Effects Of Astragali Radix On Drug Metabolizing Enzymes And Efflux Transporters And The Mechanism Underlying

ObjectiveThe phenomenon of drug-drug interaction(DDI)and adverse reactions as a result of Chinese medicine co-administrated with other medicines has attracted more and more serious attention of scholars both at home and abroad.According to the statistics,over 30%of all adverse drug reactions are caused by DDI.The pharmacokinetics of a drug in the body,drug-metabolizing enzymes(DMEs)and efflux transporters(ETs)are involved in themetabolism,detoxification,and elimination of xenobiotics,which play a crucial role in protecting human body from xenobiotics.However,these DMEs and ETs are easily affected by their own substrates,potentially affecting their pharmacokinetics,bioavailability,toxicity,and therapeutic response.Therefore,DMEs and ETs is the important targets causing DDI.Nowdays,more and more research about the regulation of Chinese medicine on DMEs and ETs has been reported.Astragali radix(HQ)is a widely used herbal medicine in Chinese clinical practice.Modern pharmacological studies and clinical practices indicate that HQ possesses various biological functions,such as potent immunostimulant effects,hepatoprotective properties,anti-cancer effects,neuroprotective effects,anti-aging properties,antioxidative effects,antimicrobial and antiviral.It has been widely used in fields of dietary supplements,health care product and drugs.However,it remains unknown that the regulation of HQ and its main bioactive compounds on DMEs and ETs.It is unclear that whether the co-administration of HQ with other substrate drugs will cause potential DMEs-or ETs-mediated DDLThe current study aims to investigate the regulation of HQ and its main bioactive compounds,including astragaloside ?(AS-?),calycosin(CS),and formononetin(FMNT)on the expression of DMEs and ETs in HepG2 cells.The results of this study would help predict the potential risk of DDI between HQ and their co-therapy drugs.This study might provide beneficial information for the correct clinical application of HQ.Methods1.Regulation of drug-metabolizing enzymes and efflux transporters by HQ,AS-?,CS,and FMNTHepG2 cells were treated with HQ,AS-?,CS,and FMNT at different concentration for 96 h.The protein and mRNA levels of Phase ? DMEs(CYP3A4,CYP2B6,and CYP2E1),Phase ? DMEs(UGT1A,UGT1A6,SULT1A1,and SULT1A3)and ETs(MDR1,BCRP,MRP2,MRP1,and MRP3)were measured using Western blot and real-time PCR,respectively.2.Regulation of P-gp and mechanism by HQ,AS-?,CS,and FMNTThe activity of P-gp and the mitochondrial mass were measured by fluorescent substrates and flow cytometry.The ATP concentration in the HepG2 cells exposed to HQ,AS-IV,CS,FMNT were detected by fluorescein chemiluminescence method.Reporter assay was used to study the ARE-luciferin activity by using HepG2-C8 cells.Results1.Regulation of the protein and mRNA levels of Phase ? DMEs by HQ,AS-IV,CS,and FMNTCompared with the control group,the HQ treatment for 96 h strikingly increased the protein and mRNA levels of CYP3A4,CYP2B6,and CYP2E1 in a dose-dependent manner.AS-IV treatment,specifically at 100 ?M concentration,markedly increased the mRNA levels of CYP3A4 and CYP2E1.CS gradually increased the protein levels of CYP3A4 and CYP2B6 with a rising dose.Exposure to CS at the tested doses significantly increased the mRNA levels of the three enzymes.FMNT significantly up-regulated the CYP3A4 levels;but markedly down-regulated the CYP2E1 levels at the doses of 50 and 100 ?M.FMNT induction significantly increased the mRNA levels of CYP3A4 and CYP2E1 at the tested doses.2.Regulation of the protein and mRNA levels of Phase ? DMEs by HQ,AS-IV,CS,and FMNTThe cells exposed to HQ strongly increased the protein levels of UGT1A with respect to the control cells.However,HQ significantly down-regulated the protein levels of UGT1A6 and SULT1A1.A significant dose-dependent increase in UGT1A levels was observed when AS-IV was used to treat the HepG2 cells.The treatment of CS significantly increased the UGT1A and SULT1A1 protein levels and decreased the SULT1A3 protein levels,up-regulated the mRNA levels of UGT1A,SULT1A1 and SULT1A3.An increase was observed only in the UGT1A protein levels in the cells treated with FMNT;FMNT significantly decreased the protein levels of SULT1A1 and SULT1A3.HQ and FMNT treatment strikingly up-regulated all the tested mRNA levels of Phase ? DMEs in a dose-dependent manner,with respect to the control cells.3.Regulation of the protein and mRNA levels of ETs by HQ,AS-IV,CS,and FMNTCompared with the control cells,exposure to HQ significantly increased the protein and mRNA levels of P-gp,MRP2,BCRP,and MRP3,but decreased the MRP1 protein levels,up-regulated the MRP1 mRNA levels in a dose-dependent manner.AS-? markedly up-regulated the protein levels of P-gp,MRP1,and MRP3.AS-IV also significantly decreased the BCRP protein levels in the 25 and 50 ?M groups.CS treatment increased protein and mRNA levels of P-gp,MRP2,and MRP3 levels at the same time;the BCRP protein levels were significantly decreased at 50 and 100 ?M CS treatments.FMNT significantly increased the protein and mRNA levels of P-gp,MRP2,MRP1 and MRP3.4.Regulation of P-glycoprotein expression and activity by HQ,AS-IV,CS,and FMNTHQ,AS-?,CS,FMNT decreased the intercellular accumulation of Rh123 in HepG2 cells,that is,HQ,AS-IV,CS,FMNT induced the efflux activity of P-gp.Moreover,HQ,AS-IV,CS,FMNT increased the ATP levels in HepG2.HQ increased the mitochondrial mass,that is,HQ may increase the mitochondrial function of HepG2.HQ,CS and FMNT induced ARE-luciferase activity in HepG2-C8 cells,which indicated that HQ,CS and FMNT could activate the Nrf2 signaling pathway.ConclusionsHQ,AS-?,CS,and FMNT significantly regulated the expression of the major DMEs and ETs.HQ produced stronger regulations(induction or inhibition)on DMEs and ETs than AS-IV,CS,or FMNT alone.HQ,AS-?,CS,and FMNT significantly increased the efflux activity of P-gp and intracellular ATP levels.Among,HQ induced the P-gp efflux activity may through increae the the mitochondrial function.The induction of HQ,CS,and FMNT on P-gp may be related to Nrf2 pathway.Conclusively,we predicted that the co-administration of HQ with other substrate drugs will cause potential DMEs-or ETs-mediated DDI.Careful monitoring is essential for the concomitant use of HQ with associated substrate drugs to avoid adverse interactions.