CHANGES OF BILIARY EXCRETORY FUNCTION AND CANALICULAR CARRIES IN INTRAHEPATIC CHOLESTASIS

从肝细胞到肝管膜的转运是胆汁酸和有机阴离子在肝内转运的限速步骤，分别由胆汁盐输出泵（Bsep）和多药耐药蛋白2 （Mrp2）介导。在肝内胆汁淤积症中，胆汁酸和有机阴离子的胆汁排泄受损，在实验性胆汁淤积症中也得到了类似的结果。因此，我们研究了这些转运体在phalloidin和脂多糖诱导的具有代表性的胆汁淤积症中的变化。通过将Bsep和Mrp2转运体的c端肽偶联到KLH上，产生了抗Bsep和Mrp2的抗体。抗Bsep抗体的特异性受转染Bsep的Sf9细胞的质膜限制，抗mrp2抗体的特异性受Sprague-Dawley大鼠及其高胆红素突变体EHBR的胆管膜限制。静脉注射phalloidin (0.5 mg/kg) 90分钟或静脉注射脂多糖（2.5 mg/kg） 18小时后，取肝脏，匀浆，经蔗糖梯度离心得到的膜组分进行Western blotting。phalloidin治疗后，Bsep和Mrp2的量均显著降低，表明这些转运蛋白对小管膜的靶向性受损。脂多糖处理后，Mrp2的表达量明显降低至<5%，而Bsep的表达量则适度降低（60%）。这些结果可以解释脂多糖治疗对有机阴离子排泄的选择性损害，以及脓毒症中仅伴有轻度胆汁淤积的明显高胆红素血症。

Transport from the hepatocytes to the canalicular membrane is a rate-limiting step of hepatic transport of bile acids and organic anions, which is mediated by bile salt export pump (Bsep) and multidrug resistance protein 2 (Mrp2), respectively. In intrahepatic cholestasis, biliary excretion of bile acids and organic anions is impaired, and similar results were obtained in experimental cholestasis. Therefore, the changes of these transporters in representative cholestasis induced by phalloidin and lipopolysaccharide was studied. Antibodies against Bsep and Mrp2 were raised by using C-terminal peptides of these transporters coupled to KLH. The specificity of anti-Bsep antibody was confined by plasma membrane of Sf9 cells transfected with Bsep, and that of anti-Mrp2 antibody was confined by bile canalicular membrane of Sprague-Dawley rats and their hyperbilirubinemic mutant, EHBR. Ninety minutes after intravenous phalloidin injection (0.5 mg/kg) or 18 hours after intravenous lipopolysaccharide injection (2.5 mg/kg), the liver was removed, homogenated, and then, the membrane fraction obtained by a sucrose gradient centrifugation was subjected to Western blotting. Both the amount of Bsep and Mrp2 were markedly decreased by phalloidin treatment, suggesting an impaired vesicular targeting of these transporters to the canalicular membrane. By lipopolysaccharide treatment, the amount of Mrp2 was markedly decreased to <5%, whereas the decrease of Bsep was moderate (60%). These results may explain the selective impairment of organic anion excretion by lipopolysaccharide treatment, as well as a marked hyperbilirubinemia only with mild cholestasis in sepsis.