板蓝根及其所含靛蓝和靛玉红对小鼠肾有机阳离子转运体OCT1和OCT2的影响

Effects of Radix Isatidis and contained indigo and indirubin on organic cation transporters OCT1 and OCT2 in mouse kidney

目的探讨板蓝根饮片水煎液（DRI）、板蓝根颗粒（GRI）及其所含成分靛蓝、靛玉红对小鼠肾有机阳离子转运体（OCT）中2个主要亚型OCT1和OCT2的影响。方法 NIH小鼠每组60只,分别ig给予DRI 1.6和6.4 g·kg^-1（生药量）,GRI 0.615和2.460 g·kg^-1,靛蓝0.008和0.640 mg·kg^-1,靛玉红0.0192和1.5360 mg·kg^-1,每天2次,连续5 d。同时设纯水和0.5%羧甲纤维素钠（CMC）为对照组,糊精加蔗糖（各1.5 g·kg^-1）添加剂组和奎尼丁（0.025 g·kg^-1）阳性对照组。末次给药后60 min静脉注射二甲双胍（Met）5 mg·kg^-1,给Met后1.0,2.5,5.0,7.5,10.0和20.0 min时每组分别各取10只小鼠处死,收集全血并摘取双肾,右肾匀浆后测定Met蓄积量,左肾用于检测OCT m RNA表达。另取NIH小鼠每组10只,同法给药,摘取左肾制备肾切片进行Met摄取实验。用高效液相色谱法测定血清、肾组织及肾切片匀浆液中Met浓度;药动学软件（DAS 2.0）分析血清及肾组织中Met的主要药动学参数;实时定量PCR法测定小鼠肾OCT1和OCT2m RNA的表达。结果与纯水对照组比,0.5%CMC组和蔗糖加糊精组各项检测指标均无显著性差异;DRI6.4 g·kg^-1、GRI 2.460 g·kg^-1、靛蓝0.640 mg·kg^-1和靛玉红1.5360 mg·kg^-1组血液药动学参数均出现显著变化（P〈0.05,P〈0.01）：t1/2β延长13%-97%,Vd减少13%-72%,Cl降低9%-65%,AUC0-20 min增加13%-135%;各供试物组肾组织Met蓄积量显著升高（P〈0.01）;肾切片Met摄取量均明显降低（P〈0.05,P〈0.01）;肾组织OCT1和OCT2 m RNA表达水平均不同程度下调（P〈0.05,P〈0.01）。结论 DRI、GRI、靛蓝和靛玉红在所用剂量下对小鼠肾OCT1和OCT2均有明显抑制作用,DRI和GRI的这种抑制作用可能主要来自其所含的靛蓝和靛玉红。

OBJECTIVE To investigate the effect of Radix Isatidis and its constituents indigo and in-dirubin on two principal subtypes of organic cation transporters （OCT） OCT1,OCT2 in vivo in mice.METHODS Decoction of Radix Isatidis （DRI）1.6 and 6.4 g · kg^-1,granules of Radix Isatidis （GRI）0.615 and 2.460 g·kg^-1,indigo 0.008 and 0.640 mg·kg^-1 and indirubin 0.0192 and 1.536 mg·kg^-1 were ig given to NIH mice （60 mice per group）,twice a day for 5 d.Four control groups were set up,including the vehicle of water,0.5% sodium carboxymethyl cellulose （CMC）,additives of sucrose plus dextrin （1.5 g·kg^-1） and positive control quinidine （0.025 g·kg^-1）.Sixty minutes after the last dosing,all the mice were iv given metformin （Met）5 mg·kg-1,and at 1.0,2.5,5.0,7.5,10.0 and 20.0 min after Met iv,10 mice in each group were sacrificed to collect whole blood and kidneys respectively.The right kidney was homogenized for Met accumulation test and the left one used to extract total RNA for analysis of OCT1 and OCT2 mRNA expressions by real-time PCR.The contents of Met in sera and kidneys were quantified by HPLC.Major pharmacokinetic parameters of Met in sera were analyzed by pharmacokinetic software （DAS 2.0）.RESULTS There was no significant difference between water control group,0.5% CMC group and sucrose plus dextrin group in any examined item.Compared with vehicle control group （water and 0.5% CMC group）,all the related pharmacokinetic parameters in DRI 6.4 g · kg^-1,GRI 2.46 g · kg^-1,indigo 0.640 mg · kg^-1 and indirubin 1.536 mg · kg^-1 groups were changed significantly （P〈0.05,P〈0.01）.The elimination half time （t1/2β） was prolonged 13%-97%,volume of distribution reduced by 13%-72%,clearance （Cl） reduced by 9%-65%,and the area under the concentration-time curve （AUC0-20 min） increased by 13%-135%.AUC0-20 min obtained from renal Met accumulations was significantly increased （P〈0.01） while Met uptake by kidney slices was reduced （P〈0.05,P〈0.01）.The expressions of OCT1 and OCT2 mRNA were obviously down-regulated （P〈0.05,P〈0.01）.CONCLUSION The mouse renal OCT1 and OCT2 are significantly inhibited by DRI,GRI,indigo and indirubin.The inhibitory effect of Radix Isatidis on OCT1 and OCT2 probably arises from indigo and indirubin contained.