| Malaria remains by far one of the most prevalent infectious diseases worldwide,posing a major threat to global public safety.Artemisinin(ART)is a highly effective anti-malarial compound extracted from Artemisia annua.ART and its derivatives are the first-line drugs for malaria treatment and have saved thousands of lives worldwide.However,in recent years,there have been numerous reports of resistance to ART by Plasmodium,and the resistance mechanism is complicated and unclear,and no small molecule inhibitor can reverse the resistance of ART.Our group screened chrysosplenetin(CHR),the most abundant polymethoxyflavonol compound,from the acetone layer of ART industrial waste,and found that chrysosplenetin itself has no antimalarial activity,but has a sensitizing effect on ART and is more effective against ART-resistant malaria parasites.It also inhibited the ART metabolism-related enzyme CYP3A4,down-regulated P-gp gene and protein levels,and regulated the abnormal metabolism of heme associated with ART resistance,thus improving the antimalarial activity of ART.It has been shown that the ABC transporter can transport various substrates such as drugs across the membrane through the energy generated by ATP hydrolysis,which affects drug efflux and induces multidrug resistance(MDR),suggesting that the development of ART resistance in Plasmodium is related to the multidrug resistance mechanism mediated by ABC transporter.Meanwhile,the peroxide bridge of ART was broken by Fe2+catalytic in heme,reactive oxygen species(ROS)were released,and ROS can be lethal to Plasmodium at all stages by alkylating proteins necessary for Plasmodium growth and by disrupting oxidative defense systems,suggesting that the development of Plasmodium resistance to ART is related to the specific drug resistance mechanism mediated by heme and ROS.It is conjectured that CHR can play a dual regulatory role on both ABC transporter-mediated ART multidrug resistance mechanism and heme-and ROS-mediated ART-specific resistance mechanism,and has potential application as an ART clinical chaperone drug.In summary,this project aims to investigate the regulatory role of CHR on the ABC transporters involved in the induction of ART multidrug resistance,as well as the regulatory role of CHR on ROS-mediated ART specific drug resistance,to clarify the important family members of ABC transporters involved in the regulation of ART resistance by CHR and their functions,and to clarify the role of the classical ABC transporter P-gp and explore its upstream regulatory mechanism for subsequent,the theoretical basis for the further study of the mechanism is laid.The main contents of this topic are as follows:P-gp-encoding gene wild type(WT)and P-gp-encoding gene knock out(KO)mouse models were used to construct the pathological models of ART-sensitive/resistant Plasmodium berghei infected WT/KO mice by intraperitoneal injection of ARTsensitive/resistant Plasmodium berghei,respectively.0.5%CMC-Na,ART,ART-CHR(1:2)was given by gavage administration for 7 consecutive days,with rifampicin(RIF)as a P-gp positive inducer.1.Differences in the symptoms of mice in different models under the synergistic effect of CHR on ARTART alone had killing effect on both sensitive and resistant Plasmodium,but the effect on resistant Plasmodium was weaker,and ART-CHR(1:2)could slow down the process of infection of erythrocytes by resistant Plasmodium;the sign indexes and survival curve of mice showed that the status of mice in ART-CHR(1:2)group was better than that of ART group alone,and the survival rate of KO mice was higher than that of WT mice.It was suggested that CHR as a potential ART chaperone drug could enhance the antimalarial activity of ART,and the classical ABC transporter P-gp had significant significance in the antimalarial effect of ART.2.Regulation of CHR on MDR caused by the main ABC transporter members MRP1,2,4,5 and BCRPTo find the main members of the ABC transporter involved in CHR regulation of MDR,we used RT-qPCR to determine the expression of MRP 1,2,4,5 and BCRP mRNA in small intestinal tissue,liver tissue and peripheral blood of each group.In the small intestine,ART-CHR(1:2)downregulated BCRP mRNA expression in the WT-resistant group and upregulated MRP1,2,and 5 mRNA in the WT-resistant group compared with ART alone.ART alone downregulated MRP4 in the KO-resistant group,and ART-CHR(1:2)showed different pharmacological effects.comparison between WT and KO groups showed that BCRP mRNA expression in the KO-resistant group was lower than that in the WT-resistant with ART alone,and the opposite trend was observed for the ARTCHR(1:2).In liver,ART-CHR(1:2)downregulated MRP1,4 mRNA expression in the WTresistant group and MRP2 and BCRP mRNA expression in the KO-resistant group compared with ART alone;ART-CHR(1:2)shifted differences in the expression of MRP 1 in the sensitive/resistant groups,MRP2 in the resistant group and MRP4 in the sensitive group between WT and KO groups after treatment with ART alone.In peripheral blood,compared with ART alone,ART-CHR(1:2)downregulated the expression of MRP1,2,4,5 and BCRP mRNA in peripheral blood of WT resistance group;ART upregulated the expression of BCRP mRNA in KO resistance group alone,and ART-CHR(1:2)downregulated its expression level,this result is consistent with the trend of regulation of heme by ART alone or ART-CHR(1:2)in KO resistance group found by our group previously.The above results suggest that CHR,as an ART chaperone drug,may affect the absorption,distribution,metabolism,and excretion of ART by regulating the expression of MRP1,2,4,5,and BCRP,MRP1,2,4,5and BCRP have different functions in different tissues and their expression is tissue-differentiated.playing their respective functions in improving the utilization of ART and the protection of the organism by exocytosis of toxins.3.The role of CHR in the regulation of ROS and GSHTo investigate the role of CHR in the regulation of ROS-mediated ART-specific resistance,the levels of ROS and GSH in small intestinal tissue,liver tissue and plasma were measured in each group using a multifunctional enzyme-labeled instrument.The results showed that in the small intestine,compared with ART alone,ART-CHR(1:2)upregulated ROS content in the WT-resistant group,GSH content in the WT-sensitive group and elevated GSH content in the KO-sensitive/resistant group.In liver,ART alone down-regulated ROS content in WT-resistant group,ART-CHR(1:2)up-regulated ROS content,and the regulation of ART and combination in WT-sensitive group was opposite to that in WT-resistant group;the comparison between WT and KO groups showed that the regulation of ROS in each group was opposite to that of GSH.In plasma,ART-CHR(1:2)up-regulated ROS content in the WT sensitive/resistant group and GSH content of WT resistant group;ROS/GSH content in the KO resistant group was lower than that in the WT resistant group,which was consistent with the trend of the results that the heme content in the KO resistant group was generally lower than that in the WT resistant group in each group found by the subject group earlier.Taken together,we suggest that CHR can regulate the abnormal ROS/GSH levels induced by ART,and this regulation may be associated with improving the antimalarial activity of ART.4.Research on the mechanism of action based on PI3K/AKT/m-TOR,MAPK signaling pathway,TNF-α,IFN-γ,IL-1βPI3K/AKT/m-TOR and MAPK signaling pathways are closely related to the development of cellular resistance to drugs,while the abnormal expression of inflammatory factors such as TNF-α,IFN-γ and IL-1β caused by oxidative stress in the host due to Plasmodium invasion can affect PI3K,AKT and m-TOR,and can also participate in the tertiary kinase cascade reaction of MAPK signaling pathway as a stimulating factor.induce the development of drug resistance,and can also trigger cytokine cascade effects to release ROS,which further damage Plasmodium.Therefore,to investigate the role of PI3K/AKT/m-TOR,MAPK signaling pathway,TNF-α,IFN-γ,and IL-1β in the dual regulation of CHR on the ABC transporter that induces multidrug resistance and ROS that mediates ART-specific drug resistance,RT-qPCR was used to measure PI3K,AKT,m-TOR,and ERK in small intestine tissue,liver tissue and peripheral blood of each group.m-TOR,ERK,JNK,P38,TNF-α,IFN-γ,IL-1β mRNA expression in small intestine tissues,liver tissues and peripheral blood of each group by RTqPCR to lay the foundation for the in-depth study of the subsequent mechanism.The results showed that:In small intestine tissues,ART could up-regulate PI3K and AKT protein expression in WT resistance group alone,and its expression could be down-regulated by ART-CHR(1:2),and the exact opposite trend appeared after P-gp knockdown,indicating that CHR could affect P-gp through PI3K/AKT pathway,thus regulating ART resistance situation.ART could down-regulated ERK,JNK,and P38 mRNAs in the WT-resistant group alone,and ART-CHR(1:2)could up-regulated their expression,and the result in the KO-resistant group is opposite.In liver tissue,ART could down-regulated AKT,m-TOR and ERK mRNA expression in the WT-resistant group and up-regulated their expression in the ART-CHR(1:2)alone,the trend of regulation of P38 by ART alone or in combination is opposite.In peripheral blood,ART alone up-regulated AKT,m-TOR,ERK,JNK,and P38 mRNA in the WT-resistant group,and ART-CHR(1:2)down-regulated their expression,whereas the trend of results in the WT-sensitive group was opposite.in the WT and KO group comparison,the expression of PI3K,AKT,m-TOR,ERK,JNK,and P38 mRNAs were differentially expressed after ART alone and in combination,and the regulation of PI3K,AKT,m-TOR,ERK,JNK,and P38 by CHR may be related to P-gp.Taken together,the regulation of major members of the ABC transporter by CHR may be related to the PI3K/AKT/m-TOR,MAPK signaling pathway.In small intestine tissues,ART-CHR(1:2)down-regulated IL-1β mRNA expression in the WT-resistant group,up-regulated TNF-α and IFN-γ mRNA expression in the WTresistant group;ART alone and ART-CHR(1:2)showed different pharmacological effects on IFN-γ in WT and KO groups.In the liver,ART-CHR(1:2)had almost no effect on TNFα,IFN-γ,and IL-1β mRNA in the WT-resistant group and up-regulated IL-1β mRNA in KO group;in the comparison of WT and KO groups,ART had opposite pharmacological effects on TNF-α and IFN-γ in the sensitive/resistant group alone,and ART-CHR(1:2)had consistent effects on their modulation.In peripheral blood,ART-CHR(1:2)down-regulated IL-1β mRNA expression in WT-resistant group,up-regulated IFN-γ mRNA expression in the WT-resistant group.ART-CHR(1:2)down-regulated TNF-α,IFN-γ,IL-1β mRNA expression in the KO-resistant group;in the comparison of WT and KO groups,the trend of regulation of IFN-γ in the control group was consistent with that of treatment with ART alone,the regulation of IFN-γ by ART alone and ART-CHR(1:2)was opposite,which indicates that the regulation of abnormal IFN-γ expression caused by ART by CHR may be related to P-gp.In summary,CHR may affect the pharmacological effects of ART by influencing the expression of important members of the ABC transporter and ROS levels through the regulation of TNF-α,IFN-γ,and IL-1β.ConclusionChrysosplenetin has no antimalarial activity,but the combination with ART can sensitize the antimalarial effect of ART,and the killing effect on ART-resistant Plasmodium is more obvious.The mechanism may be through the regulation of PI3K/AKT/m-TOR and MAPK signaling pathways,which are closely related to the development of drug resistance,as well as TNF-α,IFN-γ,IL-1β,which can induce the development of drug resistance and trigger cytokine cascade effects,thus further exerting dual regulation of ABC transporters involved in the induction of multidrug resistance and ROS content that induces ART specific insecticide,and thus improving ART resistance. |
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