肝损伤与血脑屏障上P-GP和MRP2等ABC类转运体功能/表达改变及其对脑内药物处置的影响

Hepatic encephalopathy is a main neuropsychiatric complication of both acute and chronic liver failure. Our previous study showed that liver injury induced by thioacetamide impaired function and expression of P-glycoprotein(P-GP), but enhanced function and expression of multidrug resistance-assocaited protein 2(MRP2) at blood brain barrier(BBB) of rats. The aim of the project was to investigate:1)function and expression of ABC(ATP-binding cassette) transporters including P-GP, MRP2 and breast cancer resistance protein(BCRP) in BBB of rats suffered from liver injury using their specific substrates, western blot and QT-PCR; their alteration in ABC trnasporter-dependent manner; effect of the alterations in function and expresssion of the ABC transporters on neurosteroid accumulations and dispostion of substrate drugs in brain of rats suffered from liver injury. In vitro, effect of serum derived from rats suffered from liver injury and their abnormal components such as ammonemia on expression and function of the ABC transporters in rat brain microvessel endothelial cells(rBMECs).At the same time, the signaling proteins involved in the regulation of ABC transporters were also documented. Transport characters of neurosteroids across rBMEC monolayer were also measured to investigate contribituion the ABC transporters to transport of the neurosteroids across BBB.Some results obtained in the cells were further confirmed using gene silence and gene transfection. The results may highlight the roles of ABC transporter alteration at BBB in development of hepatic encephalopathy as well as guidance for drug use and prediction of drug adverse effects in patents suffered liver injury.

肝性脑病是肝损伤主要并发症。我们前期发现肝损伤大鼠血脑屏障（BBB）上P-糖蛋白（P-GP）功能与表达受损，而多药耐药相关蛋白2（MRP2）功能与表达加强。用其特异性底物以及western blot和QT-PCR等研究肝损伤大鼠BBB上P-GP、MRP2和乳腺癌耐药蛋白（BCRP）等ABC转运体功能与表达改变及机制；这种改变对脑内神经类固醇蓄积和苯巴比妥等底物药物在脑内处置影响；高血氨等对大鼠BBB上P-GP、MRP2和BCRP等ABC转运体功能与表达影响。用脑血管内皮细胞研究肝损伤大鼠血清或铵盐等血清异常成分对P-GP、MRP2和BCRP等ABC转运体功能与表达影响、相关的信号通路及其脑内神经类固醇在细胞中转运，其结果进一步用基因沉默和基因转染细胞验证。其成果对于阐明BBB上P-GP、MRP2和BCRP等ABC运体在肝性脑病中作用、临床合理用药和不良反应预测有重要意义。

肝性脑病是肝损伤主要并发症。用硫代乙酰胺(TAA)、胆管结扎（BDL）和门脉结扎(PVL)等三种方法诱导肝损伤动物模型，结合体外血脑屏障（BBB）细胞模型，研究：1) 肝损伤时BBB上P-糖蛋白（P-GP）、多药耐药相关蛋白2(MRP2) 和乳腺癌耐药蛋白(BCRP)等ABC转运体功能和表达；2) 引起这些ABC 转运体功能和表达改变可能机制；3) BBB 上P-GP和BCRP等功能和表达改变对齐多夫定（AZT）和苯巴比妥等药物在脑内处置和中枢活性/毒性影响。结果显示不同机制引起的肝损伤，对BBB上P-GP、BCRP和MRP2的功能和表达影响是不同的。TAA诱导的肝损伤下调P-GP和BCRP功能和表达，而上调MRP2功能和表达。血氨、胆红素和胆酸盐水平升高可能是引起这些转运体功能和表达改变主要因素。TAA诱导的肝损伤和腹腔注射醋酸铵引起高血氨均显著下调BCRP功能和表达，这种下调作用与氨-ROS-ERK1/2通路激活有关。尽管高血氨是肝损伤主要特征，但急性高血氨显著上调P-GP和MRP2功能和表达，而不是下调P-GP功能和与表达，这一点不同于TAA引起的肝损伤。体外结果显示高浓度氨诱导P-GP和MRP2功能和表达与NF-B信号通路激活有关。醋酸铵饮食或醋酸铵饮食加PVL引起的慢性高血氨也能上调P-GP功能和表达，但这种诱导作用与ROS-ERK1/2通路激活有关。低浓度氨或慢性高血氨通过ROS-ERK1/2通路激活促进P-GP膜转导。高胆红素血症是胆汁淤积肝损伤引起BCRP功能和表达下调主要原因，而高鹅去氧胆酸可能是TAA引起肝损伤下调P-GP功能和表达的主要因素。BBB上P-GP和BCRP功能下调，增加苯巴比妥和齐多夫定（AZT）等脑内分布，增加药物的中枢活性和毒性。其成果对于阐明BBB上P-GP、MRP2和BCRP等ABC运体在肝性脑病中作用、临床合理用药和不良反应预测有重要意义。