Pleiotropic Transcription Factors As Target For Intracerebral Hemorrhage Treatmen

描述（由申请人提供）：鉴于脑出血（ICH）中细胞保护治疗的所有临床试验均失败，新的候选治疗应具有多效性效应，与ICH发病机制的复杂性相对应。在这里，我们提出，核因子-红细胞E2相关因子2（Nrf 2），一种多效性蛋白，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ICH治疗的一个有前途的目标。初步数据显示：（1）细胞保护：啮齿类动物脑出血后Nrf 2的激活剂可减轻氧化应激、炎症，保护脑细胞免受损伤，而Nrf 2基因缺失则具有相反的作用;（2）血肿消退：Nrf 2的激活剂可促进脑小胶质细胞/巨噬细胞的吞噬作用，加速小鼠脑出血后血肿消退。这一过程与Nrf 2介导的CD 36清道夫受体表达有关，因为阻断Nrf 2和CD 36可抑制小胶质细胞的吞噬作用。此外，Nrf 2介导的神经毒性血红蛋白的清除可能是通过Nrf 2驱动的触珠蛋白（Hp）表达，因为Nrf 2激活改善，严重的低触珠蛋白血症会延迟ICH后血红蛋白的清除。因此，总体假设是Nrf 2是在ICH病理学的早期和晚期阶段保护脑免受损伤的可行靶标。包括三个特定目的（SA）：（SA 1）在ICH样体外模型中研究Nrf 2作为脑出血后细胞保护和炎症的调节剂。我们将初级神经元或小胶质细胞置于“ICH样”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，我们将通过使用来自Nrf 2缺陷小鼠的细胞、抑制Nrf 2的DNA诱饵、过表达Nrf 2的基因转移和激活Nrf 2的药理学试剂来确定Nrf 2的细胞保护作用（包括吞噬功能的保存）。(SA2)研究Nrf 2作为促进ICH后血肿消退的机制。我们将使用Nrf 2，CD 36和Hp缺陷小鼠（或从这些小鼠中分离的小胶质细胞）来探索Nrf 2及其下游靶点，以及CD 36和Hp在血肿消退中的作用及其对继发性脑损伤的影响。(SA3)研究莱菔硫烷（Nrf 2激活剂）治疗ICH的临床效用。我们将采用小鼠脑出血模型，优化SF治疗脑出血的条件。我们将首先根据最高效率、安全性、治疗持续时间和有效治疗小鼠ICH的最长时间窗确定最佳治疗剂量。我们的长期目标是探索减少ICH发病机制和改善功能恢复的药理学和分子治疗。公共卫生相关性：脑出血（ICH）占所有卒中的10 - 15%，1年死亡率大于50- 60%。目前尚无FDA批准的ICH有效治疗方法。在我们的研究中，我们将确定转录因子Nrf 2的激活是否可能代表ICH治疗的可行靶点，转录因子Nrf 2调节许多基因的表达，这些基因增加脑细胞对ICH诱导的损伤的抵抗力。我们的目标是探索Nrf 2有益作用的潜在分子机制，并建立基于Nrf 2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.