| ObjectiveExploring the potential molecular mechanism of meridian-guiding effect of Vinegar-baked Radix Bupleuri(VBRB)by determining the effects of VBRB and its components on the uptake activities,protein and mRNA expression of drug transporters.Methods1.Test dosages of VBRB and its components using in BRL3A and HEK293 cells were determined by MTT assay.2.Cytoplasmic cisplatin(co-substrate of Oct2 and Mrp2)contents in BRL3A cells were detected by HPLC,and sample protein concentrations were determined by BCA method.Substrate uptake rate was normalized by protein concentration.The mRNA expression of Oct2 and Mrp2 in BRL3A cells was detected by real-time quantitative PCR(RT-PCR).The relationship between meridian-guidingeffect of VBRB with Oct2 and Mrp2 were analyzed.3.Cytoplasmic Sodium taurocholate(TCANa,co-substrate of Ntcp and Oatp2)contents in BRL3A cells were detected by HPLC,and sample protein concentrations were determined by BCA method.Substrate uptake rate was normalized by protein concentration.The protein and mRNA expression of Ntcp and Oatp2 in BRL3A cells was detected by Western Blot and RT-PCR,respectively.And then the relationship between meridian-guidingeffect of VBRB with Ntcp and Oatp2 were analyzed.4.Colchicine is co-substrate of MRP1 and Pgp.For excluding the interference effect of MRP1 on Pgp,VBRB and its components used with MRP1 inhibitor MK571.Cytoplasmic colchicine contents were detected by HPLC,and sample protein concentrations were determined by BCA method.Substrate uptake rate was normalized by protein concentration.The protein and mRNA expression of Pgp in HEK293 was detected by Western Blot and RT-PCR,respectively.Then,the relationship between meridian-guidingeffect of VBRB with Pgp were analyzed.5.Colchicine used as MRP1 substrate.For excluding the interference effect of Pgp on MRP1,VBRB and its components used with Pgp inhibitor verapamil.And determination of cytoplasmic substrate contents,peotein and mRNA expression of MRP1 in HEK293 cell were measured by HPLC,Western Blot and RT-PCR,respectively.Then,the effect of VBRB and its components on Pgp were analyzed.As MRP1 function depends on GSH,reduced glutathione(GSH,2 mM)was added in HEK293 cells to stimulate MRP1 function.And the effect of VBRB and its components on colchicine uptake,and MRP1 protein expression and mRNA levels in GSH stimulating HEK293 cells(high MRP1 expression)were measured by HPLC,Western and RT-PCR,respectively.And then,by comparing different effect on normal cells with high MRP1 expression cells,we analyzed the relationship between MRP1 and meridian-guidingeffect of VBRB.6.After treated with VBRB and its components,mRNA expression of Bsep,Oat1,Oat2 and Oct1 in BRL3A cell were measured by RT-PCR.7.hOCT2-HEK293,hMRP2-HEK293 and hPgp-HEK293 cells with high expression of OCT2,MRP2 and Pgp gene were stable transfected,by using plasmid DNA with a green fluorescent protein reporter gene system.Which genes introduced to HEK293 cells by using the chemical transfection reagent Mega Tranl.0.And aminoglycoside antibiotic(G418)was added to make these genes express stably.After treated with G418 for 8 weeks,the transfection efficiency of cells,protein and mRNA levels were detected by flow cytometry,Western Blot and RT-PCR,respectively.And the relationship between meridian-guidingeffect of VBRB with Oct2 and Mrp2 were analyzed.8.Cisplatin was used as substrate of OCT2.The effects of VBRB and its components on the uptake activity in hOCT2-HEK293 cell,and OCT2 protein and mRNA expression were determined by HPLC,Western Blot and RT-PCR,respectively.And the relationship between meridian-guiding effect of VBRB with OCT2 were determined.9.Cisplatin was used as substrate of MRP2.The effects of VBRB and its components on the uptake of cisplatin in hMRP2-HEK293 cell,and MRP2 protein and mRNA expression were determined by HPLC,Western Blot and RT-PCR,respectively.And the relationship between meridian-guiding effect of VBRB with MRP2 were determined.10.Rhodamine B was used as substrate of Pgp,and the cytoplasmic substrate content in hPgp-HEK293 cells were detected flow cytometry.Then protein expression was detected by Western blot,and mRNA level was detected by RT-PCR.And the relationship between meridian-guiding effect of VBRB with Pgp were determined.Results1.According to the results of MTT assay,the test dosages of VBRB and its components.For BRL3A cells,VBRB(10 mg.ml-1),PSS(10 mg.ml-1),Buf(50 mg.ml-1),MHE(50 mg.ml-1),SSa(20 μ g.ml-1),SSb(100 μg.ml-1),SSb2(100 μg.ml-1),SSc(50 μg.ml-1),SSd(10 μg.ml-1),Cle(10 μg.ml-1)were used.And for HEK293 cells,VBRB(10 μg.ml-1),PSS(50 mg.ml-1),Buf(50 mg.ml-1),MHE(50 mg.ml-1),SSa(20 μg.ml-1),SSb(50 μg.ml-1),SSb2(50 μg.ml-1),SSc(50μg.ml-1),SSd(10 μg.ml-1),Cle(50 μg.ml-1)were used.2.VBRB and its components did not transported cisplatin in the same direction.After BRL3A cells treated with VBRB and its components for 24 h,the results were as follows:when compared with the control group,VBRB(10 mg.mL-1),Buf(50 mg.mL-1),SSc(50 μg.ml-1),SSd(10 μg.ml-1)increased the cellular uptake of cisplatin by 123.8%,146.6%,164.1%and 49.7%(P<0.01),respectively.While PSS(10 mg.mL-1),SSb(100 μg.ml-1),SSb2(100 μg.ml-1)decreased the cellular uptake of cisplatin by 44.6%,45.2%,53.20%(P<0.05),and MHE(50 mg.mL-1),Cle(10 μg.ml-1)decreased by 56.1%,54.8%(P<0.01).Besides,when compared with the control group,VBRB(10 mg.mL-1),SSd(10μg.ml-1),Cle(10 μg.ml-1)increased the expression of Oct2 mRNA by 98.4%,60.3%and 78.0%(P<0.05),and VBRB,PSS,Buf,MHE,SSb2 and Cle increased the Mrp2 mRNA levels by 184.1%,194.4%,169.1%,181.9%,33.5%and 262.9%(P<0.01).While SSa and SSb increased the Mrp2 mRNA levels by 60.4%and 141.6%(P<0.05)。3.In addition to SSa,SSd and MHE,When treated with VBRB and its components for 24 h,other drugs increased the uptake activity of TCANa in BRL3A cells.When compared with the control group,VBRB(10 mg.mL-1),PSS(10 mg.mL-1),Buf(50 mg.mL-1),SSb2(100 μg.ml-1),SSc(50 μg.ml-1)and Cle(10 μg.ml-1)increased the uptake of TCANa by 11.4%,17.8%,21.2%,29.6%,27.9%and 24.9%(P<0.05).While MHE(50 mg.mL-1)decreased it by 23.0%(P<0.05),and SSa(20μg.ml-1),SSd(10 μg.ml-1)did not change the uptake of TCANa.The effect of vinegar Bupleurum on expression of Ntcp gene was associated with the composition and concentration of them.When compared with the control group,SSb2(100 μg.ml-1)increased the protein expression of Ntcp in BRL3A cells by 18.3%(P<0.05).While PSS(10 mg.mL-1),MHE(50mg.mL-1),SSa(20μg.ml-1),SSc(50 μg.ml-1)、SSd(10 μg.ml-1),Cle(10 μg.ml-1)decreased it by 29.4%,62.0%,28.8%,40.2%and 66.1%(P<0.05).And Buf(50mg.mL-1),SSb(100 μg.ml-1),SSb2(100 μg.ml-1),SSc(50 μg.ml-1)and Cle(10 μg.ml-1)increased the mRNA expression of Ntcp by 34.9%,41.4%,146.3%,105.8%and 73.4%(P<0.05).And PSS(10 mg.mL-1),MHE(50 mg.mL-1)decreased it by 44.2%and 52.3%(P<0.05).Besides,the effect of vinegar Bupleurum on expression of Oatp2 gene was associated with the composition and concentration of them as well.When compared with the control group,SSb2(100 μg.ml-1),SSc(50 μg.ml-1),SSd(10μg.ml-1)upregulated the protein expression of Oatp2 by 248,3%,270.8%and 97.2%(P<0.05),and SSb(100 μg.ml-1)upregulated by 481.5%(P<0.01).While MHE(50 mg.mL-1)downregulated the protein expression of Oatp2 by 17.2%(P<0.05).VBRB(10 mg.mL-1),Buf(50 mg.mL-1),SSb(100 μg.ml-1),SSb2(100 μg.ml-1),Cle(10 μg.ml-1)increased the mRNA expression of Oatp2 by 66.2%,86.5%and 42.0%(P<0.05),116.8%and 176.0%(P<0.01).4.VBRB and its components decreased the uptake of colchicine in HEK293 cells.Comparing with control group,after treated with VBRB and its components for 24 h,VBRB(10 mg.ml-1),PSS(50 mg.ml-1),Buf(50 mg.ml-1),MHE(50 mg.ml-1),SSa(20 μg.ml-1),SSb(50 μg.ml-1),SSb2(50 μg.ml-1,SSc(50 μg.ml-1),SSd(10μg.ml-1)and Cle(50 μg.ml-1)decreased the uptake of colchicine significantly(P<0.05).When combined used with MRP1 inhibitor MK571,PSS and SSa increased the uptake of colchicine by 31.2%(P<0.05)and 12.8%.And other groups promoted the activity of Pgp and showed decreased uptake:VBRB(10 mg.ml-1),Buf(50 mg.ml-1),SSb(50 μg.ml-1),SSd(10 μg.ml-1)decreased the uptake fo colchicine by 14.8%,11.7%,21.1%,28.9%(P<0.05).Different components of VBRB showed a difference effect on Pgp.Compared with control group,VBRB(10 mg.mL-1),SSc(50 μg.ml-1,SSd(10 μg.ml-1)decreased the protein expression of Pgpby 37%,34.6%and 45.1%(P<0.05).While SSa(20 μg.ml-1),SSb(50 μg.ml-1),SSb2(50 μg.ml-1)and Cle(50μg.ml-1)increased it by 31.9%,111.0%,68.0%and 82.8%(P<0.05).5.When combined with Pgp inhibitor verapamil,the effect that different components from VBRB on Pgp were diffenert.Compared with control group,after treated with VBRB and verapamil for 48 h in HEK293 cells:Buf(50 mg.mL－1)、SSa(20 μg.ml-1)、SSb2(50 μg.ml-1)decreased the uptake od colchicine by 34.0%,21.4%and 24.8%(P<0.05),and PSS(50 mg.mL－1)increased the protein expression of MRP1 by 15.6%(P<0.05).But it had no biological change on the mRNA expression of MRP1.Under GSH status,the effect of VBRB on MRP1 was more significant.And compared with control group,after treated with VBRB and GSH(2 mM)for 48 h in HEK293 cells:VBRB(10 mg.ml-1)and MK571(50 μM)enhanced the uptake of colchicine by 92.7%and 75.6%(P<0.01),and MHE(50 mg.ml-1),SSa(20 μg.ml-1),SSb2(50 μ g.ml-1),SSd(10 μg.ml-1)enhanced it by 30.6%,38.0%,51.4%and 40.5%(P<0.05).Besides,VBRB and SSd reduced protein expression of MRP1 by 45.6%(P<0.05)and 78.9%(P<0.01).But it had no biological change on the mRNA expression of MRP1.6.After treated with VBRB and its components in BRL3A for 24 h:(1)VBRB enhanced the mRNA expression of Bsep.Compared with control group,VBRB(10rmg.mL-1),PSS(10mg.mL-1),Buf(50mg.mL-1),MHE(50mg.mL-1),SSb(100 μg.ml-1)and SSb2(100 μg.ml-1)downregulated the mRNA expression of Besp in BRL3A cells(P<0.05),and SSa(20 μg.ml-1)and SSc(50 μg.ml-1)reduced it significantly without biological change.(2)VBRB enhanced the mRNA expression of Oatl.Compared with control group,VBRB(10 mg.mL-1),Buf(50 mg.mL-1),MHE(50 mg.mL－1),SSb(100 μg.ml-1),SSb2(100 μg.ml-1),SSc(50 μg.ml-1)and Cle(10 μg.ml-1)upregulated the mRNA expression of Oatl in BRL3A cells by 49.9%,86.9%,65.4%,92.7%,136.5%,77.43%and 95.3%(P<0.05).(3)VBRB enhanced the mRNA expression of Oat2.Compared with control group,BRB(10mg.mL-1),PSS(10mg.mL-1),Buf(50 mg.mL-1),SSa(20 μg.ml-1),SSc(50 μg.ml-1)and Cle(10 μg.ml-1)upregulated the mRNA expression of Oat2 in BRL3A cells by 245.7%,177.7%,291.2%,96.4%,77.6%(P<0.05).(4)The effect of components from VBRB on Octl was not the same.Compared with control group,Buf(50 mg.mL－1)and SSa(20 μg.ml-1)reduced the mRNA expression of Octl in BRL3A cells by 57.2%and 51.3%(P<0.05).While SSb(100 μg.ml-1)and Cle(10 μg.ml-1)enhenced it by 130.4%and 104.6%(P<0.05).7.Construction and identification of stable high expression of human OCT2,MRP2 and Pgp gene in HEK293 cell linesThe transfection efficiency of hOCT2-HEK293,hMRP2-HEK293,hPgp-HEK293 cells,Vector cells(which transfected with pCMV6-AC-GFP plasmid cells)which screened by G418 for 8 weeks detected by flow cytometry were as follows:76%,69%,66%,82%.Western Blot results indicated that:stable transfected hOCT2-HEK293,hMRP2-HEK293,hPgp-HEK293 cells showed high expression of OCT2,MRP2 and Pgp protein,respectively,and Vector cells scarcely express the corresponding OCT2,MRP2 or Pgp protein.RT-PCR results showed that:compared to the vector cells,OCT2,MRP2 and Pgp mRNA expression of hOCT2-HEK293,hMRP2-HEK293,hPgp-HEK293 cells were 3.21,4.07,2.99 flod as the Vector’ s,respectively.8.VBRB increased the uptake of cisplatin in hOCT-HEK293 cells.Compared with control group,VBRB(10 mg.ml-1),MHE(50 mg.ml-1),SSa(20 μg.ml-1),SSb2(50 μg.ml-1,SSc(50 μg.ml-1,SSd(10 μg.ml-1)and Cle(50 μg.ml-1)increased the cellular uptake of cisplatin by:54.7%,40.8%,15.0%,61.3%,49.2%,54.6%and 53.2%(P<0.05),and PSS(10 mg.ml-1)and SSb(50 μg.ml-1)increased by:101.9%and 140.9%(P<0.01).VBRB increased the protein expression of OCT2 in hOCT2-HEK293 cells.Compared with control group,Western Blot results showed VBRB,PSS,Buf,MHE,SSb,SSb2 and SSd increased OCT2 protein expression by:190.9%,98.3%,53.2%,56.4%,13.8%,49.7%,and 44.2%(P<0.05).Besides,VBRB increased the mRNA expression of OCT2 in hOCT2-HEK293 cells.Compared with control group,VBRB,PSS,Buf SSa,SSb,SSb2,SSc,SSd and Cle increased mRNA expression of OCT2 by:91.8%,188.1%,166.0%,61.8%,47.1%,111.3%,67.0%and 121.0%,and 132.4%(P<0.05);and MHE group increased by 101.3%(P<0.01).9.VBRB increased the uptake of ciaplatin in hMRP2-HEK293 cells.Compared with control group,VBRB(10 mg.ml-1),PSS(50 mg.ml-1),MHE(50 mg.ml-1),SSb(50 μg.ml-1),SSb2(50 μg.ml-1),SSc(50 μg.ml-1)and MK571(50 μM)increased the uptake of cisplatin by:35.7%,49.9%,36.2%,58.5%,35.8%,44.3%and 19.5%(P<0.05).And VBRB,SSb,SSb2 and SSd reduced protein expression of MRP2 by:63.8%,42.5%,40.6%and 57.8%(P<0.05),while SSc(50 μg.ml-1)increased 16.7%of MRP2 protein expression(P<0.05).VBRB and its components had little effect on MRP2 mRNA.10.VBRB increased the uptake of Rhodamine B in hPgp2-HEK293 cells.Compared with control group,cellular uptake of rhodamine B were significantly increased,PSS(50 mg.mL-1)increased by 18.7%(P<0.05);and VBRB(10 mg.mL-1),Buf(50mg.mL-1),MHE(50mg.mL-1),SSa(20 μg.ml-1),SSb(50 μg.ml-1),SSb2(50 μg.ml-1),SSc(50 μg.ml-1),SSd(10 μg.ml-1),Cle(50 μg.ml-1)and Verapamil(50i μM)increased by:30.2%,27.7%,27.62%,17.8%,28.9%,42.4%,32.5%,41.6%,31.0%and 20.5%(P<0.01).Western Blot results showed that SSa,SSc,MHE significantly reduced Pgp protein expression by 54.3%,49.0%and 67.8%(P<0.01),and SSd reduced Pgp protein expression by 59.1%(P<0.05).But VBRB and its components had no effect on Pgp mRNA expression.Conclusion1.VBRB enhanced the uptake of cisplatin in BRL3A cells,which might be associated with upregulation of Oct2 mRNA expression by SSd and downregulation of Mrp2 mRNA expression by SSc.This indicated that upregulation Oct2 and downregulation Mrp2 might be the mechanism of meridian-guiding effect of VBRB.Whereas PSS,MHE,SSb,SSb2 and Cle inhibited the uptake of cisplatin might be associated with downregulation of Mrp2 mRNA expression,and it indicated that PSS,MHE,SSb,SSb2 and Cle were associated with low liver toxicity of platinum anticancer drugs.2.VBRB enhanced the uptake of bile salts in BRL3A cells,which might be associated with Buf,SSb2,SSc and Cle which upregulated the expression of Ntcp and SSb2,SSc,Cle upregulated the expression of Oatp2.And it indicated that upregulation of Ntcp and Oatp2 might be the mechanism of meridian-guiding and synergism effect of VBRB,and SSb2,SSc and Cle were the effective components from VBRB.Although PSS promoted the uptake of substrate,it had nothing to do with Ntcp and Oatp2.MHE decreased the uptake of bile salts,and downregulated the expression of Ntcp and Oatp2.It indicated that MHE was advantageous to prevent the occurrence of intrahepatic cholestasis.3.After combined used with MK571,VBRB reduced the uptake of colchicine in normal cells,and it might be associated with SSb,SSb2,and Cle upregulated the protein expression of Pgp.Which also indicated that VBRB protected normal cells from anticancer drugs,and upregulation of Pgp was one of the mechanisms associated with meridian-guiding and attenuated effect of VBRB.And the inhibition of SSd and promotion of PSS had nothing to do with Pgp.4.Without GSH,VBRB reduced the uptake of colchicine,and showed a protection of normal cells.But it had nothing to do with the protein and mRNA expression of MRP1,and indicated that VBRB protected normal cells from other pathway.While under GSH ststus,VBRB increased uptake og colchicine and might be associated with downregulation of MRP1 by PSS,Buf,MHE,SSb2 and SSd.It indicated that downregulation of high expression of MRP1 might be one of the mechanism of meridian-guiding and attenuated effect of VBRB.5.VBRB enhanced the mRNA expression of Oatl and Oat2,while did not change the mRNA expression of Bsep and Octl a lot.6.Stable overexpression of OCT2,MRP2 and Pgp in hOCT2-HEK293,hMRP2-HEK293,hPgp-HEK293 cells were successfully constructed,and can be used in the efficacy screening experiments.7.When OCT2 overexpress,VBRB enhanced the uptake of cisplatin in hOCT2-HEK293 cells.Which might be associated with the upregulation of OCT2 protein or mRNA expression by PSS,Buf,MHE,SSa,SSb,SSb2,SSc,SSd and Cle.That indicated upregulation of OCT2 might be one of the mechanism of the meridian-guiding effect of VBRB.8.When MRP2 overexpress,VBRB enhanced the uptake of cisplatin in hMRP2-HEK293 cells.Which might be related to the upregulation of MRP2 protein or mRNA expression by SSb,SSb2 and SSd.That indicated downregulation of MRP2 was one of the mechanism of the meridian-guiding effect of VBRB.While the fact that PSS,MHE and SSc enheanced the uptake of cisplatin was not associated with MRP2 expression.9.When Pgp overexpress,VBRB enhanced the uptake of Rhodamine B in hPgp2-HEK293 cells.Which might be associated with the downregulation of Pgp protein expression by MHE,SSa,SSc and SSd.It indicated downregulation of Pgp might be one of the mechanism of the meridian-guiding effect of VBRB.In summary,VBRB increased the liver uptake of substrates by upregulating the expression of OCT2,Ntcp and Oatp2,and decreased the uptake of substrates by downregulating the expression of overexpressed MRP1,MRP2 and Pgp.The meridian-guiding effect of VBRB might be associated with drug transporters,which took part in drug distribution.The meridian-guiding effect of VBRB might be a common result of various components form VBRB affecting the transporters,and the regulation of VBRB on drug transporters is a possible mechanism of meridian-guiding. |
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