Qiu Xi, Zhang Yueping, Liu Tongtong, Shen Hong, Xiao Yongling, Bourner Maureen J, Pratt Jennifer R, Thompson David C, Marathe Punit, Humphreys W Griffith, Lai Yurong
Life Science and Technology Center, Sigma-Aldrich , St. Louis, Missouri 63103, United States.
Mol Pharm. 2016 Apr 4;13(4):1206-16. doi: 10.1021/acs.molpharmaceut.5b00659. Epub 2016 Mar 8.
In the present study, we characterized in vitro biosynthesis and disposition of bile acids (BAs) as well as hepatic transporter expression followed by ABCB11 (BSEP) gene knockout in HepaRG cells (HepaRG-KO cells). BSEP KO in HepaRG cells led to time-dependent BA accumulation, resulting in reduced biosynthesis of BAs and altered BA disposition. In HepaRG-KO cells, the expression of NTCP, OATP1B1, OATP2B1, BCRP, P-gp, and MRP2 were reduced, whereas MRP3 and OCT1 were up-regulated. As a result, BSEP KO altered the disposition of BAs and subsequently underwent adaptive regulations of BA synthesis and homeostasis to enable healthy growth of the cells. Although BSEP inhibitors caused no or slight increase of BAs in HepaRG wild type cells (HepaRG-WT cells), excessive intracellular accumulation of BAs was observed in HepaRG-KO cells exposed to bosentan and troglitazone, but not dipyridamole. LDH release in the medium was remarkably increased in HepaRG-KO cultures exposed to troglitazone (50 μM), suggesting drug-induced cellular injury. The results revealed that functional impairment of BSEP predisposes the cells to altered BA disposition and is a susceptive factor to drug-induced cholestatic injury. In total, BSEP inhibition might trigger the processes but is not a sole determinant of cholestatic cellular injury. As intracellular BA accumulation is determined by BSEP function and the subsequent adaptive gene regulation, assessment of intracellular BA accumulation in HepaRG-KO cells could be a useful approach to evaluate drug-induced liver injury (DILI) potentials of drugs that could disrupt other BA homeostasis pathways beyond BSEP inhibition.
在本研究中，我们对胆汁酸（BAs）的体外生物合成和代谢以及肝脏转运蛋白表达进行了表征，随后在HepaRG细胞（HepaRG-KO细胞）中敲除了ABCB11（BSEP）基因。HepaRG细胞中的BSEP基因敲除导致胆汁酸随时间积累，从而减少了胆汁酸的生物合成并改变了胆汁酸代谢。在HepaRG-KO细胞中，NTCP、OATP1B1、OATP2B1、BCRP、P-糖蛋白和MRP2的表达降低，而MRP3和OCT1上调。结果，BSEP基因敲除改变了胆汁酸代谢，随后对胆汁酸合成和稳态进行适应性调节，以实现细胞的健康生长。尽管BSEP抑制剂在HepaRG野生型细胞（HepaRG-WT细胞）中未引起胆汁酸增加或仅引起轻微增加，但在暴露于波生坦和曲格列酮的HepaRG-KO细胞中观察到胆汁酸在细胞内过度积累，而双嘧达莫则不会。在暴露于曲格列酮（50 μM）的HepaRG-KO培养物中，培养基中的乳酸脱氢酶释放显著增加，表明药物诱导的细胞损伤。结果表明，BSEP的功能障碍使细胞易发生胆汁酸代谢改变，是药物性胆汁淤积损伤的敏感因素。总的来说，BSEP抑制可能触发这些过程，但不是胆汁淤积性细胞损伤的唯一决定因素。由于细胞内胆汁酸积累由BSEP功能和随后的适应性基因调节决定，评估HepaRG-KO细胞中细胞内胆汁酸积累可能是一种有用的方法，用于评估可能破坏BSEP抑制以外的其他胆汁酸稳态途径的药物的药物性肝损伤（DILI）潜力。
