α1-抗胰蛋白酶通过抑制铁超载减轻早产脑白质损伤的机制研究

The life quality of preterm infants are seriously threatened by white matter damage and its long-term neurological sequelae. Our previous clinical studies showed that plasma alpha 1-antitrypsin (AAT) level in premature infants with white matter damage was significantly lower than that in preterm infants without brain injury, and was associated with long-term outcomes. It is reported that iron chelating agents could reduce oligodendrocyte injury by inhibiting microglia activation in vitro. AAT could inhibit transferrin receptor, and decrease inflammatory cytokines when bound to cell surface receptor CD36 in monocytes. We hypothesize that AAT protects oligodendrocytes against lipid peroxidation and cell death in premature brain by inhibiting iron overload mediated oxidative stress and release of inflammatory cytokines. In this study, we aim at investigating the role of AAT in preterm white matter injury by testing the effects on a 5-day-old mouse cerebral hypoxia-ischemia white matter injury model, supplemented intranasally or intraperitoneally with AAT to assess its potential short-term and long-term protective effects. In addition, transgenic AAT deficiency mice will be used to verify the effects of AAT, the association between AAT and rion overload, and the cellular and molecular mechanisms of AAT. This study will supply a new method to prevent preterm white matter damage and serious neurological sequelae.

早产儿脑白质损伤及其远期神经系统后遗症是导致儿童伤残的首位原因，目前尚无特效防治措施。我们前期临床研究显示脑白质损伤早产儿血浆α1-抗胰蛋白酶（AAT）水平显著降低并与长期预后有关。体外研究显示铁螯合剂能减轻小胶质细胞活化介导的少突胶质细胞损伤。AAT能够抑制单核细胞转铁蛋白受体的铁转运，且与细胞表面受体CD36结合后能够抑制炎症因子的产生。因此我们假设，AAT通过抑制铁超载介导的小胶质细胞氧化应激和炎症因子的释放，减轻少突胶质细胞脂质过氧化损伤和细胞死亡。本课题拟采用新生5日龄小鼠和AAT缺陷转基因小鼠制备早产脑白质损伤模型结合不同的给药途径，应用免疫组化、蛋白印迹、ELISA、Luminex、流式细胞术、动物神经行为学实验技术与方法，探索AAT对早产脑白质损伤的近期和远期神经保护作用，及其与铁超载的关系和细胞分子机制，为早产儿脑白质损伤及严重神经系统后遗症的防治提供新的措施。

早产脑损伤是导致儿童认知和运动发育障碍的常见原因之一，针对其有效的治疗方法目前也很有限。α1抗胰蛋白酶是一种内源性丝氨酸蛋白酶抑制剂，具有抗炎、抗细胞凋亡和细胞保护作用，可能有助于治疗早产脑损伤。本研究的目的是研究α1抗胰蛋白酶在小鼠早产脑损伤模型中是否具有神经保护作用。在出生第5天的C57BL/6J小鼠幼崽中，通过动脉结扎的方式阻断小鼠幼崽的右侧颈总动脉血流，然后进行缺氧诱导。免疫组织化学染色和核磁共振成像评估脑损伤的程度。Fluoro-Jade B化学荧光染色和免疫组织化学染色以研究神经元细胞死亡和血脑屏障通透性。CatWalk运动步态分析和矿场实验监测运动功能和焦虑样行为。实验结果发现，在缺氧缺血脑损伤后的小鼠中给予α1抗胰蛋白酶治疗可减轻小鼠的脑损伤，并伴有血脑屏障通透性降低、神经元细胞死亡减少，caspase-3激活减少以及小胶质细胞激活被抑制。此外，α1抗胰蛋白酶治疗显着改善了小鼠缺氧缺血脑损伤后引起的运动功能障碍，并且发现α1抗胰蛋白酶的神经保护作用在雄性小鼠中更为显著。总之，研究结果表明，α1抗胰蛋白酶治疗对缺氧缺血诱导的新生小鼠早产脑损伤具有神经保护作用，并且这种作用在雄性小鼠中更为明显。本研究为早产儿脑白质损伤的治疗提供了一种新的有前景的药物，有利于改善早产脑损伤后的长期预后，提高生存质量。