| Background:Shengmai Formula（SMF）,is derived from the classic Shengmai Powder,which has the effect of nourishing qi and Yin,and is widely used for the treatment of cardiovascular diseases.Our previous study found that the uptake transporter OATP1B1/1B3,NTCP and efflux transporter BCRP all mediate the interaction of the main effective components in SMF.Organic anion transporter 1（OAT1）is a very important renal uptake transporter,which can mediate the transmembrane transport of many endogenous and exogenous substances.In recent years,drug-drug interaction based on OAT1 has attracted great attention of scholars.The interaction between the main effective components of SMF and OAT1 is still unclear,which is worthy of further study.Objectives:In this study,we used HEK293 cell models stably expressing OAT1 to study the OAT1 mediated transport of the main effective components of SMF and their effects on the transport function of OAT1,as well as the interaction among the components based on OAT1.Meanwhile,we used the rat in vivo model to investigate the effects of ginsenoside Re,ginsenoside Rd and methylophiopogonanone A and other active components of SMF on the in vivo process of schizandrin B.The interaction mechanism and compatibility mechanism of complex components of traditional Chinese medicine based on OAT1 were aimed to deeply reveal from the perspective of pharmacokinetics.Methods:1.Firstly,we established a sensitive and reliable LC-MS method for the determination of ginsenoside Re,ginsenoside Rd,ginsenoside Rb1,ginsenoside Rg1,schizandrin A,schizandrin B,schizandrol A,schizandrol B,ophiopogonin D,ophiopogon Dˊ,methylophiopogonanone A and methylophiopogonanone B in HEK293 cells,and the concentration of schizandrin B in rat plasma and urine.2.Secondly,by using HEK293-OAT1 and HEK293-MOCK cell models,we studied the OAT1-mediated transport of the main effective parts and active components in SMF and their effects on the uptake and transport of 6-CF,the classic substrate of OAT1,as well as the interaction and compatibility based on OAT1.3.Thirdly,the effects of ginsenoside Re,ginsenoside Rd and methylophiopogonanone A on the in vivo process of schizandrin B were investigated by using rat in vivo model,and the interaction and compatibility mechanism of the main effective components of SMF in rats were discussed from the perspective of pharmacokinetics.Results:1.The quantitative analysis of ginsenoside Re,ginsenoside Rd,ginsenoside Rb1,ginsenoside Rg1,schizandrin A,schizandrin B,schizandrol A,schizandrol B,ophiopogonin D,ophiopogon Dˊ,methylophiopogonanone A,methylophiopogonanone B in cells and schizandrin B in rat plasma and urine by LC-MS showed good specificity,precision and accuracy.2.In the 12 main active components of SMF,only the uptake and transport of schizandrin B in HEK293-OAT1 cells was significantly higher than that in HEK293-MOCK cells.Probenecid,a classical inhibitor of OAT1,significantly inhibited the uptake and transport of schizandrin B in HEK293-OAT1 cells.It is suggested that schizandrin B may be a potential substrate of OAT1.The Km and Vmaxof schizandrin B for OAT1 were 30.19±4.21μM,50.39±2.65 pmol/mg protein/min。3.In HEK293-OAT1 cells,km（μM）and Vmax（pmol/mg protein/min）of 6-CF were 10.02±1.01 and 222.60±4.71 respectively.4.The inhibition rates of ginseng total saponins（GTS）,ophiopogon total saponins（OTS）,ophiopogon total flavonoids（OTF）,and fructus schisandrae total lignans（STL）on 6-CF at 50μM were 40.2%,34.9%,48.3%and 48.7%,respectively.The results showed that ginsenoside Re,ginsenoside Rd and methylophiopogonanone A had significant inhibitory effects on the uptake of 6-CF.The inhibitory rates were52.4%,58.5%and 59.9%at 50μM,and the IC50 values were 49.21±1.10μM,23.83±1.10μM and 38.71±1.20μM,respectively.Ginsenoside Re,ginsenoside Rd and methylophiopogonanone A may inhibit the uptake of 6-CF by OAT1 through noncompetitive inhibition.Under the action of 50μM of ginsenoside Re,ginsenoside Rd and methylophiopogonanone A,the Km of 6-CF was almost unchanged,and the Vmax decreased from 222.60±4.71 pmol/mg protein/min to 170.30±1.95 pmol/mg protein/min,123.20±2.48 pmol/mg protein/min and 149.00±3.43 pmol/mg protein/min,respectively.5.GTS,OTS,OTF,STL,ginsenoside Re,ginsenoside Rd and methylophiopogonanone A significantly inhibited the uptake of schizandrin B in HEK293-OAT1 cells,with inhibition rates of 49.9%,68.1%,48.9%,63.1%,39.8%,46.7%and 21.3%at 50μM,respectively.6.In rats,the coadministration of ginsenoside Re,ginsenoside Rd and methylophiopogonanone A significantly increased the AUC0-t,AUC0-∞and Cmax of schizandrin B,and decreased the CL and the 12 h cumulative urine excretion of schizandrin B.Conclusions:1.Ginsenoside Re,ginsenoside Rd and methylophiopogonanone A can inhibit OAT1-mediated 6-CF uptake and transport in varying degrees.They are inhibitors of OAT1 and may interact with clinical drugs based on OAT1.2.Schizandrin B may be a potential substrate of OAT1.Ginsenoside Re,ginsenoside Rd and methylophiopogonanone A inhibit the uptake and transport of schizandrin B through OAT1 in cells and its pharmacokinetics in rats.Suggesting that the main effective components of Shengmai formula are based on OAT1 mediated interaction and compatibility mechanism. |
