Botanical Phenolics on Oxidative/Nitrosative Signaling Pathways/Grace Sun

脑内过量的氧化和亚硝酸盐(O/N)活性被认为是许多神经退行性疾病(包括中风)下的神经元兴奋毒性和神经胶质细胞炎症反应的基础。最近的研究发现，NADPH氧化酶是脑细胞中活性氧物种(ROS)的重要来源。NADPH氧化酶的激活与许多下游信号通路有关，包括诱导iNOS和产生一氧化氮(NO)的NF-KB通路。在其他反应中，NO可以通过与蛋白质反应性半胱氨酸硫醇残基相互作用形成S亚硝硫醇来调节蛋白质的结构和功能。许多与NADPH氧化酶信号通路相关的蛋白质似乎被S亚硝化修饰。虽然机制尚不清楚，但有相当多的证据表明，许多植物化合物可能会保护大脑免受O/N伤害，包括那些由脑缺血造成的伤害。为了与该中心的中心目标保持一致，项目2将发现新的植物药物，并验证中风介导的神经元兴奋毒性和神经胶质炎症反应部分归因于NADPH氧化酶产生的ROS以及随后NADPH氧化酶信号通路中关键蛋白的S亚硝酸基。目的1.利用神经元和星形胶质细胞/小胶质细胞筛选植物提取物和从提取物中分离出的纯化合物，以研究它们抑制NMDA介导的神经元兴奋毒性和细胞因子诱导的神经胶质细胞炎症反应的能力。植物药物对神经元和神经胶质细胞中蛋白质的S亚硝化以及差异表达的基因和蛋白质的影响将使用新开发的NitroDIGE程序和相互作用核心中的mRNA大测序设备来研究。目的2采用小鼠大脑中动脉闭塞造成的局灶性脑缺血模型，研究饮食中添加特定植物药对脑缺血/再灌注损伤的保护作用。这些研究将包括使用缺乏N0X2和iNOS的小鼠来测试植物药对行为结果、神经元存活、神经胶质细胞激活和炎症反应的影响。目的3将验证这样的假设：已鉴定的植物通过激活Nrf2/Keap1抗氧化信号通路来减少脑缺血/再灌注损伤，从而提供神经保护作用。在这些研究中，将使用星形胶质细胞或神经元靶向的条件性Keapl缺失小鼠。在目标2和目标3中，我们还将确定不同脑区中与氧化五和心钠素-氧化相关的五条信号通路的S硝化和炎性蛋白及其m RNA的变化。项目2的体外和体内方法，以及该中心独特和最先进的核心设施的支持，将结合确定特定的细胞和分子机制，通过这些机制，新的植物药物和/或其活性成分可以发挥有益的作用，防止中风损害。

Excessive oxidafive and nitrosative (O/N) activifies in the brain have been recognized as the basis for the neuronal excitotoxicity and glial cell inflammatory responses that underiie many neurodegenerative diseases, including stroke. Recent studies have discovered the role of NADPH oxidase as an important source of reactive oxygen species (ROS) in brain cells. Activafion of NADPH oxidase is associated with a number of downstream signaling pathways, including the NF-KB pathway that induces INOS and produces nitric oxide (NO). Among other reactions, NO can regulate protein structure and function by interacfing with protein reactive cysteine thiol residues to form S-nitrosothiol. Many proteins associated with NADPH oxidase signaling pathways appear to be modified by S-nitrosylation. While the mechanisms are unknown, there is considerable evidence to suggest that a number of botanical compounds may protect the brain against O/N insults, including those resulfing from cerebral ischemia. In keeping with the central goal ofthis Center, Project 2 will identify new botanicals and test the hypothesis that stroke-mediated neuronal excitotoxicity and glial inflammatory responses are due in part to ROS from NADPH oxidase and subsequent S-nitrosylafion of key proteins in the NADPH oxidase signaling pathways. In Aim 1. neurons and astrocytes/microglia will be used to screen botanical extracts and pure compounds isolated from the extracts for their ability to suppress NMDA-mediated neuronal excitotoxicity and cytokine-induced inflammatory responses in glial cells. The effects of botanicals on S-nitrosylation of proteins and differentially-expressed mRNA and proteins in neurons and glial cells will be invesfigated using the newly developed NitroDIGE protocol and mRNA megasequencing facilities in the Interactions Core. In Aim 2, the mouse focal cerebral ischemia model induced by occlusion of the middle cerebral artery will be used to investigate whether dietary supplementation with specific botanicals offers protective effects against cerebral ischemia/reperfusion damage. These studies will include using mice deficient in N0X2 and iNOS to test the effects of botanicals on behavioral outcome, neuronal survival, glial activation, and inflammatory responses. Aim 3 will test the hypothesis that identified botanicals offers neuroprotective effects by reducing cerebral ischemia/reperfusion damage through activation of the Nrf2/Keap1 antioxidant signaling pathway. In these studies, astrocyte- or neuron-targeted conditional Keapl-null mice will be used. In Aims 2 and 3, we will also identify changes in S-nitrosylated and inflammatory proteins and mRNA associated with oxidafive and anfi-oxidafive signaling pathways in disfinct brain regions. The in vitro and in vivo approaches of Project 2, together with support from the unique and state-of-the-art core facilities of this Center, will combine to identify specific cellular and molecular mechanisms through which novel botanicals and/or their active components may exert beneficial effects and protect against stroke damage.