Interactions And Compatibility Mechanism Of The Major Effective Components In Shengmai Formula Mediated By BCRP At The Cellular And Vesicular Levels

Backgroud:Shengmai Formula（SMF）is composed of Radix ginseng,Radix ophiopogonis,and Fructus schisandrae.Shengmai injection is commonly used for the treatment of hypotensive shock,viral myocarditis,and ischemic heart and brain diseases.Our previous studies have shown that OATP1B1/1B3 and NTCP all mediate the interaction and compatibility mechanism of the complex components of SMF.Breast cancer resistance protein（BCRP）is an important efflux transporter and plays an important role in the drug treatment process in vivo.The interactions and adverse reactions between herb and chemical medicines mediated by BCRP have attracted great attention of many scholars.It is worth to explore whether BCRP participates in the transport process of the complex components of SMF,and whether BCRP-mediated interactions and compatibility mechanisms among different components.Objective:The purpose was to deeply study the effect of the main active components in SMF on the transport functions of BCRP and the interaction and compatibility mechanism among the complex components in SMF based on BCRP using LLC-PK1,LLC-PK1/BCRP cell models and BCRP vesicle model.It is helpful to provides experimental basis for the clinical application of SMF,and also expands new ideas for the research of modernization of traditional Chinese medicine.Methods:1.A rapid and sensitive LC-MS method was establisehed for the detection of MTX,ginsenoside Rb1,Rd,Rg1,Re,ophiopogonin D,ophiopogon Dˊ,methylophiopogonanone A,methylophiopogonanone B,schizandrin A,schizandrin B,schizandrol A and schizandrol B in cell and vesicle samples.2.By using the LLC-PK1 cell model that stably expresses BCRP,the accumulation and transport of the main active fractions and active components in SMF was examined and the inhibition of BCRP classic substrate MTX transport,and BCRP-mediated interactions and compatibility mechanisms among different components was also investigated.3.By using the vesicle model that stably expresses BCRP,the transport of the main active fractions and active components in SMF was examined and the inhibition of BCRP classic substrate lucifer yellow transport,and BCRP-mediated interactions and compatibility mechanisms among different components was also investigated.Results:1.The LC-MS quantitative analysis methods for the determination of MTX,ginsenoside Rb1,Rd,Rg1,Re,ophiopogonin D,ophiopogon Dˊ,methylophiopogonanone A,methylophiopogonanone B,schizandrin A,schizandrin B,schizandrol A and schizandrol B have good specificity,precision and accuracy in this experiment,which accord with the relevant requirements.2.In the BCRP cell model,the accumulation of ginsenoside Re,Rg1,methylophiopogonanone B,and schizandrin A in LLC-PK1/BCRP cells among the12 active components in SMF was significantly lower than that in LLC-PK1 cells.After adding the inhibitor Ko143,the amount of accumulation was increased.In the bi-directional transported study,the ER of the 12 active components in SMF in LLC-PK1 cells were all less than 2.But the ER of ginsenoside Re,Rg1,methylophiopogonanone B,schizandrin A in LLC-PK1/BCRP cells were 2.23,2.54,4.10 and 5.51 respectively,all greater than 2,and then decreased to 1.67,1.43,2.01,2.70 after the addition of BCRP inhibitor Ko143.In the cell accumulation study,schizandrol B increased the uptake of MTX by 2.13-fold at 100μM,and the ER of MTX was decreased from 7.89 to 3.83 in the bi-directional transported study among the four main active fractions and the 12 main effect components of SMF.100μM of schizandrol B can also increase the uptake of methylophiopogonanone B and schizandrin A by 1.14-and 1.43-fold in the cell accumulation study,respectively.When adding 100μM of schizandrol B,the ER of methylophiopogonanone B was decreased from 3.91 to 2.97,and schizandrin A from 5.82 to 4.02 in the bi-directional transported study.But the effect was not observed for ginsenoside Rg1,Re.3.In the BCRP vesicle model,the uptake kinetic parameters K_m（μM）of ginsenoside Re,Rg1,methylophiopogonanone B and schizandrin A for BCRP were determined to be 111.9±31.26,82.01±16.72,57.06±8.789 and 37.19±6.512,Vmax（pmol/mg protein/min）were determined to be 39.37±5.197,40.84±3.467,131.2±8.692 and 168.3±10.82,respectively.Their uptake in BCRP vesicles was all significantly inhibited by the classical inhibitor Ko143.The main active fractions GTS and STL in SMF all exhibit an obvious inhibitory effect on BCRP-mediated uptake of lucifer yellow,and they inhibited the transport activity by 89%,70%at 100μM,respectively.And ginsenoside Rd,Rb1 and schizandrol B also exhibited an obvious inhibitory effect on the uptake of lucifer yellow among the 12 active components in SMF,they inhibited lucifer yellow uptake by 78%,59%and 74%at100μM,respectively.The IC₅₀0 values of ginsenoside Rd,Rb1 and schizandrol B were20.46±1.47μM,42.02±2.31μM,27.13±5.49μM,respectively.Ginsenoside Rd,Rb1 and schizandrol B may inhibit the BCRP-mediated uptake of lucifer yellow by noncompetitive inhibition mode,and the V_maxax values were decreased from 254.8±8.322 pmol/mg protein/min to 180±4.751pmol/mg protein/min,from 254.8±8.322pmol/mg protein/min to 207.8±6.381pmol/mg protein/min,from 254.8±8.322pmol/mg protein/min to 202.3±4.232pmol/mg protein/min,respectively.while the K_m values remained unchanged.Besides,STL,ginsenoside Rd and schizandrol B can significantly inhibited the uptake of ginsenoside Re in BCRP vesicles,this uptake was decreased by 29%,21%and 17%at 50μM,respectively.Ginsenoside Rb1 has no significant effect on the transport of ginsenoside Re.STL,ginsenoside Rd,Rb1 and schizandrol B significantly inhibited the uptake of ginsenoside Rg1,and this uptake was decreased by 52%,30%,19%and 18%at 50μM,respectively.GTS,STL,ginsenoside Rd,Rb1 and schizandrol B significantly inhibited the uptake of methylophiopogonanone B,and this uptake was decreased by 71%,55%,31%,22%and 26%at 50μM,respectively.GTS,ginsenoside Rd,Rb1 and schizandrol B significantly inhibited the uptake of schizandrin A,and this uptake was decreased by85%,54%,33%and 32%at 50μM,respectively.Conclusions:1.Ginsenoside Re,Rg1,methylophiopogonanone B and schizandrin A are the potential substrates of BCRP in LLC-PK1/BCRP cells.Schizandrol B can inhibit the transport of MTX mediated by BCRP.Schizandrol B is a potential inhibitors of BCRP,which may interact with clinical drugs based on BCRP.Schizandrol B has no inhibitory effect on BCRP-mediated transport of ginsenoside Rg1,Re.There are interaction and compatibility mechanisms of the main complex components in SMF based on BCRP.2.Ginsenoside Re,Rg1,methylophiopogonanone B and schizandrin A are the potential substrates of BCRP in the BCRP vesicle model.GTS,STL,ginsenoside Rd,Rb1 and schizandrol B are potential inhibitors of BCRP,which can inhibit BCRP-mediated transport of lucifer yellow by different degrees,and may interact with clinical drugs mediated by BCRP.GTS,STL,ginsenoside Rd,Rb1 and schizandrol B have different degrees of inhibitory effects on the transport of ginsenoside Rg1,Re,methylophiopogonanone B and schizandrin A mediated by BCRP.There are interaction and compatibility mechanisms of the main complex components in SMF based on BCRP.