Studies On Screening Of Components Inducing Drug Metabolizing Enzymes Expression Mediated By Nuclear Receptors In Artemisia Annua L.Extracts

Malaria is a vector-borne infectious disease mainly caused by the anopheles mosquitoes carrying malaria parasites which has serious impact on human body and organs and mainly in Africa and subtropics.Artemisinin was approved by the World Health Organization(WHO)as the drug of choice for the treatment of cerebral malaria falciparum malaria worldwide.To counter the threat of resistance of P.falciparum to monotherapies,artemisinin combination(ACT)therapy was recommended by WHO.Nevertheless,the considerable production costs and inadequate availability made ACT practically inaccessible to many patients in developing countries,which limited its present usefulness in the campaign against malaria.Thus,some researchers began to study on Artemisia annua L.,including tea and dry leaves,which also called plant artemisinin combination therapy.Researches had showed that Artemisia annua L.contain constituents and components which have antimalarial activity or synergistic effect with artemisinin.Although the uncontrollable doses as well as the complexity of the components do not support the tea to be used as the treatment of malaria,it had been used for the prevention and treatment of malaria in some malaria epidemic of developing countries.However,some questions occurred as the usage of Artemisia annua L.tea,such as the effect was significant after single dose,but high recrudescence after multiple dose and curative effect reappeared if discontinuation in a period time.Therefore,we speculated that metabolic interaction may occur between various chemical constituents of Artemisia annua L.tea agent.Cytochrome P450 enzymes are the most important drug metabolizing enzymes of the human body.CYP3A4 and CYP2B6 exhibit high inducibility.Nuclear receptors are important transcription regulatory factors in vivo as well as CYP3A4 and CYP2B6 are mainly mediated by PXR and CAR.It is necessary to consider the mechanism of metabolic interaction based on the regulation of nuclear receptors.In this study,the inductions of CYP3A4/2B6 mediated by PXR and CAR by some components in Artemisia annua L.were considered.It will provide some theoretical and experimental basis for artemisinin combination therapy based on Artemisia annua L..The summary is as follows:1.Construction of pGL3-CYP2B6-Luc plasmidIn this chapter,the pGL3-CYP2B6-Luc plasmid was constructed.Firstly,the sequences of the distal and proximal promoter of human CYP2B6 were synthesized,then inserted the sequences into the upstream of the pGL3-basic reporter gene to obtain the pGL3-CYP2B6-Luc plasmid.2.Establishment and optimization of dual luciferase reporter gene systemIn this chapter,the constructed pGL3-CYP2B6-Luc and the existed plasmids in our laboratory were used to establish in vitro reporter gene screening models based on hPXR/hCAR3.Expression plasmids,reporter gene plasmids and internal reference plasmids weretransient transfected into HepG2 cells.Rifampicin,a classic agonist of hPXR,and CITCO,a classical agonist of hCAR3,were used to evaluate the inductive multiple of CYP3A4/2B6.The amount of plasmids was optimized by hPXR-CYP3A4 pathway and applied to the other three pathways.Finally,the screening models of four pathways were constructed,which would be used to detect the induction of hPXR/hCAR3-CYP3A4/2B6 by crude extract and refined extract of Artemisia annua L.3.Studies on the regulation of CYP3A4/2B6 mediated by hPXR/hCAR by extracts of Artemisia annua L.The established and optimized dual-luciferase reporter gene models were adopted to study the extracts of Artemisia annua L..The activity of dual-luciferase was determined after 48 hours with stimulation by the extracts of Artemisia annua L..The results showed that several segmental extracts,including 50%,85%and Total,could induce the expression of CYP3A4 by PXRwt to some extent,the extracts of EtoAc and PE also had a strong activation effect.This activation effect was also affected by the matrix of the crude extracts.The screening of refined extracts showed that Arteannuin A exhibited a strong activation effect as well as Arteannuin B had a weak activation effect.Further studies showed that Arteannuin A and Arteannuin B had different inducing effects on CYP3A4/2B6 through the five mutants of PXR which indicated that gene polymorphisms of PXR may affect the regulation of CYP3A4/2B6 by PXR.In addition,the CYP3A4/2B6 study based on CAR showed that Arteannuin A and Arteannuin B could activate CAR to some extent,thus induce the expression of CYP3A4/2B6.4.Verification of Arteannuin A and Arteannuin B by protein expression,gene level and computer simulationThe determination of gene expression and protein expression of CYP3A4/2B6 were used to verify the induction effects of Arteannuin A and Arteannuin B on CYP3A4/2B6.After treated by drugs for 48h,cell proteins and RNAs were extracted,and the changes of CYP3A4/2B6 protein and gene expression were detected by Western blot and real-time PCR under instructions.The results showed that Arteannuin A and Arteannuin B had different inductive effects on CYP3A4/2B6,similarly to those of the reporter gene screening method.The results of molecular docking indicated that both Arteannuin A and Arteannuin B could be accommodated in the ligand binding cavity of PXR/CAR LBD.The difference of hydrogen bond and hydrophobic force may contribute to the difference of activation effect between them.