites, and sepsis-induced hepatic cell death using NOS2 gene titration. 1B. Determine if AMPK activation through NO-dependent CREB/NRF-1 activity promotes mitochondrial biogenesis and inhibitory phosphorylation of pro-apoptotic Bad and BNIP3, preventing apoptosis in sepsis. Aim 2: To determine if specific strategies to activate or inhibit AMPK independently of NOS2 regulate hepatic mitochondrial biogenesis and cell survival and lessen NO-dependent cell damage in sepsis. 2A. Determine if the pharmacological activation of AMPK independently of NOS2 promotes mitochondrial biogenesis and/or inhibits apoptosis in sepsis. We will also use high-throughput screening to identify one or more novel selective agonists of AMPK. 2B. Compare NO-dependent and NO-independent AMPK activation for effects on the transcriptional regulation of mitochondrial biogenesis in sepsis using CREB and PGC-1� as key readouts. These studies will provide new molecular data on AMPK regulation of mitochondrial biogenesis in sepsis, and on the extent to which NO production is regulatory. Proof-of-concept would lead to rational pharmacological approaches to activate and support mitochondrial biogenesis while minimizing NO-induced collateral damage. Understanding these fundamental regulatory mechanisms is needed to design forward-thinking therapeutic approaches to protect mitochondrial quality control during sepsis.