Effect of Inducible Antioxidants on Hemoglobin Toxicity

描述(申请人提供)：出血合并许多中枢神经系统的创伤性损伤和大约20%的中风。在接下来的几个小时里，红细胞溶解并将其内容物释放到血管外空间。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Hb的氧化毒性参与了出血性中枢神经系统损伤的发病机制。此外，由于其时间较长，Hb毒性可能是治疗干预的理想靶点。因此，进一步了解这种毒性的细胞机制和预防似乎是可取的。 培养的神经元对HB非常脆弱，但星形胶质细胞通过一种需要蛋白质合成的机制来抵抗。初步实验表明，这种差异可能部分是由两种可诱导的抗氧化剂：血红素加氧酶(HO)和铁蛋白的影响造成的。前者受Hb快速诱导，可能促进星形胶质细胞合成富含L的铁蛋白。相反，Hb减少了富含L的铁蛋白在神经元中的表达；作为血红素分解的产物释放的铁可能是有毒的。 这个项目将解决HO和铁蛋白在细胞培养和体内模型中的作用。HO-1的过度表达将通过基因转移在胶质细胞、神经元或混合培养中完成；活性、血红素介导的活性氧形成和细胞死亡之间的关系将被建立。然后将比较野生型、HO-1基因敲除和HO-2基因敲除小鼠准备的培养物中细胞对Hb或氯化血红素的易感性。使用专门识别H-或L-铁蛋白的抗体，铁蛋白的亚基含量将在基线时进行评估，并对这些培养物中的Hb做出反应。还将测定HasA的表达，HasA与血红素铁结合并可能促进血红素铁的吸收。富含H和L的铁蛋白杂化聚合物将由重组的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异聚体，将有助于研究H：L比率对细胞对血红素介导的损伤的易感性的影响。最后，将野生型、HO-1或HO-2基因敲除的壳核和过度表达HO-1的转基因小鼠注射Hb或胶原酶，以诱导内源性出血。然后将在注射后12-96小时的特定时间点对周围神经元的丢失、DNA切割和caspase-3激活进行量化。

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.