P-gp Inhibitor Chrysosplenetin Regulates Abnormal Heme Metabolism In Artemisinin-Resistant Plasmodium And Its Mechanism

Malaria,as a serious parasitic infectious disease,remains a major threat to global public security.With the spread of resistance to antimalarial drugs such as Chloroquine（CQ）in the last century,malaria was once in a state of unavailability until the discovery of Artemisinin（ART）by professor Tu Youyou’s team.However,drug resistance has been reported in Thailand,Myanmar and other regions with high malaria incidence.The drug resistance mechanism is complex and unclear,and there is no natural small molecule inhibitor as its partner drug to solve the clinical bottleneck problem.In the previous systematic separation of the acetone layer from the artemisinin industrial extraction waste,our research group screened a kind of Polymethoxylated Flavones（PMFs）,namely,Chrysosplenetin（CHR）.The study showed that although CHR was ineffective against the sensitive Plasmodium berghei,it showed a specific killing effect against the resistant Plasmodium.The in vivo bioavailability and antimalarial activity of ART were improved by inhibiting the activity of CYP3 A4,an enzyme related to ART metabolism,and down-regulating the protein and gene expression of P-gp,which mediates Multi-drug Resistant（MDR）.Studies have shown that the active center of cytochrome P450（CYP450）is also heme.P-gp is a key carrier involved in the transport of hydrophobic heme.At the same time,P-gp and C YP3 A4 have similar distribution positions in the body and huge numbers of overlapping substrates and inhibitors,and there is a complex synergistic effect and dialogue mechanism between the CYP3A4 and the P-gp.Heme may be one of the key regulatory factors.However,the anti-malarial and drug-resistant mechanisms of ART are directly related to the heme metabolism of Plasmodium.Therefore,as a double inhibitor of P-gp and CYP3A4,CHR has the potential to become an ART partner drug and an application prospect.In summary,this paper further investigates whether CHR regulates the abnormal heme metabolism associated with ART resistance through the P-gp/CYP3A4 dialogue mechanism,thereby reversing its resistance,and clarifies its upstream regulatory mechanism.The main contents were as follows:1.Differences in the potentiation of ART by CHR based on P-gp/CYP3A4 dialogue mechanism under different models（1）The Mdr1+/+（wild type,Wide Type,WT）and Mdrl-/-（P-gp-encoded gene Knock Out,Knock Out,KO）mice were taken as the research subjects to be infected with ART sensitive/resistant Plasmodium respectively,then the pathological models of WT and KO mice infected with the sensitive/resistant strain were established.Then CMC-Na（blank control group）,rifampin（orphan nuclear receptor PXR/CAR positive inducer）,ART alone and ARTCHR（1:2）combination were respectively administered by gavage for consecutive 7 days to investigate the differences of related physical signs and indexes of different plasmodiuminfected mice treated with different drugs and to clarify the correlation between P-gp and CHR synergistic effect.The experimental results showed that in the four kinds of mouse animal models,ART alone still had a killing effect on each plasmodium,but the effect in the resistantplasmodium group was reduced,and the compound administration therapy could specifically slow the progression of artemisinin-resistant plasmodium infection and reduce the severity of the disease in the same course of disease.At the same time,the survival curve and mouse physical signs also reflected that the survival state of mice in the combination group was superior to that of other groups,and the survival rate of KO mice was significantly higher compared to WT mice.These results suggested that CHR could be used as a potential partner drug in combination with ART to enhance the activity of antimalarial drugs,and P-gp,as a classical ABC transporter,would also participate in the anti-malarial effects of ART.（2）To explore whether the sensitization of CHR is mediated by P-gp/C YP3 A4 dialogue,we used the RT-qPCR method to determine the differences of Cyp3a11 mRNA expression in the liver,kidney and small intestine of mice in the above groups.The results showed that,in the blank control group,the expression level of Cyp3a11 mRNA in mice infected with resistant strains were higher than that in mice infected with sensitive strains,indicating that the two pathological models inherently had certain differences in the expression of artemisinin-related metabolic enzymes,which led to different sensitivities to drugs.Co-administration could improve the expression of Cyp3a11 mRNA in the resistance group,and reduce the induction and metabolism of artemisinin on itself,so that the blood concentration was increased,and the therapeutic effect was further enhanced.That indicating that CHR could solve the factor of plasmodium resistance mediated by cytochrome P450.At the same time,compared with WT mice,KO mice showed a smaller decrease in Cyp3a11 mRNA expression after administration,showing that P-gp was involved in regulating effect of ART on Cyp3a11,that is,there was a certain dialogue mechanism between P-gp and CYP3A,through which CHR could sensitize ART.2.Regulation function of CHR on abnormal heme metabolism of PlasmodiumIn order to explore the effect of CHR on the level of heme-hemozoin in mice of each pathological model group,the content of free heme in peripheral blood and the deposition of hemozoin in liver,spleen and lung of mice in each group were determined by coupled enzyme reaction method and ultraviolet spectrophotometer method.The results showed that in the WT and KO mouse model groups,the content of hemozoin in the sensitive group was higher than that in the resistant group.The results also showed that ART administration alone increased the content of hemozoin in WT mice and decrease that in KO mice.However,the compound administration therapy could reduce the content of hemozoin in each model group.In the WT and KO mouse model groups,the heme content of WT mice was higher than that of KO mice.ART treatment alone increased heme levels in all subgroups except the KO mouse-sensitive group;the compound administration therapy increased heme levels in all subgroups except the KO mouse resistant group.In conclusion,these results suggested that ART played an anti-malarial role by reducing the production of heme,resulting in the large accumulation of free heme,which damaged the membrane structure of the worms and led to their death.Besides,CHR could be used as a chaperone of ART with synergistic effect.At the same time,the heme-hemozoin results in WT and KO mice suggested that P-gp was an important node for ART to regulate abnormal heme-hemozoin metabolism of worms,indicating that CHR could regulate heme-hemozoin metabolism of worms through P-gp/CYP3A4 dialogue mechanism synergy with ART.3.Study on the mechanism of action based on NF-κB-PXR/CAR regulatory pathway（1）The PXR/CAR pathway regulates the expression of various drug metabolizing enzymes and transporter proteins/genes,therefore,to investigate the sensitizing effect of PXR/CAR pathway on ART in CHR based on P-gp/CYP3A4 dialogue mechanism,we first assumed that the anti-malarial effect of CHR was also exerted through the PXR/CAR pathway.To verify this hypothesis,RT-qPCR was used to determine the difference in PXR/CAR mRNA expression in the liver and small intestine of mice in each group.PXR mRNA results showed that the intervention had the greatest impact on the resistant group of KO mice,and treatment with ART alone or in combination had opposite pharmacological effects on the resistant group of KO and WT mice,and the same was true for the sensitive groups of WT mice and KO mice.CAR mRNA results showed that ART administration alone had the same pharmacological effect for each subgroup,but compound administration showed different pharmacological effects in WT and KO mice.At the same time,RIF is used as inducer,and the expression of both proteins can be significantly upregulated in the liver,but the induction effect in the small intestine is not significant.In conclusion,these results suggest that after coadministration of CHR and ART,they can be regulated by PXR/CAR pathway,and then affect the anti-malaria effect of ART through Pgp/CYP3A4 dialogue mechanism.（2）It has been reported NF-kappa B（NF-κB）,as an important inducible transcription factor,can regulate the expression of CYP450.At the same time,MDR1,as a stress-responsive gene,is regulated by a variety of transcription factors,and NF-κB is one of the most important ones.In summary,NF-κB is a common upstream pathway of the P-gp/CYP3A4 dialogue mechanism and it also interacts with PXR/CAR.Therefore,in order to explore the regulatory role of NF-κB pathway in the anti-malarial effect of CHR based on the P-gp/C YP3A4 dialogue mechanism,we used the RT-qPCR method to determine the difference in NF-κB mRNA expression in heart,liver,spleen,kidney,small intestine and brain of mice in the above groups.The experimental results showed that compared with the KO pathological model,the WT pathological model exhibited the opposite expression of NF-κB mRNA in the some tissues.In addition,ART intervention alone reduced the expression of NF-κB mRNA in some organs in the sensitive group and the resistant group,and the compound administration also reduced the expression.In summary,ART inhibits the activation of NF-κB.Moreover,CHR promotes the artemisinin activity and further inhibits the activation of NF-κB.In addition,P-gp is involved in the inhibition of NF-κB by ART.Therefore,after being compounded with ART,CHR can regulate the P-gp/CYP3A4 dialogue mechanism through the NF-κB pathway to better exert the pharmacological effects of ART.Conclusion:Although CHR itself has no direct antimalarial activity,its antimalarial activity can be enhanced after being mixed with ART,especially it is more effective for artemisinin-resistant Plasmodium,and has a specific killing effect.The mechanism may be that the expression of NF-κB mRNA is inhibited to inhibit the activation of NF-κB pathway.At the same time,PXR/CAR is activated as a pathway that mutually inhibits NF-κB pathway in inflammationrelated reactions,and the transcriptional activity of relevant genes is enhanced,so that CHR can jointly regulate the metabolism of heme-malaria pigment through P-gp/CYP3A4 dialogue mechanism,thereby affecting artemisinin resistance.