Study On The Effect Mechanism Of Platycodon Grandiflorum And ?-spinasterol Based On Drug Metabolism Enzyme And Drug Transporter

Objective:under the guidance of TCM medicinal guide theory and based on the previous research results on the effects of TCM medicinal guide on Balloonflower,this study was investigated on Platycodon grandiflorum and its monomer alpha spinasterol.Using cocktail probe drugs,realtime fluorescence quantitative PCR,Western blot and immunohistochemical methods,Platycodon grandiflorum and alpha spinasterol were studied in different dose group,which treated on the SD rats.The metabolizing enzymes of 5 types of enzyme activity of the rats lung,liver and kidney were studied.The expression of mRNA,enzymesproteinand the expression of the mRNA of regulation of nuclear receptorPXR,CAR,AhR from the metabolic enzymes,the effects of protein expression,and the effects of mRNA,protein expression on the efflux transporter P-pg type and PEPT2 type uptake transporter OAPT oflung,liver and kidney.The expression of Platycodon and alpha spinasterol on lung tissue of rats with MicroRNA spectrum was studied preliminarily.By the above study,to explore the mechanism of the effect of"loading up"in distribution,metabolism and excretion linksof Platycodon grandiflorum and alpha spinach.Methods:?1?SD rats?180g+20g?,male,were randomly divided into 6 groups,which including blank control group,low,medium and high dose groups of Platycodon grandiflorum?2.5 g/kg,5 g/kg,10 g/kg?,low and high dose groups of alpha spinach?5mg/kg,15 mg/kg?.After one week of adaptive feeding,each group was administrated with gavage.The Chinese medicine group was given three doses respectively while the monomer group was given by two doses of alpha spinach sterol,and the blank group was given equal volume of normal saline.During the experiment,the rats had a normal diet and drinking water.The rats in each group were given the drug in 7 days continuously,and the rats were given normal drinking water and fasting after the administration of the drug in the 6^th day.The rats were sacrificed at 2 h afteradministration by ether anesthesia and treated on the neck.Bloodsamples were collected andthe liver,lung and kidney tissues were collected which were cleaned by normal saline.Anatomy and whole process for sampling of sterilized surgical scissors,prevent the temperature resulting in sample degradation.Samples were labeled,packaged and preservated.Other samples which not need for timely treatment were immediately frozen at the-80 C refrigerator for spare.Cocktail probe drugs,realtime fluorescence quantitative PCR,Western blot and immunohistochemistry method were used to determine the different change of drug metabolizing enzymes and drug transporters and nuclear receptorof lung,liver and kidney of rats;?2?SD rats?180g+20 g?,male,were randomly divided into 3 group,which were blank control group,Platycodon grandiflorum group?10 g/kg?,alpha spinach sterol group?15 mg/kg?.The MicroRNA spectrumofrat lung tissues which treated with Platycodon and alpha spinasterol were studied using Illumina high-throughput sequencing from the Younuoda miRNA sequencing products.Results:?1?the enzyme activity,mRNA expression and protein content of the 5metabolite subtypes of CYP1A2,CYP2E1,CYP2C11,CYP2D6 and CYP3A1 in liver and kidney were increased by treated with Platycodon grandiflorum and its monomer spinach sterol.?2?the mRNA expression and protein content of three nuclear receptors of RXR,CAR and AhR were increased by treated with Platycodon grandiflorum and its monomer spinach sterol.?3?the expression of P-pg,OATP2,PEPT2 and mRNA in lung were inhibitedand the mRNA,protein expression of PEPT2 and P-pg in liver and kidney were promoted by treated with Platycodon grandiflorum and its monomer spinach sterol.?4?a target genes,ENSRNOG00000033101,were predicted by the analysis of the differential expression of miRNA in the lung by treated with Platycodon grandiflorum and its monomer spinach sterol.Conclusion:?1?Platycodon grandiflorus and?-spinasterol induce the expressions of mRNAs and proteins for the five subtypes of the drug-metabolizing enzymes,i.e.,CYP1A2,CYP2E1,CYP2C11,CYP2D6,and CYP3A1,in the liver and kidney tissues of the middle and lower burners and upregulate the enzyme activity in vitro,thereby accelerating the metabolism of drugs in the liver and kidney tissues of the lower burner and thus lowering the concentration of drugs in the liver and kidney.This is one of the mechanisms through which they exert the role of“guiding action”.?2?Platycodon grandiflorus and?-spinasterol mainly induce the expressions of mRNAs and proteins of PXR,CAR,and AhR in the liver and kidney,thereby changing the expressions of downstream CYP450s target gene and protein and upregulating the expressions of mRNAs and proteins of lower burner liver CYP450s.?3?Platycodon grandiflorus and?-spinasterol downregulate the expressions of mRNAs and proteins of lung P-pg,liver OATP2,and kidney PEPT2 and upregulate the expressions of mRNAs and proteins of lung PEPT2 and liver and kidney P-gp mRNA.This is one of the mechanisms through which they exert the role of“guiding action”.