| Background and ObjectivesChina is the second largest country of organ transplantation in the world. The long-term surviving and the survival quality of organ transplant recipients have aroused a widespread concern. The major problem of improving the quality of life is that establishing an effective and rational anti-rejection immunosuppression plan.Cyclosporine A(CsA), as a kind of the calcineurin immunosuppressants, has become the first choice of the immunosuppressants for the organ transplantation because of it’s better antirejection effect. It’s a great economical burden for the patients taking expensive CsA during lifetime.During the practice of clinical pharmaceutical care, we found that berberine (BBR) can significantly increase the plasma concentration of CsA. BBR not only greatly reduced the doses of CsA but also not increased the CsA toxicity reaction. Thus, combination therapy was expected to become an approach of saving the cost of CsA.The induction or inhibition of CYP enzyme and drug transporter P-gp in liver or intestine is main reason for the interaction of drugs in clinic. Drugs can affect the CYP enzyme and P-gp in vivo and vitro experiments, resulting in changing the function of durgs. CYP3A is an important oxidative metabolism enzymes of drug, a member of cytochrome P450 (CYP) superfamily, involving in a variety of endogenous and exogenous compounds metabolism. Because of that, the efficacies and toxicities of durgs are influenced by the activities of CYP3A. The expression of CYP3A can be induced or inhibited by a variety of drugs, and the metabolism of substrate catalyzed by CYP3A can be stimulated or decelerate, which may result in the potential interaction of drugs. P-glycoprotein (P-gp) is an important protein of the cell membrane that pumps many foreign substances out of cells. It is an ATP-dependent efflux pump which encoded by MDR1 gene. The expresson of P-gp is rich in tissues and organs, such as liver, intestines and tumor. It also plays an important role in absorption, distribution and metabolism of most drugs. The expression of P-gp can also be induced and inhibited. The transporter level interaction of chemicals can be mediated by P-gp when substrates are coupled with the inhibitors or inducers for the wide substrate range of P-gp.During investigating the mechanism of the synergy of BBR, our laboratory had found that:1.Berberine had a bidirectional regulation effect on CYP3A4 and P-gp. Under relatively low doses, berberine had induction effect on CYP3A4 and P-gp, but it had inhition in relatively high dose. Compared with positive control group, the mechanisms of regulation was probably through PXR ways.2.BBR could inhibit the metabolism in vivo of Rh123 in a dose-dependent manner, which was associated with the suppression of the activity of CYP3A.3. BBR could inhibit the metabolism of MDZ in a dose-dependent manner in vivo, which is associated with the suppression of the activity of P-gp.4. In vitro, cytochrome P450 enzyme of CYP3A was inhibited by BBR in rats and mice liver, small intestine.5.The combined application of BBR and CsA was shown significantly inhibiting the expression of CYP3A1,CYP2E1, AMD, MDR1A and MDR1B genes of the liver in mice and rats, resulting to decreasing the catabolic elimination of CsA in the liver.6.The CsA pharmacokinetic study showed that BBR had synergy to CsA in kidney transplant patients and healthy subjects.7.The clinical studies had demonstrated the effectiveness of BBR using as CsA synergist, which made it was one of the drugs with highly security and economic value in kidney transplant patients. In our previous studies, we can inferred that the synergy of CsA was related with the inhibition of CYP3A and P- gp by BBR.The regulating function of pregnane X receptor (PXR) is one of the hotspot of the research on drug interactions. PXR is a key mediating drug metabolizing enzymes and transporters, as a ligand-dependent transcription factor and an important member of the nuclear receptor family. It is widely expressed in the liver, kidney, intestine, lung, brain, placenta, pancreas and other vital of tissues and organs. Many drug metabolic enzymes and transporter are regulated by PXR. As the critical regulatory element, PXR can mediate drug interactions and regulate the drug reactions, and then adjust the drug metabolism and drug transport. The mechanism of PXR regulatory as follows:when actived by the chemicals in the nucleus, PXR forms a heterodimer with the retinoid X receptor, and binds to hormone response elements on DNA which elicits expression of gene products. PXR can also directly involve in the regulation of MDR1 gene expression by binding the promoter regions of DR4/ER6 of MDR1 gene, resulting in change of the P-gp function.It’s unclear that whether BBR regulates the expression of CYP3A and P-gp through PXR signal pathway. This study has investigated the mechanism of BBR based on our previous studies. We had constructed the transfected model using the PXR knockdown hepatoma cells and confirmed BBR can affect the CYP3A and P-gp activity at the cellular level. Our fouding showed that the molecular mechanisms of BBR regulating the CYP3A and P-gp and provided experimental evidence for BBR as CsA synerist in a wide range of clinical application.During the practice of clinical pharmaceutical care, we were looking for mechanisms that BBR increased the plasma concentration of CsA in this project, and then we had found that BBR inhibited bioactivity of CYP3A and P-gp.From the inhibition of CYP3A and P-gp, we were looking for the cellular singal pathways of BBR’s regulation.To collect the related references, the regulating function of PXR is one of the hotspot of the research on drug interactions.To study the link between drug interactions by PXR and CAR signal pathways and BBR influencing the expression of CYP3A and P-gp, we delved into mechanism of BBR synergry for CsA. Because of time constraints in this project, we haven’t further studied CAR signal pathway. In the future, the more comprehensive experiment basis of BBR as CsA synergistic agent will be provided by research on CAR.Research methods1. Construction of RNAi lentivirus vectorAccording to methods from Addgene company, pLKO.1, pMD2.G, psPAX2 plasmid’s bacterias were cultured and identified. PLKO.1 plasmid was digested by restriction enzyme, and then purificated using gel electrophoresis. The digested pLKO.1 and two specificity of synthetic interference fragments of PXR shRNA were ligated, and then the products were transformated to competent cells. Colonies were cultured from the resistance screening and the sequenced for verification.2. Construction silent PXR cellAccording to the results of sequencing, the recombinant plasmid was extracted. The recombinant plasmids, psPAX2 and pMD2.G, were transfected into 293T cells to produce coate viruses, and then the virus were transfected into HepG2 cells. The best silening PXR cell lines were choosen by analyzed PXR protein.3. The protein expressionThe protein expression was analyzed with the Western-blot. Total proteins were extracted from cells. The total protein concentration was determined using coomassie brilliant blue. The target protein was detected by immunofluorescence imager and analyzed by Imagine J software. The levels of protein expression were determined by relative quantitative analysis.4. Gene expressionThe mRNAs were extracted from cells after treated for 48h, and reversed to cDNA. After RTQ-PCR was performed, the levels of gene expression were analyzed using the 2-ΔΔCt computational method and the GAPDH as internal standard control.5. Statistical analysis methodsAll data were analyzed using SPSS 20.0 statistical software, student’s t-test was applied to compare two group, One-way ANOVA was applied to compare among mutriple groups. P value was taken two sides, and the difference was statistically significant with P<0.05 or P<0.01.Result1. The two Synthetic fragments of PXR shRNA were successfully inserted into the pLKO.1 plasmid which were verificated by sequencing.2. The PXR protein expression of cell silence was significantly reduced (.P<0.01). The stablity transfected cell lines were successfully screened out, which were named as PXRi 19#, PXRi 21#. The PXR protein expression of PXRi 21# was lower than PXRi# 19 (P< 0.05), indicating that silence of PXRi 21# was more efficient. The PXRi 21# was choosen for subsequent experiments as the silent cells. The CYP3A4 and P-gp protein expression had no significant difference in PXRi 21# cell line, compared in HepG2 as control.3. In HepG2 cells and PXRi 21# cells, BBR had showed the inhibition of protein levels of CYP3A4 and P-gp in a dose-dependent manner. The CYP3A4 and P-gp protein expression of PXRi 21# were significantly higher than that of HepG2 in BBR 40μg/ml (P<0.01)4. In HepG2 cells and PXRi 21# cells, BBR had showed the inhibition of mRNA levels of CYP3A4 and P-gp in a dose-dependent manner. The CYP3A4 and P-gp mRNA expression of PXRi 21# were significantly higher than that of HepG2 in BBR 10μg/ml (P<0.01).Conclusions1.The expression lentiviral vector of pLKO.1-PXR-siRNA was constructed, successfully obtained the cell line of PXR silence.2. The CYP3A4 and P-gp protein expression in silent cells had no difference between unsilent cell and silent cell, indicating CYP3A4 and P-pg protein expression were not affected in silent HepG2 cells.3. In HepG2 cells and silent cells, BBR had showed the inhibition of CYP3A4 and P-gp for the reduced mRNA and protein levels, and the more concentration of berberine caused the lower levels. On cell silence, the inhibition of BBR was significantly lower than that in HepG2 cells, indicating that the sensitivity of BBR was weaker in cell silence.These results had showed that the berberine regulate the expression of CYP3A4 and P-gp via PXR nuclear receptors. |
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