Effect of Inducible Antioxidants on Hemoglobin Toxicity

描述(申请人提供)：出血伴随着最严重的脑创伤，是大约15%的中风的主要事件。越来越多的证据表明，溶解的红细胞释放的血红蛋白可能会对血肿周围的组织造成氧化损伤。先前的研究表明，Hb的毒性是通过将其血红素部分转移到附近的细胞而介导的，这有利于其氧化为高铁血红蛋白。亚铁血红素是亚铁血红素的氧化形式，然后被亚铁血红素加氧酶分解为铁、一氧化碳和胆绿素。如果铁隔离不足，后两种产品的细胞保护作用可能会被压倒。然而，神经元和星形胶质细胞的铁解毒策略在很大程度上仍然不清楚。初步实验表明，神经元输出细胞外铁，必须将其隔离以防止氧化膜损伤；星形胶质细胞诱导铁蛋白，与细胞内存储一致。该项目的目标是确定铁蛋白和转铁蛋白在减轻血红素介导的神经元和星形胶质细胞损伤中的作用，并设计一种策略来优化这一过程。我们的实验目的如下：1)通过腺病毒转导H或L铁蛋白亚基在培养的神经元和星形胶质细胞中特异性地增加其表达。或者，培养铁调节蛋白(IRP)基因敲除小鼠的星形胶质细胞和神经元，它们显著增加了细胞铁蛋白。确定铁蛋白基因转移或IRP基因敲除对Hb或氯化血红素暴露后培养物活氧物种(ROS)的形成、蛋白质氧化和细胞活力的影响。2)用[55Fe]氯化血红素处理培养的神经元和星形胶质细胞。定量随后的~(55)Fe释放到培养基中或沉积在细胞铁蛋白中。评估载脂蛋白和全转铁蛋白对细胞对血红素介导的损伤的易感性的影响。3)立体定向向低转铁蛋白血症小鼠或非突变小鼠纹状体内注血。或者，通过腺病毒将铁蛋白基因或shRNA转移到IRP，增加注射血液的小鼠中铁蛋白的表达。在72小时和144小时，确定对细胞活力、蛋白质和脂肪氧化以及周围组织轴突损伤的影响。从这个项目中获得的信息可能会为出血性中风和头部创伤的受害者带来新的治疗方法。最终目标是减少血液凝块周围组织中的脑损伤，从而提高生存的可能性，并恢复独立的、富有成效的生活。

DESCRIPTION (provided by applicant): Hemorrhage accompanies most significant brain trauma, and is the primary event in about 15% of strokes. A growing body of evidence suggests that hemoglobin (Hb) release from lysing erythrocytes may contribute to oxidative injury to tissue surrounding a hematoma. Prior studies have demonstrated that Hb toxicity is mediated by transfer of its heme moieties to nearby cells, which is favored by its oxidation to methemoglobin. Hemin, the oxidized form of heme, is then catabolized by the heme oxygenase enzymes to iron, carbon monoxide, and biliverdin. The cytoprotective effects of the latter two products may be overwhelmed if iron sequestration is inadequate. However, the iron detoxification strategies of neurons and astrocytes remain largely undefined. Preliminary experiments suggest that neurons export extracellular iron, where it must be sequestered to prevent oxidative membrane injury; astrocytes induce ferritin, consistent with intracellular storage. The goal of this project is to define the role of ferritin and transferrin in mitigating heme-mediated injury to neurons and astrocytes, and to devise a strategy to optimize this process. Our experimental aims are as follows: 1) Specifically increase expression of H or L-ferritin subunits in cultured neurons and astrocytes by adenoviral transfer of their genes. Alternatively, culture astrocytes and neurons from iron regulatory protein (IRP) knockout mice, which have markedly increased cell ferritin. Determine the effect of ferritin gene transfer or IRP knockdown on culture reactive oxygen species (ROS) formation, protein oxidation, and cell viability after Hb or hemin exposure. 2) Treat cultured neurons and astrocytes with [55Fe]hemin. Quantify subsequent 55Fe release into the culture medium or deposited in cell ferritin. Assess the effect of apotransferrin and holotransferrin on cell vulnerability to heme-mediated injury. 3) Stereo- tactically inject blood into the striata of hypotransferrinemic mice or non-mutant littermates. Alternatively, increase ferritin expression in blood-injected mice by adenoviral transfer of ferritin genes or shRNA to IRP's. At 72 and 144 hours, determine the effect on cell viability, protein and lipid oxidation, and axonal injury in surrounding tissue. The information gained in this project may lead to new treatments for victims of hemorrhagic stroke and head trauma. The ultimate goal is to reduce brain injury in tissue surrounding a blood clot, and to thereby improve the likelihood of survival and return to an independent, productive life.