Effect of Sepsis on Cytochrome aa3 Oxidation State

 描述(由申请人提供)：这是一份职业发展奖的申请书，旨在为PI提供综合生物科学和生物医学工程方面的培训，为成为一名专注于炎症疾病(如败血症)对亚细胞能量学影响的独立调查员做准备。脓毒症导致ATP耗竭和细胞色素Aa3(电子传递链的末端成分)减少。PI将调查未控制的脓毒症(大鼠盲肠结扎和穿孔)和已有的脓毒症治疗对细胞色素Aa3氧化状态(Cytox)、组织PO2、ATP/ADP比率和存活率的影响，以更好地了解这种已观察到的能量“静止”(SA1)的影响。我假设这种“静止”是适应性的，无法关闭新陈代谢将与发病率有关。组织氧在炎症中的作用尚不清楚。氧气可以是有益的(允许有氧呼吸)，也可以是有害的(产生活性氧类[ROS])。在有/没有选择性细胞色素Aa3抑制的情况下，脓毒症时PI将改变组织O2 同时检测组织氧分压、细胞毒性、氧化应激标志物、三磷酸腺苷：腺苷二磷酸比率、低氧诱导因子1(HIF-1)的表达和存活率，以确定组织氧在炎症过程中是否具有保护作用，以及阐明其影响的潜在机制(S)(SA2)。I假设败血症引起的静息减少了低组织PO2的影响，而异常组织PO2会导致ROS的产生和发病率。预适应(PC)在多种病理状态下提供细胞保护。虽然药物PC对脓毒症有保护作用，但它 在临床上是不切实际的，因此PI将测试缺氧和缺血PC作为保护机制的有效性。在短暂的控制性低氧或远程缺血预适应(RIPC)后，将会导致大鼠的脓毒症。检测细胞毒性、缺氧诱导因子-1、血红素加氧酶-1(HO-1)、诱导型一氧化氮合酶的表达和存活率(SA3)。我推测RIPC将导致HIF-1上调，减弱ROS的产生。有效性的证明将为高危患者提供一种“先发制人”的实用方法。HIF-1抑制ROS的产生并诱导线粒体生物合成(通过HO-1)，这可能是有机体的生存，但可能限制T细胞的效率(ROS具有杀菌作用)。HIF-1下调对细胞能量平衡和免疫活性的影响尚不清楚。PI将比较野生型和缺乏HIF-1 I.1亚型的“敲除”小鼠的脓毒症耐受性，特别是观察HIF-1的表达、细胞毒作用、氧化应激和存活率(SA4)。 虽然抑制淋巴细胞中的HIF-1可以改善脓毒症患者的存活率(通过增加杀菌活性)，但我假设全球范围内HIF-1的下调将增加由于有害ROS的过度产生而导致的死亡率。这些特定目标的完成将增加我们对脓毒症对细胞色素Aa3氧化状态、亚细胞能量学和HIF-1的影响的理解，最终为进一步的工作提供基础，为这一毁灭性疾病过程开发新的治疗策略。

 DESCRIPTION (provided by applicant): This is an application for a career development award to provide the PI with training in integrative biosciences and biomedical engineering in preparation for a career as an independent investigator focused on the impact of inflammopathies (e.g. sepsis) on subcellular energetics. Sepsis leads to ATP depletion and cytochrome aa3 (terminal component of the electron transport chain) reduction. The PI will investigate the impact of uncontrolled sepsis (rat cecal ligation and puncture) and established sepsis therapies on cytochrome aa3 oxidation state (Cytox), tissue pO2, ATP:ADP ratio, and survival in order to better understand the impact of this observed energetic "quiescence" (SA1). I hypothesize that this "quiescence" is adaptive and that inability to shut down metabolism will be associated with morbidity. The role of tissue O2 in inflammation is not clear. O2 can be both helpful (allowing for aerobic respiration) and harmful (reactive oxygen species [ROS] production). The PI will alter tissue O2 with/without selective cytochrome aa3 inhibition in septic rats while measuring tissue pO2, Cytox, markers of oxidative stress, ATP:ADP ratios, hypoxia-induced factor 1(HIF-1) expression, and survival to determine if tissue O2 is protective during inflammation, as well as elucidate the underlying mechanism(s) for its impact (SA2). I hypothesize sepsis-induced quiescence diminishes the impact of low tissue pO2, and that supranormal tissue pO2 leads to ROS production and morbidity. Preconditioning (PC) provides cellular protection in a variety of pathologies. While pharmacologic PC is protective in sepsis, it is clinically impractical, thus the PI will test the efficacy of hypoxic and ischemic PC as a protective mechanism. After a brief period of controlled hypoxia or remote ischemic preconditioning (RIPC), sepsis will be induced in rats. Cytox, expression of HIF-1, heme-oxygenase-1 (HO-1), inducible nitric oxide synthetase, and survival will be measured (SA3). I hypothesize that RIPC will lead HIF-1 upregulation, attenuating ROS production. Demonstration of efficacy would suggest a practical means of "pre-empting" sepsis in high-risk patients. HIF-1 attenuates ROS production and induces mitochondrial biogenesis (via HO-1) which are likely prosurvival for the organism but may limit T cell efficacy (ROS are bactericidal). The impact of HIF-1 downregulation on the balance of cellular energetics and immunocompetence is not clear. The PI will compare sepsis tolerance in wild-type and "knockdown" mice deficient in the HIF-1 I.1 isoform, specifically looking at HIF-1 expression, Cytox, oxidative stress, and survival (SA4). While HIF-1 suppression in lymphocytes improves survival in sepsis (by increasing antibactericidal activity), I hypothesize that global HIF-1 downregulation will increase mortality due to overproduction of harmful ROS. Completion of these Specific Aims will increase our understanding of the impact of sepsis on cytochrome aa3 oxidation state, subcellular energetics, and HIF-1, ultimately providing a foundation for additional work to develop novel therapeutic strategies for this devastating disease process.