NEONATAL BILIRUBIN NEUROTOXICITY AND P-GLYCOPROTEIN

未结合的高胆红素血症是新生儿时期最常见的临床疾病，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，近期出院的婴儿核黄疸的再次出现证明了这一点。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通过血脑屏障（BBB）进入中枢神经系统（CNS）。最近的研究表明胆红素是ATP依赖的整体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，并限制了各种亲脂化合物的脑流入。我们观察到：(1)P-gp缺陷的成年转基因小鼠脑胆红素摄取显著增加；(2)与成年小鼠相比，胎儿和新生野生型小鼠脑中P-gp的表达（mRNA和蛋白）显著降低。这些发现表明P-gp在阻止胆红素流入中枢神经系统中起重要作用，并且在新生儿脑微血管中有限的P-gp表达可能会增加该年龄组脑胆红素的摄取。提出的实验旨在验证三个假设：1)胆红素与P-gp相互作用并由P-gp运输；2)大脑P-gp的表达、水平和功能受时间和空间方式的调节，3)代谢抑制、缺氧和/或缺氧/缺血联合损害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了人或小鼠P-gp基因的lc - pk1细胞）和体内（野生型和P-gp缺陷小鼠）模型来表征1)胆红素和P-gp之间的相互作用，ii)中枢神经系统P-gp的个体发生和区域表达，以及iii)代谢抑制对P-gp的影响。除了我们的初步研究外，没有关于P-gp在中枢神经系统中的发育表达的信息，只有关于胆红素和P-gp相互作用以及影响P-gp功能因素的有限数据。建议的研究结果将充分检验我们上述的假设。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血脑屏障P-gp表达的模式，从而增强对新生儿胆红素神经毒性的保护。

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.