| Background:Berberine,an isoquinoline alkaloid extracted from natural medicine,is commonly used in the treatment of gastrointestinal diseases.Recent studies have confirmed that it can reduce the levels of low density lipoprotein and triglyceride by increasing the expression of LDL-C receptor,activating AMP kinase and blocking MAPK/ERK pathway.Statins mainly reduce the level of low-density lipoprotein cholesterol by inhibiting the synthesis of mevalonic acid and reducing the synthesis of cholesterol in the liver.Based on the different mechanisms of BBR and statins in regulating plasma lipids,BBR and statins are often used in clinical practice for the treatment of hyperlipidemia.It was reported that about 60%of the side effects of statin rhabdomyolysis were caused by drug interactions caused by combined drugs changing the activity of metabolic enzymes or transporters,so the effects of BBR on drug metabolic enzymes and transporters have attracted much attention.Studies have found that the concentrations of BBR,its metabolites berberrubine and demethylene berberine in liver were much higher than those in plasma,and can affect the function of drug metabolic enzymes and transporters.However,it is still unknown whether drug interactions occur due to their effects on drug metabolizing enzymes and transporters,and should be systematically investigated.It is worth mentioning that many studies have shown nuclear receptors play a vital role in regulating drug metabolism and transport related target genes.Berberine could increase the expression of ABCA1 transporter in macrophages,reduce cholesterol accumulation and foam cell formation by activating LXR?nuclear receptor.However,there is no research report on the effect of BBR on the metabolism and transport of lipid-regulating drugs around the nuclear receptor-metabolic enzyme/transporter pathway.Therefore,an in-depth and systematic study on the role of BBR in the nuclear receptor-CYP450/OATPs pathway can provide a scientific basis for the rational combination of BBR and statins.The aim of this subject was to investigate the effects of BBR and its metabolites BRB and DBB on the expression of CYP450enzyme and OATP1B1 transporter and metabolic transport of statins in HepG2 cell,and to study the pharmacokinetics of atorvastatin and rosuvastatin after long-term administration of BBR in rat model,to analyze the relationship between the changes of pharmacokinetic characteristics and the activity of metabolic enzymes and transporters.Finally,in the HepG2 cell,the molecular mechanism of drug regulation in nuclear receptor-CYP450/OATPs pathway was explored by molecular biological techniques such as RT-qPCR,Western-blot and double luciferase reporter gene.Objectives:1.Systematically explore the effects of BBR,BRB and DBB on the expression of CYP1A2,3A4,2B6 mRNA,protein and the activity of enzyme metabolic substrate in HepG2 cells.2.Systematic study the effects of BBR,BRB and DBB on OATP1B1 mRNA,protein expression and transport of atorvastatin and rosuvastatin in HepG2 cells.3.SD rat model was used to study the effects of long-term administration of BBR on the pharmacokinetics of atorvastatin and rosuvastatin,and to explore the relationship between the effect and metabolic enzymes/transporters4.Based on the regulatory pathways of nuclear receptors AhR,PXR and RXR?,the effects of BBR and BRB on the regulation of metabolic enzymes CYP1A2,3A4and transporter OATP1B1 were studied,and the molecular mechanisms of BBR and BRB on AhR,PXR and RXR?-CYP450/OATP1B1 pathways were revealed.Methods:1.RT-qPCR assay was used to investigate the effects of BBR,BRB and DBB on the expression of CYP1A2,3A4,2B6 and OATP1B1 mRNA in HepG2 cells.2.Western blot assay was used to study the effects of BBR and BRB on the expression of CYP1A2,3A4,2B6,OATP1B1,AhR,PXR,RXR?,FXR and LXR?in HepG2 cells.3.LC-MS/MS method was established to determine the concentrations of phenacetin,midazolam and atorvastatin to evaluate the effects of long-term induction of BBR and BRB on the activities of CYP1A2 and 3A4 in HepG2 cells.The concentrations of atorvastatin and rosuvastatin in cell samples and plasma were determined to investigate the effects of long-term induction of BBR and BRB on the uptake of atorvastatin and rosuvastatin by HepG2 cells,and the effects of long-term administration of BBR on the pharmacokinetics of atorvastatin and rosuvastatin in rats.4.The double luciferase reporter genes of pGL3-AhR-CYP1A2-luc and pGL3-PXR-CYP3A4-luc were constructed to explore the role of AhR and PXR in the regulation of CYP1A2 and CYP3A4 expression by BBR and BRB.The double luciferasereportergenesofpGL3-FXR-OATP1B1-lucand pGL3-LXR?-OATP1B1-luc were constructed to study the role of RXR?,FXR and LXR?in the regulation of OATP1B1 transporter by BBR and BRB.5.AhR,PXR,RXR?,FXR,LXR?were silenced by RNA interference technique in HepG2,to explore the role of BBR and BRB in the regulation of CYP1A2 and CYP3A4 pathway by AhR and PXR,and the role of BBR and BRB in the regulation of OATP1B1 pathway by RXR?,FXR and LXR?.Result:1.Effects of BBR,BRB and DBB on the expression of CYP1A2,3A4,2B6and the activity of enzyme metabolic substrates1.1 BBR and BRB promote the expression of CYP1A2 and 3A4 mRNA in HepG2 cellsRT-qPCR results showed that 550?M BBR and 560?M BRB could promote the expression of CYP1A2 mRNA in a concentration-dependent manner,the EC50was 11.2?M and 14.3?M,respectively,and 1580?M DBB could also slightly induce the expression of CYP1A2 mRNA,but there was no significant difference compared with the control group?P>0.05?.550?M BBR and 560?M BRB could promote the expression of CYP3A4 mRNA in a concentration-dependent manner,and the EC50 was 8.2?M and 9.6?M,respectively.1580?M DBB had no significant effect on the expression of CYP3A4 mRNA?P>0.05?.However,550?M BBR?560?M BRB and 1580?M DBB had no significant induction effect on the expression of CYP1B6 mRNA.1.2 BBR and BRB induced the expression of CYP1A2 and 3A4 proteins in HepG2 cellsThe results of Western blot showed that the positive control 50?M omeprazole could significantly induce the expression of CYP1A2 protein about 3.5 times,while25,50?M BBR and 30,60?M BRB could induce the expression of CYP1A2 protein,which were 1.6,2.7,1.4,1.9 times compared with the blank control group,respectively.the difference was statistically significant?P<0.05?.After treated with 10?M rifampicin,50?M BBR and 60?M BRB for 48 h,the induction ratio of CYP3A4protein expression was 3.1,2.5,2.7 times compared with the blank control group,respectively.1.3 BBR and BRB increase the activities of CYP1A2 and 3A4 in HepG2 cellsThe results showed that compared with the blank control group,the positive controls 50?M omeprazole,50?M BBR and 60?M BRB increased the metabolism of phenacetin by 175%,165%and 157%compared with the blank control group,respectively.BBR at the concentration of 5?M and 50?M could increase the ability of CYP3A4 to metabolize midazolam and atorvastatin,and its EC50 was 9.8?M and48.2?M,respectively.The EC50 of 560?M BRB on the metabolism of midazolam and atorvastatin were 26.7?M and 56.3?M,respectively.2.Effects of BBR,BRB and DBB on OATP1B1 expression and transport function in HepG2 cells2.1 Effects of BBR,BRB and DBB on OATP1B1 mRNA and protein expression in HepG2 cellsRT-qPCR results showed that 550?M BBR and 560?M BRB could promote the expression of OATP1B1 mRNA in a concentration-dependent manner,and the EC50 was 21.4?M and 12.5?M,respectively,while there was no significant difference in mRNA expression of OATP1B1 in different concentrations of DBB.The results of Western blot showed that after HepG2 cells were treated with 25?M and 50?M BBR for 24 hours,the expression of OATP1B1 protein was up-regulated by 1.5,2.3 times compared with the control group,while its main metabolite BRB could up-regulate the expression of OATP1B1 protein at 3060?M,which was statistically significant compared with the blank control group.60?M BRB could up-regulate the expression of OATP1B1 protein about 3.4 times.However,there was no significant difference in the expression of OATP1B1 in the concentration range of 1580?M?P>0.05?.2.2 BBR and BRB increase the uptake of atorvastatin and rosuvastatin by HepG2 cells.The LC-MS/MS method established in this study for the determination of atorvastatin and rosuvastatin in cell samples has a good linear relationship in the range of 5640nmol/L and 2144nmol/L standard curves,respectively,and the precision and accuracy meet the requirements.The results of uptake assay showed that 550?M BBR could increase the uptake of RSV and ATV in HepG2 cells in a concentration-dependent manner,and their EC50 values were 19.01?M and 24.16?M,respectively.560?M BRB could also increase the uptake of RSV and ATV in a concentration-dependent manner in HepG2 cells.The EC50 values were 13.95?M and27.59?M,respectively.3.Effects of long-term administration of BBR on the pharmacokinetics of atorvastatin and rosuvastatin and its correlation with metabolic enzyme transporter in SD rats.Long-term administration of high-dose BBR significantly affected the pharmacokinetics of atorvastatin and rosuvastatin in rats.The results showed that after continuous administration of 300 mg/kg BBR,the maximum plasma concentration of atorvastatin in the control group and the experimental group was9.04±0.97 ng/mL and 6.51±0.45 ng/mL,respectively,which was about 28%lower than that in the control group.The area under the dose-time curve?AUC0-t?was40.87±13.91 ng/mL·h and 28.72±17.15 ng/mL·h,respectively,which was about30%lower than that in the control group,which may be due to the increase of OATP1B1 liver uptake of statins and the decrease of systemic circulation dose.The elimination half-life?T1/2?decreased from 8.2±2.32 h to 5.5±1.49 h,a decrease of about 33%,while the clearance rate?CLz/F?increased by about 140%.Obviously,this was due to the induction of BBR,which accelerated the metabolism of atorvastatin and shortened the elimination process of atorvastatin by CYP3A4.Similarly,in the blank control group and the experimental group given 300mg/kg BBR continuously,the Cmax of rosuvastatin in rat plasma was 5.83±0.95?g/mL and 3.78±0.13?g/mL,decreased by about 35%,the AUC0-t was 36.80±11.91?g/L·h and 28.88±11.79?g/L·h,respectively,which was still due to the increase of OATP1B1 activity by BBR.However,the CLz/F of rosuvastatin was 2.86±0.83 L/h and 3.08±0.74 L/h,T1/2 was 4.71±1.03 h and 4.91±1.28 h,respectively,which was not significantly different from that of the control group,suggesting that the change of CYP3A4 activity had no effect on the metabolism of rosuvastatin,which was consistent with the literature report that rosuvastatin was hardly metabolized by CYP450.4.Mechanism of BBR and BRB regulating CYP1A2/CYP3A4 based on AhR/PXR-CYP1A2/3A4 pathway.4.1 BBR and BRB activate AhR/PXR to increase the activity of CYP1A2/CYP3A4 reporter gene.The results of double luciferase reporter genes showed that compared with the control group,the luciferase activity of 10,25,50?M BBR to pGL3-AhR-CYP1A2-luc was 1.3,2.4 and 3.4 times higher than that of pGL3-AhR-CYP1A2-luc,and that of 15,30,60?M BRB to pGL3-AhR-CYP1A2-luc was 1.1,1.9 and 2.4 times higher,respectively.The luciferase activity of pGL3-PXR-CYP3A4-luc was up-regulated 1.2,1.4,and 2.3 fold at 10,25,and 50?M BBR,respectively;the luciferase activity of pGL3-PXR-CYP3A4-luc was up-regulated 1.1,1.3,and 1.8 fold at 15,30,and 60?M BRB,respectively.The results showed that BBR and BRB induced the activities of pGL3-AhR-CYP1A2-luc and pGL3-PXR-CYP3A4-luc reporter genes in a concentration-dependent manner.The results showed that BBR and BRB could activate the CYP1A2/CYP3A4promoter and induce the expression of CYP1A2/CYP3A4 protein by acting on AhR/PXR,respectively.4.2 Silencing AhR/PXR reduced the induction of CYP1A2/CYP3A4 protein expression by BBR and BRB.In normal HepG2 cells,the expression of CYP1A2 protein was up-regulated by5 nM TCDD,50?M BBR and 60?M BRB by 3.5,2.4 and 1.9 times,respectively.After transfected with shRNA-hAhR plasmid,the expression of CYP1A2 protein was up-regulated by 5 nM TCDD,50?M BBR and 60?M BRB by 1.2,0.96,0.89 times.In normal HepG2 cells,compared with the control group,10?M RIF,50?M BBR and 60?M BRB up-regulated CYP3A4 expression by 2.7,1.8 and 2.3 times,respectively,while 10?M RIF,50?M BBR and 60?M BRB up-regulated CYP3A4expression by 0.95,0.92,0.97 times when transfected with shRNA-hPXR plasmid.It can be seen that silencing AhR/PXR significantly reduces the induction of CYP1A2/CYP3A4 protein expression by BBR and BRB.That further confirmed the induction of CYP1A2/CYP3A4 by BBR and BRB was mediated by AhR/PXR.4.3 BBR and BRB induce AhR/PXR nuclear translocationWestern blot showed that 50?M BBR and 60?M BRB upregulated AhR nucleoprotein 2.6 fold and 2.5 fold,50?M BBR and 60?M BRB upregulated PXR nucleoprotein 3.4 fold and 1.9 fold,the difference was statistically significant compared with the blank group.It was suggested that BBR and BRB could promote the transfer of AhR/PXR from cytoplasm to nucleus.5.Mechanism of BBR and BRB regulating OATP1B1 based on RXR?,FXR and LXR?pathways5.1 BBR and BRB activated RXR?to increase the activity of OATP1B1 reporter geneThe double luciferase reporter genes showed that both BBR and BRB could induce the activities of pGL3-FXR-OATP1B1-luc and pGL3-LXR?-OATP1B1-luc reporter genes in a concentration-dependent manner.After treated with a classical inhibitor UVI3003,the reporter gene activity of BBR and BRB decreased significantly compared with the group without RXR?inhibitor.The results suggest that nuclear receptor RXR?plays a key role in the regulation of OATP1B1 expression by BBR and BRB.5.2 Silencing FXR,LXR?and RXR?reduced the induction of OATP1B1protein expression by BBR and BRB.The results showed that compared with the blank control group,50?M BBR could up-regulate the expression of OATP1B1 2.1 times,while after silencing FXR or LXR?,the induction was decreased to 1.5,1.4 times,respectively.After silencing RXR?,the induction effect was only 0.94 times that of the blank control group.Compared with the blank control group,60?M BRB could up-regulate the expression of OATP1B1 by 1.9 times.After silencing FXR or LXR?,the induction fold decreased by 1.4,1.2 times,respectively.After silencing RXR?,the induction effect of BRB was only 0.86 times that of the blank control group,and there was significant difference compared with the experimental group without silencing nuclear receptors?P<0.05?.These results further suggested that RXR?is the key upstream nuclear receptor of BBR and BRB in the regulation of OATP1B1.5.3 BBR and BRB induce nuclear translocation of FXR,LXR?and RXR?The results of Western blot showed that 25,50?M BBR increased FXR nucleoprotein by 1.4,1.6 times,LXR?nucleoprotein by 1.3,2.0 times,and RXR?nucleoprotein by 1.7,1.9 times,respectively.30,60?M BRB increased FXR nucleoprotein 1.6,1.8 times,LXR?nucleoprotein 1.2,1.5 times,and RXR?nucleoprotein 1.4,1.8 times as much as the control group.The results showed that BBR and BRB promoted the transfer of FXR,LXR?and RXR?from cytoplasm to nucleus.Conclusion:1.BBR and BRB induced the expression of CYP1A2,3A4 mRNA and protein and increased the metabolic activity of the enzyme in a dose-dependent manner.2.BBR and BRB increased the expression of OATP1B1 mRNA and protein in a dose-dependent manner,and significantly promoted the uptake of Atorvastatin and rosuvastatin by HepG2 cells.3.Continuous administration of 300 mg/kg BBR significantly decreased the Cmax and AUC0-t of atorvastatin and rosuvastatin in rat plasma,which was closely related to the expression of OATP1B1 induced by BBR,which significantly increased the liver uptake of atorvastatin or rosuvastatin.At the same time,BBR also induced CYP3A4 activity,which accelerated the metabolism of atorvastatin in rats.Therefore,If BBR was used in combination with statins,based on the effect of BBR on its distribution and metabolism,it may be considered to reduce the dose of statins as appropriate and avoid the occurrence of adverse reactions such as rhabdomyolysis as much as possible.4.The results further proved that BBR and BRB promoted the nuclear translocation of AhR and PXR,activated CYP1A2 and CYP3A4 promoters,thus significantly increased the activity of CYP1A2 and CYP3A4,promoted the nuclear translocation of FXR,LXR?and RXR?,activated the OATP1B1 promoter,thus significantly increased the activity of OATP1B1.5.In summary,in the regulatory pathway of AhR and PXR-CYP450/RXR?-OATP1B1,BBR and BRB could promote the nuclear translocation and activation of AhR,PXR,FXR,LXR?and RXR?,and significantly improve the metabolism and transport ability of statins,which provided more experimental basis for the correct combination of berberine and statins in the treatment of regulating blood lipids,and added new content to the regulatory mechanism of AhR,PXR,RXR?-CYP450/OATP1B1. |
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