小檗碱、巴马汀和药根碱在“三明治”培养大鼠原代肝细胞中的胆汁外排特征
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摘要
目的研究小檗碱、巴马汀和药根碱在大鼠原代细胞的胆汁外排特征。方法建立"三明治"培养大鼠原代肝细胞(sandwich cultured rat hepatocytes,SCRH)模型,分别加入小檗碱、巴马汀、药根碱,在含Ca~(2+)和无Ca~(2+)缓冲液中孵育,采用UPLC-MS/MS分别测定3种生物碱的细胞蓄积量,计算胆汁排泄指数和胆汁清除率,评价小檗碱、巴马汀、药根碱在大鼠原代细胞的胆汁外排特征;并考察P-gp、Mrp2抑制剂对3种活性成分在SCRH细胞模型中的外排转运影响。结果随着孵育时间的延长,3种生物碱的细胞蓄积量增加,且含Ca~(2+)条件下的细胞蓄积量与无Ca~(2+)条件下相比具有明显差异;P-gp抑制剂环孢素A、维拉帕米均能减少小檗碱、巴马汀、药根碱的胆汁排泄,且呈浓度依赖性,Mrp2抑制剂MK571、丙磺舒对3种生物碱的胆汁排泄无明显影响。结论小檗碱、巴马汀、药根碱均经过胆汁外排,P-gp介导了3种生物碱的胆汁外排,Mrp2未参与其胆汁排泄。
Aim To study the biliary excretion characteristics of berberine,palmatine and jateorhizine in rat hepatocytes. Methods Berberine,palmatine and jateorhizine were incubated with the sandwich-cultured rat hepatocytes( SCRH) in standard Ca~(2+)buffer or Ca~(2+)free buffer. The accumulation of the three compounds under different conditions were measured by UPLC-MS/MS. The biliary excretion index and biliary clearance were calculated,and the effect of P-gp or Mrp2 inhibitor on the transport of three compounds was also investigated. Results While the incubation time increased,the accumulation of the three compounds also increased. There were obvious differences in accumulation of berberine,palmatine and jateorhizine in incubations treated with standard buffer and calcium-free buffer. The P-gp inhibitors ciclosporin A and verapamil could inhibit the biliary excretion of berberine,palmatine and jateorhizine. However,the Mrp2 inhibitors MK571 and probenecid had no effect on biliary excretion of the three compounds. Conclusions The biliary excretion of berberine,palmatine and jateorhizine is mainly through an active process. They are all the P-gp substrates other than Mrp2 substrates.
Aim To study the biliary excretion characteristics of berberine,palmatine and jateorhizine in rat hepatocytes. Methods Berberine,palmatine and jateorhizine were incubated with the sandwich-cultured rat hepatocytes( SCRH) in standard Ca~(2+)buffer or Ca~(2+)free buffer. The accumulation of the three compounds under different conditions were measured by UPLC-MS/MS. The biliary excretion index and biliary clearance were calculated,and the effect of P-gp or Mrp2 inhibitor on the transport of three compounds was also investigated. Results While the incubation time increased,the accumulation of the three compounds also increased. There were obvious differences in accumulation of berberine,palmatine and jateorhizine in incubations treated with standard buffer and calcium-free buffer. The P-gp inhibitors ciclosporin A and verapamil could inhibit the biliary excretion of berberine,palmatine and jateorhizine. However,the Mrp2 inhibitors MK571 and probenecid had no effect on biliary excretion of the three compounds. Conclusions The biliary excretion of berberine,palmatine and jateorhizine is mainly through an active process. They are all the P-gp substrates other than Mrp2 substrates.
引文
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[13]Matsunaga N,Nunoya K,Okada M,et al.Evaluation of hepatic disposition of paroxetine using sandwich-cultured rat and human hepatocytes[J].Drug Metab Dispos,2013,41(4):735-43.
[2]高纯颖,陈笑,钟大放.转运体在药物经肝脏清除过程中的作用[J].药学学报,2012,47(5):565-72.[2]Gao C Y,Chen X,Zhong D F.Role of transporters in hepatic drug disposition[J].Acta Pharm Sin,2012,47(5):565-72.
[3]梁瑞峰,石科,刘雪,等.小檗碱、巴马汀和药根碱在大鼠原代肝细胞中的摄取转运特征[J].中国药学杂志,2017,52(14):1251-6.[3]Liang R F,Shi K,Liu X,et al.Uptake characterizations of berberine,palmatine and jateorhizine in rat primary hepatocytes[J].Chin Pharm J,2017,52(14):1251-6.
[4]Seglen P O.Preparation of isolated rat liver cells[J].Methods Cell Biol,1976,13:29-83.
[5]Mohamed L A,Kaddoumi A.Tacrine sinusoidal uptake and biliary excretion in sandwich-cultured primary rat hepatocytes[J].JPharm Pharm Sci,2014,17(3):427-38.
[6]Lee J K,Paine M F,Brouwer K L.Sulindac and its metabolites inhibit multiple transport proteins in rat and human hepatocytes[J].J Pharmacol Exp Ther,2010,334(2):410-8.
[7]Giacomini K M,Huang S M,Tweedie D J,et al.Membrane transporters in drug development[J].Nat Rev Drug Discov,2010,9(3):215-36.
[8]聂昊,王晖.P糖蛋白在不同组织中的分布与功能研究进展[J].广东药学院学报,2012,28(4):456-60.[8]Nie H,Wang H.Advances in the expression and function of p-glycoprotein in different human tissues[J].J Guangdong Pharm Univ,2012,28(4):456-60
[9]牛璐,金晶,陈攀,等.黄芩素对胆汁外排转运体MRP2和BSEP的影响[J].中国药理学通报,2015,31(1):147-8.[9]Niu L,Jin J,Chen P,et al.Effect of baicalein on expression of MRP2 and BSEP in Hep G2 cells[J].Chin Pharmacol Bull,2015,31(1):147-8.
[10]Shimizu K,Sano T,Kubota R,et al.Effects of the amino acid constituents of microcystin variants on cytotoxicity to primary cultured rat hepatocytes[J].Toxins(Basel),2013,6(1):168-79.
[11]Shin D H,Park S H,Jeong S W,et al.Hepatic uptake of epirubicin by isolated rat hepatocytes and its biliary excretion after intravenous infusion in rats[J].Arch Pharm Res,2014,37(12):1599-606.
[12]Zeigerer A,Wuttke A,Marsico G,et al.Functional properties of hepatocytes in vitro are correlated with cell polarity maintenance[J].Exp Cell Res,2017,350(1):242-52.
[13]Matsunaga N,Nunoya K,Okada M,et al.Evaluation of hepatic disposition of paroxetine using sandwich-cultured rat and human hepatocytes[J].Drug Metab Dispos,2013,41(4):735-43.