水通道蛋白8在胆汁流障碍引起的肝内胆汁淤积中的作用及机制研究

Intrhepatic cholestasis was caused by bile flow impairment，but it was not clear to impairment of bile flow. Plasma (canalicular) membrane of hepatocytes was rate-limiting step for bile flow into canaliculi. Aquaporin8(AQP8), it was water channel protein which can transport water across cell membrane dependent on osmotic pressure in fluid, was expressed in canaliculi membrane of hepatocytes. The high level of expression in canaliculi membrane suggested that an important role for AQP8 in the pathophysiological function of liver. So we investigated the possible role of AQP8 in water transport during bile flow formation and obstruction and how mechanisms by AQP8 deficient mice.In the previous study, we collected bile from AQP8 deficient mice and wild type mice. It showed that bile and bile flow were significantly decreased in AQP8 deficient mice under both basal and bile salt-stimulated conditions. Bile composition analysis showed water was decreased in bile of AQP8 deficient mice and indicated that AQP8-facilitated canalicular membrane water transport was involved in hepatic bile flow formation. To further confirm the mechanism of reduced hepatic bile flow formation in liver of AQP8 deficient mice, we would make intrahepatic cholestasis model by estrogen in wild type and AQP8 deficient mice. We would first collect bile in two group mice and bile composition also would be assayed including bile salt, glutathione and membrane phospholipid phosphatidylcholine (PC). To assay density and diameter and area of bile canaliculi of liver and AQP8 expression, using immunostaining and fluorescence and western blotting in tissue level and cell level. It would indicate that damage of membrane structure of bile canaliculi of liver was key cause for bile flow impairment and it was associated with AQP8 expression of liver in mice. Moreover, we would examine bile salt export pump(BSEP) excretion assay and compare the water permeability of canaliculi membrane of hepatocytes with AQP8 deficient mice and wild type mice. To evaluate that AQP8 deficient affect water transport across canalicular membrane of hepatocytes into canaliculi,whereas increase BSEP extration activity. So the high level of bile salt in bile contribute to damage of lipid of canalicular membrane. Together, those studies would support the new notion that deficient of AQP8 would affect structure of canalicular membrane of hepatocytes resulting from high level of bile salt in bile which decreased water permeability of canalicular membrane and leading in damage of bile canaliculi and impairment of bile flow.

胆汁流障碍是引起胆汁淤积的重要因素。在胆汁流形成过程中，毛细胆管膜对胆汁流具有限速作用，但胆汁流障碍的形成机制尚不明确。水通道蛋白8（Aquaporin-8，AQP8）大量表达于毛细胆管膜上，是选择性高效转运水分子的特异性孔道。在前期工作中，通过对AQP8基因敲除小鼠胆汁分泌量与成分的测量，发现AQP8敲除明显减少了胆汁分泌量并增加胆盐分泌量。本申请拟利用AQP8敲除小鼠建立肝内胆汁淤积模型，进一步揭示是否AQP8敲除影响了水的跨膜转运，反馈性增强毛细胆管膜上胆盐输出泵(BSEP)的表达和分泌活性，使胆汁中胆盐和磷脂酰胆碱形成混合微粒的比例失衡，破坏了毛细胆管膜上磷脂成分，继而影响了毛细胆管膜的结构稳定性，引起毛细胆管形成障碍，导致小鼠胆汁流速和分泌量减少，形成胆汁流障碍造成胆汁淤积的分子机制。可能为胆汁流障碍引起的胆汁淤积的研究提供新的理论依据和药物治疗的新靶点。

肝内胆汁淤积是临床常见的肝脏基础病变，其发生机制十分复杂。近年来国内外对肝内胆汁淤积的发生机制研究虽取得很大进展。但具体的机制仍需深入探究。水通道蛋白8（Aquaporin-8，AQP8）高表达于肝细胞的毛细胆管膜上，影响了毛细胆管膜上水的转运。我们前期利用AQP8基因敲除小鼠，发现AQP8参与了小鼠的胆汁流速和分泌；同时还影响了胆盐的分泌。进而我们提出假说：由于AQP8基因敲除，可能改变毛细胆管膜的水通透性，影响了水的跨膜转运。反馈性增强胆盐输出泵（BSEP）的表达与分泌活性，使胆汁中胆盐与磷脂酰胆碱形成混合微粒的比例失衡，破坏毛细胆管膜上磷脂成分，继而影响了毛细胆管膜结构稳定性，引起毛细胆管形成障碍，导致胆汁流速和分泌量减少，形成胆汁流障碍，最终造成肝内胆汁淤积。通过实验证实：AQP8 敲除减少了毛细胆管形成的数量和减小了毛细胆管腔的面积；.降低了肝细胞的胆汁分泌。在小鼠胆汁淤积模型中，AQP8敲除破坏了毛细胆管的管状结构，同时伴有肝细胞毛细胆管膜上的Mrp2蛋白的表达量降低，导致AQP8敲除小鼠出现明显黄染症状。从而明确AQP8维持了毛细胆管膜结构稳定性，稳定毛细胆管膜上胆盐输出泵（BSEP）的表达与活性，并保证毛细胆管的正常形成，从而维持胆汁流的正常生成。