HMG1 SIGNALING IN INFLAMMATION FOLLOWING BRAIN ISCHEMIA

描述（由申请人提供）：在缺血性卒中中，神经血管单元中的多种类型的脑细胞参与一系列复杂的信号机制，这些信号机制在脑损伤期间进化并导致炎症和细胞死亡。核蛋白HMG-1是炎症的主要调节因子。一旦从细胞中释放出来，HMG-1就会上调一系列炎症细胞因子，并激活许多不同的炎症细胞。已知HMG-1的下游受体靶点是RAGE、TLR2和TLR4。然而，对于这种上游信号分子HMG-1是否在神经炎症中发挥作用，特别是缺血性中风后，所知甚少。在小鼠短暂性脑缺血模型中获得的初步数据表明，缺血性脑损伤后，HMG-1既从细胞核中易位，又从细胞质中迅速释放。因此，我们假设HMG-1一旦在缺血性中风后从脑细胞中释放出来，就会通过上调炎症介质和招募白细胞到缺血区域来触发炎症反应。我们将探索由HMG-1引起的炎症反应有助于神经血管单元内的级联损伤通路，从而加剧缺血性损伤的新假设。因此，阻断HMG-1信号可能是减少缺血性损伤的一种新的有效方法。我们提出三个目标来探索这一假设。目的1将证实和扩展初步数据，即脑缺血导致HMG-1从细胞核转运到细胞质，然后释放到细胞外空间。目的2将根据其他系统的结果，在体外描述脑细胞中HMG-1的信号通路。这将包括评估HMG-1推定受体（RAGE、TLR2和TLR4）的表达，以及细胞因子（TNF-a、IL-1¿）和其他与MAPK和NF刺激相关的炎症相关分子的上调。重组HMG-1处理和单克隆抗HMG-1阻断后，特别是在内皮细胞内。目的3将验证抑制HMG-1信号调节小鼠MCAo后的炎症反应并可能减少脑损伤大小的假设。因此，我们建议探索生物化学和分子方法来证明HMG-1在缺血性脑损伤中的关键作用，并通过这样做，研究新的信号级联和中风的治疗靶点。脑缺血引起的炎症反应与脑损伤有因果关系。核蛋白HMG-1已被证明在某些情况下（如全身性败血症）是炎症的主要调节剂，但尚不清楚它是否在脑缺血后的炎症中发挥任何作用。本研究旨在探索HMG-1在缺血性脑损伤中的关键作用，从而探索新的信号级联反应和脑卒中治疗靶点。

DESCRIPTION (provided by applicant): In ischemic stroke, multiple types of brain cells in the neurovascular unit engage in a complex array of signaling mechanisms evolving during brain injury and leading to inflammation and cell death. The nuclear protein HMG-1 is a master regulator of inflammation. Once released from cells, HMG-1 upregulates a host of inflammatory cytokines and activates a number of different inflammatory cells. The known downstream receptor targets for HMG-1 are RAGE, TLR2 and TLR4. Very little is known, however, about whether this upstream signaling molecule HMG-1 plays a role in neuroinflammation, in particular following ischemic stroke. Preliminary data, obtained in a mouse model of transient brain ischemia, suggest that HMG-1 is both translocated from the nucleus and released from the cytosol rapidly after ischemic brain injury. We therefore hypothesize that HMG-1, once released from brain cells after ischemic stroke, triggers an inflammatory response by upregulating inflammatory mediators and recruiting leukocytes to the ischemic territory. We will explore the novel hypothesis that the inflammatory response elicited by HMG-1 contributes to a cascade of injury pathways within the neurovascular unit exacerbating ischemic damage. Blockade of HMG-1 signaling may therefore be a new and effective approach to reducing ischemic injury. We propose 3 aims to explore this hypothesis. Aim 1 will confirm and extend preliminary data establishing that brain ischemia causes HMG-1 translocation from the nucleus to the cytoplasm and then its release into the extracellular space. Aim 2 will characterize the signaling pathways of HMG-1 in brain cells, in vitro, based on results from other systems. This will include assessing the expression of HMG-1 putative receptors (RAGE, TLR2 and TLR4), as well as the up-regulation of cytokines (TNF-a, IL-1¿) and other inflammatory-associated molecules associated with stimulation of MAPK and NF?B upon treatment with recombinant HMG-1 and blockade by monoclonal anti-HMG-1, particularly within the endothelium. Aim 3 will test the hypothesis that inhibition of HMG-1 signaling modulates the inflammatory response and possibly reduces brain lesion size after MCAo in mouse. We therefore propose to explore biochemical and molecular approaches to demonstrate a pivotal role for HMG-1 in ischemic brain injury and by so doing, investigate novel signaling cascades and treatment targets for stroke. The inflammatory response to brain ischemia has been causally linked to brain damage. The nuclear protein HMG-1 has been shown to be a master regulator of inflammation in some situations like systemic sepsis, but it is not known whether it plays any role in inflammation following brain ischemia. This application proposes to explore biochemical and molecular approaches to demonstrate a pivotal role for HMG-1 in ischemic brain injury and by so doing, investigate novel signaling cascades and treatment targets for stroke.