NEONATAL BILIRUBIN NEUROTOXICITY AND P-GLYCOPROTEIN

未结合型高胆红素血症是新生儿期最常见的临床症状，严重时可导致神经损伤和长期的不良神经发育后遗症，在早期出院的近期婴儿中再次出现核黄体症就是明证。高胆红素血症脑病的发病机制尚不清楚，但胆红素穿过血脑屏障(BBB)进入中枢神经系统(CNS)是其发展的核心。最近的研究表明，胆红素是ATP依赖的完整质膜外排泵磷酸糖蛋白(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内流入。我们观察到：1)成年P-gp基因缺陷转基因小鼠的脑胆红素摄取显著增加；2)P-gp在胚胎和新生野生型小鼠脑中的表达(mRNA和蛋白)明显低于成年小鼠。提示P-gp在阻止胆红素进入中枢神经系统起重要作用，新生儿脑微血管中P-gp表达受限可能增强该年龄组的脑胆红素摄取。这些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转运；2)脑内P-gp的表达、水平和功能受时间和空间的调节；3)代谢抑制、缺氧和/或缺氧/缺血的组合损害P-gp的功能。我们将使用体外(放射配基结合和光亲和标记)、细胞(小鼠脑毛细血管内皮细胞和转人或小鼠P-gp基因的LLC-PK1细胞)和体内(野生型和P-gp缺陷小鼠)模型来研究I)胆红素与P-gp的相互作用，ii)CNS P-gp的个体发育和区域表达，以及iii)代谢抑制对P-gp的影响。除我们的初步研究外，目前尚无P-gp在中枢神经系统发育过程中表达的信息，只有有限的数据显示胆红素与P-gp之间的相互作用以及影响P-gp功能的因素。拟议研究的结果将充分检验我们的上述假设。所获得的信息将为P-gp在减弱脑胆红素摄取中所起的作用提供新的见解，并推动开发增加新生儿BBB P-gp表达的方法，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.