| Background: Warfarin is a widely used anticoagulant drug in clinical work,mainly in the prevention and treatment of atrial fibrillation,valvular disease,valve replacement and venous thrombotic disease,and is irreplaceable for patients with prosthetic valve replacement.Panax notoginseng saponin(PNS)is the main active ingredient of pseudo-ginseng.With the widespread use of dietary supplements,we found in our clinical work that there is a potential interaction between pseudo-ginseng and warfarin in combination.However,as herbal medicine to activate blood circulation and remove blood stasis,pseudo-ginseng shows antagonistic effects in combination with warfarin,while no relevant studies have been reported.NR1I3 is a member of the nuclear receptor superfamily,and its role in regulating drug metabolism is crucial.Some studies have shown that NR1I3 acts as a transcription factor for CYP2C9,which is the main drug metabolizing enzyme of warfarin.Therefore,whether NR1I3 is involved in the effect of Panax ginseng total saponin on the efficacy and pharmacokinetics of warfarin needs to be further investigated.NR1I3 is a ligand-dependent transcription factor that is dephosphorylated and translocated to the nucleus after ligand activation to play a transcriptional regulatory role.Meanwhile,in addition to the ligand activation pathway,micro RNAs are also present in transcription factors to regulate transcriptional activity.However,whether micro RNA acting on NR1I3 is involved in the drug interaction between Panax ginseng total saponin and warfarin remains to be demonstrated.In this study,we intend to systematically investigate the mechanism of interaction between Panax notoginseng saponins and warfarin,the main active ingredient of pseudo-ginseng,in terms of pharmacodynamics,pharmacokinetics and related drug metabolism genes,to provide a theoretical basis for the rational clinical use of warfarin.Part Ⅰ.Effect of PNS on the pharmacodynamics and pharmacokinetics of warfarin Objectives:1.To observe the effects of PNS on the pharmacodynamics,pharmacokinetics and drug metabolizing enzymes of warfarin in rats.2.To observe the effects of PNS on the pharmacodynamics and drug metabolizing enzymes of warfarin in HepG2 cells.Methods:1.Qualitative analysis of PNS by high performance liquid chromatography mass spectrometry(HPLC-MS).2.To establish high performance liquid chromatography-mass spectrometry(HPLC-MS/MS)method to determine the concentration of warfarin in rats and to observe the effects of PNS on their coagulation function,coagulation factors,blood concentration and related drug metabolizing enzymes CYP2C9,CYP1A2 and CYP3A4.3.Elisa was performed to detect the effect of PNS on coagulation factors in HepG2 cells.Western Blot and QRT-PCR were performed to detect its effect on drug metabolizing enzymes.Results:1.High performance liquid chromatographic method showed that good separation could be achieved for Panax ginseng saponin R1,ginsenoside Rg1,ginsenoside Re,ginsenoside Rb1,and ginsenoside Rd,and the resulting high performance liquid chromatograms were graphically consistent with the total saponins of Panax ginseng included in the national drug standards,which were identified as Panax notoginseng saponins.2.The results of in vivo experiments in rats showed that PNS was able to reduce the pharmacodynamics of warfarin.Compared with the warfarin alone group,the International normalized ratio(INR)and the activated partial thromboplastin time(APTT)were significantly lower,and the coagulation factor II and coagulation factor X were significantly higher in the combination group,and the differences were statistically significant.At the same time,the area under the drug-time curve(AUC0-t)and mean residence time(MRT0-t)were significantly lower,and the apparent volume of distribution(Vz/F)and clearance rate(CLz/F)were significantly higher in the combination group compared with the warfarin group alone,and the differences were all statistically significant.The results of Western Blot and QRT-PCR showed that PNS significantly induced CYP2C9 and NR1I3,the major drug metabolizing enzymes of warfarin,while there was no significant change in CYP1A2,CYP3A4 and VKORC1,the target of warfarin.The results of immunohistochemical study also confirmed the induction of CYP2C9 and NR1I3 by PNS.3.The results of cellular assays showed that coagulation factor II and coagulation factor X were significantly increased in HepG2 cells in the combination group compared with the warfarin group alone.Western blot and QRT-PCR results showed that PNS showed significant induction of CYP2C9 and NR1I3,the main drug metabolizing enzymes of warfarin,with statistically significant differences.Conclusion:PNS accelerates warfarin drug metabolism and reduces its efficacy by inducing the drug metabolizing enzyme CYP2C9.Part Ⅱ.PNS induces NR1I3 to affect the pharmacodynamics and pharmacokinetics of warfarin Objective:To investigate the role and mechanism of NR1I3 in the effect of PNS on the pharmacodynamics and pharmacokinetics of warfarin.Methods:1.Cellular experiments: stable transgenic cell lines with NR1I3 interference and overexpression were constructed,and the interference and overexpression efficiency were detected using QRT-PCR and Western Blot.After the cells were treated with drugs,qRT-PCR and Western Blot were used to detect the effect of interference and overexpression of NR1I3 on the drug metabolizing enzyme CYP2C9.Elisa was used to detect the changes of coagulation factor II and coagulation factor X after interference and overexpression of NR1I3.2.Animal experiments: Interfering and overexpressing adeno-associated virus injection virus AAV9-NR1I3 was constructed to specifically infect rat liver tissues via tail vein injection,and the infection efficiency was verified by Western Blot and QRT-PCR.After the drug treatment,the changes of coagulation function and coagulation factors of rats were detected,the changes of warfarin blood concentration and pharmacokinetic parameters were detected by HPLC-MS/MS,and the effects of interfering and overexpressing NR1I3 on rat liver drug metabolizing enzyme CYP2C9 were detected by qRT-PCR and Western Blot.Results:1.qRT-PCR and Western Blot results showed that mRNA and protein levels were significantly reduced after interference with NR1I3 in HepG2 cells,while mRNA and protein levels were significantly increased after overexpression of NR1I3.The expression of drug-metabolizing enzyme CYP2C9 was downregulated after interference with NR1I3,and the elevation of coagulation factor II and coagulation factor X due to coadministration of PNS was decreased,while the expression of drug-metabolizing enzyme CYP2C9 was increased after overexpression of NR1I3,and the Elisa results showed that the elevation of coagulation factor II and coagulation factor X due to PNS was more significant and the difference was statistically significant.2.qRT-PCR and Western Blot results showed that mRNA and protein levels were significantly reduced after interference with NR1I3 in rat liver tissues,while mRNA and protein levels were significantly increased after overexpression of NR1I3.The expression of CYP2C9,the drug-metabolizing enzyme of warfarin,was down-regulated in the liver tissues of rats after NR1I3 interference,and the INR and APTT decreased and the coagulation factor II and coagulation factor X increased due to the coadministration of PNS were partially restored.Meanwhile,the drug concentration and pharmacokinetic parameters of warfarin in rats showed that the metabolism of warfarin was slowed down after the interference of NR1I3,and the accelerated metabolism of warfarin caused by the coadministration of PNS was partially restored.After overexpression of NR1I3,the expression of drug metabolizing enzyme CYP2C9 in rat liver tissues increased,and the decrease of INR and APTT,and the increase of coagulation factor II and coagulation factor X due to the coadministration of PNS were more significant.Meanwhile,the drug concentration and pharmacokinetic parameters of warfarin in rats showed that the metabolism of warfarin was accelerated after overexpression of NR1I3,and the accelerated metabolism of warfarin due to the coadministration of PNS was more significant,and the differences were statistically significant.Conclusion:PNS affects the pharmacodynamics and pharmacokinetics of warfarin through the induction of NR1I3.Part Ⅲ PNS regulating miR-214-3p/NR1I3 affects the pharmacodynamics and pharmacokinetics of warfarin Objective:To screen the key micro RNAs that regulate NR1I3 in the effect of PNS on the pharmacodynamics and pharmacokinetics of warfarin,and to demonstrate that PNS affects the pharmacodynamics and pharmacokinetics of warfarin by regulating miR-214-3p/NR1I3.Methods:1.Micro RNAs with potential regulation of NR1I3 mRNA 3’-UTR and high expression in the liver were predicted by Target Scan,an online prediction software to further screen for micro RNAs that may regulate NR1I3.2.HepG2 cells were treated with dosing to detect changes in the expression abundance of micro RNAs and to screen for miR-214-3p with reduced expression.3.Construct wild-type(psi CHECK2-NR1I3-Wt)and mutant(psi CHECK2-NR1I3-Mut)reporter gene plasmids and confirm the regulatory effect of miR-214-3p on NR1I3 mRNA 3’-UTR using dual luciferase reporter gene assay.4.miR-214-3p mimic and miR-214-3p inhibitor were constructed and transfected into HepG2 cells respectively,and the changes of coagulation factor II and coagulation factor X were detected by Elisa after drug treatment,and the changes of NR1I3 and CYP2C9 protein levels were detected by Western Blot.5.HepG2 cells overexpressing NR1I3 were simultaneously transfected with miR-214-3p mimic,and the changes of coagulation factor II and coagulation factor X were detected by Elisa,and the changes of NR1I3 and CYP2C9 protein levels were detected by Western Blot.6.HepG2 cells interfering with NR1I3 were transfected with miR-214-3p inhibitor,and the changes of coagulation factor II and coagulation factor X were detected by Elisa,and the changes of NR1I3 and CYP2C9 protein levels were detected by Western Blot,and the reversion experiment further verified the regulatory effect of miR-214-3p on NR1I3.7.HepG2 was treated with Panax ginseng total saponin adjuvant for immunofluorescence staining,and the changes of NR1I3 cellular localization were observed by fluorescence microscopy.Results:1.A total of four micro RNAs that may regulate NR1I3 were screened by software prediction: miR-214-3p,miR-125a-3p,miR-25-3p,miR-155-5p.2.After the dosing treatment of HepG2 cells,the expression of miR-214-3P decreased,the expression of miR-125a-3p increased,and the difference was statistically significant,miR-25-3p and miR-155-5p expression abundance did not change significantly.3.Dual luciferase reporter gene results showed that overexpression of miR-214-3p significantly inhibited psi CHECK2-NR1I3-Wt reporter gene luciferase activity with statistically significant differences,while the reporter gene system mutated at the NR1I3 mRNA 3’UTR miR-214-3p binding site(psi CHECK2-NR1I3-Mut),overexpression of miR214-3p had no effect on reporter gene luciferase activity.4.Mi R-214-3p mimic transfection of HepG2 cells resulted in downregulated protein levels of NR1I3 and CYP2C9 compared to control,with partial reversion of elevated coagulation factor II and coagulation factor X due to coadministration of PNS.Mi R-214-3p inhibitor transfection of HepG2 cells resulted in downregulated protein levels of NR1I3 and The protein levels of CYP2C9 were upregulated,and the elevation of coagulation factor II and coagulation factor X due to PNS was more significant and the difference was statistically significant.5.After transfection of miR-214-3p mimic in HepG2 stable-transfected cell line overexpressing NR1I3,the elevation of coagulation factor II and coagulation factor X due to overexpression of NR1I3 was partially restored,and the upregulation of protein levels of NR1I3 and CYP2C9 was also partially restored.6.After transfection of miR-214-3p inhibitor in HepG2 stable-transformed cell line with NR1I3 interference,the decrease in coagulation factor II and coagulation factor X due to NR1I3 interference was partially restored,and the downregulation of protein levels of NR1I3 and CYP2C9 was also partially restored.7.Immunofluorescence results showed that the activation of NR1I3 and its entry into the nucleus increased after treatment with PNS,demonstrating the regulatory effect of PNS on the activation of NR1I3.Conclusion:Mi R-214-3p targets NR1I3 mRNA 3’-UTR,there is a regulatory relationship,and PNS affects the pharmacodynamics and pharmacokinetics of warfarin by regulating miR-214-3p/NR1I3. |
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