基于NTCP/Ntcp和BSEP/Bsep介导的胆汁酸转运受阻导致托伐普坦药物性肝损伤机理研究

Drug-induced liver injury (DILI) has become a serious issue in the practice of clinical medicine and DILI is responsible for the post-marketing withdrawal of many drugs. In January 2013, FDA issued a drug warning for tolvaptan with potential risk of liver injury. Vasopressin II receptor antagonist-related hepatic impairment was also reported in several previous studies. However, the mechanisms underlying vasopressin II receptor antagonist-associated DILI are poorly understood. In the present project, cell transfection technique will be adopted to investigate the effect of tolvaptan on sodium taurocholate cotransporting peptide (NTCP/Ntcp) and bile salt export pump (BSEP/Bsep) mediated cellular uptake and excretion of bile acids, such as taurocholate and chenodeoxycholate. In vitro sandwich-cultured rat hepatocyte and in vivo animal models will be also employed to determine the correlation between tolvaptan induced liver injury and changes of Ntcp- and Bsep-mediated bile acid hepatobilliary transport. Subsequently, bile acid homostasis as well as toxicity associated biomarkers will be analyzed in mice following oral dosing of 180 mg/kg/day of tolvaptan for 30 days. Finally, FXR/Fxr- and SHP/Shp-mediated molecular regulation of NTCP/Ntcp and BSEP/Bsep will be studied and discussed in both cell and mouse models, which helps to clarify the mechanism of tolvaptan induced liver injury. In summary, the research results will not only provide a practical interpretation for tolvaptan induced liver injury, but also offer a theoretical understanding of vasopressin II receptor antagonist-related hepatic impairment.

药物性肝损伤（DILI）已成为困扰临床安全用药不容忽视的重要问题。2013年1月FDA发布了关于托伐普坦引发DILI的风险警告，另有文献报道多种血管加压素II型受体拮抗剂也存在肝毒性。但此类药物导致DILI的机制尚不清楚。本项目以托伐普坦为研究对象，采用细胞转染技术研究药物转运体NTCP与BSEP在胆汁酸摄取与分泌中的作用；选用"三明治"培养大鼠肝细胞模型与整体动物模型探讨Ntcp与Bsep介导的胆汁酸肝胆转运改变与托伐普坦引起肝毒性的相关性，寻找与肝毒性相关的生物标志物；并从细胞与整体动物两个层面深入研究托伐普坦对FXR介导的NTCP/Ntcp与BSEP/Bsep功能与表达分子调控机制，阐明托伐普坦产生DILI的作用机理。研究结果将为托伐普坦所致DILI提供实验依据，并为揭示此类药物肝毒性机制提供理论依据，具有重要的临床用药指导意义。

托伐普坦（Tolvaptan, TVP）是一种选择性精氨酸加压素(AVP)V2受体拮抗剂，用于治疗高容或等容性低钠血症。2013年，美国FDA发布安全警告，长期使用TVP可能引发致死性肝损伤。本项目研究了TVP对主要胆汁酸转运体钠离子/牛磺胆酸共转运多肽(Sodium taurocholate co-transporting polypeptide, NTCP)和胆汁酸盐输出泵(Bile salt export pump, BSEP)转运功能及小鼠体内胆汁酸稳态的影响，并探讨其诱发药物性肝损伤的可能机制。.本研究建立hNTCP-HEK293转染细胞，并选用BSEP膜囊泡模型，以10种酰胺化胆汁酸作为探针底物，考察TVP对NTCP和BSEP功能的影响。结果表明，TVP对T-CA细胞摄取的抑制作用最强，IC50值为34.20 ± 3.16 µM；G-CA与G-UDCA次之；对其它胆汁酸的摄取抑制IC50值均远高于50 µM。BSEP膜囊泡转运试验结果表明，除G-LCA和T-UDCA外，托伐普坦对其它8种酰胺化胆汁酸IC50值都接近或小于25 µM，说明TVP可明显抑制BSEP转运功能。同时考察了100 mg/kg 和1000 mg/kg TVP灌胃后昆明小鼠肝胆胆汁酸转运及体内胆汁酸稳态的变化。结果显示，小鼠连续口服TVP 30 d后血清ALT和AST较对照组有所升高，给药3 d仅高剂量组有显著变化。高剂量组小鼠血清ALP明显升高。高低剂量组小鼠血清和肝脏中TBA均显著升高。切片结果显示长期口服TVP后，小鼠肝脏细胞骨架模糊并出现空泡病变。此外，给药3 d后，胆汁中12种胆汁酸浓度显著降低，肝脏中7种胆汁酸及血浆中CA浓度显著升高；给药30 d后，胆汁中14种胆汁酸浓度显著降低，肝脏中T-CDCA和T-MCA以及血浆中CDCA浓度显著升高。小鼠给药3 d后，主要胆汁酸外排转运体蛋白表达上调，部分胆汁酸外排转运体代偿性上调。长期给药后，胆汁酸外排转运体蛋白表达下调，胆汁酸外排减少；胆汁酸酰胺化酶表达降低，同时，TVP负反馈下调胆汁酸合成酶Cyp7a1的表达水平。.综上，本研究采用过表达模型研究TVP对主要胆汁酸转运体NTCP与BSEP功能的影响。并用整体动物模型研究了TVP对小鼠体内胆汁酸稳态的影响。本研究结果为TVP肝毒性机制提供理论依据，为临床安全用药提供指导意义。