肉碱/有机阳离子转运体对元胡止痛方中主要药性成分的转运机制研究

Mechanism of transmembrane transport of main medicinal components in Yuanhu Zhitong Prescription by OCTNs

目的研究肉碱/有机阳离子转运体(carnitine/organic cation transporter)N1、N2(OCTN1、OCTN2)对元胡止痛方中主要药性成分的跨膜转运机制,预测OCTN1和OCTN2在各药性成分跨膜转运过程中的作用。方法应用OCTN1、OCTN2高表达细胞株S2-OCTN1、S2-OCTN2,测定元胡止痛方中主要苦味药性成分延胡索乙素、巴马汀、原阿片碱和辛味药性成分欧前胡素、异欧前胡素对OCTN1、OCTN2的抑制作用及半数抑制浓度(IC50)。通过测定OCTN1、OCTN2及空白载体转染细胞Mock对不同药性成分的摄取能力,判断各药性成分是否为OCTN1和OCTN2的底物,进一步测定延胡索乙素在OCTN1和OCTN2中的米氏常数(Km)和细胞摄取底物最大转运速率(Vmax)。结果苦味药性成分延胡索乙素、巴马汀、原阿片碱和辛味药性成分欧前胡素、异欧前胡素对OCTN1和OCTN2的转运活性有显著抑制作用,对OCTN1的IC50分别是15.43、14.06、21.23、4.91、23.29μmol/L;对OCTN2的IC50分别是19.08、14.24、8.90、17.06、13.98μmol/L。OCTN1对延胡索乙素、巴马汀和原阿片碱的摄取活性分别是Mock的3.46、8.72和2.88倍,OCTN2对延胡索乙素、巴马汀和原阿片碱的摄取活性分别是Mock的2.58、5.86和9.75倍,OCTN1和OCTN2对欧前胡素和异欧前胡素的摄取活性与Mock均无显著差异。转运动力学实验结果显示,延胡索乙素在OCTN1上的Km和Vmax为6.39μmol/L、120.92 pmol/(mg·min),在OCTN2上的Km和Vmax为25.66μmol/L、637.03 pmol/(mg·min)。结论元胡止痛方中的药性成分延胡索乙素、巴马汀、原阿片碱、欧前胡素和异欧前胡素对OCTN1和OCTN2的转运活性具有较强的抑制作用,其中延胡索乙素、巴马汀和原阿片碱是OCTN1和OCTN2的底物,因此在不同药性成分跨膜转运过程中存在由OCTN1和OCTN2介导的药物-药物相互作用。

Objective To study the transmembrane transport mechanism of carnitine/organic cation transporters 1 and 2(OCTN1 and OCTN2)on the main medicinal components of Yuanhu Zhitong Prescription(元胡止痛方),and predict the role of OCTN1 and OCTN2 in the transmembrane transport of various medicinal components.Methods The OCTN1,OCTN2 highly expressed cell lines S2-OCTN1,S2-OCTN2 were used to detect the inhibitory effects and half inhibitory concentrations(IC50)of main bitter medicinal components tetrahydropalmatine,palmatine,protopine and pungent medicinal components imperatorin and isoimperatorin on OCTN1 and OCTN2 in Yuanhu Zhitong Prescription.By measuring the uptake ability of OCTN1,OCTN2 and blank vector transfected cell Mock to different drug components,it was judged whether the drug components were the substrate of OCTN1 and OCTN2.The michaelis constant(Km)and the maximum transport rate(Vmax)of the substrate uptake by the cell of tetrahydropalmatine in OCTN1 and OCTN2 were further determined.Results Bitter medicinal components tetrahydropalmatine,palmatine,and protopine and pungent medicinal components imperatorin and isoimperatorin significantly inhibited the transport activity of OCTN1 and OCTN2,the IC50 of inhibitory effect of which on OCTN1 was 15.43,14.06,21.23,4.91,and 23.29μmol/L,respectively;And the IC50 of inhibitory effect on OCTN2 was 19.08,14.24,8.90,17.06,and 13.98μmol/L,respectively.The uptake activity of OCTN1 to tetrahydropalmatine,palmatine and protopine was 3.46,8.72,and 2.88 times higher than that of Mock,and the uptake activity of OCTN2 to tetrahydropalmatine,palmatine and protopine was 2.58,5.86,and 9.75 times higher than that of Mock,respectively.The uptake activity of OCTN1 and OCTN2 on imperatorin and isoimperatorin was not significantly different from Mock.The Km and Vmax of tetrahydropalmatine on OCTN1 were 6.39μmol/L and 120.92 pmol/mg protein/min,on OCTN1 were 25.66μmol/L and 637.03 pmol/mg protein/min.Conclusion Tetrahydropalmatine,palmatine,protopine,imperatorin,and isoimperatorin in Yuanhu Zhitong Prescription have strong inhibitory effects on the transport activity of OCTN1 and OCTN2,in which tetrahydropalmatine,palmatine and protopine are the substrates of OCTN1 and OCTN2,so there is drug-drug interaction mediated by OCTN1 and OCTN2 in the process of transmembrane transport of different medicinal components.