Role of Dual Oxidase in post-stroke brain inflammation and injury

项目摘要 缺血性脑损伤仍然是导致长期残疾和死亡的主要原因，治疗选择有限。 脑缺血损伤与过量产生活性氧自由基(ROS)密切相关 导致内皮功能障碍、血脑屏障破坏、神经细胞死亡和脑恶化 损坏。因此，减少ROS的努力对改善卒中预后有重大影响。双氧合酶(DUOX)是 还原烟酰胺二核苷酸磷酸氧化酶家族的新成员。Duox1的主要功能是 产生过氧化氢(H2O2)/ROS。Duox1处于低水平，参与基本的细胞功能， 抗菌宿主防御、甲状腺激素产生、呼吸道上皮细胞迁移和损伤。然而， Duox1的过度产生和激活可能导致包括炎症在内的病理事件， 细胞凋亡、高血压、癌症和组织损伤。呼吸道上皮细胞中Duox1与ROS的直接联系 细胞在我们之前的工作中得到了展示。促炎细胞因子和错乱的钙信号增加了 DUOX在呼吸道上皮细胞和甲状腺组织中的活性和表达有趣的是，缺血性中风会导致 异常的钙离子内流。尽管有这些令人信服的观察，DUOX在大脑和大脑中的具体作用 缺血在很大程度上是未知的。我们最近发现了啮齿类动物的局灶性脑缺血，并在体外 缺氧缺糖快速诱导血管内皮细胞和神经细胞表达Duox1 与ROS产量的增加有关。然而，用Duox1特异性小分子药物预处理神经细胞 干扰RNA可降低Duox1的表达和ROS水平。这些数据让我们假设大脑 缺血引起Duox1的过度表达，进而增加大脑中的ROS，导致脑缺血加重 导致脑损伤恶化的促炎和细胞凋亡过程。我们进一步提出Duox1抑制 在减轻脑缺血后的脑损伤和神经功能障碍方面具有巨大的潜力。因此，我们的 具体目的是确定局灶性脑内Duox1表达的时空变化 脑缺血/再灌注；确定Duox1抑制或基因缺失是否降低ROS，以及 减少缺血性脑损伤，从而促进卒中后功能恢复；探讨 Duox1是脑缺血时炎症和细胞凋亡过程的关键驱动因素。我们将实现这些目标 在活体动物和体外细胞中使用广泛的分子、细胞和生化方法 文化典范。总体而言，这项“概念验证”研究将确定Duox1在 脑缺血。这些研究为更好地理解Duox1介导的ROS的作用铺平了道路 和神经炎症机制，并可能开辟一种有前途的治疗脑部疾病的新方法。 血管疾病。

Project Summary Ischemic brain damage remains a leading cause of long-term disability and death with limited treatment options. Cerebral ischemic injury is strongly associated with excessive production of reactive oxygen species (ROS) that contributes to endothelial dysfunction, blood brain barrier disruption, neuronal cell death, and worsened brain damage. Thus, efforts to curtail ROS have major impact on improving stroke outcome. Dual oxidases (Duox) are novel members of reduced nicotinamide dinucleotide phosphate oxidases family. The main function of Duox1 is to generate hydrogen peroxide (H2O2)/ROS. Duox1 at low levels, is involved in essential cellular functions, antimicrobial host defense, thyroid hormone production, and airway epithelial cell migration, and injury. However, excessive production and activation of Duox1 may contribute to pathological events including inflammation, apoptosis, hypertension, cancer, and tissue damage. The direct link between Duox1 and ROS in airway epithelial cells was shown in our previous work. Pro-inflammatory cytokines and deranged calcium signaling increase the activity and expression of Duox in airway epithelial cells and thyroid tissues. Interestingly, ischemic stroke causes aberrant Ca2+ influx. Despite these compelling observations, the specific roles of Duox in the brain and cerebral ischemia are largely unknown. We have recently identified that focal cerebral ischemia in rodents, and in-vitro oxygen glucose deprivation rapidly induce the expression of Duox1 in endothelial and neuronal cells in association with increased ROS production. However, pre-treatment of neuronal cells with Duox1 specific small interfering RNA decreased Duox1 expression and ROS levels. These data led us to hypothesize that cerebral ischemia evokes Duox1 over-expression which in turn increases ROS in the brain, leading to exacerbation of pro-inflammatory and apoptotic processes that worsen the brain injury. We further propose that Duox1 inhibition has a great potential to mitigate post-ischemic brain damage and neurological dysfunction. Accordingly, our specific Aims are; To determine the spatiotemporal changes in the expression of Duox1 in brain following focal cerebral ischemia/reperfusion; To determine if Duox1 inhibition or genetic loss of Duox1 decreases ROS, and reduces ischemic brain damage, and thus improves post-stroke functional recovery; To investigate the role of Duox1 as a key driver of inflammatory and apoptotic processes in ischemic brain. We will address these Aims using a wide array of molecular, cellular, and biochemical approaches in both in vivo animal, and in vitro cell culture models. Overall, this ‘proof of concept’ study will determine a previously unidentified role for Duox1 in ischemic brain. These studies pave the way towards better understanding of the role of Duox1 mediated ROS and neuro-inflammatory mechanisms in brain and may open up a promising new approach for treating cerebro- vascular diseases.