Study On The Effect And Mechanism Of Danshensu On The Uptake Of Rosuvastatin By Organic Anion Transporting Polypeptide 1B1

Background:Danshensu( salvianic acid A) is a phenolic aromatic acid compound isolated from salvia miltiorrhiza. The prophase researches of our research group show that danshensu is the substrate of organic anion transporting polypeptide 1B1 or the homologous transporter of rat oatplb2.Rosuvastatin is also the substrate of OATP1B1/ oatplb2,danshensu and rosuvastatin are often used together in clinical practice,whether danshensu show the competitive inhibition on the transport and uptake of rosuvastatin by OATP1B1,and then weaken the lipid-lowering efficacy of rosuvastatin after decreasing the drug concentration of targets? However, that was not the case of clinical effect,the reason is paid more attention to!So this topic will carry out the study around the influence of the SLCO1B1 m RNA which is the coding genes of transporter OATP1B1 and the function expression of OATP1B1 protein caused by danshensu,It will provide theoretical and experimental basis on lipid lowering therapy to use danshensu and rosuvastatin together.Objectives:Based on the successfully constructed Hep G2 cell model with high expression of SLCO1B1 m RNA and OATP1B1,we explored the influence of danshensu on the transcription of SLCO1B1 gene and the expression of OATP1B1 by RT-PCR and Western-blot.LC-MS is used to study the influence caused by danshensu on the uptake of rosuvastatin by OATP1B1,To clarify the effect and mechanism of danshensu on the uptake of rosuvastatin by OATP1B1.Methods:1. Cultivate Hep G2 cells regularly, Observe the shape and status of cells and draw the cell growth curve.2. Test the toxicity of danshensu,rifampicin, rosuvastatin and BSP on the Hep G2 cells by MTT. Choose the conditions under which the cell viability was over 80% for the following experiments.3. Incubate Hep G2 cells with different concentrations of rifampicin for 48 h, thendetect the expression of SLCO1B1 m RNA and OATP1B1 of Hep G2 cells,to estimate the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cell model.4.Conduct the parallel controlled trial using rifampicin as probe of the inductor of OATP1B1,incubate Hep G2 cells with different concentrations of danshensu for48 h, then detect the expression level of SLCO1B1 m RNA and OATP1B1 of Hep G2 cells by RT-PCR and Western-blot, to estimate the influence of danshensu on the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells,and compare the strength of induction effect between danshensu and rifampicin.5.The influence caused by danshensu on the uptake of rosuvastatin by OATP1B1① Explore and establish methods of detecting and sample treatment of rosuvastatin in the cell samples of Hep G2 cells.② Determine the optimum condition for the uptake by exploring the influence of time and drug concentration on the uptake of rosuvastatin,and choose the optimum conditions for the following experiments.③ Incubate Hep G2 cells with different concentrations of rifampicin for the optimum time chossed,detect the influence of danshensu on the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells.and conduct the parallel controlled trial using BSP as probe of the potent inhibitor of OATP1B1, calculate the inhibition parameters IC50 of danshensu and BSP,and compare the strength of inhibition effect between danshensu and BSP.Results:1.Compared with blank group,incubate Hep G2 cells with 10~100μM rifampicin for 48 h,10μM,25μM,50μM,100μM rifampicin make the expression of SLCO1B1 m RNA increase by 58.7%,82.6%,97.8%,130.4%;and make the expression of OATP1B1 increase by 44.4%,57.4%,90.7%,131.5%;the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells could be up-regulated after incubation with10~100μM rifampicin for 48 h, the difference has statistical significance(P<0.05). and the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells could be up-regulated after incubation with 10~100μM,and the induction was increased with the concentration(R2=0.7947;R2=0.9187).2.Compared with blank group, 20μM,40μM,80μM,120μM danshensu make theexpression of SLCO1B1 m RNA increase by 20.5%,30.7%,42.0%,45.5%;and make the expression of OATP1B1 increase by 15.2%,55.7%,84.9%,89.9%;the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells could be up-regulated after incubation with 20~120μM danshensu for 48 h, the difference has statistical significance(P<0.05). and the expression of SLCO1B1 m RNA and OATP1B1 in Hep G2 cells could be up-regulated after incubation with 20~100μM,and the induction was increased with the concentration(R2=0.8409;R2=0.8296).Induction parameter EC50 of danshensu is 39.0μM compared with 10.3μM of rifampicin as the parallel control group.3.Incubate Hep G2 cells with danshensu for different time(0~48h),the results show that after incubated Hep G2 cells with danshensu for 12 h and 24 h,RVS intake the uptake of RVS decrease by 22.02% and 12.20%;while the uptake of RVS increase by 1.83% and 85.39% when incubated for 36 h and 48 h.Incubate Hep G2 cells with0~120μM danshensu for 48 h,then conduct the 30μM RVS uptake experiments using58.55μM Danshensu as substrate,compared with blank group,10μM,20μM danshensu make the uptake of RVS decrease by 6.89%,22.10%;and 40μM,80μM,120μM danshensu make the uptake of RVS increase by 1.23%,79.93%,87.64%.4.Conduct the parallel controlled trial using BSP as probe of the potent inhibitor of OATP1B1,results show that,under the same experimental conditions,inhibition parameter IC50 of BSP is 17.92μM compared with 58.55μM of danshensu,the ability of danshensu inhibiting the uptake of RVS in Hep G2 cells is weaker than BSP.Conclusions:1.Danshensu could induce the expression of SLCO1B1 m RNA and OATP1B1 and enhance the ability on uptake of Hep G2 cells.2.When incubate Hep G2 cells with danshensu of low concentration for short time,the uptake of RVS performance as competitive inhibition,when the induction time extending and the concentration increasing,the uptake of RVS performance as induction.