Pleiotropic Transcription Factors As Target For Intracerebral Hemorrhage Treatmen

描述(申请人提供)：鉴于所有脑出血(ICH)细胞保护疗法的临床试验都失败了，新的候选疗法应该具有与脑出血复杂发病机制相对应的多效性效应。在这里，我们认为核因子-红系E2相关因子2(Nrf2)是一种多效性蛋白，它调节一系列关键的细胞保护、解毒和吞噬调节基因的转录，是脑出血治疗的一个有希望的靶点。初步数据提示：(1)细胞保护：啮齿类动物脑出血后Nrf2激活剂可减轻氧化应激、炎症反应，保护脑细胞免受损伤；(2)血肿溶解：Nrf2激活剂促进小鼠脑出血后小鼠脑内小胶质细胞/巨噬细胞的吞噬功能，加速血肿溶解。这一过程与Nrf2介导的CD36清道夫受体的表达有关，因为阻断Nrf2和CD36会抑制小胶质细胞的吞噬作用。此外，随着Nrf2活化的改善，Nrf2介导的神经毒性血红蛋白的清除可能通过Nrf2驱动的结合珠蛋白(HP)的表达来实现，而严重的低结合球蛋白血症会延缓脑出血后的血红蛋白清除。因此，总体假设是，在脑出血病理的早期和晚期，Nrf2是保护大脑免受损害的一个可行的靶点。包括三个特定的目的(SA)：(SA1)在类似脑出血的体外模型中研究Nrf2作为脑出血后细胞保护和炎症的调节因子。我们将使原代神经元或小胶质细胞处于类似脑出血的环境中，并确定其后果：细胞死亡、氧化应激、炎症反应(小胶质细胞)。接下来，我们将通过使用Nrf2缺陷小鼠的细胞、DNA诱饵抑制Nrf2、基因转移过表达Nrf2以及药物激活Nrf2来确定Nrf2的细胞保护作用(包括保持吞噬功能)。(SA2)探讨Nrf2促进脑出血后血肿消退的作用机制。我们将使用Nrf2、CD36和Hp缺陷小鼠(或从这些小鼠分离的小胶质细胞)来探索Nrf2及其下游靶点以及CD36和Hp在血肿解决中的作用及其对继发性脑损伤的影响。(SA3)研究萝卜硫醚(Nrf2激活剂)治疗脑出血的临床应用。采用小鼠脑出血注血模型，优化SF治疗脑出血的条件。我们将首先在小鼠脑出血的最高效率、安全性、治疗持续时间和最长有效治疗时间窗口方面确定最佳治疗剂量。我们的长期目标是探索药物和分子疗法，以减少脑出血的发病机制，促进功能恢复。公共卫生相关性：脑出血(ICH)占所有中风的10%至15%，一年死亡率超过50%至60%。目前还没有FDA批准的治疗脑出血的有效方法。在我们的研究中，我们将确定转录因子Nrf2的激活是否可能代表脑出血治疗的一个可行的靶点。Nrf2调节许多基因的表达，这些基因增加了脑细胞对脑出血诱导的损伤的抵抗力。我们的目标是探索NRF2的S有益作用的潜在分子机制，并建立以NRF2为基础的治疗方法的初步药理学标准。

DESCRIPTION (provided by applicant): Given the failure of all clinical trials with cytoprotective therapy in intracerebral hemorrhage (ICH), new candidate therapy should have a pleiotropic effect which corresponds to the complex profile of ICH pathogenesis. Here, we propose that nuclear factor-erythroid E2-related factor 2 (Nrf2), a pleiotropic protein that regulates transcription of a battery of key cytoprotective, detoxifying, and phagocytosis-regulating genes represents a promising target for ICH therapy. Preliminary data suggest: (1) Cytoprotection: Activators of Nrf2 after ICH in rodents reduce oxidative stress, inflammation, and protect brain cells from damage, while Nrf2 gene deletion has the opposite effect; (2) Hematoma resolution: Activators of Nrf2 promote phagocytosis by brain microglia/macrophages and accelerate hematoma resolution in mice after ICH. This process is linked to Nrf2-mediated CD36 scavenger receptor expression, since blocking Nrf2 and CD36 inhibits phagocytosis by microglia. In addition, Nrf2-mediated clearance of neurotoxic hemoglobin could be through Nrf2- driven haptoglobin (Hp) expression, as Nrf2-activation improves, and severe hypohaptoglobinemia retards hemoglobin clearance after ICH. Thus, the overall hypothesis is that Nrf2 is a viable target in protecting the brain from damage during the both early and late stages of ICH pathology. Three Specific Aims (SA) are included: (SA1) investigates Nrf2 as a regulator of cytoprotection and inflammation after intracerebral hemorrhage in an ICH-like in vitro model. We will subject primary neurons or microglia to "ICH-like" environment and establish its consequences: cell death, oxidative stress, inflammatory response (microglia). Next, we will establish the cytoprotective role of Nrf2 (including preservation of phagocytotic functions) by using cells from Nrf2 deficient mice, DNA decoy to inhibit Nrf2, gene transfer to overexpress Nrf2, and pharmacologic agents to activate Nrf2. (SA2) Investigate Nrf2 as a mechanism to promote hematoma resolution after ICH. We will use Nrf2-, CD36-, and Hp-deficient mice (or microglia isolated from these mice) to explore the role of Nrf2 and its downstream targets, as well as CD36 and Hp, in hematoma resolution and their impact on secondary brain damage. (SA3) Investigate the clinical utility of sulforaphane (Nrf2 activator) as a treatment for ICH. We will use blood injection model of ICH in mouse to optimize conditions for the treatment of ICH with SF. We will first establish an optimal therapeutic dose with respect to the highest efficiency, safety, duration of treatment, and longest time window for effective treatment for ICH in mice. Our long term goal is to explore pharmacological and molecular therapies that will reduce ICH pathogenesis and improve functional recovery. PUBLIC HEALTH RELEVANCE: Intracerebral hemorrhage (ICH) accounts for 10 to 15% of all strokes and has a one-year mortality rate greater than 50-60%. There is no FDA approved effective treatment for ICH. In our study, we will determine if activation of transcription factor Nrf2, which regulates expression of many genes that increase resistance of brain cells to ICH- induced damage, may represent a viable target for ICH treatment. Our goal is to explore the underlying molecular mechanism of Nrf2's beneficial effect, and to establish the initial pharmacologic criteria for therapies using Nrf2-based approach.