| Background:Fluvastatin,a widely used lipid regulating agent in clinic,which exhibits a lipid-lowering effect after it was transferred to the liver by OATP1B1.Studies have shown that the OATP1B1 gene polymorphism has a direct impact on the pharmacokinetic parameters of statins.Thus,it is worth our effort to investigate whether gene polymorphism could impact the transport of FV,and subsequently influence the lipid-lowering effect.At present,the combination of statins and danshen preparation is used to treat hyperlipidemia in China,which showed good therapeutic effect.The pharmacodynamic mechanism of these two drugs has been reported,but the interaction and the pharmacokinetics mechanism of these two drugs are not clear.Therefore,it is necessary to study the effect of OATP1B1 gene polymorphism on the transport of FV,the effects of danshen related components on the transport of FV and its mechanism to lay the theoretical and experimental foundation for FV individualized therapy and clinical application of the combination with danshen preparation.Objectives:To study the difference of FV transport and the effect of danshen related components on the uptake of FV based on OATP1B1 genetic polymorphism.To further explore the potential mechanism of lipid lowering and adverse drug reaction reducing effect so as to lay a scientifical foundation for drug individualized therapy.Methods:1.To explore the impact of time and concentration on the uptake of FV in OATP1B1*1a ? OATP1B1*5 and OATP1B1*15-HEK293 T cells to obtain the optimum time and concentration of the uptake of FV in different genotypes OATP1B1-HEK293 T cells.To compare the uptake kinetics and dynamics parameters of FV(Km,Vmax and CLint)in different genotypes OATP1B1 cells to further analyse the influence of OATP1B1*5 and OATP1B1*15 on FV transport.2.To incubate different genotypes OATP1B1-HEK293 T cells with danshen related components at different concentrations for 48 h to further investigate the changes in the uptake of FV in different genotypes OATP1B1-HEK293 T cells.To screen out three components of danshen which significantly impact the uptake of establish methods of detecting and sample treatment of FV in the cell samples of HEK293 T cells.3.To conduct the positive controlled trial using 10?M rifampicin and incubate the wild type OATP1B1-HEK293 T cells with ursolic acid,cryptotanshinone and dihydrotanshinone at different concentrations for 48 h.To detect the expression level of SLCO1B1 mRNA and OATP1B1 by RT-qPCR and Western-blot to analyze the mechanism of action of these 3 components on FV transport.Results:1.The dynamic parameter Km(?M)and Vmax(pmol/mg protein·min-1)of FV in OATP1B1*1a?OATP1B1*5 and OATP1B1*15 cells are 17.59± 2.33,8.57±0.42;12.52±1.43(P<0.5),4.42±0.21(P<0.5)and 18.49± 2.06,9.13±0.61,respectively.And the value of CLint in OATP1B1*1a?OATP1B1*5 and OATP1B1*15 cells are 0.49,0.36(P<0.5)and 0.50,respectively.2.The uptake of FV in different genotypes OATP1B1-HEK293 T cells was increased to different degree by incubating with rifampin,ursolic acid,cryptotanshinone and dihydrotanshinone I at different concentrations.The uptake rate of FV was increased with the increase of concentration.However,the uptake of FV in various genotypes OATP1B1-HEK293 T cells were inhibited by Tanshinone I at high concentrations.And danshensu,protocatechuic acid,protocatechuic aldehyde,salvianolic acid B and tanshinone IIA had no obvious impact on FV transport in different genotypes OATP1B1-HEK293 T cells.3.The expression of SLCO1B1 mRNA and OATP1B1 in wild type OATP1B1-HEK293 T cells could be up-regulated after incubation with various concentrations of ursolic acid,cryptotanshinone and dihydrotanshinone I for 48 h,and the induction was increased with the increase of concentration.The ursolic acid have the strongest induction and cryptotanshinone comes second,while the induction of dihydrotanshinone I was weaker than 10?M rifampicin.Conclusion:1.The transport activity of FV in OATP1B1*5-HEK293 T is lower compared to that of OATP1B1*1a-HEK293T(wild type),while the transport activity of FV in OATP1B1*15-HEK293 T and OATP1B1*1a-HEK293T(wild type)bears no significant difference.2.The uptake of FV in different genotypes OATP1B1-HEK293 T cells were increased to different extent by ursolic acid,cryptotanshinone and dihydrotanshinone I.However,the uptake of FV in different genotypes OATP1B1-HEK293 T cells was decreased to varying extent by Tan I at high concentrations.Other components showed no considerable impact on the uptake of FV in different genotypes OATP1B1-HEK293 T cells.3.Ursolic acid,cryptotanshinone and dihydrotanshinone I could induce the expression of SLCO1B1 mRNA and OATP1B1 and enhance their transport function.Thus,these three components could further increase the uptake of FV,reduce its plasma concentration,strenthen its lipid-lowering efficacy and reduce the incidence of adverse reactions. |
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