| Since the late 1980s the tuberculosis epidemic situation has been rising clearly around the world. The main reasons of which lie in growing and spread of drug-resistant strains, especially the sharp rise of multidrug-resistant ones.So far, the research on drug-resistance mechanism in MDR-TB(multidrug-resistant tuberculosis) has focused on the field in drug resistant of mycobacterium tuberculosis, with a little regard for the perspective of host. According to the research results of the mechanism of multi-drug resistance of anti-cancer that the occurrence of multi-drug resistance is mainly caused by the role of P-gp and other active MDR-associated efflux pump, the present study consists of two parts:molecular biology experiment and animal experiment. From the perspective of host, we studied the effects of four anti-tuberculosis drugs on P-gp in vitro, and further researched on the effects of different therapeutic regimens and different treatment durations on the tissue distributions of anti-tuberculosis drugs and the mdrla mRNA expression level in various tissues of Kunming mice in vivo.OBJECTIVEStudy on the effects of therapeutic doses of five anti-tuberculosis drugs (rifampicin(RFP), isoniazid(INH), ethambutol (EMB), pyrazinamide(PZA), levofloxacin (LVX)) on the function and expression of P-gp in Caco-2 cells, to investigate whether there are significant interaction between these major anti-tuberculosis drugs and P-gp, and to study the effect of different therapeutic regimen and different treatment durations on the distributions of anti-tuberculosis drugs and the mdrla mRNA expression level in various tissues of Kunming mice, to primarily explore the effects of the factors of host on drug resistance of anti-tuberculosis drugs. In addition, research on the potential factors of drug resistance in clinical anti-tubercular treatment standard regimen.METHODS1. Establishment of Caco-2 cell model and Cytotoxicity Studies In VitroCaco-2 cell and ECV304 cell were cultured with MEM (Modified Eagle's Medium) and were applied to study the effects of four anti-tuberculosis drugs on the function and expression of P-gp. The expression of P-gp in the cells was identified by immunofluorescence assay.MTT (Methyl thiazolyl tetrazolium) assay was applied to determine the maximum non-cytotoxic dose of each tested drugs to ensure the activity of cells during the test and to consult the safe concentration of each drugs for follow-up research. The dosage at which the survival rate of cells is above 90% was considered as the highest non-cytotoxic dosage.2. Rh-123 uptake experiments assay the function of P-gpECV304 cell was used as negative cell control. RFP was used as positive control of the inductive effect on the function of P-gp. VER was used as positive control of the inhibitory effect on the function of P-gp. Flow cytometry were used to study the effects of four anti-tuberculosis drugs on the uptake of rhodamine-123 which is a substrate of P-gp in Caco-2 cell.3. Flow cytometry assay the expression of P-gpRFP was used as positive control of up-regulation effect on the expression of P-gp and VER was used as positive control of down-regulation effect on the expression of P-gp. Caco-2 cells without drug treatment were used as negative control. Flow cytometry (FCM) was applied to study the effects of four anti-tuberculosis drugs on the expression of P-gp in Caco-2 cells.4. Real-time PCR assay the expression of MDR1 mRNARFP was used as positive control of up-regulation effect on the expression in mRNA level of MDRl gene, and VER was used as positive control of down-regulation effect on the expression in mRNA level of MDR1 gene. Caco-2 cell without drug treatment was used as negative control. FQ-PCR (real-time fluorescent quantitative Polymerase Chain Reaction) was used to measure the expression of MDR1 mRNA in Caco-2 cells and to study the effects of four anti-tuberculosis drugs on the expression of MDR1 mRNA.5. Determination the concentrations of three anti-tuberculosis drugs in mouse plasma and tissues by LC-MS/MSChromatographic separation of the analytes was performed on a C4 column (250 mm x4.6 mm,5.0μm, Welch materials,USA) with a column temperature at 25℃. The mobile phase was a gradient of a mixture of 0.05% formic acid in water and methanol. The flow-rate was 1.0 ml/min and an injection volume was 20μL. The ESI source was operated in positive mode, quantification was achieved under multiple reactions monitoring (MRM) mode.6. The distribution of three anti-tuberculosis drugs in various tissues and plasma of KunMing mouse by different therapeutic durations and regimens168 Kunming mice (30±3g) were randomly divided into seven groups of 24 animals each. Each group was injected intraperitoneally with physiological saline (A), INH alone (45 mg·kg-1) (B), RFP alone (67.5mg·kg-1) (C), LVX alone (45mg-kg-1) (D), INH +RFP(45mg·kg-1,67.5mg·kg-1,respectively) (E), LVX+RFP (45mg-kg-1 67.5mg-kg-1,respectively) (F) and RFP+INH+LVX(67.5mg·kg-1, 45mg-kg-1,45mg-kg-1, respectively) (G) once daily. Drugs volume of injection is 0.2 ml in each mouse. The animals of each experimental group were respectively taken a half to receive following treatment 2 hours after the first intraperitoneal injection. The orbital blood of each mouse was taken was treated with anticoagulant Heparin and centrifugation at 6000 rpm for 10min.And then sacrificed the mouse, quickly took tissue samples, washed by physiological saline and weigh each tissues. Plasma, brain, liver, kidney, lung and small intestine samples were collected and were kept in refrigerator at-70℃. The remaining animals in each experimental group were sacrificed and were taken the above samples 2 hours after the 10th intraperitoneal injection.7. Study on effects of different therapeutic regimens for continuous 10 days on mdrla mRNA expression level in various tissues of KunMing mice21 Kunming mice (30±3 g) were randomly divided into seven groups of 3 animals each. Grouping and administration dosage were the same as chapter 6.2 hours after the 10th intraperitoneal injection, sacrifice the mice and take brain, kidney, lung and small intestine samples. All samples were stored in liquid nitrogen. Real-time fluorescence quantitative RT-PCR was used to determine the expression of mdrla mRNA in various tissues.RESULTS1. Cytotoxicity Studies In Vitro:For 3days'MTT assay, RFP at 100μmol/L, INH at 150umol/L, LVX 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), LVX(2μmol/L), EMB(30μmol/L), PZA(100μmol/L) and VER(10μmol/L) were chosen to take MTT assay for 10days,20days,30days,40days and 50days, and all compounds were non-toxic to Caco-2 and ECV304 cells.2. Effects of each drug on the function and expression of P-gp and the expression of MDR1 mRNA:When the intervention time is 1 hour, the four drugs groups, positive inductive control group(RFP) and positive inhibitory control group(VER) were all significantly enhance P-gp function but no effect on the expression of P-gp and MDR1 mRNA, the role were not selective. When the intervention time is 3 days, the four drugs groups, RFP group and VER group were all significantly reduce the function of P-gp and down-regulated effect on the expression of P-gp and MDR1 mRNA, the role was not selective either. When the intervention time is 10 days, the RFP, INH and EMB groups were all significantly up-regulated effect on the function and expression of P-gp and the expression of MDRl mRNA, which showed induction; the VER and LVX groups down-regulated effect on the function and expression of P-gp and the expression of MDR1 mRNA, which showed inhibition; compared with the negative control, PZA group was non-significance difference. When the intervention time is 20 days, the drugs groups and positive control groups showed the same trend as the situation when the intervention time is 10 days. When the intervention time is 30 days, the drugs groups and positive control groups showed the same trend on the function of P-gp as the situation when the intervention time is 10 days and 20 days, but on the expressions of P-gp and MDR1 mRNA were all significantly higher than that of negative control group (P<0.05), showing a non-selective up-regulated the expression of P-gp and MDR1 mRNA. When the intervention time is 40,50 days, the drugs groups and positive control groups were all significantly up-regulated effect on the function and expression of P-gp and the expression of MDR1 mRNA, which showed a non-selective induction.3. Determination the concentrations of three anti-tuberculosis drugs in mouse plasma and tissues by LC-MS/MS:The established method was successfully applied to simultaneous determination of INH, RFP and LVX in mouse plasma and different mouse tissues. Calibration ranges for INH were 0.11-5.42μg/g in tissues and 0.18-9.04μg/ml in plasma, for RFP were 0.12-1200μg/g in tissues and 4.0-200μg/ml in plasma, and for LVX were 0.13-26.2μg/g in tissues and 0.09-4.53μg/ml in plasma. The lower limits of quantification (LLOQs) for INH, RFP and LVX in mouse tissues were 0.11,0.12 and 0.13μg/g and for those in mouse plasma were 18.1,20.0 and 21.8ng/ml, respectively. The limits of detection (LODs) for INH, RFP and LVX in mouse tissues were 0.04,0.05 and 0.05μg/g and for those in mouse plasma were 5.5,6.0 and 6.6ng/ml, respectively.4. The distribution of three anti-tuberculosis drugs in various tissues and plasma of KunMing mouse by different therapeutic durations and regimens:In plasma, the drugs concentrations of each experimental group treated with intraperitoneal injection for continuous 10 days were significantly higher than that of group treated with intraperitoneal injection only one time. In tissues, the drugs concentrations in brain, liver, kidney and lung of each experimental group treated with intraperitoneal injection for continuous 10 days were significantly lower than that of group treated with intraperitoneal injection one time, and LVX can obviously slow the decreasing trend of the tissue distribution of drugs combined with itself. But in small intestine, there were no difference in the drugs concentrations between experimental group treated with intraperitoneal injection for continuous 10 days and group receiving intraperitoneal injection one time.5. The mdrla mRNA expression level in various tissues of KunMing mice by different therapeutic regimens for continuous 10 days: Contrasted with control group, the effects of the mdrla mRNA expression in group treated with intraperitoneal injection for continuous 10 days were significantly different in various tissues in the same group, and were different in the same tissue among different groups too. As a whole, INH, RFP and INH+RFP group can significantly up regulate the expression of mdrla mRNA. Especially, the expression of mdrla mRNA was up-regulated 5-70 folds in brain and small intestine, and 8-12 folds in kidney. While LVX can obviously down-regulate the expression of mdrla mRNA in brain and kidney(0.8-0.9 folds), and the up-regulated level of mdrla mRNA in brain, kidney and small intestine for LVX+RFP and LVX+RFP+INH group were lower than RFP and RFP+INH group, respectively. The mdrla mRNA level of each experimental group in lung tissue presented down-regulated trend, which may relate to lung tissue itself with less distribution of P-gp. In lung tissue, P-gp only distribute into its secretory cells. This experiment determined mdrla mRNA expression level in the entire lung tissue, so the whole mdrla mRNA level was relatively low, and the influence of drugs was not obvious either.CONCLUSIONS1. In vitro cell experiments, we verified elementarily that INH and EMB may be the inducers of P-gp, LVX may be the inhibitor of P-gp and PZA may have no obvious interaction with P-gp, but it needs further verification.2. Animal experiments have revealed that different therapeutic regimens have obvious influence on the distribution of drugs and the mdrla mRNA level of expression in tissues. The overall trend is that the expression of mdrla mRNA was up-regulated, the drugs concentrations of tissues decrease, and therapeutic regimens using LVX has significant antagonistic effect on this phenomenon.3. According to the results of our experiments in vitro and vivo, long-term administration with anti-tuberculosis can up-regulate the function and expression of P-gp and the expression of mdrla mRNA thus reduce the drugs concentrations in tissues, which is probably one of the important mechanisms of anti-tuberculosis resistance.
|
PDF Full Text Request