| Cardiovascular and cerebrovascular diseases have become the first disease that leads to human death.With the increasing incidence of cardiovascular and cerebrovascular diseases in recent years,the use of antiplatelet drugs has played an important role in the prevention and treatment of thrombosis.Clopidogrel bisulfate(also known as clopidogrel)is clinically used for the treatment of a variety of diseases such as atherosclerosis,which can effectively reduce the activation and aggregation of ADP induced platelets.Clopidogrel is a substrate of P glycoprotein(P-gp).After absorption in gastrointestinal tract,some drugs are re discharged by P-gp efflux.As a precursor drug,clopidogrel itself is inactive,and about 85%of the clopidogrel after oral esterase(mainly CES-1)is hydrolyzed into an inactive carboxylic acid metabolite derivative.After absorption into the body circulation,only less than 15%of the drug is converted into its active product through the metabolism of the liver cytochrome CYP450 enzyme line(cytochrome P450).The enzymes involved in the activation of clopidogrel include CYP1A2,CYP2B6,CYP2C9,CYP2C19,CYP3A4 and so on.Clopidogrel has definite adverse reactions such as gastrointestinal bleeding,and some patients do not produce antiplatelet agglutination,that is,the presence of clopidogrel resistance.All these factors limit the application scope and clinical efficacy of the drug.Panax Notoginsen is a common Chinese medicine for activating blood and removing blood stasis,and the Panax notoginsen saponins(PNS)are the important material basis for exerting blood circulation and removing blood stasis.Xuesaitong and Xuesaitong are the most important two major varieties of the Panax notoginsen saponins.Studies have shown that Xuesaitong and clopidogrel have synergistic effects in two groups,and Xuesaitong can improve clopidogrel resistance in rats.However,the effect of Panax notoginsen saponins on the absorption of clopidogrel and the activity of metabolic activator were not reported after the combination of two drugs.Therefore,MDCK-MDR1 cells were used to study the absorption characteristics of clopidogrel and the effect of Panax notoginsen saponins on its transmembrane absorption,and from mRNA and protein expression levels,the effects of clopidogrel and Panax notoginsen saponins on the main metabolic enzymes of CYP450 and CES-1 were studied through the grouping of L02 normal hepatocytes and SD rats,and the possible regulatory mechanism of the possible interaction was further predicted.1 Experimental study on MDCK-MDR1 monolayer transport of clopidogrel and its compatibility with Panax notoginsen saponins(1)The MDCK-MDR1 cell culture method was established.(2)In the transmembrane transport experiment of MDCK-MDR1 cells,the safe concentration range of Panax notoginsen saponins,clopidogrel and combination group was determined:the safe concentration of DMSO,clopidogrel sulfate,Panax notoginsen saponins and clopidogrel with Panax notoginsen saponins was in the range of 0-0.2%,0-10μg·mL-1,0-200μg·mL-1 and 1-10μg·mL-1,respectively.(3)The integrity of cell monolayers was examined by measuring resistance values.The standard substance,atenolol and propranolol,was used to investigate the monolayer transport model of MDCK-MDR1 cells and the permeability of cell membrane.The results showed that the integrity and permeability of the monolayer were in accordance with the model conditions.(4)The P-gp exo activity of the MDCK-MDR1 transport model was examined by digoxin and verapamil.and the results showed that the cell transport model showed high P-gp activity and could be used to study the transport characteristics and mechanism of P-gp efflux substrates.(5)The results of the clopidogrel transport experiment showed that the different concentrations of clopidogrel(0.4,5μg·mL-1)in the outer row of the MDCK-MDR1 cell monolayer were significantly different,and the reason for the difference was related to the saturation of the outer row protein.Panax notoginsen saponins had no significant effect on transmembrane transport of clopidogrel bisulfate.2 The effects of clopidogrel on the level of mRNA and protein of CYP450 and CES-1 in L02 cells after single use and combined with Panax notoginsen saponinsThe effects of drugs on the level of mRNA and protein expression of clopidogrel active metabolic enzymes were studied with L02 human normal hepatocyte model.(1)The L02 cell culture method was established.(2)The safety concentration range of L02 cell administration was determined by MTT.the safe concentration of DMSO,clopidogrel sulfate,Panax notoginsen saponins and clopidogrel with Panax notoginsen saponins(1:4)was in the range of 0-0.5%,0-20μg·mL-1,0-50μg·mL-1 and 0-10μg·mL-1,respectively.(3)The total RNA was extracted and the target genes were detected by real time fluorescence PCR,and the relative quantitative results were calculated.The total protein was extracted and the target protein was measured by Western Blot method.The relative gray value of each group was calculated by Actin as the internal reference correction.The results of RT-PCR and WB showed that Panax notoginsen saponins and clopidogrel bisulfate had a certain regulatory effect on P450 and CES-1 protein levels.It is worth noting that the levels of CYP1A2,CYP2C9,CYP3A4 mRNA and protein were significantly improved compared with the 10μg·mL-1 single group of clopidogrel sulfate and Panax notoginsen saponins,but the level of CES-1 mRNA and protein in the combined group had no significant effect on the clopidogrel single group.It is suggested that the combined use of clopidogrel and Panax notoginsen saponins may promote the activation of clopidogrel sulfate by up regulation of the protein level of the related enzymes related to clopidogrel sulfate,and thus improve the concentration of active drugs and ultimately improve the therapeutic effect.3 Effects of clopidogrel,Panax notoginsen saponins alone and two combinations on liver CYP450 and CES-1 mRNA and protein level in SD rats.Based on the SD rat model,we investigated the effects of drugs on the expression of mRNA and protein of clopidogrel related enzymes.SD rats were randomly divided into four groups:blank control group,clopidogrel group,Panax notoginsen saponins,clopidogrel group combined with Panax notoginsen saponins.After 14 days of intragastric administration,liver samples were taken for testing.The target genes in rat liver were detected by real time fluorescence PCR method,and the relative quantitative results were calculated.The total protein,Panax notoginsen saponins and clopidogrel had a certain regulatory effect on the level of P450 and CES-1 protein in the liver of SD rats.The levels of CYP1A2,CYP2C9,CYP3A4 mRNA and protein were significantly increased after the combined use of clopidogrel sulfate and Panax notoginsen saponins,and significantly down regulation of CES-1 mRNA levels caused by the single use of clopidogrel,and had no significant effect on the level of CES-1 protein.It is suggested that the combined use of clopidogrel and Panax notoginsen saponins may increase the protein level of the related enzymes related to the activation of the clopidogrel sulfate,and then promote the activation of the hepatic clopidogrel precursor,thus improving the concentration of active drugs and ultimately improving the therapeutic effect. |
