| Malaria,one of the three major infectious diseases in the world,is a major disease that endangers human health.The World Malaria Report 2019 published by WHO shows that the most common malaria parasites in the world are Plasmodium falciparum and Plasmodium vivax.Plasmodium falciparum is the most prevalent malaria parasite in the WHO Africould Region,accounting for 99.7%of estimated malaria cases in 2018.Thus,a reasonable solution to the problem of malaria remains a major challenge facing humanity.Plasmodium is the pathogen of malaria,it could parasitize in Anopheles and vertebrates.The stage of parasitizing in human red blood cells is called erythrocytic stage.Glycolysis is the main energy supply mode of Plasmodium during the erythrocytic stage,and the productivity by the oxidative phosphorylation pathway is less.Because erythrocytic stages of Plasmodium consumes a large amount of glucose,it will increase the protein expression of its own glycolysis related enzymes and change the permeability of the red blood cell membrane.Plasmodium is similar to tumor cells,the rapid demand for energy makes it choose glycolysis to supply energy.The glycolytic pathway has a short reaction process and is completed in the cytoplasm,which could provide energy in a short time.Although the oxidative phosphorylation pathway provides less energy,the mitochondria of Plasmodium play a vital role in the entire transmission process.As the first-line antimalarial drug in the world,the antimalarial mechanism of artemisinin is still unknown,but the role of mitochondria is mentioned in many hypotheses.There is also a hypothesis that mitochondria activate artemisinin and play an antimalarial effect.Therefore,mitochondria may be potential targets for antimalarial drugs.This article aims to find a method that could measure the energy metabolism pathways of Plasmodium and investigate the effects of oxidative phosphorylation and glycolysis of erythrocytic stages of Plasmodium.At present,extracellular flux analysis has become a mainstream method for detecting biological energy in cells,which could monitor the energy metabolism of cells in real time without interference from the outside and without destroying the structure of cells.Seahorse XF Analyzers directly measure real time extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)of cells-indicators of the two major energy-producing pathways:glycolysis and oxidative phosphorylation.The Seahorse XFe96 analyzer used in this article could measure up to 96 samples at one time,its high-throughput characteristics overcome the limitations of the traditional Clark oxygen electrode,making the measurement more efficient,flexible and easy to repeat.Seahorse XF analysis is mostly used for the analysis of tumor cells,fibroblasts,neuronal cells,and T cells.There is less analysis of red blood cells and plasmodium,and there is no mature method.Therefore,this paper integrates the existing theories and methods,and screens and determines various influencing factors according to the characteristics of red blood cells and Plasmodium.Finally,a novel method for real-time in situ detection of Plasmodium erythrocyte energy metabolism is established.Using the established method to investigate the effects of existing international first-line antimalarial drugs,drugs under development and related inhibitor compounds on the energy metabolism of Plasmodium.The effects of various compounds on oxidative phosphorylation and glycolysis of erythrocytic stages of Plasmodium were evaluated separately,providing basic data for further research.1.Based on Seahorse XF Analyzer——A novel method for real-time in situ detection energy metabolism of erythrocytic stages of Plasmodium falciparumP.falciparum 3D7 was cultured in vitro,and the trophozoite stage plasmodium was more uniform after multiple synchronization treatments.High purity Plasmodium trophozoites were separated and enriched by the magnetic principle of magnetic bead separation.The enrichment purity was up to 90.55%by flow cytometry.Enriched trophozoite stage Plasmodium for seahorse XF analysis.Firstly,four adhesives were screened,Poly,C1010 and Rat-0.12 were initially determined.Three adhesives were subsequently screened simultaneously with the cell density.During the simultaneous screening of adhesive and cell density,the detection procedures and media components were optimized.The detection procedure was increased from the original three Cycles to four Cycles.The original Mix process of the instrument was couldcelled.In order to ensure that the detection value could be in the optimal detection range of 20-160 pmol/min,AM was initially changed to UB.The results showed that high cell density(9-10M)would lead to inadequate nutrition of the Plasmodium,resulting in reduced activity and death,and the OCR value detected would be less than 20 pmol/min.At low cell density,due to the small number of Plasmodium,the optimum detection ranges of the instrument couldnot be met,and the OCR value was only about 20 pmol/min.Therefore,after combining the results of the two experiments,the optimal cell density of Plasmodium was 5M.Then found the best adhesive at the optimal cell density of 5M,and finally determine that Poly was the adhesive by dual indicators of the number of supernatant cells and OCR value.At this time,the OCR value could reach 78.18 pmol/min.Therefore,the optimal adhesion agent Poly and the optimal cell density 5M were determined for subsequent index screening.The composition of four kinds of media including Plasmodium growth medium was compared,and it was found that compared with the other three types of media,AM lacked some buffer components and was not suitable for the growth of Plasmodium.Compared with the pH value of the entire process of the machine,UB and UBP could maintain the growth environment of the malaria parasite on the machine,the active status of Plasmodium was better,and the OCR value is correspondingly higher.The comparison of OCR values showed that the OCR value of the entire detection process under AM was less than 20 pmol/min,and the initial OCR values of UB and UBP were about 40 pmol/min.The OCR value had an upward trend during the detection process,of which OCR of UBP the upward trend was more obvious.Combining the two factors of pH and OCR.UBP was finally determined as the medium for detection of Plasmodium.After determining several conditions such as adhesive,cell density,and medium,Oligo and FCCP were screened.The results showed that Oligo does not decrease the OCR value of the system at low concentrations(0-5 μM),and the system doesd not respond.After gradually increasing the concentration of Oligo.the OCR value decreases at a concentration above 10 μM,10-15 μM the concentration effect was similar.Based on the combination of the system’s OCR value response and drug action trends,two concentrations were obtained:Oligo concentration of 10 μM,FCCP concentration of 0.5 μM,and Oligo concentration of 12.5 μM,FCCP concentration of 1 μM.After further comparison of the two concentrations,the final Oligo concentration of 10 μM and FCCP concentration of 0.5 μM were selected.After screening and confirming the conditions of adhesive,cell density,medium,and inhibitors of mitochondrial complexes,a general program suitable for the detection of Plasmodium erythrocytes was prepared,and the mitochondrial respiration diagram of P.falciparum 3D7 under normal conditions was drawn.2.Effects of antimalarial drugs on the proliferation of Plasmodium falciparum under the same experimental conditionsTen major international antimalarial drugs,including artemisinin and its derivatives,and three major international drugs under development were evaluated for antimalarial activity under the same experimental conditions,and IC50 values of 24 compounds were obtained.The results showed that for international first-line antimalarial drugs,the IC50 value of most drugs was nM level,and the IC50 value of only a few drugs was μM level.The drugs with better antimalarial activity than ART were CQ(IC50=14.10 nM),Amo(IC50=4.48 nM),Mef(IC50=10.50 nM),Lum(IC50=11.22 nM),ARE(IC50=7.88 nM),ARM(IC50=5.47 nM),ATS(IC50=5.00 nM),DHA(IC50=3.80 nM),Ato(IC50=0.37 nM)and Mal(IC50=4.27 nM),As a known inhibitor of ETC complex III,Ato was more effective than DHA.In addition,the three drugs under development all had good antimalarial activity,and their IC50 values were lower than most international first-line antimalarial drugs.IC50 values were DSM(IC50=7.84 nM),Cip(IC50=1.19 nM),Blue(IC50=5.93 nM).The antimalarial activity of all three drugs was better than ART,and the efficacy of Cip was better than DHA.In the following experiments,the two antimalarial drugs Sul and Cli with larger IC50 values were excluded,and the remaining antimalarial drugs were examined at the same IC50 level.3.Effects of antimalarial drugs and related inhibitors on mitochondrial respiration of Plasmodium falciparumThe mitochondrial stress test kit was selected and the established method was used to evaluate the bioenergy of P.falciparum 3D7 mitochondria at different times.A line chart was drawn to visualize the real-time effects of drugs on Plasmodium.The results showed that artemisinin and its derivatives had no significouldt effect on the mitochondrial respiration of P.falciparum 3D7.Drawing a histogram that could represent the function of mitochondria showed that ARM had a significouldt effect on increasing mitochondrial maximum respiration and proton leak(P≤0.001),which promotes mitochondrial respiration to some extent,but doesd not substantially destroy mitochondrial ETC.The results of antimalarial drugs and drugs under development showed that a total of 22 compounds in 13 major categories had no significouldt effect on mitochondrial respiration of P.falciparum 3D7 at the concentrations of 5×IC50 and 10 x IC50,and most antimalarial drugs did not damage the mitochondrial ETC of Plasmodium.The results of artemisinin and its derivatives were the same as those of the single study.Artemisinin and its derivatives significouldtly increased the mitochondrial maximal respiration with the increase of drug dose.CQ and QN,at a concentration of 10 x IC50,were significouldt for increasing mitochondrial proton leak and reducing spare capacity(P≤0.001),but the entire process did not completely destroy the function of Plasmodium mitochondrial ETC.With the increase of the drug dose,Dap inhibited mitochondria.The concentration of 10 x IC50 could inhibit mitochondrial respiration,and it could significouldtly increase the basal respiration level of mitochondria(P≤0.0001).As the dose of the drug increased,the inhibitory effected of Azy on mitochondria increased,and the maximum respiration of mitochondrial was significouldtly reduced at a concentration of 10 x IC50(P≤0.001).Pro only inhibited mitochondrial respiration at high concentrations of 10×IC50,which could significouldtly reduce maximum respiration of mitochondrial(P≤0.0001).As an inhibitor of mitochondrial ETC complex III,Ato could significouldtly change the curve under the concentration of 5 x IC50 and 10×IC50.It showed that the OCR value was reduced and the oxygen consumption rate was reduced.It could obviously inhibit mitochondrial ETC and mitochondrial respiration of Plasmodium.DSM and Cip increased the maximum respiration of mitochondrial significouldtly at a concentration of 5×IC50(P≤0.01),and Blue significouldtly reduced the maximum respiration of mitochondrial at a concentration of 10×IC50(P≤0.01),but the three drugs did not completely disrupt the function of Plasmodium mitochondrial ETC.Therefore,under the conditions and methods of this experiment,except for individual drugs,most of the antimalarial drugs had no significouldt effect on the mitochondrial respiration of P.falciparum 3D7.Based on the results of the above experiments,it was speculated that most antimalarial drugs did not initiate the death mechanism of mitochondria at first when they played the role of antimalarial drugs,which also reflected that mitochondria should not be an early event in the process of malaria death.Subsequently,the iron death inducer and ion channel inhibitors related to iron ion transport were investigated.The results showed that Era and Sor could inhibit the mitochondrial respiration of Plasmodium at a concentration of 10×IC50,and RSL3 did not significouldtly inhibit the mitochondrial respiration of Plasmodium.However,all three iron death inducers could significouldtly reduce mitochondrial basal respiration and reduce proton leak(P≤0.01).After the ion channel inhibitor at a concentration of 20 × IC50 was applied to Plasmodium,Baf and EIPA had no significouldt effect on the mitochondrial respiration of P.falciparum 3D7,and Ami had a significouldt inhibitory effect on the mitochondrial respiration of P.falciparum 3D7.DHA was used in combination with Ami,Baf,and EIPA,and the results showed that the combination had inhibitory effects on the mitochondrial respiration of P.falciparum 3D7,and the combination of two drugs had a certain synergistic effect on the mitochondrial inhibitory effect of Plasmodium.4.Effects of artemisinin compounds and iron death inducers on glycolysis of Plasmodium falciparumThe glycolysis rate analysis kit was selected and the established method was used to evaluate the glycolysis of P.falciparum 3D7.In order to rule out the impact of RBC on the glycolysis of Plasmodium,the glycolysis of RBC and iRBC was first investigated.The results showed that under normal circumstances,the ECAR value of iRBC glycolysis was around 50-70,the ECAR value of RBC glycolysis was near the baseline,and the glycolytic intensity of iRBC was about 50-70 times that of RBC.The results of artemisinin and its derivatives showed that the five compounds had a slight inhibitory tendency on glycolysis at a high concentration,and the inhibitory effect was not obvious.Under the conditions and methods of this experiment and the selected concentration,artemisinin compounds had no significant effect on the glycolysis of P.falciparum 3D7.The percentage of PER that could reflect the glycolytic contribution rate showed that the percentage of glycolytic PER and basal PER in different dosed groups of ART and the blank group could reach more than 96%,and the ratio of mitochondrial OCR value to glycolytic PER was less than 0.1.This showed that Plasmodium falciparum used glycolysis as its main energy supply pathway,and mitochondria produce little energy.In addition,the results of the iron death inducer showed that the system’s long detection time led to a decrease in the activity of the Plasmodium,and the curve of the blank group showed a gradual decline,which suggested that when the use of instruments to analyze the energy metabolism of Plasmodium,the overall detection time of the system should not be too long,and it is best to control it within 3 hours to ensure the activity of Plasmodium and the accuracy of the system detection.Therefore,the effect of iron death inducer on glycolysis of P.falciparum 3D7 needs to be further inspection. |
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