胆汁酸介导S1PR2/SphK2信号级联激活促进慢性胆管病变分子机制研究

Cholestasis is characterized by dysregulation of the balance between cholangiocyte growth and loss, inflammation and obstruction of bile ducts. The defective intrahepatic bile circulation induces the accumulation of bile in the liver and damage of the liver cells and ultimately leads to end-stage liver disease. The molecular mechanism is unclear. It has been reported that sphingosine 1-phosphate receptor 2 (S1PR2) and sphingosine kinase 2 (SphK2) play important role in cholangiocyte proliferation. Our preliminary studies showed that taurocholic acid (TCA), one of the conjugated bile acids (CBAs), dose-dependently increased the mRNA levels of S1PR2 and SphK2 in mouse large cholangiocytes (MLE). S1P- and TCA-induced cell proliferation and cell migration were inhibited by S1PR2 shRNA in MLE. Bile duct ligation (BDL) significantly up-regulated the mRNA levels of both S1PR2 and SphK2 in mouse primary cholangiocytes. BDL up-regulated S1PR2 and SphK2 in a wild type mouse liver. BDL-induced bile duct proliferation and liver fibrosis were significantly reduced in S1PR2-/- mice as indicated by cytokeratin-19 (CK-19) staining and sirius red staining. BDL significantly reduced the expression of both FXRα and ASBT in mouse primary cholangiocytes. In order to explore the potential cellular/molecular mechanisms by which conjugated bile acids promote chronic cholangiopathy in cholestatic liver disease. We hypothesize that conjugated bile acids-mediated activation of S1PR2/SphK2 signaling cascades plays a critical role in promoting chronic cholangiopathy. In order to validate the hypothesis, we will use many molecular and biochemical methods including overexpression of S1PR2 and SphK2 in cultured cells and in vivo using adenovirus associated virus, knockdown gene expression using lentiviral shRNA, quantitation of the mRNA levels of key genes involved in the regulation of cholangiocyte proliferation, bile acid and S1P transporters, inflammatory response, and liver fibrosis, immunohistochemistry staining, and sirus red staining, et al. In addition, Mass levels of S1P in serum, bile and liver will be determined by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). For in vitro studies, mouse and human cholangiocytes and primary mouse cholangiocytes will be used to determine the signaling pathways of bile acid-induced activation of S1PR2/SphK2. For in vivo studies, common BDL of S1PR2-/-, SphK2-/-, and appropriate wild-type mice will be used to define the role of S1PR2 and SphK2 in bile acids-induced cholangiocyte proliferation and liver fibrosis under cholestatic conditions. Completion of this study will not only identify the potential cellular/molecular mechanisms involved in the initiation and progression of cholestatic liver diseases, but also establish a novel theory in bile acid and S1P biology, which will likely lead to new therapeutic approaches for cholestatic liver diseases and a reduction of morbidity and mortality in patients with cholangiopathies.

胆汁淤积可致胆管细胞增殖、胆管病变和肝细胞损伤，最终引起胆汁淤积性肝病，其发病机理尚不清楚。研究表明鞘氨醇1-磷酸受体2（S1PR2）和鞘氨醇激酶2（SphK2）在胆管细胞增殖中起重要作用。预实验结果显示，胆汁酸诱导的离体胆管细胞和胆汁淤积小鼠的胆管细胞，细胞膜上S1PR2和细胞核内SphK2的表达水平均升高；而胆汁淤积S1PR2-/-小鼠胆管增殖和肝纤维化显著改善。我们提出假说，胆汁酸介导S1PR2/SphK2信号级联激活，促进胆管细胞增殖，引起慢性胆管病变和肝损伤。本项目拟通过野生型、S1PR2-/-和SphK2-/-小鼠胆汁淤积体内模型及胆汁酸诱导的离体胆管细胞体外模型，采用重组腺相关病毒转染、免疫组化、qPCR和Western blot等方法，研究胆管细胞增殖标志基因、相关受体和激酶等的变化趋势，阐明胆汁酸在促进胆管增殖及肝损伤的分子机制。为胆汁淤积性肝病的治疗提供新的靶点和方法。

背景：胆汁淤积引起肝内胆汁循环异常，导致胆汁在肝脏蓄积、胆管和肝脏细胞损伤，最终导致晚期肝病。对于胆汁酸在胆汁淤积性肝内胆管损伤中促进慢性胆管病变的潜在细胞/分子机制仍然未知。.目的：探讨兰索拉唑致药源性肝内胆管损伤的机制，为胆汁淤积性肝内胆管损伤致肝胆病变的治疗和药物发现提供新的思路与依据。.方法：本研究从体内、体外两方面探讨兰索拉唑导致肝内胆管损伤的机制。ICR小鼠兰索拉唑灌胃给药6个月后，使用血生化、HE染色和免疫组化等检查判断小鼠肝内胆管损伤的机制以及程度。使用EDU细胞增殖试剂盒研究兰索拉唑对肝内胆管上皮细胞（HIBEC细胞）功能的影响，使用激光共聚焦和流式细胞仪研究经兰索拉唑处理后细胞内钙离子的变化情况，利用HIBEC细胞进行Western blot和Real-time PCR实验等。.结果：小鼠血生化结果显示随着小鼠给药剂量增加，与空白溶媒对照组相比，LPZ给药组LW/BW较对照组明显增加；HE染色显示，对照组和低剂量组小鼠无明显损伤，高剂量组小鼠出现肉芽肿和炎症浸润，主要分布在肝脏门静脉区；Masson三色染色显示高剂量组纤维化，对照组和低剂量组无明显损伤。在HIBEC细胞实验中，CCK8以及EDU实验结果显示兰索拉唑可促进细胞增殖；激光共聚焦实验结果显示当加入兰索拉唑后，会出现异常的[Ca2+]i信号升高；Real-time PCR实验显示，LPZ能显著提高IP3R3和CK-19 mRNA的表达（P<0.05）；Western blot实验显示ERK1/2、p-ERK1/2和STAT-3、p-STAT3的磷酸化程度增加，IP3R3、CK-19和PCNA的表达也随着药物浓度的增加而增加。.结论：本研究揭示了Ca2+在LPZ诱导的肝内胆管损伤中起关键作用，表明LPZ通过激活P2RX4和P2RY2，促进IP3的形成，IP3激活IP3/Ca2+引起ERK1/2和STAT-3通路活化，导致肝内胆管损伤。