Effects Of Radix Glycyrrhiza And Its Three Main Components On The Function And Expression Of P1-glycoprotein In Caco-2 Cells

OBJECTIVETo initially explore a new detoxication mechanisms of radix glycyrrhiza by evaluating the effects of radix glycyrrhiza and its three main components (glycyrrchizin, glycyrrhetinic acid and liquiritin) on the function and expression of p-glycoprotein in Caco-2 cell monolayers.METHODS1 Cells Culture and Morphological Validated of CellsCaco-2 cells and ECV304 cells were cultured with MEM (Eagle's minimum essential medium) contains 10% FBS (Fetal Bovine Serum) in a humidified atmosphere of 95% air and 5% CO2 at 37℃.The expression of P-glycoprotein in the cells was identified by immunofluorescence assy.2 Cytotoxicity Studies In VitroMTT (diphenyltetrazolium bromide) assay was used to find the non-cytotoxicity dosage of verapami,rifampicin,radix glycyrrhiza and its three main components in Caco-2 and ECV304 cells. The dosage at which the survival rate of cells is above 90% was considered as the highest non-cytotoxic dosage.3 Effects of radix glycyrrhiza and its three main components on the function of P-glycoprotein Verapamil was used as positive control of a inhibitors on the function of P-glycoprotein. Caco-2 cells without drug dealing were used as negative control. The effect of radix glycyrrhiza and its three main components on P-glycoprotein function was analyzed using Rh-123 assay, flowcytometry was used to determine the intracellular Rh-123 concentration.4 Effects of radix glycyrrhiza and its three main components on the expression of P-glycoproteinRifampicin was used as positive control of up regulation effect on the expression of P-glycoprotein. Caco-2 cells without drug dealing were used as negative control. Flowcytometry was used to measure the expression of P-glycoprotein in Caco-2 cells.RESULTS1 Cells Culture and Morphological Validated of CellsThe expression of P-glycoprotein is plentiful in Caco-2 cells but sparse in ECV304 cells. Caco-2 cells is suitable for studying radix glycyrrhiza and its three main components on the function and expression of P-glycoprotein, and ECV304 cells is fit to be the negative control.2 Cytotoxicity Studies In VitroFor 3days' MTT assay, radix glycyrrhiza, glycyrrchizin and liquiritin at concentrations of 1～100μg·mL-1, glycyrrhetinic acid at concentrations of 0.1～10μg·mL-1, verapamile and rifampicin at concentrations of 10μg·mL-1 were found to be non-cytotoxic towards the Caco-2 cells. These compounds were non-toxic to ECV304 at the same concentrations too. Then choose the concentrations as the experimental concentrations 3 Effects of radix glycyrrhiza and its three main components on the function of P-glycoproteinWhen the concentration of radix glycyrrhiza, glycyrrchizin and liquiritin was between 100～1μg·mL-1, the intracellular fluorescence of radix glycyrrhiza, glycyrrchizin and liquiritin groups was lower than that of negative control group (P<0.05). It showed the rhodamine 123 uptake in Caco-2 cells were was significantly decreased 34.66%,28.90%,24.56%,27.89%,26.29%,37.33%,19.51%,21.86%和19.03% respectively by concomitant of radix glycyrrhiza, glycyrrchizin and liquiritin with the low, middle and high concentration (1,10, 10μg·mL-1) after short term (60min) co-incubation.But the intracellular fluorescence of glycyrrhetinic acid groups at concentrations of 0.11～0μg·mL-1 were not significant different with the negative group. Glycyrrhetinic acid in experimental concentrations (0.1-10μg·mL-1) doesn't modulate the activity of P-glycoprotein in Caco-2 cells.4 Effects of radix glycyrrhiza and its three main components on the expression of P-glycoproteinThe positive rate of P-gp in Caco-2 cells were significantly increased 31.18%,61.67%,70.32%,77.43%,37.58%和49.14% by concomitant of middle concentration(10μg·mL-1) of radix glycyrrhiza, low, middle and high concentration (100,10,1μg·mL-1) of glycyrrchizin, middle and high concentration of glycyrrhetinic acid after longer term (72h) co-incubation.CONCLUSIONThe study evaluated the effect of radix glycyrrhiza and its main components on the function and expression of P-glycoprotein in Caco-2 cell monolayers for the first time.Our findings provide experimental that radix glycyrrhiza and some of its main components such as glycyrrchizin, glycyrrhetinic acid and liquiritin may affect the function or expression of P-glycoprotein in Caco-2 cell monolayers. Radix Glycyrrhiza could induce the function and up-regulate the expression level of P-gp in Caco-2 cells, and the effective constituents might be glycyrrchizin. These findings suggested that radix glycyrrhiza could induce the efflux of toxics in cells mediated by P-gp, leading to a decrease in cells levels of toxics and the consequent prevention of toxicity, seems to be a complementary of the detoxication mechanisms of glycyrrhiza that should be further studied.In addition, our results suggest that radix glycyrrhiza and some of its main components could affect the the function and expression of P-glycoprotein in Caco-2 cell monolayers, and caution should be exercised when radix glycyrrhiza is to be used together with other drugs that are P-gp substrate.