| ObjectiveP-glycoprotein(P-gp)is the mainly factor of clinical multidrug resistant(MDR),and there is not any ideal inhibitor so far.To investigate the effect of several terpenoids on P-gp,efficacy was analysed by drug-drug interations.And transport activity,P-gp protein and mRNA expression,ATPase activity and pharmacokinetics were carried out,at last used the molecular simulation for further analysis.MethodsThis dissertation firstly analyzed the mode of interaction of combined administration of terpenoids with mitoxantrone hydrochloride to screen out the active compounds which had synergistic interaction as the candidate terpenoids for further research.Then studied the effect of active terpenoids on P-gp transport activity,protein and mRNA expression,and ATPase activity in cellsto discuss the mechanism.The change of P-gp substrate pharmacokinetics was determined after combined administration with partial candidate terpenoids to discuss the effect of active terpenoids on oral absorbtion.With the computer molecular simulaiton,the molecular combined mechanism between terpenoids and P-gp were illustrated.The detailed procedures was as shown below:1.Human sarcoma of uterus MES-SA and its drug-resistant type MES-SA/MX2 cells were used as the cell model for investigating.Their growth morphology was observed.The experimental density was confirmed by analysis of cells growth curve.Compare the P-gp protein and mRNA expression,transport activity between normal and drug-resistant type to ensure the MDR phenomenon was induced by P-gp.2.Test dose of terpenoids on MES-SA/MX2 cells were determined by MTT assay through analyzing the proliferation inhibition.3.MTT methods was used to measure the growth inhibition rate of terpenoids combined with different concentration of mitoxantrone hydrochloride,then calculated the IC50 and resistant fold(RF),Compounds had synergistic interaction as the candidate active terpenoids.4.TMRM was used as P-gp fluorescent probe substrate.Flow cytometer was used to detect the TMRM fluorescence intensity in MES-SA/MX2 cells to illustrate the transport ability of P-gp affected by active terpenoids.5.Western blot and RT-PCR method were used to detect the P-gp protein and MDR1 expression in cells after administration candidate active terpenoids,to determine the effect of the candidate on the P-gp expression.6.ATPase kit was used to detect the ATPase activity in MES-SA/MX2 cells after administration candidate terpenoids.7.SD rats were used to carry out the pharmacokinetics experiment.The change of puerarin pharmacokinetics was detected after administration partial active compounds to explore the interaction between test compounds and P-gp.8.Computer molecular simulation technique was used to find out the binding site between P-gp and terpenoids to explore the molecular mechanism.Results1.According to the growth morphology observation experiment knew that MES-SA cells were fusiform and.good adherent while MES-SA/MX2 cells were both roundness and fusiform and bad adherent.Mitoxantrone hydrochloride shown different proliferation inhibition in two cells.The IC50 of mitoxantrone hydrochloride in MES-SA cells was 1.23 ?M while in MES-SA/MX2 cells was 97.5 ?M.The result shown the drug-resistant of MES-SA/MX2 cells was stronger than MES-SA cells.The WB and RT-PCR results suggested P-gp hardly expressed in MES-SA cells while a large amount of P-gp expressed in MES-SA/MX2 cells.The flow cytometer result shown that higher fluorescence in the MES-SA cells than MES-SA/MX2 cells,suggestting that MES-SA/MX2 cells had stronger transport ability than MES-SA cells in P-gp sbustrate.Comprehensively,MES-SA/MX2 cells owned higher drug-resistant which was induced by P-gp.2.MTT assay shown different terpenoids had proliferation inhibition vary in degree in MES-SA/MX2 cells.The concentration under IC20 was chosen for further combination administration research.At last the experimental concentration of terpenoids were as follows:?monoterpenes:1-borneolum(10 ?M,50 ?M),borneolum(10?M,50 ?M),borneolum syntheticum(10 ?M,50 ?M),paeoniflorin(20 ?M).?sesquiterpenes:artemisinin(20 ?M),dihydroartemisinin(20 ?M),patchoulic alcohol(20 ?M).? diterpene:andrographolide(5 ?M,20?M),isoandrographolide(20 ?M,50 ?M),andrographolic acid(20 ?M,50 ?M),debydroandrographol i de(20 ?M,50 ?M),debydroandrographol i de sodium hydrogen sulfite(20 ?M,50 ?M).? triterpenoid:ginsenoside Rbl(20 ?M),ginsenoside Rb3(20 ?M),ginsenoside Rhl(20 ?M),ginsenoside Rh2(20 ?M),ginsenoside Rd(20 ?M),ginsenoside Rc(20?M),notoginsenoside Rbl(20 ?M),Panax Notoginseng Saponins(20 ?M),saikosaponin A(5 ?M),saikosaponin B2(5 ? M),saikosaponin C(5 ? M),saikosaponin D(2 ?M),oleanolic acid(5 ?M),ursolic acid(5 ?M),glycyrrhetinic acid(5 ?M),betulinic acid(5 ?M),limonin(5 ?M),Asperosaponin ?(5 ?M),ginsengnoside(5 ?M).3.Study on the chemosensitivity effect of terpenoisds:none MDR cells RF was 1.0,while the MDR cells RF was 79.39.The resistatnt fold of posistive drug verapamil was 4.16 revealing significant reversing effect on MDR.The experiment demonstrated terpenoids which can increase the sensitivity of mitoxantrone hydrochloride and decrease the RF listed below:(Dmonoterpenes:RF of 1-borneolum(10 ?M,50?M)were 27.33 and 9.66 respectively;RF of borneolum(10 ?M,50 ?M)were 65.89 and 15.72 respectively;RF of borneolum syntheticum(10 ?M,50 ?M)were 78.74 and 22.81 respectively.?diterpene:RF of andrographolide(5 ?M)was 56.27;RF of isoandrographolide(20 ?M)was 37.41;RF of debydroandrographolide(20 ?M)were 35.02;RF of debydroandrographolide sodium hydrogen sulfite(20 ?M)were 37.30.4.The P-gp transport activity was valued by fluorescence intensity of TMRM in MES-SA/MX2 cells:The relative fluorescence intensity of 10000 cells in control group was 100%.The verapamil positive group revealed increased TMRM retention rate in cells in various concentration,after the concentration higher than 50 ? M,the fluorescence intensity tend to stable,the relative fluorescence intensity up to 172%.The relative fluorescence intensity of candidate terpenoids listed below:?monoterpenes:1-borneolum(1,10,50 ?M)were 111.5%(P<0.01),116.22%(P<0.01),123.0%(P<0.01);Borneolum(1,10,50 ?M)were 100.0%,104.7%(P<0.01),104.4%(P<0.01);Borneolum syntheticum(1,10,50 ?M)were 103.3%(P<0.05),103.9%(P<0.01),104.2%(P<0.01).?diterpene:Andrographolide(1,10,25,50,100 ?M)were100.8%,101.4%,106.5%(P<0.05),107.9%(P<0.05),108.0%(P<0.05);Isoandrographolide(1,10,25,50,100 ?M)were 98.9%,98.7%,104.0%(P<0.05),104.4%(P<0.05),122.9.0%(P<0.01);Debydroandrographolide(1,10,25,50,100?M)were103.9%,104.0%,113.1%(P<0.01),122.6%(P<0.01),148.8%(P<0.01);Debydroandrographolide sodium hydrogen sulfite(1,10,25,50,100?M)were101.1%,102.6%,110.0%(P<0.01),108.5%(P<0.05),115.7%(P<0.01).5.The effect of candidate terpenoids on P-gp protein expression results as shown:The P-gp protein expression of control group was 100%.?monoterpenes:The P-gp protein expression of 1-borneolum(10,20,50?M)were 71.6(P<0.01),90.4%(p<0.05),114.5%;The P-gp protein expression of borneolum(10,20,50 ?M)were 78.9%(P<0.05),74.4%(P<0.05),70.7%(P<0.05);The P-gp protein expression of borneolum syntheticum(10,20,50 ?M)were 66.2%(P<0.01),79.0%(P<0.05),97.8%.?diterpene:The P-gp expression of andrographolide(1,5,10 ?M)were 87.4%(P<0.05),68.5%(P<0.01),40.7%(P<0.01);The P-gp protein expression of isoandrographolide(1,10,100 ?M)were 74.5%(P<0.01),76.4%(P<0.01),81.8%(P<0.01);The P-gp protein expression of debydroandrographolide(1,10,100 ?M)were 86.1%,85.0%(P<0.05),70.2%(P<0.05);The P-gp protein expression of debydroandrographolide sodium hydrogen sulfite(1,10,100 ?M)were 98.2%,97.6%,127.2%.6.The candidate terpenoids which regulated the P-gp protein expression were chosen to RT-PCR experiment.The MDR1 of control group was 100%while verapamil 10?M as the positive group were 89.3%.The MDR1 results of candidate terpenoids were shown as:?monoterpenes:The MDR1 of 1-borneolum 20 ?M,borneolum 20 ?M,borneolum syntheticum 20?M were 82.1%(P<0.01),101.4%,95.3%(P<0.01).?diterpene:Andrographolide 5 ?M,isoandrographolide 20 ?M,debydroandrographolide 20 ?M,debydroandrographolide sodium hydrogen sulfite 20 ?M were 94.7%(P<0.01),83.1%(P<0.01),91.0%,93.6%.7.The activity of ATPase in cells affects the level of P-gp transport function.Hence ATPase activity was chosen for further research at the ATPase activity in cells.It found that ATPase activity of control group was 1.0494 U/mgProt.The verapamil positive group did not affect the ATPase activity.The results of terpenoids were shown as:?monoterpenes:1-borneolum(10,20,50 ?M)were 0.9376(P<0.05),0.9402(P<0.05),0.9322(P<0.05)U/mgProt;Borneolum(10,20,50 ?M)werel.084,1.066,0.9665(P<0.05)U/mgProt;Borneolum syntheticum(10,20,50 ?M)were 1.087,0.9874,0.9348(P<0.05)U/mgProt.?diterpenes:Andrographolide 5 ?M,Isoandrographolide 20 ?M,Debydroandrographolide 20 ?M,Debydroandrographolide sodium hydrogen sulfite 20 ?M were 0.9786(P<0.05),1.0904,1.1006,1.0520 U/mgprot.8.Comprehensive Analysis of pharmacological effects of terpenoids in cells,partial of monoterpenes and diterpenes were chosen for further pharmacokinetic research in rats.The results shown:?The monoterpenes 1-borneolum had a stronger power than borneolum:1-borneolum(100 mg · Kg-1)increased the puerarin AUC(0-t)71.9%(P<0.01)?AUC(0-?)62.4%,enhanced the Cmax 374.4%(P<0.01)compare to the control group,shorten the Tmax 50.05%(P<0.05);Borneolum(100 mg · Kg-1)increased the puerarin Cmax to some extent and shorten the Tmax,suggesting the effect on puerarin,but the results was not significant with control group,and its effect weaken than 1-borneolum.?Both of Andrographolide and dehydroandrographolide had a stronger power than isoandrographolide:Andrographolide(60 mg · Kg-1)and dehydroandrographolide(60 mg · Kg-1)increased the AUC(0-t)were 30.77%and 27.3%?AUC(0-?)was 27.2%and 67.3%(P<0.05),enhanced the Cmax were 11.5%(P<0.05)and 26.9%(P<0.05).Isoandrographolide(60 mg · Kg-1)decresed the puerarin AUC(0-?)to 71.4%(P<0.01),MRT(0-?)decresed to 40.2%,prolonged the Tmax to 332.7%.9.Took advantage of molecular simulation,reliable human P-gp model was got.The amino acid binding sites of human P-gp were got after binding with P-gp substrates,inhibitors and candidate terpenoids.It was found that TM1,TM5,TM6,TM7,TM11 and TM12 of transmembrane domain were the binding region of P-gp.The most binding amino acid were MET192,GLN195,SER196,THR199,ILE306,TYR307,TYR310,PHE336,LEU339,ILE340,PHE343,SER344,GLN347,GLN725,PR0726,PHE728,ALA729,PHE732,PHE759,GLN946,MET949,TYR950,TYR953,IEU975,PHE978,SER979,VAL982,PHE983,MET986,MET68,MET69,LEU65.Conclusion1.The 1-borneolum((-)-borneol)?borneolum((+)-borneol).borneolum Syntheticum(borneol)could reversed the MDR induced by P-gp which mainly affected the expression of P-gp and transport activity.This related to its spacew structure.It showed that left-handed structure((-)-borneol)had stronger inhibitory activity than right-handed structure((+)-borneol)in vitro and vivo.2.The five compounds from herba andrographitis revealed different degrees of inhibiting function on MDR.Isoandrographolide was the stereoisomerism of andrographolide,these two compounds mainly affected the P-gp protein expression and transport activity,however andrographolide revealed more powerful inhibition ability.Andrographolide could influence the ATPase activity of cells.The vivo pharmacokinetic experiment showed andrographolide had stronger power on reversing P-gp substrate transport than isoandrographolide.It demonstrated that space sturcture differecence was a main influece factor on inhibiting P-gp function.Debydroandrographolide showed the strongest power on inhibiting P-gp among five compounds.It could inhibite the expression of P-gp and transport activity.3.Molecular simulation results showed that the partial amino acids on TM1,TM5,TM6,TM7,TM11 and TM12 screw parts of P-gp transmembrance region had singficant relationship between P-gp and inhibitor/substrate.It also found that candidate terpenoids had a close relationship with these amino acids.Hence,study on binding situation between these amino acids and experimental compound may good to screen the terpenoids P-gp inhibitor.Comprehensively,several terpenoids revealed the reversing function on P-gp,but their function way were not the same.The mechanism may associated with the combined structure between P-gp and terpenoids compounds. |
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