| Objective:To evaluate the effects of high-altitude hypoxia on the activities of four cardiovascular system drugs bosentan,simvastatin,sildenafil and nifedipine as well as the expression of CYP3A4.To explore the regulatory role of CYP3A4 by hypoxia mediated by nuclear receptors PXR and CAR and miRNA-522-3p.To provide theoretical support for clinical rational drug use in the plateau areas.Methods:To evaluate the effects of high-altitude hypoxia on the pharmacokinetics of four cardiovascular system drugs,male Sprague Dawley（SD）rats were randomly divided into 4 groups,namely,plain control group,acute hypoxic group,chronic hypoxic group,and chronic hypoxic back to plain group,which were further divided into bosentan,simvastatin,sildenafil and nifedipine groups,with 8 rats in each group.Blood sample was collected from the eye sockets of rats at the corresponding time points after drug administration.The concentration of four cardiovascular system drugs and their metabolites were determined in the plasma of rats by UPLC-MS.Liver tissues were collected for protein and mRNA expression measurements of CYP3A1 by Western blot and RT-q PCR.To further evaluate the impact of high-altitude hypoxia on CYP3A1expression,different altitude groups of rats and cell experiments were added and validated using different experimental methods.SD rats were randomly divided into plain control group,moderate-altitude acute hypoxic group,moderate-altitude chronic hypoxic group,high-altitude acute hypoxic group,and high-altitude chronic hypoxic group.Half male and half female,each group containing 10 rats.ELISA and RT-q PCR were used to determine the protein and mRNA expression of PXR,CAR,and CYP3A1 in rat liver.Treatment of HepG2 cells with gradient oxygen concentration and different hypoxic culture times.ELISA and RT-q PCR were used to determine the protein and mRNA expression of PXR,CAR,and CYP3A1 in HepG2 cells.To investigate the transcriptional regulation of CYP3A4 mediated by nuclear receptors PXR and CAR in hypoxic,HepG2 cells were treated with hypoxia,and the promoter activity of CYP3A4 was detected by double-luciferase reporter assay.Silence of PXR and CAR of HepG2 cells by RNAi inhibited,and the mRNA expression of Cyp3a4 were determined by RT-q PCR.HepG2 cells were treated with PXR inhibitors（KCZ）and CAR inhibitors（RA）,and the promoter activity of CYP3A4 was detected by double-luciferase reporter assay.HepG2 cells were treated with PXR agonist（Rif）and CAR agonist（CITCO）,and the mRNA expression of Cyp3a4 were determined by RT-q PCR.HepG2 cells were treated with 2%,5%and 21%oxygen concentrations and Rif and CITCO,and the promoter activity of CYP3A4 was detected by double-luciferase reporter assay.To investigate the transcriptional regulation of CYP3A4 mediated by nuclear receptors PXR and CAR in hypoxic,HepG2 cells were treated with hypoxia,and the promoter activity of CYP3A4 was detected by double-luciferase reporter assay.Silence of PXR and CAR of HepG2 cells by RNAi inhibited,and the mRNA expression of Cyp3a4 were determined by RT-q PCR.HepG2 cells were treated with PXR inhibitors（KCZ）and CAR inhibitors（RA）,and the promoter activity of CYP3A4 was detected by double-luciferase reporter assay.HepG2 cells were treated with PXR agonist（Rif）and CAR agonist（CITCO）,and the mRNA expression of Cyp3a4 were determined by RT-q PCR.HepG2 cells were treated with 21%O2,2%O2,5%O2 and normoxic and Rif and CITCO,and the promoter activity of CYP3A4was detected by double-luciferase reporter assay.To investigate the transcriptional regulatory effect of miRNA mediated hypoxia on CYP3A4,high-throughput sequencing analyzed the differentially expressed miRNAs of HepG2 cells after hypoxic treatment and predicted bioinformatics targets.Over-and under-expression of miRNA-522-3p in HepG2 cells,and the protein and mRNA expression of CYP3A4 were determined by Western-blot and RT-q PCR and verify the regulatory effect of miRNA-522-3p on CYP3A4.Results:There were significant changes in the pharmacokinetic properties of the drugs in rats exposed to high-altitude hypoxia.Compared with the plain control group,the CL/F and V/F of bosentan in the chronic hypoxic group were significantly decreased,while AUC（0-24）,AUC（0-∞）,MRT（0-24）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and V/F of hydroxybosentan in the chronic hypoxic group were significantly decreased,while AUC（0-24）,AUC（0-∞）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and Ke of simvastatin in the chronic hypoxic group were significantly decreased,and t1/2,Tmax,AUC（0-∞）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and V/F of simvastatin hydroxy acid in the chronic hypoxic group were significantly decreased,and AUC（0-24）,AUC（0-∞）,MRT（0-24）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and V/F of sildenafil in the acute and chronic hypoxic group were significantly decreased,while AUC（0-24）,AUC（0-∞）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and V/F of N-desmethyl sildenafil in the chronic hypoxic group were significantly decreased,and AUC（0-24）,AUC（0-∞）,MRT（0-24）,and Cmax were significantly increased.Compared with the plain control group,the CL/F and Ke of nifedipine in the acute and chronic hypoxic group were significantly decreased,and AUC（0-24）,AUC（0-∞）,t1/2,and MRT（0-∞）were significantly increased.Compared with the plain control group,the CL/F of dehydronifedipine in the acute and chronic hypoxic group were significantly decreased,the MRT（0-∞）,AUC（0-∞）,MRT（0-24）,and AUC（0-24）were significantly increased.The study showed a significant decrease in the expression of CYP3A1 in rats after exposure to acute and chronic hypoxia at both the protein and mRNA levels.There was a 23.95%,36.46%,and 29.59%decrease in the protein expression of CYP3A1 in the acute hypoxia,chronic hypoxia,and chronic hypoxia to plain groups,respectively,compared with the plain control group.Additionally,the expression of CYP3A1 protein of the chronic hypoxia group decreased by 16.45%compared to the acute hypoxia group;however,there was no significant difference between the other groups.Compared to the plain control group,there was a 52.23%,64.74%,and 57.59%decrease in the Cyp3a1 mRNA expression levels in the acute hypoxia,chronic hypoxia,and chronic hypoxia to plain groups.Further research on protein and mRNA expression showed that the expression of PXR,CAR,and CYP3A1 decreased in moderate-altitude hypoxic group.There was a significant decrease in the protein expression of PXR,CAR and CYP3A1 in the hepatic tissues of rats exposed to hypoxic conditions compared with the plain control group.There was a 41.01%,47.36%,50.63%and 63.56%decrease in the protein expression of PXR in the moderate-altitude acute hypoxic group,moderate-altitude chronic hypoxic group,high-altitude acute hypoxic group and high-altitude chronic hypoxic group,respectively,compared with the plain control group.There was a 43.05%and 47.38%decrease in the protein expression of CAR in the high-altitude acute hypoxic group and high-altitude chronic hypoxic group,respectively,compared with the plain control group.There was a 56.54%and 41.84%decrease in the protein expression of CYP3A1 in the moderate-altitude chronic hypoxic group and high-altitude chronic hypoxic group,respectively,compared with the plain control group.There was a significant decrease in the mRNA expression of Nr1i2,Nr1i3 and Cyp3a1 in the hepatic tissues of rats exposed to hypoxic conditions compared with the plain control group.There was a 29.41%and 37.25%decrease in the mRNA expression of Nr1i2 in the moderate-altitude chronic hypoxic group and high-altitude acute hypoxic group,respectively,compared with the plain control group.There was a 18.87%and 33.02%decrease in the mRNA expression of Nr1i3 in the high-altitude acute hypoxic group and high-altitude chronic hypoxic group,respectively,compared with the plain control group.There was a 37.25%,59.80%,65.69%,and 64.71%decrease in the mRNA expression of Cyp3a1 in the moderate-altitude acute hypoxic group,moderate-altitude chronic hypoxic group,high-altitude acute hypoxic group,and high-altitude chronic hypoxic group,respectively,compared with the plain control group.Compared with the normoxia group,the protein expression of PXR,CAR and CYP3A4 in HepG2 cells changed significantly in each hypoxic group.There was a46.57%,24.58%,31.69%,40.75%,and 28.46%decrease in the protein expression of PXR in the 2%O2-24 h,5%O2-6 h,5%O2-12 h,5%O2-24 h and 5%O2-48 h groups,respectively,compared with the normoxia group.There was a 35.41%,36.22%and38.92%decrease in the protein expression of CAR in the 2%O2-24 h,5%O2-2 h and5%O2-6 h groups,respectively,compared with the normoxia group.There was a50.98%,66.59%and 52.69%decrease in the protein expression of CYP3A4 in the 2%O2-24 h,5%O2-2 h and 5%O2-6 h groups,respectively,compared with the normoxia group.Compared with the normoxia group,the expression of Nr1i2,Nr1i3 and Cyp3a4 mRNA in HepG2 cells changed significantly in each hypoxia group.There was a 50.00%and 48.00%decrease in the mRNA expression of Nr1i2 in the 5%O2-2h and 5%O2-6 h groups,respectively,compared with the normoxia group.There was a 55.00%and 66.00%decrease in the mRNA expression of Nr1i3 in the 2%O2-24 h and 10%O2-24 h groups,respectively,compared with the normoxia group.There was an 85.00%,78.00%,57.00%,56.00%and 50.00%decrease in the mRNA expression of Cyp3a4 in the 10%O2-24 h,5%O2-2 h,5%O2-6 h,5%O2-12 h and 5%O2-48 h groups,respectively,compared with the normoxia group.Regulation of high-altitude hypoxia on the transcription of CYP3A4mediated by PXR and CAR.The luciferase activity of CYP3A4 decreased by59.28%under the condition of 2%O2.Hypoxia significantly reduced the promoter activity of CYP3A4.After treatment of HepG2 cells with si RNA-PXR+CAR,the relative expression of Cyp3a4 mRNA was decreased by 53.33%.After treatment with nuclear receptor inhibitor KCZ,RA and KCZ+RA,the promoter activities of CYP3A4 were decreased by 23.35%,38.48%,and 16.81%,respectively.After treatment with PXR agonist RIF 24 h,the mRNA expression of Nr1i2 and Cyp3a4 was increased by 108.00%and286.00%,respectively.After treatment with CAR agonist CITCO 2 h,the mRNA expression of Nr1i3 and Cyp3a4 was increased by 202.00%and263.00%,respectively.After treatment with RIF and CITCO 24 h,the mRNA expression of Nr1i2,Nr1i3 and Cyp3a4 was increased by 85.00%,52.00%and84.00%,respectively.At 2%O2 and 5%O2,nuclear receptor agonists had no significant effect on the activity of CYP3A4 promoter.At normoxic oxygen concentration,the activity of CYP3A4 promoter in the agonist RIF+CITCO group increased significantly by 53.46%.High-throughput sequencing found that 16 miRNAs were down-regulated and 14 miRNAs were up-regulated in HepG2 cells under hypoxia.Bioinformatics target prediction showed that miRNA-522-3p was associated with CYP3A4.The expressions of protein and mRNA of CYP3A4 increased after the treatment of HepG2 cells with an inhibitor of miR-522-3p and decreased in the mimic group.The results showed that miRNA-522-3p had a negative regulatory effect on CYP3A4.Conclusion:High-altitude hypoxia significantly altered the pharmacokinetic characteristics of bosentan,simvastatin,sildenafil,nifedipine and the corresponding metabolites hydroxybosentan,simvastatin acid,N-desmethyl sildenafil,and dehydronifedipine,mainly manifesting as slower clearance and longer retention time in vivo.The expression of drug metabolizing enzyme CYP3A4 and nuclear receptors PXR and CAR significant changed in high-altitude hypoxic environments.PXR and CAR mediate the transcriptional regulation of CYP3A4 by hypoxia.Mi RNA-522-3p was identified as an important factor mediating CYP3A4 changes under hypoxic conditions.Hypoxia affects the in vivo metabolism of bosentan,simvastatin,sildenafil and nifedipine by inhibiting the expression of PXR,CAR and miRNA-522-3p,resulting in altered expression of downstream CYP3A4.When the above four cardiovascular drugs are used clinically in high-altitude hypoxic areas,the pharmacokinetic characteristics should be re-evaluated,and the dose administered should be adjusted appropriately for rational use. |
