HMG1 SIGNALING IN INFLAMMATION FOLLOWING BRAIN ISCHEMIA

描述(申请人提供)：在缺血性中风中，神经血管单位中的多种类型的脑细胞参与了一系列复杂的信号机制，在脑损伤期间演变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就会上调一系列炎性细胞因子，并激活一些不同的炎性细胞。已知的HMG-1下游受体靶点是RAGE、TLR2和TLR4。然而，关于这种上游信号分子HMG-1是否在神经炎症中发挥作用，特别是在缺血性中风后，人们知之甚少。在小鼠短暂性脑缺血模型中获得的初步数据表明，在缺血性脑损伤后，HMG-1既从细胞核移位，又从胞浆中迅速释放。因此，我们假设HMG-1一旦在缺血性卒中后从脑细胞中释放出来，通过上调炎症介质和招募白细胞到缺血区而触发炎症反应。我们将探索这一新的假设，即HMG-1引起的炎症反应有助于神经血管单位内的一系列损伤途径，从而加剧缺血性损伤。因此，阻断HMG-1信号通路可能是减轻缺血损伤的一种新的有效途径。我们提出3个目的是为了探索这一假说。目的1将证实和扩展初步数据，证实脑缺血导致HMG-1从细胞核移位到细胞质，然后释放到细胞外空间。目的2将基于其他系统的结果，在体外表征HMG-1在脑细胞中的信号通路。这将包括评估HMG-1可能的受体(RAGE、TLR2和TLR4)的表达，以及细胞因子(TNF-α、IL-1β)和其他炎症相关分子的上调，这些分子与重组HMG-1治疗和单抗HMG-1阻断后MAPK和NF？B的刺激有关。目的3验证抑制HMG-1信号转导调节炎症反应，并可能缩小小鼠大脑中动脉闭塞后病变范围的假说。因此，我们建议探索生化和分子方法来证明HMG-1在缺血性脑损伤中的关键作用，并通过这样做，研究新的信号级联反应和中风的治疗靶点。对脑缺血的炎症反应与脑损伤有因果关系。核蛋白HMG-1已被证明是某些情况下炎症的主要调节因子，如全身性脓毒症，但尚不清楚它是否在脑缺血后的炎症中发挥作用。这项申请建议探索生化和分子方法，以证明HMG-1在缺血性脑损伤中的关键作用，并通过这样做，研究新的信号级联反应和中风的治疗靶点。

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.