Effects Of Radix Isatidis And Its Constituents On Major Organic Cantion Transporters In Mice Kidneys

ObjectiveTo investigate the effects of Radix Isatidis and its major constituents (indigo and indirubin) on kidney organic cantion transporters (OCTs, Oct1 and Oct2) and liver/intestinal microsomal cytochrome P450 enzymes (CYP450, Cyp3a4 and Cyp2e1) in vivo in mice.MethodsDecoction 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 the NIH mouse (60 mice per group), twice a day for 5 days, while four control groups were set up, including vehicle of water,0.5% sodium carboxymethyl cellulose (CMC), additives of sucrose plus dextrin (1.5 g·kg-1 each) and positive control quinidine (0.025 g·kg-1) groups. After last dosing of the test samples, all the mice were iv given Metformin (Met) 5 mg·kg-1 and 1.0,2.5,5.0,7.5,10.0,20.0 min later,10 mice per group were euthanized to collect whole blood and kidneys were quickly removed at each time point respectively. Each right kidney was homogenized for Met accumulation test.Met uptake for kidney slices were performed on another group of NIH mice, the test samples were the same as before, while four control groups were set up, including vehicle of water,0.5% CMC, ketoconazole (0.024 g·kg-1), rifampin (0.040 g·kg-1). After last dosing of the test samples,10 mice per group were euthanatized, kidneys, liver and intestine were quickly removed respectively. Each right kidney was used to quantitate Met uptake for kidney slices and each left kidney was used to extract total mRNA for analysis of Octl, Oct2 gene expressions by the real-time PCR. Livers and intestines were used to detect the activities of Cyps in microsomes.The concentrations of Met in sera and in kidney homogenates were determined by High Performance Liquid Chromatography (HPLC). Major pharmacokinetic parameters of Met in sera and kidney homogenates were calculated by pharmacokinetic software (DAS 2.0). The activities of Cyps in microsomes were determined by ultraviolet spectrophotometry. The proteins in kidney homogenates and microsomes were quantified by Folin-phenol method.ResultsThere was no significant difference among water control group,0.5% CMC group and sucrose plus dextrin group in all examined items. Compared with vehicle control groups (water and 0.5% CMC group), pharmacokinetic parameters in DRI high dosage, GRI high dosage, indigo high dosage and indirubin high dosage groups were all changed:elimination half time (t1/2β) was prolonged 13～97%, volume of distribution (Vd) reduced 13～72%, the clearance (CL) reduced 9～65%, the area under the curve (AUC0-20 min) increased 13-135%; AUC0-2Omin obtained from renal Met accumulations was significantly increased (P< 0.01); Met uptake by kidney slices were lower (P< 0.05); the mRNA expressions of Octl and Oct2 were down-regulate(P< 0.05).The results of Cyp3a4 activites in liver microsome assay:compared with control groups, when erythromycin is the substrate, all investigational groups were significantly higher than control groups (P< 0.01); when aminopyrine is the substrate, both low and high dosage of DRI and indigo groups, as well as the high dosage of GRI and indirubin groups were significantly higher than control groups (P< 0.01).The results of Cyp3a4 activites in intestinal microsome assay:compared with control groups, when erythromycin is the substrate, both low and high dosage of GRI groups, as well as high dosage of DRI, indigo and indirubin groups were significantly higher than control group(P< 0.01); when aminopyrine is the substrate, all investigational groups were significantly higher than control groups (P< 0.01).The results of Cyp2el activites in liver microsome assay:compared with control groups, both low and high dosage of GRI and indigo groups, as well as the high dosage of DRI and indirubin groups were obviously higher than control groups (P< 0.05, P< 0.01).ConclusionDecoction of Radix Isatidis, granules of Radix Isatidis, indigo and indirubin inhibit the renal Octl/Oct2, while induce the activites of Cyp3a4/Cyp2el in liver/intestinal microsomes of mice. The inhibition/induction of Radix Isatidis probably stems from indigo and indirubin contained in it.