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

项目摘要 缺血性脑损伤仍然是长期残疾和死亡的主要原因，治疗选择有限。 脑缺血性损伤与活性氧（ROS）的过度产生密切相关， 导致内皮功能障碍、血脑屏障破坏、神经元细胞死亡和脑功能恶化。 损害因此，减少ROS的努力对改善卒中结局具有重大影响。双氧化酶（Duox）是 还原型烟酰胺二核苷酸磷酸氧化酶家族的新成员。Duox 1的主要功能是 以产生过氧化氢（H2 O2）/ROS。低水平的Duox 1参与基本的细胞功能， 抗微生物宿主防御、甲状腺激素产生和气道上皮细胞迁移以及损伤。然而，在这方面， Duox 1的过度产生和活化可能导致病理事件包括炎症， 凋亡、高血压、癌症和组织损伤。Duox 1与气道上皮细胞活性氧的直接联系 细胞在我们以前的工作中被展示。促炎细胞因子和紊乱的钙信号传导增加了 Duox在气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 异常Ca 2+内流尽管有这些令人信服的观察结果，Duox在大脑和脑组织中的特定作用仍然存在。 局部缺血在很大程度上是未知。我们最近发现，在啮齿动物局灶性脑缺血， 缺氧缺糖可迅速诱导内皮细胞和神经细胞表达Duox 1， 增加ROS的产生。然而，用Duox 1特异性小的神经元细胞预处理， 干扰RNA降低Duox 1表达和ROS水平。这些数据让我们假设大脑 缺血引起Duox 1过度表达，这反过来又增加了脑中的ROS，导致缺血性脑损伤的恶化。 促炎和凋亡过程，使脑损伤恶化。我们进一步提出Duox 1抑制 具有减轻缺血后脑损伤和神经功能障碍的巨大潜力。因此，我们的 具体目的是：确定局灶性脑缺血后Duox 1表达的时空变化 确定Duox 1抑制或Duox 1的遗传缺失是否会降低ROS， 减少缺血性脑损伤，从而改善卒中后功能恢复; Duox 1是缺血性脑中炎症和凋亡过程的关键驱动因素。我们将实现这些目标 在体内动物和体外细胞中使用广泛的分子、细胞和生物化学方法， 文化模式总的来说，这项“概念验证”研究将确定Duox 1在以下方面的一个先前未被确定的作用： 脑缺血这些研究为更好地理解Duox 1介导的ROS的作用铺平了道路。 和神经炎症机制，并可能开辟一个有前途的新途径，治疗脑- 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.