NEONATAL BILIRUBIN NEUROTOXICITY AND P-GLYCOPROTEIN

新生儿胆红素神经毒性和 P-糖蛋白

• 批准号: 6188292
• 负责人: JON F WATCHKO
• 金额: $ 19.27万
• 依托单位: MAGEE-WOMEN'S HOSPITAL OF UPMC
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-17 至 2003-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6188292
• 关键词: P glycoprotein; adenosinetriphosphatase; affinity labeling; autoradiography; azides; bilirubin; biological transport; blood brain barrier; brain circulation; brain mapping; genetic regulation; genetically modified animals; glycoprotein biosynthesis; hypoxia neonatorum; intermolecular interaction; kernicterus; laboratory mouse; neonatal transient jaundice; neuroprotectants; neurotoxins; porphyrin metabolism; radiotracer; tissue /cell culture; vascular endothelium permeability

Unconjugated hyperbilirubinemia is the most common clinical condition in the newborn period and when severe can result in neurologic injury with long term adverse neurodevelopment sequelae as evidenced by the reemergence of kernicterus in near-term infants subject to early hospital discharge. The pathogenesis of hyperbilirubinemic encephalopathy remains unclear but central to its development is the passage of bilirubin across the blood-brain barrier (BBB) into the central nervous system (CNS). Recent studies suggest that bilirubin is a substrate for the ATP dependent integral plasma membrane efflux pump phosphoglycoprotein or P-gp. P-gp is expressed in abundance on the luminal aspect of brain capillary endothelial cells and limits the brain influx of a variety of lipophilic compounds. We have observed i) that brain bilirubin uptake is significantly increased in adult P-gp deficient transgenie mice, and ii) that P-gp expression (mRNA and protein) is markedly lower in the fetal and neonatal wild type mouse brain as compared with adults. These findings suggest that P-gp plays an important role in preventing the influx of bilirubin into the CNS and that limited P-gp expression in the newborn brain microvasculature may enhance brain bilirubin uptake in this age group. The proposed experiments are designed to test three hypotheses: 1) that bilirubin interacts with and is transported by P-gp; 2) that brain P-gp expression, levels, and function are regulated in a temporal and spatial fashion, and 3) that metabolic inhibition, hypoxia, and/or a combination of hypoxia/ischemia impairs P-gp function. We will use in vitro (radioligand binding and photoaffinity labeling), cellular (mouse brain capillary endothelial cells, and LLC-PK1 cells transfected with the gene for human or mouse P-gp), and in vivo (wild type and P-gp deficient mice) models to characterize the i) interaction between bilirubin and P-gp, ii) the ontogeny and regional expression of CNS P-gp, and iii) the effects of metabolic inhibition on P-gp. Other than our preliminary studies, there is no information regarding the developmental expression of P-gp in the CNS, and only limited data on the interaction between bilirubin and P-gp and the factors that impact P-gp function. Results of the proposed studies will fully test our above stated hypotheses. The information obtained will provide novel insights regarding the role P-gp plays in attenuating brain bilirubin uptake and serve as an impetus towards developing modalities that increase BBB P-gp expression in newborns thereby enhancing protection against neonatal bilirubin neurotoxicity.

非结合高胆红素血症是新生儿期最常见的临床病症，严重时可导致神经损伤，并伴有长期不良的神经发育后遗症，如早期出院的近足月婴儿再次出现核黄疸即可证明。 高胆红素血症性脑病的发病机制尚不清楚，但其发展的核心是胆红素穿过血脑屏障（BBB）进入中枢神经系统（CNS）。 最近的研究表明，胆红素是 ATP 依赖性积分质膜外排泵磷酸糖蛋白或 P-gp 的底物。 P-gp 在脑毛细血管内皮细胞的管腔方面大量表达，并限制多种亲脂性化合物的脑流入。 我们观察到 i) 成年 P-gp 缺陷转基因小鼠的脑胆红素摄取显着增加，ii) 与成人相比，胎儿和新生野生型小鼠大脑中 P-gp 表达（mRNA 和蛋白质）显着降低。这些发现表明，P-gp 在防止胆红素流入中枢神经系统方面发挥着重要作用，新生儿脑微血管中有限的 P-gp 表达可能会增强该年龄组的脑胆红素摄取。 所提出的实验旨在检验三个假设：1）胆红素与 P-gp 相互作用并由 P-gp 转运； 2) 大脑 P-gp 表达、水平和功能以时间和空间方式受到调节，3) 代谢抑制、缺氧和/或缺氧/缺血的组合会损害 P-gp 功能。 我们将使用体外（放射性配体结合和光亲和标记）、细胞（小鼠脑毛细血管内皮细胞和转染人或小鼠 P-gp 基因的 LLC-PK1 细胞）和体内（野生型和 P-gp 缺陷型）小鼠）模型来表征 i）胆红素和 P-gp 之间的相互作用，ii）CNS P-gp 的个体发育和区域表达，以及 iii）代谢抑制的影响在 P-gp 上。除了我们的初步研究之外，没有关于中枢神经系统中 P-gp 发育表达的信息，关于胆红素和 P-gp 之间的相互作用以及影响 P-gp 功能的因素的数据也很有限。 拟议研究的结果将充分检验我们的上述假设。所获得的信息将提供关于 P-gp 在减弱脑胆红素摄取中的作用的新见解，并推动开发增加新生儿 BBB P-gp 表达的方式，从而增强对新生儿胆红素神经毒性的保护。