异鼠李素和阿魏酸在大鼠原代肝细胞的肝摄取性质

Uptake characterizations of isorhamnetin and ferulic acid in rat primary hepatocytes

目的建立大鼠原代肝细胞摄取模型,评价异鼠李素和阿魏酸的肝细胞摄取性质。方法应用RT-qPCR技术测定0,0.5,2和4h悬浮培养原代大鼠肝细胞上摄取转运体有机阴离子转运多肽(Oatp)1和Oatp2,有机阴离子转运体(Oat)2和有机阳离子转运体(Oct)1的mRNA表达。应用已知底物非索非那定、普伐他汀、甲氨蝶呤和阿昔洛韦评价转运体的功能。将已知底物或受试药物在4℃和37℃分别与肝细胞孵育不同时间,LC-MS/MS定量测定细胞摄取量,考察温度和时间对底物摄取的影响。由浓度依赖的摄取实验,计算得到Km、Vmax和主动摄取率等参数。在肝细胞模型上,将转运体的阳性抑制剂与异鼠李素10μmol·L-1和阿魏酸50μmol·L-1共孵育2min,观察抑制剂对受试药转运的影响。结果悬浮培养大鼠原代肝细胞上表达有转运体Oatp1,Oatp2,Oat2和Oct1,并能介导已知底物的主动摄取。转运体的mRNA表达水平随着时间的延长而快速下降,4h的表达水平约为0h的10%。转运体对已知底物摄取活性也随时间呈下降趋势,但程度显著低于表达水平,4h的活性约为0h的28.7%～71.4%。在经验证的肝细胞模型上,异鼠李素的4℃和37℃摄取无显著差异,主动转运率为14.6%。阿魏酸在37℃的摄取量显著高于4℃,主动转运率为84.1%;Oct阳性抑制剂奎尼丁100μmol·L-1能显著抑制阿魏酸的肝摄取,抑制率为64.9%。结论成功建立了大鼠原代肝细胞摄取模型,并用底物进行了验证。异鼠李素通过被动扩散进入肝细胞,阿魏酸的肝细胞摄取则以主动转运为主。

OBJECTIVE To establish hepatic uptake model with rat primary hepatocytes,and to investigate the uptake characterizations of isorhamnetin and ferulic acid.METHODS The mRNA expressions of uptake transporters organic anion transporting polypeptide（Oatp）1,Oatp2,organic anion transporter（Oat）2 and organic cation transporter（Oct）1 on rat primary hepatocytes were measured using reverse transcriptase-quantitative polymerase chain reaction assays（RT-qPCR） after the cells were cultured for 0,0.5,2 and 4 h,respectively.The activity of these transporters was assessed using a panel of known substrates,including aciclovir,methotrexate,pravastatin and fexofenadine.The substrates or testing drugs were incubated with rat hepatocytes at 4℃ or 37℃,in the presence of cytochrome P450 enzyme inhibitor ABT for different periods of time.The intracellular concentrations of the substrates and the drugs were determined using a validated LC-MS/MS method to calculate the uptake amounts.The effect of cultural time and temperature on the hepatic uptake of these compounds were assessed.The kinetic parameters of Km,Vmax and the uptake rates（Clactive and Cluptake） were obtained from concentration dependent uptake tests.The effects of the known transporter inhibitors（rifampin,ritonavir,probenecid and quinidine） on the hepatic uptake of ferulic acid were also evaluated by pre-incubation of these inhibitors with the rat hepatocytes for 15 min,followed by co-incubation with ferulic acid（50 μmol·L-1） for 2 min.RESULTS The mRNA expression of the target transporters（Oatp1,Oatp2,Oat2 and Oct1） on the rat primary hepatocytes was demonstrated by RT-qPCR.The activities of these transporters were confirmed by active uptake of known substrates in the rat hepatocytes.The expression levels of these transporters were decreased rapidly.The expression levels detected at 4 h were only about 10% of that for the freshly isolated cells.However,the uptake activities of the transporters at 4h were kept at the levels from 28.7% to 71.4% of that measured at 0 h.The hepatic uptake of isorhamnetin in the validated rat hepatocyte model was assessed at 4℃ and 37℃ and no difference was observed.In contrast,the hepatic uptake of ferulic acid showed a significant difference at 4℃ and 37℃.The ratios of the active uptake to the total uptake for isorhamnetin and ferulic acid were 14.6% and 84.1%,respectively.The known Oct1 inhibitor quinidine（100 μmol·L-1） could remarkably reduce the hepatic uptake of ferulic acid with the inhibition rate of 64.9%.CONCLUSION A hepatic uptake model is established successfully with freshly isolated rat hepatocytes in suspension.The model is validated by using known substrates and inhibitors.The transport of isorhamnetin into hepatocytes is via a passive diffusion process.The uptake of ferulic acid across the sinusoidal membrane is mainly through an active process,which may be associated with Oct1 transporter.