| Background:Statins were usually used to treat hyperlipidemia and its complications such as coronary heart disease, the treatment of atherosclerosis. Danshen, as traditional Chinese medicine, was used for the treatment of cardiovascular and cerebrovascular diseases.Danshen was often combined with statins for the treatment of hyperlipidemia, cardiovascular and cerebrovascular diseases.But whether danshen effect the pharmacokinetics of rosuvastatin is not clear. We found that water-soluble components danshensu and ursolic acid of danshen were the substrates of OATP1B1 or rat homologous transporter Oatplb2.At the same time, most statins are the substrates of OATP1B1/Oatp1b2.Therefore, whether danshensu and ursolic acid effect the transport of statins by OATP1B1/Oatplb2, and effect the pharmacokinetics of statins..It is worth to explore.This papers explore the effect of danshensu and ursolic acid on the pharmacokinetics of rosuvastatin in rats.Then, study the effects of danshensu and ursolic acid on the transport of rosuvastatin in Caco-2 cell model, and explore the effects of danshensu and ursolic acid on uptake of rosuvastatin in primary hepatocytes. Finally, constructe the HEK293T cell model that can stably express OATP1B1*1a and OATP1B1*5 gene. Then take liver drug transporter of OATP1B1 as a starting point, to study the effect of danshensu and ursolic acid on the mediated transport of rosuvastatin by wild-type and mutant OATP1B1 based on the phenomenon of genetic polymorphism of OATP1B1. Explore the effect mechanism of danshensu and ursolic acid on the OATP1B1-mediated rosuvastatin uptake in vitro and vivo.Objective:1.To explore the effect of danshensu and ursolic acid on the pharmacokinetics of rosuvastatin in rats.2.To explore the effects of danshensu and ursolic acid on the transport of rosuvastatin in Caco-2 cell model. 3.To explore the the effects of danshensu and ursolic acid on uptake of rosuvastatin in primary hepatocytes.4. To constructe the HEK293T cell model that can stably express OATP1B1* 1a and OATP1B1* 5 gene.At the molecular level, to explore the effect of danshensu and ursolic acid on uptake of rosuvastatin in OATP1B1*1a and OATP1B1*5 HEK293 cell, and so as to prove the molecular mechanism that the effect of danshensu, ursolic acid on rosuvastatin pharmacokinetics.Methods:1. Male SD rats (220±15g) were randomly divided into groups as rosuvastatin control group, the combination danshensu with rosuvastatin group and the combination ursolic acid with rosuvastatin group,6 rats in each group. Oral administration dose of rosuvastatin, danshensu and ursolic acid were 100mg/kg, 100mg/kg and 80mg/kg. Danshensu (or ursolic acid) have been fed before rosuvastatin when drug combination was applied, and interval was 15mins. Blood samples were also collected from the orbital venous at 0,0.5,1,1.5,3,5,8,10,12.24 h following an oral administration of rosuvastatin.Blood samples were centrifuged and the obtained plasma samples were stored at -20℃.LC-MS was applied to determine the concentration of rosuvastatin. Rosuvastain plasma-concentration data were analysed using DAS 2.1 Microsoft. Statistical analysis was conducted using SSPS12.0. P value less than 0.05 was considered as statistical significance.2. The establishment of Caco-2 monolayer cell mode, the TEER value and activity of alkaline phosphatase were used to inspect integrity and polarity of model. Explore the cell toxicity of verapamil, rosuvastatin, danshensu and ursolic acid on Caco-2 cell.Then study the uptake feature of rosuvastatin in Caco-2 cell and explore the effects of danshensu and ursolic acid on transport of rosuvastatin in Caco-2 monolayer mode cell that study verapamil, danshensu and ursolic acid on the effect of rosuvastatin'apparent permeability coefficient (PappBL-AP and PappAP-BL, respectively. Statistical analysis was conducted using SSPS12.0. P value less than 0.05 was considered as statistical significance.3. Rat primary liver cells were isolated by In situ collagenase perfusion, cultured. Explore the liver cell toxicity of rosuvastatin, danshensu and ursolic acid. Studying the effect of time, drug concentration on uptake of rosuvastatin in primary hepatocytes. Then research the effect of danshensu and ursolic acid on the uptake of rosuvastatin in liver cells, respectively:Liver cell will be diluted to 1×106ml with DMEM, then added to 24 well culture plate,2ml each well. Experiment was designed as blank group, rosuvastatin control group, rosuvastatin combined with a series concentrations of ursolic acid or danshensu group. Drug concentration of rosuvastatin was 20uM. Series concentrations of danshensu were 20,40 and 80 uM. Series concentration of ursolic acid were 4,8 and 16 uM. Cells were hatched in Incubator at 37℃for 40sec, slow suction medium culture containing the drug, then cells were washed four times with 4℃culture medium and 0.5ml sterile water was added. The sample was palced at ultra-low temperature freezer at-80℃, repeated freezing solution 3 times. Samples was determined by LC-MS after treatment. Statistical analysis was conducted using SSPS12.0. P value less than 0.05 was considered as statistical significance.4. Using PCR (polymerase chain reaction) to take target gene OATP1B1*5 from the OATP1B1*5 Plasmid (pcDNA3.1 (-)/Zeo:Invitrogen), and use target gene as a template cloned another gene OATP1B1*1a by point mutation, then both were holded DNA sequencing. The pGC-FU GFP lentiviral vector transfer system was used as gene transmit medium to constructe the recombinant lentiviral vector of OATP1B1*1a-GFP fusion gene and OATP1B1*5-GFP fusion gene.The fluorescence detection and Westernblot were used to observe the GFP protein expression. Real-time fluorescence quantitative PCR (Real-time quantitative PCR, RTQ-PCR) was applied to detect virus titlers of lentiviral. The OATP1B1-GFP fusion gene lentivirus (Lenti-OATP1B1*1a, Lenti-OATP1B1*5) and GFP lentivirus (Lenti-GFP) were transfected HEK293T. Observing the fluorescence and Westernblot were used to detect the gene expression of OATP1B1*1a and OATP1B1*5. Study the cell toxicity of rosuvastatin, danshensu and ursolic acid on HEK293T and study the uptake feature of rosuvastatin on OATP1B1*1a and OATP1B1*5 HEK293 cell. Then study the effect of danshensu and ursolic acid on uptake of rosuvastatin in OATP1B1*1a and OATP1B1*5 HEK293 cell, the experiment was designed as control group, rosuvastatin group, rosuvastatin combined a series of concentrations of danshensu (0.1,1 and 10 uM) or ursolic acid (0.18,1.8 and 18 uM) group. The rosuvastatin concentration was 10 uM. The samples of HEK293T was determined by LC-MS. Statistical analysis was conducted using SSPS12.0. P value less than 0.05 was considered as statistical significance.Results:1. Both ursolic acid and danshensu could significantly effect the pharmacokinetic features of rosuvastatin in rats. Given a single dose of danshensu or ursolic acid, pharmacokinetic parameters of Cmax, AUC0-t and AUC0-∞were significantly increased, while the clearance rate CLz/F was significantly reduced. Compared with the control group, combined with the danshensu, rosuvastatin pharmacokinetic parameters values of Cmax. AUCo-t and AUC0-∞were increased by about 122.59%,193.62% and 195%, while CLz/F values decreased by 60%; While combination with ursolic acid rosuvastatin pharmacokinetic parameters values of Cmax, AUC0-t and AUC0-∞were increased by about 163.02%,145.99% and 157.55%, while the CLz/F value is 57.84% lower.2. Caco-2 cells grew well, with clear border, tight junctions and integrity. As the culture time, TEER values of Caco-2 cells also increased and after 21-24 days the TEER was more than 500Ω.cm-2.At the same time, Caco-2 cells there was clear polarization. Therefore, a reliable Caco-2 cell monolayers model was established. Uptake of rosuvastatin in Caco-2 cell associated with the time and concentration, but no effect on HBSS PH value. The P-gp inhibitors (verapamil) and P-gp substrates (danshensu and ursolic acid) were no cause effect on the transport of rosuvastatin in Caco-2 monolayer cell model, the apparent permeability coefficient (Papp) and the apparent permeability (PDR) were all no statistical difference.3.The method of separation of primary rat liver cell was successfully established. The uptake of rosuvastatin calcium in primary hepatocytes increased rapidly in the time of 0-40sec, afte 80sec gradually reached a steady state.Uptake of rosuvastatin in primary hepatocytes was linear increase during the concentration 5-20uM and the uptake of rosuvastatin reached saturation at 60uM.The uptake kinetic parameters Km and Vmax of rosuvastatin in primary hepatocytes were 25.11±8.49 uM and 16.97± 2.19 nmol. min-1.mg-1 protein. The reduction of 20,40 and 80uM danshensu on the hepatocytes uptake of rosuvastatin were 3.13%,41.15% and 74.62%. The concentration of 40uM and 80uM can significantly effect the rosuvastatin uptake in liver cells, and inhibition parameter of IC50 was 53.04±2.43uM.The reduction of 4, 8 and 16uM ursolic acid on the hepatic uptake of rosuvastatin were 1.70±0.94%, 47.58±1.80% and 71.16±0.19%. Rosuvastatin uptake in liver cells was significantly inhibited at concentration of 8uM and 16uM, but at 4uM did not show serious inhibition. The inhibitory parameters of IC50 was10.88±0.29uM.4.The OATP1B1*1a-GFP fusion gene and OATP1B1*5-GFP fusion gene lentiviral vector can normally express in HEK293T cells. The virus titlers of recombinant lentivirus (Lenti-OATP1B1*1a, Lenti-OATP1B1*5) was 2x10'TU/ml. After transfected by Lenti-OATP1B1*1a, Lenti-OATP1B1*5, fluorescence expression increased in HEK293T with increasing of time and MOI(Multiplicity of infection). The optimal MOI is 50. Transfection rate of Lenti-OATP1B1*1a, Lenti-OATPIB1*5 was about 80% when MOI was 50. The study showed that the transport of rosuvastatin by OATP1B1 was related with OATP1B1 genotypes. Compared with OATP1B1*1a,mutant OATP1B1*5 significantly decreased the transport capacity. The uptake of OATP1B1*5-HEK293T was significantly less than OATP1B1*1a-HEK293T (P<0.05). The uptake kinetic parameters Km and Vmax of OATP1B1*1a-HEK293T on rosuvastatin were 19.87±5.96 uM and 6.63±0.70 pmol min-1 mg-1 protein, while the uptake kinetic parameters Km and Vmax of kinetic parameters Km and Vmax of OATP1B1*1a-HEK293T on rosuvastatin were 10.05±4.02 uM and 2.58±0.30pmol min-1 mg-1 protein. The inhibition of danshensu on OAT1B1 transport of rosuvastatin had relation with OATP1B1 genotypes. Danshensu (1 and 10uM) can show as competitive inhibitor of the mutant OATP1B1*5 to the transport of rosuvastatin, and could reduce the uptake of rosuvastatin about 39.11±4.94% and 63.61±3.94%,respevtively.The parameters of IC50 was 3.26±0.54uM.However, as for the wild-type OATP1B1*1a gene, danshensu showed less inhibition.When danshensu concentrations were 0.1,1 and 10uM, reducing the transport of rosuvastatin were about 3.96±3.40%,8.22±2.40%,11.56±3.04%. When the concentration of ursolic acid were 1.8uM and 18uM, ursolic acid showed significant competitive inhibition to both OATP1B1*la and OATP1B1*5 transporter. The reducing of the OATP1B1*1a transport on rosuvastatin were 34.60±2.99% and 66.08±1.83%, and for OATP1B1*5 were 34.27±7.08% and 66.95±1.14%. Inhibitory parameters were IC50 were6.25±0.42uM and 6.07±0.57uM for OATP1B1*1a and OATP1B1*5, respectively.Conclusions:1. Both danshensu and ursolic acid can effect rosuvastatin pharmacokinetics, they both significantly increased the pharmacokinetic parameters of Cmax and AUC, while clearance rate CLz/F showed significantly lower (P<0.05).2.Active transport and passive diffusion absorption existed in uptake of Rosuvastatin in Caco-2 cell model. Rosuvastatin is not a substrate of P-gp. Danshensu and ursolic acid had no effect on the intestinal uptake of rosuvastatin.3. Danshensu and ursolic acid can compete to inhibit the uptake of rosuvastatin in hepatocytes.4. The HEK293T cell model were successfully constructed and stably expressed gene of OATP1B1*1a and OATP1B1*5.Ursolic acid showed a competitive inhibition on OATP1B1*1a and OATP1B1*5-mediated rosuvastatin transport. Danshensu showed competitive inhibition on OATP1B1*5-mediated rosuvastatin transport, while no significant inhibition on OATP1B1*1a.
|
PDF Full Text Request