Initial Study On Interaction Between Four Anti-tuberculosis Drugs And P-glycoprotein

OBJECTIVETo study the effects of four anti-tuberculosis drugs on the function and expression of P-glycoprotein,make further validation for the inducement of rifampicin on P-glycoprotein function and express,and study initially on interaction between four anti-tuberculosis drugs and P-glycoprotein.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 perospirone and verapamile 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 four anti-tuberculosis drugs on the function of P-glycoproteinThe effect of four anti-tuberculosis drugs on P-glycoprotein function was analyzed using Rh-123 assay,flowcytometry was used to determine the intracellular Rh-123 concentration.4 Effects of four anti-tuberculosis drugs on the expression of P-glycoproteinRifampicin was used as positive control of up regulation effect on the expression of P-glycoprotein and verapamil was used as positive control of down 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.5 Effects of four anti-tuberculosis drugs on the expression of MDR1 mRNARifampicin was used as positive control of up regulation effect on the expression of MDR1 mRNA and verapamil was used as positive control of down regulation effect on the expression of MDR1 mRNA. FQ-PCR(real-time fluorescent quantitative Polymerase Chain Reaction) was used to measure the expression of MDR1 gene mRNA 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 four anti-tuberculosis drugs 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,RFP at 100μmol/L,INH at 150μmol/L,LVFX at 5μmol/L,EMB at 150μmol/L,PZA at 200μmol/L and VER at 10μmol/L were found to be non-cytotoxic towards the Caco-2 cells.Then,RFP(10μmol/L),INH(80μmol/L),LVFX(2μmol/L),EMB(30μmo l/L),PZA(100μmol/L) and VER(10μmol/L) were chosen to take 10days' and 20days' MTT assay,and all compounds were non-toxic to Caco-2 and ECV304 cells.3 Effects of four anti-tuberculosis drugs on the function of P-glycoproteinWhen the intervention time is 1 hour,the intracellular fluorescence of the four drugs was significantly lower than that of negative control group (P＜0.05).When the intervention time is 3 days,the intracellular fluorescence of the four drugs was all significantly higher than that of negative control group(P＜0.05).When the intervention time is 10 days,the intracellular fluorescence of the three drugs(INH,EMB,PZA) was all significantly lower than that of negative control group(P＜0.05),and the intracellular fluorescence of the LVFX group was significantly higher than that of negative control group(P＜0.05).When the intervention time is 20 days,the intracellular fluorescence of the two drugs(INH,EMB) was all significantly lower than that of negative control group(P＜0.05),and the intracellular fluorescence of the LVFX group and PZA group was significantly higher than that of negative control group(P＜0.05).4 Effects of four anti-tuberculosis drugs on the expression of P-glycoproteinWhen the intervention time is 1 hour,there was no difference between the four drugs and negative control group in the expression of P-glycoprotein(P＞0.05).When the intervention time is 3days,the intracellular fluorescence of the four drugs was all significantly highly than that of negative control group(P＜0.05).When the intervention time is 10 days,the intracellular fluorescence of the three drugs(INH,EMB,PZA) was all significantly higher than that of negative control group(P＜0.05);the intracellular fluorescence of LVFX was all significantly lower than that of negative control group (P＜0.05).When the intervention time is 20 days,the intracellular fluorescence of the two drugs(INH,EMB and PZA) was all significantly higher than that of negative control group(P＜0.05);the intracellular fluorescence of LVFX was all significantly lower than that of negative control group (P＜0.05).5 Effects of four anti-tuberculosis drugs on the expression of MDR1 mRNAWhen the intervention time is 1 hour,there was no difference between the four drugs and negative control group in the expression of MDR1 mRNA(P＞0.05).When the intervention time is 3days,the MDR1 mRNA expression of the four drugs was all significantly lower than that of negative control group(P＜0.05).When the intervention time is 10 days,the MDR1 mRNA expression of the three drugs(INH,EMB,and PZA) was all significantly higher than that of negative control group(P＜0.05);the MDR1 mRNA of LVFX was all significantly lower than that of negative control group(P＜0.05).When the intervention time is 20 days,the MDR1 mRNA expression of the three drugs(INH,EMB and PZA) was all significantly higher than that of negative control group(P＜0.05);the MDR1 mRNA of LVFX was all significantly lower than that of negative control group(P＜0.05).CONCLUSIONOur findings provide experimental that the four anti-tuberculosis drugs interfere directly with the function and indirectly with the expression of P-gp.LVFX is probably the inhibitor of P-glycoprotein. INH and EMB may be the inducers of P-glycoprotein,but it needs further verification.What's more,PZA is probably no obvious interaction between PZA and P-glycoprotein,but it also needs further verification. Therefore,when used together with other drugs which are P-gp substrates, they may increase or decrease the plasma concentration of these drugs.