胆汁淤积下肝脏水通道蛋白AQP10下调的效应及分子机制研究

Cholestasis is a common clinical syndrome caused by bile formation, secretion and excretion disorders, which can lead to liver fibrosis,cirrhosis, liver failure, etc, while its pathogenesis still not clear.The main components of bile are water and bile acid.Previous studies mainly focused on cholic acid transporters, while few studies on the molecular basis and regulatory mechanisms of hepatocyte water transport during bile formation.Recently, it has been reported that the high expression of aquaporin8 can promote bile excretion and fight against liver damage caused by cholestasis.Our preliminary experiments found that the expression of aquaporin10 in human obstructive cholestasis liver was significantly lower than that in control, and TCA was found to significantly inhibit the expression of AQP10.AQP10 can increase the membrane water permeability, the low expression in hepatic may lead to water permeability decreased and the bile secretion decreased, resulting in bile secretion dysfunction, increasing liver damage caused by cholestasis.Based on this, we speculated that cholic acid in cholestasis can inhibit the expression of AQP10 and cause bile excretion dysfunction to aggravate cholestasis.In this project we will intends to use the biological techniques of AQP10 gene knockin mice and primary hepatocyte culture, to clarified the molecular mechanism and functional role of low expression of AQP10 in cholestasis,and this work is expected to provide a new theory for the mechanism of cholestasis liver disease and contribute to a new clue for clinical treatment.

胆汁淤积是由胆汁形成、分泌和排泄障碍引起的一种常见临床综合征，易导致肝纤维化、肝硬化、肝衰竭等，但其发病机制不清楚。胆汁最主要成分是水和胆汁酸，前期研究主要聚焦于胆酸转运蛋白，但对水通道蛋白的研究甚少。近期有文献报道，水通道蛋白AQP8高表达可以促进胆汁排泄而对抗胆汁淤积肝损害。我们预实验提示在胆汁淤积患者肝脏中AQP10低表达，同时发现牛磺胆酸TCA可以明显抑制水通道蛋白AQP10的表达。AQP10能增加细胞膜水的渗透性，肝脏中低表达可能导致胆管面膜水渗透性降低引起胆汁分泌减少，从而导致胆汁分泌功能障碍，引起胆汁淤积肝损害加重。据此我们推测胆汁淤积下胆酸可以通过抑制AQP10的表达，而引起胆汁排泌障碍加重胆汁淤积。本项目拟运用AQP10基因敲入小鼠、原代肝细胞培养等生物学技术，阐明AQP10在胆汁淤积下低表达的分子机制及效应，为胆汁淤积肝病发生的机制提供新的理论，为临床治疗提供新的线索。

胆汁形成和分泌受损会导致胆汁淤积，其特征是肝脏和血清中胆汁酸的过度集聚。现大多数研究揭示了胆汁淤积下的适应性反应，包括抑制胆汁酸合成和增强胆汁酸外排。然而值得注意的是，水占胆汁的95%，水通道蛋白（AQPs）对胆汁形成至关重要。最近研究报道，AQP8和AQP9在胆管结扎(BDL)大鼠肝脏中表达下调，且在大鼠肝脏中过表达人AQP1可减轻胆汁淤积。然而，水通道蛋白10（AQP10）在人胆汁淤积肝脏中的表达及其调控机制尚不清楚。为明确水通道蛋白10在人胆汁淤积肝脏中的表达，我们收集了34例梗阻性胆汁淤积患者和12例对照组的血清和肝脏样本，检测肝脏中AQP10 mRNA及蛋白表达水平。为探讨胆汁淤积下AQP10发挥的生物学功能，构建了AAV8-Alb启动子-hAQP10腺相关病毒，并通过尾静脉注射构建肝细胞特异性过表达hAQP10小鼠，并造胆汁淤积小鼠模型用于功能研究。同时构建了PLC/PRF/5-Ctrl，PLC/PRF/5-ASBT，PLC/PRF/5-AQP10，以及PLC/PRF/5-ASBT-AQP10细胞系进一步研究胆汁淤积下AQP10的功能。通过荧光素酶及染色质免疫共沉淀等技术阐明胆汁淤积下AQP10下调的具体分子机制。研究结果表明，AQP10在梗阻性胆汁淤积症患者中明显下调，且与血清总胆汁酸(TBA)水平呈负相关。肝细胞特异性过表达hAQP10可显著减轻BDL小鼠胆汁淤积性肝损伤和肝内胆汁酸积累。结合胆汁酸，如TCA及炎症因子TNFα，能显著抑制AQP10的表达。此外，通过荧光素酶及染色质免疫共沉淀证实NFκB p65可直接结合AQP10启动子，降低其转录活性。然而，加入NFκB信号通路特异性抑制剂(BAY11-7082)这些变化被抑制了。在胆汁淤积发生时，结合胆汁酸及TNFα可通过激活 NFκB信号通路刺激转录因子p65表达上调，从而抑制肝细胞中水通道蛋白AQP10 的表达。肝细胞特异性过表达AQP10显著减轻了BDL小鼠的胆汁淤积性肝损伤和肝内胆汁酸积聚。因此，AQP10在肝脏中的过表达可能是治疗胆汁淤积症的新策略。