Effect of Inducible Antioxidants on Hemoglobin Toxicity

描述（由申请人提供）： 出血使许多CNS创伤性损伤和约20%的中风复杂化。 在随后的几个小时内，红细胞溶解并将其内容物释放到血管外空间中。 释放的最丰富的蛋白质是血红蛋白（Hb）。 越来越多的实验证据表明，细胞外血红蛋白的氧化毒性有助于出血性中枢神经系统损伤的发病机制。 此外，由于其延长的时间过程，Hb毒性可能是治疗干预的理想目标。 因此，进一步深入了解细胞机制和预防这种毒性似乎是可取的。 培养的神经元对Hb非常脆弱，但星形胶质细胞通过需要蛋白质合成的机制具有抵抗力。 初步的实验表明，这种差异可能是部分解释的两种诱导型抗氧化剂的影响：血红素加氧酶（HO）和铁蛋白。 前者是快速诱导的血红蛋白，并可能促进合成富含L-铁蛋白的星形胶质细胞。 相比之下，血红蛋白减少了神经元中富含L的铁蛋白的表达;作为血红素分解产物释放的铁可能是有毒的。 本项目将探讨HO和铁蛋白在细胞培养和体内模型中的作用。 HO-1的过表达将通过基因转移在神经胶质、神经元或混合培养物中完成;将建立活性、血红素介导的活性氧形成和细胞死亡之间的关系。 然后在从野生型、HO-1敲除和HO-2敲除小鼠制备的培养物中比较细胞对Hb或氯化血红素的脆弱性。 使用特异性识别H-或L-铁蛋白的抗体，将在基线和这些培养物中对Hb的反应中评估铁蛋白的亚基含量。 还将测定结合并可促进血红素铁摄取的HasA的表达。 富含H和L的铁蛋白杂聚物将由重组H或L-铁蛋白构建。 神经元和神经胶质细胞通过受体介导的内吞作用摄取这些杂聚物，将允许调查的H：L比对细胞的脆弱性血红素介导的损伤的影响。 最后，将野生型、HO-1或HO-2敲除小鼠和过表达HO-1的转基因小鼠的壳核注射Hb或胶原酶以诱导内源性出血。 然后在注射后12-96小时的规定时间点定量周围神经元丢失、DNA裂解和半胱天冬酶-3活化。

DESCRIPTION (provided by applicant): Hemorrhage complicates many traumatic injuries to the CNS and about 20% of strokes. Over subsequent hours, erythrocytes lyse and release their contents into the extravascular space. The most abundant protein released is hemoglobin (Hb). A growing body of experimental evidence suggests that the oxidative toxicity of extracellular Hb contributes to the pathogenesis of hemorrhagic CNS injury. Moreover, because of its prolonged time course, Hb toxicity may be an ideal target for therapeutic intervention. Further insight into the cellular mechanisms and prevention of this toxicity therefore seems desirable. Cultured neurons are highly vulnerable to Hb, but astrocytes are resistant via a mechanism that requires protein synthesis. Preliminary experiments suggest that this discrepancy may be explained in part by the effects of two inducible antioxidants: heme oxygenase (HO)- and ferritin. The former is rapidly induced by Hb and may facilitate synthesis of L-rich ferritin in astrocytes. In contrast, Hb decreases the expression of L-rich ferritin in neurons; iron released as a product of heme breakdown may then be toxic. This project will address the role of HO and ferritin in cell culture and in vivo models. Overexpression of HO-1 will be accomplished in glial, neuronal, or mixed cultures via gene transfer; the relationship between activity, heme-mediated reactive oxygen species formation, and cell death will be established. Cellular vulnerability to Hb or hemin will then be compared in cultures prepared from wild-type, HO-1 knockout, and HO-2 knockout mice. Using antibodies that specifically recognize H- or L-ferritin, the subunit content of ferritin will be assessed at baseline and in response to Hb in these cultures. Expression of HasA, which binds to and may facilitate heme iron uptake, will also be determined. H and L-rich ferritin heteropolymers will be constructed from recombinant H or L-ferritin. Neuronal and glial uptake of these heteropolymers via receptor-mediated endocytosis will allow investigation of the effect of the H:L ratio on cellular vulnerability to heme-mediated injury. Finally, the putamen of wild type, HO-1 or HO-2 knockout, and transgenic mice that overexpress HO-1 will be injected with Hb, or with collagenase to induce an endogenous hemorrhage. Surrounding neuronal loss, DNA cleavage, and caspase-3 activation will then be quantified at defined time points 12-96 hours after injection.