Molecular Mechanism Study Of The Hepatic Uptake Of Ophiopogonin D Mediate By Organic Anion-transporting Polypeptides

Background:Ophiopogonin D is one of the main active ingredient of Shen Mai Injection thatwas often coadministerated with other drugs for the treatment of cardiovascular andcerebrovascular diseases. Many cases about herb-drug interaction mediated bytransporters have been reported in recent years. In our previous study, we found thatthe plasma concentration of OPD in rats given shenmai injection is higher than thosegiven the same doses of OPD. We also observed that the ratio between plasmaconcentration and liver concentration of OPD in shenmai injection group is about5-7times larger than that in OPD group. Besides, the liver concentration of OPD reducedsignificantly and the plasma concentration of OPD increased after thecoadministration of OPD with OATPs/Oatps inhibitors compared with that givenOPD alone. This indicate that OPD may be the substrates of OATPs and theinteraction may occur among the complicated components in shenmai injectionmediated by OATPs/Oatps.Therefore, it is necessary to study the mechanism of theliver transport OPD by using in vitro cell model and plasmid construction model.Objectives:The pharmacokinetic characteristics and transporting mechanism of OPD in liverwill be explored at the cellular level by constructing rat primary hepatocyte model sothat we can understand the inner relationship between the uptake of OPD in liver andoatps. Finally, We construct high expression HEK293T cell model to explore theapproch of OPD transporting in liver at the molecular level, exploring and revealingthe mechanism of interaction and compatibility between herb-drug mediated byOATPs.Methods:1. Study on the uptake mechanism of OPD in rat primary hepatocyte①Explore and establish methods of detecting OPD in hepatocyte and sampletreatment.②Construct the rat primary hepatocyte model:observing the cell morphology with microscope, determining the activity and yield of hepatocytes by trypan blueexclusion experiments.③Determine the optimum condition for the uptake by exploring the influence oftime,density and drug concentration on the uptake of OPD. Calculate Vmax, Km andother pharmacokinetic parameters by exploring the pharmacokinetic characteristics ofOPD.④Explore influence of oatps non-specific inhibitor rifampin, rosuvastatin on theuptake of OPD. Determine the potential relationship between the uptake of OPD inliver and oatps.⑤Detect the concentration of OPD to analyse the main uptake access of OPD inhepatocytes by combining specific oatps inhibitors of oatps with OPD.2.Study on the molecular mechanism of OPD uptake byOATP1B1*1a-HEK293T cells①Establish and explore methods of detecting OPD in HEK293T cells andsample treatment.②Construct the high expression cell model by infecting HEK293T cells with theOATP1B1*1a-GFP plasmid.③Induce the expression of OATP1B1genes in OATP1B1*1a-HEK293T cellswith sodium butyrate and confirm the cell model is successfully constructed by theuptake experiment of rosuvastatin.④Explore the uptake characteristics of OPD by OATP1B1*1a-HEK293T cells.Combine OPD with OATP1B1inhibitor rosuvastatin,rifampin,bromosulfalein andGlycyrrhizic acid to explore the effect of OATP1B1on the uptake of OPD.Finally,reveal the transport mechanism of OPD in OATP1B1*1a-HEK293T cells.Results：1. Study on the uptake mechanism of OPD in rat primary hepatocytes①The established LC-MS detection methods of OPD was specific and sensitiveenough to meet test requirements.②The vigor and yield of rat primary hepatocytes obtained by the two stepcollagenase perfusion method was above85%and0.8-1×108/liver,which accordedwith the requirements for the uptake experiment. ③The uptake of OPD in primary hepatocytes increased rapidly in5-10min andsaturated at10min.Besides, the uptake of OPD would also increase with the densityof primary hepatocytes and the concentration of OPD, ranging in0.25×106-2×106/ml and1-32μΜ. At the concentration of16μΜ, the uptake increased slowly andmight be saturated.According to the Michaelis-Menten equation V=Vmax×[S]/(Km+S),the uptake kinetics parameter Km was8.10μΜ, Vmax was54.39nmol-1.min-1.mg-1protein and Vmax/Km was6.72.④Different concentration of rosuvastatin(5,10,20μΜ) could inhibit the uptakeof OPD in rat primary hepatocyte to some extent. Compared with the controlgroup,the uptake of4μM OPD decreased by8.6%,17.42%,32.53%respectively,theuptake of8μM OPD decreased by9.36%、25.37%、40.18%,and the uptake of16μMOPD decreased by9.27%、23.61%、32.21%.Besides,rifampin would not inhibit theuptake of OPD in rat primary hepatocyte.The difference was not statisticallysignificant.⑤The oatp1b2specific inhibitor glycyrrhizic acid(5,10,20,40μΜ)inhibitedthe uptake of OPD in rat primary hepatocyte.The higher the concentration was,themore significant the inhibition was.The uptake of OPD in primary hepatocytedecreased by33.53%,39.92%,51.54%and57.50%,which was of great statisticalsignificance(P＜0.01).And the inhibitory parameter IC50was25.39μM. Besides,digoxin(oatp1a4specific inhibitor),BSP and ibuprofen (oatp1a1specific inhibitor)would barely inhibit the uptake of OPD in liver.2.Study on the uptake mechanism of OPD by OATP1B1*1a-HEK293T cells①The LC-MS detection methods of OPD in the HEK293T cell sample wasspecific and sensitive enough to meet test requirements..②Construct OATP1B1*1a-GFP plasmid to infect HEK293T cells.According tothe preliminary experiment,we found that the transfetion rate was about80%whenMOI was80. HEK293T cells with fluorescence could be observed after transfectedby Leni-OATP1B1*1a and the intensity of fluorescence was increased with time andMOI,which meant the cell model was successfully constructed.③The uptake of classic OATP1B1substrate rosuvastatin was2.3times largerthan that of the control group after incubating with1mM sodium butyrate for 48h,which proved that the model was successfully induced for the following uptakeexperiment.④The uptake kinetics parameter of OATP1B1*1a-HEK293T cells Km andVmax was5.50μΜ and29.07nmol.min-1.mg-1protein while that of rosuvastatin were24.42and15.02.Thus we could draw a conclusion that OPD had higher affinity withOATP1B1and transport faster in unit time compared with rosuvastatin.⑤Different concentrations of rosuvastatin(2,10,50μM) would inhibit the uptakeof OPD in OATP1B1*1a-HEK293T cells to some extent and the uptake of OPDcould decrease by8.78±6.20%,29.23±9.58%and63.94±8.04%.The inhibitoryparameter IC50was36.74μM.Besides,BSP and rifampin could also inhibit the uptakeof OPD in OATP1B1*1a-HEK293T cells.BSP(8、20、50μM) could decrease theuptake of OPD by3.93±8.63%,9.03±6.73%and32.42±9.43%whilerifampin(15,30,60μM) could decrease the uptake of OPD by4.77±6.09%,34.37±5.19%and52.90±5.56%and the inhibitory parameter IC50was55.29μM. Inaddition, Glycyrrhizic acid(30,60,120μM) could decrease the uptake of OPD by7.61±9.02%,18.03±2.53%,20.83±4.66%and the inhibitory effect is low.Conclusions:①The established LC-MS detection methods of OPD in rat primary hepatocytesand HEK293T cells samples was specific and sensitive enough to meettestrequirements.②The rat primary hepatocytes obtained by two step collagenaseperfusionmethod has high vitality,which accords with the requirements for the uptakeexperiment.③The constructed OATP1B1*1a-HEK293T cells induction model with stableexpression of OATP1B1gene and high activity can meet the requirements for thetransport and mechanism research of human hepatocyte uptake drugs.④OPD can be uptaken in rat primary hepatocyte and the uptake decreases whenincubated with rosuvastatin (oatp1b2,oatp1a1and oatp1a4competitiveinhibitor),which infers that the uptake of OPD in rat primary hepatocyte may relatewith oatps.Further study shows that the oatp1b2specific inhibitor glycyrrhizicacid(5,10,20,40μΜ) could competitively inhibit the uptake of OPD in primary hepatocyte and the higher the concentration is,the more significant the inhibitionis.However,rifampin(oatp1a4and oatp1a1inhibitor),digoxin(oatp1a4specificinhibitor),BSP and ibuprofen(oatp1a1specific inhibitor) barely inhibit the uptake ofOPD.Thus we can draw a conclusion that the uptake of OPD in rat primaryhepatocyte may be mediated by oatp1b2.⑤OPD can be uptaken in the OATP1B1*1a-HEK293T cells.Compared with rosuvastatin, OPD has higher affinity with OATP1B1and can be transportedfaster in unit time.Besides,OATP1B1competitive inhibitor rosuvastatin,BSP,rifampin,bromosulfalein and Glycyrrhizic acid can inhibit the uptake of OPD in OATP1B1*1a-HEK293T cells with different level,which further hints OPD is thesubstrate of OATP1B1and OATP1B1may be one of the main access of OPD transported into liver.