Effects Of Tetramethylpyrazine And Ginsenoside Rg1 On P-gp Function And Expression In Caco-2 Cells

OBJECTIVESTo study the effects of tetramethylpyrazine and ginsenoside Rg1 on p-glycoprotein function and expression in Caco-2 cells.METHODS1.Cell culture and morphological validated of cellsCaco-2 cells and ECV cells were cultured with DMEM(Dulbecco's Modified Eagle's Medium)which 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.The Caco-2 cells were cultured in transwell plates to establish monolayer model.The EVOM epithelial voltohmmeters,fluorescein and propranolol were used to test the function of the monolayer model.2.Cytotoxicity studies in vitroMTT(diphenyltetrazolium bromide)assay was used to find non-cytotoxicity dosage of tetramethylpyrazine and ginsenoside Rg1 in Caco-2 and ECV cells,and only when the survival rates of cells above 90%,the doses were considered as the non-cytotoxic dosage.3.Effects of tetramethylpyrazine and ginsinoside Rg1 on the efflux of rhodamine-123 in cellsECV cells were used as negative control cells.Cyclosporin A was used as positive control drug to posses the inhibitory effect on the function of p-glycoprotein.HPLC-UV was used for analysis the concentrations of rhodamine-123,which is a substrate of p-glycoprotein.4.Effects of tetramethylpyrazine and ginsenoside Rg1 on rhodamine-123 transport though Caco-2 cells monolayerCyclosporin A was used as positive control drug to posses the inhibitory effect on the function of p-glycoprotein.HPLC-UV was used for analysis the concentrations of rhodamine-123 in the study of transport.5.Measurement of expression of p-glycoprotein in cellsFlow cytometry was used to measure the expression of p-glycoprotein in Caco-2 and ECV cells.RESULTS1.Cell culture and morphological validated of cells The expression of p-glycoprotein is plentiful in Caco-2 cells but sparse in ECV cells.The Caco-2 cell monolayer model was tight and intact.Caco-2 cell monolayer model was established successfully.Caco-2 cell and monolayer model were suitable for studying tetramethylpyrazine, ginsenoside Rg1 on the function and expression of p-glycoprotein.2.In vitro cytotoxicity studiesTetramethylpyrazine at concentrations ranging from 60 to 240μg/mL, ginsenoside Rg1 at concentrations ranging from 6 to 24μg/mL,were found to be non-cytotoxic towards the Caco-2 cells and ECV cells.3.Effects of tetramethylpyrazine and ginsinoside Rg1 on the efflux of rhodamine-123 in cellsMiddle and high(120 and 240μg/mL)concentrations of tetramethylpyrazine could decrease the efflux of rhodamine-123 from Caco-2 cells.Ginsenoside Rg1 at high concentration(20μg/mL)was observed to decrease p-glycoprotein mediated efflux.To combine tetramethylpyrazine with ginsenoside Rg1 at middle concentrations showed a great synergistic inhibition effect on the efflux of rhodamine-123 from Caco-2 cells.4.Effects of tetramethylpyrazine and ginsinoside Rg1 on rhodamine-123 transport through Caco-2 cells monolayerBoth middle and high concentrations(120 and 240μg/mL)of tetramethylpyrazine could increase the transport of rhodamine-123 from the apical to basolateral.Ginsenoside Rg1 at high concentration (20μg/mL)was observed to increase the transport rate of rhodamine-123. Combining tetramethylpyrazine with ginsenoside Rg1 in middle concentrations(120 and 10μg/mL),increased rhodamine-123 level of Caco-2 cells that may be by synergistic effect.5.Measurement of expression of p-glycoprotein in cellsLonger term(72h)co-incubation of the Caco-2 cells with middle and high concentrations of tetramethylpyrazine(120 and 240μg/mL)had the down-regulation effect of cellular p-glycoprotein level,but ginsenoside Rg1 didn't have this effect.Tetramethylpyrazine and ginsenoside Rg1 in middle concentration(120 and 10μg/mL)didn't show a synergistic effect on the expression the cellular p-glycoprotein level when co-incubation with Caco-2 cells in 72h.CONCLUSIONS1.Tetramethylpyrazine may be a substrate and inhibitor of p-glycoprotein. It could significantly inhibit the function of p-glycoprotein,and seemed to act directly on the activity of p-glycoprotein or be a binding site competitor of rhodamine-123.Tetramethylpyrazine could significantly decrease the expression of p-glycoprotein after incubated with Caco-2 cells in 72h.2.Ginsenoside Rg1 may be an inhibitor of p-glycoprotein.It could significantly inhibit the activity of p-glycoprotein at high concentration. Longer term(72 h)co-incubation of the Caco-2 cell monolayer with ginsenoside Rg1 had no effect on p-glycoprotein levels.Ginsenoside Rg1 inhibited the activity of p-glycoprotein without decreasing the expression of Caco-2 cell monolayer.3.Combining tetramethylpyrazine with ginsenoside Rg1 showed a great synergistic effect on p-glycoprotein mediated efflux and transport of rhodamine-123 in the cells but no synergistic effect on p-glycoprotein's expression.4.The oral bioavailability of p-glycoprotein substrate may be influenced by tetramethylpyrazine and ginsenoside Rg1 or tetramethylpyrazine combining with ginsenoside Rg1 in the p-glycoprotein status.5.Tetramethylpyrazine may represent a naturally novel multidrug resistance reversal agent,which is singificant for the chemotherapy against human cancer.