Study On The Influence Mechanism Of Breviscapine On The Transport Process Of Statins Based On MRP2

Purpose:The subject of this experiment was to select clinically representative simvastatin,pravastatin,and atorvastatin in combination with breviscapine,to study the transport process of these three statin drugs co-administration of breviscapine and the affected of MRP2 efflux transporter function.From the perspective of drug efflux transporter,the reveal affected breviscapine on the transport process mechanism of three kinds of statins.It can provide a certain experimental basis for the rational combination of two drugs in clinical practice.To guide the rational use of drugs in clinical and provide the scientific evidence.Methods:(1)Determination of the concentration of three kinds of statins in biological samples.①Determination of simvastatin concentrationMethod for the determination of the biological samples the concentrations of Simvastatin.Diamonsil(R)C18(1 5 0mm×4.6mm)of chromatographic column,mobile phase of acetonitrile-0.15%aqueous acetic acid(64:36,v/v),the flow rate was 1.0mL/min,the detection wavelength was 238nm,the column temperature was 25 ℃.②Determination of Pravastatin concentrationMethod for the determination of the biological samples the concentrations of Pravastatin.Diamonsil(R)C 18(150mm × 4.6mm)of chromatographic column,mobile phase of methanol-0.2mol/L ammonium acetate aqueous solution(53:47,v/v),the flow rate was 1.0mL/min,the detection wavelength was 237nm,the column temperature was 30℃.③Determination of Atorvastatin concentration Study on effects of breviscapine and three kinds of statins on the gene expression and protein content of MRP2 transporters in ratsMethod for the determination of the biological samples the concentrations of A t o r v a s t at i n.Diamonsil(R)C 18(1 50mm×4.6mm)of chromatographic column,mobile phase of acetonitrile-0.0 3 mol/Lpotassium dihydrogen phosphate solution(49:51,v/v),the flow rate was 1.0mL/min,the detection wavelength was 240nm,the column temperature was 40℃.(2)Study on effects of breviscapine and three kinds of statins on the gene expression and protein content of MRP2 transporters in ratsRats were randomly divided into statin group and statin combined with bieviscapine intravenous injection group.Seven days of continuous administration after the last dose,the samples of tissue and bile were collected in different time point after administration drug,and processed all the biological samples.After treatment,concentration of three kinds of statins HPLC method for the determination of the above biological in the sample,drawing the tissue concentration time column chart,bile and urine cumulative excretion time curve,Statistical software SPSS22.0 was used to carry out statistical analysis of two groups of data.(3)Study on effects of breviscapine and three kinds of statins on the gene expression and protein content of MRP2 transporters in ratsRats were divided into four groups:control group,statin alone group,breviscapine alone group,statin combined with breviscapine group,respectively.Each group continuously administered once a day for seven days.The control group received physiological water,the eighth day took liver tissue.RNA and proteins was extracted from liver cells in each group by grinding.Reverse transcription polymerase chain reaction(RT-PCR)was used to determine the effect of drugs on the expression of MRP2 transporter gene in rat liver.The protein was extracted from liver cells in each group by grinding.The influence of drugs on the content of MRP2 transporter protein in rat liver was determined by Western blot.Statistical software SPSS22.0 was used to analyze the data of each group.Results:(1)The effect of breviscapine on the transport process of three statins in ratsCombined use of simvastatin and Breviscapine Injection,Simvastatin rapidly distributed in various tissues,it can be detected in different tissues in 0.5h.The concentration of Simvastatin in different tissues increased significantly after combined with breviscapine,and bile excretion decreased significantly,excretion decreased.Combined use of pravastatin and Breviscapine Injection,Pravastatin rapidly distributed in various tissues,it can be detected in different tissues in 0.4h.Except brain,the concentration of Pravastatin in different tissues increased significantly after combined with breviscapine,and bile excretion decreased significantly,excretion decreased.Combined use of atorvastatin and Breviscapine Injection,Atorvastatin rapidly distributed in various tissues,it can be detected in different tissues in 0.5h.The concentration of Atorvastatin in different tissues increased significantly after combined with breviscapine,and bile excretion decreased significantly,excretion decreased.(2)Effects of breviscapine and three kinds of statins on the gene expression of MRP2 transporter in ratsCompared with saline group,the gene expression and protein contentd of MRP2 transporters did not significantly change in simvastatin group,and the gene expression of MRP2 transporters decreased in breviscapine group and breviscapine combined with simvastatin group(P<0.05),protein content slightly lower,but no significant difference,and the two groups had no significant difference.Compared with saline group,the gene expression and protein contentd of MRP2 transporters did not significantly change in pravastatin group,and the gene expression of MRP2 transporters decreased in breviscapine group and breviscapine combined with pravastatin group(P<0.05),protein content slightly lower,but no significant difference,and the two groups had no significant difference.Compared with saline group,the gene expression and protein contentd of MRP2 transporters did not significantly change in atorvastatin group,and the gene expression and protein contentd of MRP2 transporters decreased in breviscapine combined with atorvastatin group(P<0.05),and the gene expression of MRP2 transporters decreased in breviscapine group(P<0.05),there was no significant difference in protein content.Breviscapine group and Breviscapine combined with atorvastatin group showed no significant difference in mRNA expression and protein content of MRP2.Conclusions:Have a better therapeutic effect in the treatment of unstable angina,heart failure and other cardiovascular and cerebrovascular diseases associated with statins and Breviscapine injection at the same time.To play the efficacy of drugs,it may be related to the effect of breviscapine on the distribution of statins、the excretion of bile and enhance the pharmacological effects of statins on the heart and cerebral vessels.Breviscapine respectively combined with Simvastatin,pravastatin and atorvastatin,gene expression of rat liver MRP2 transporter was inhibited in different degrees,reduce the protein content of MRP2 transporter and competition inhibits transporter function.The transporter substrate is three statins,which reduces the excretion of the bile and increases the concentration of the tissue.Therefore,the reason of increase the curative effect that was breviscapine competition inhibited the transporter function of MRP2,reduced statins excretion and further enhanced statins efficacy.