Study On The Mechanism Of Anti-malarial Action Of Dihydroartemisinin

According to the data from World Malaria Report in 2018,there is a total of 219 million cases of malaria in the world,and the global tally of malaria deaths reached 435 000 deaths,while 216 million cases of malaria and 435 000 malaria deaths reported in 2016.Eliminating malaria remains a serious challenge.In humans,there are five species that can cause malaria:Plasmodium falciparum,Plasmodium malariae,Plasmodium ovale,Plasmodium vivax and Plasmodium knowlesi.Among infected individuals,Plasmodium falciparum causes the most severe and fatal form of malaria in humans.According to the 2018 World Malaria Report,P.falciparum is the most prevalent malaria parasite in sub-Saharan Africa,accounting for 92%of the estimated total number of malaria cases in 2017.At present,artemisinin-based combination therapy is the main method of malaria treatment.Artemisinin and its derivatives are among the most important and effective antimalarial drugs,with proven safety and efficacy in clinical use.Artemisinin and its derivatives have saved millions of lives across the globe in Plasmodium falciparum malaria,but their action mechanism needs to be fully understood.The research on the antimalarial mechanism of artemisinin not only contributes to the rational use of artemisinin and its derivatives,but also facilitates the joint application based on mechanism synergy,reducing the resistance of artemisinins,and promoting similar screening and development of new antimalarial drugs.It is well known that the artemisinin derivatives' activation is mainly haem or ferrous iron dependent to generate reactive oxygen species(ROS)and other free radicals.Recent years,it is reported that artemisinin derivatives induce ferroptosis in tumor cells,and artemisinin and its derivatives are inducers of ferroptosis.However,the involvement of ferroptosis in malaria has not been reported.Ferroptosis is a new form of regulated cell death characterized by the iron-dependent accumulation of membrane lipid peroxide,causing cytoplasmic membrane damage.Ferroptosis is diffierent from apoptosis,necrosis and autophagy in terms of morphology,genetics,and biochemical characteristics.Since parasites and tumor cells are biologically distinct,whether ferroptosis is a death mode of Plasmodium falciparum,whether ferroptosis is a main mechanism of artemisininin in parasite killing,which links are the main targets,all of them are worth studying and discussing.Objective:To explore whether ferroptosis is a way of death in Plasmodium falciparum,whether ferroptosis is an important pathway in the antimalarial mechanism of dihydroartemisinin(DHA)and explore its deep mechanism.Method:We selected DHA,erastin(ERA),RSL3,sorafenib(SOR),liproxstatin-1(Lip-1),DFO for the study of antiparasitic effect in P.falciparum 3D7.Then,we explored the anti-malarial effects of DHA in combination with ferroptotic inducers(ERA,RSL3,SOR)and inhibitors(DFO,Lip-1)during asexual blood stages of Plasmodium falciparum 3D7.Among them,the combination of DHA and ferroptosis inhibitor(Lip-1)is the focus of research.Flow cytometry was used to detect the effects of intracellular labile iron pool(LIP)and membrane lipid peroxide on parasites after treated by dihydroartemisinin,ferroptosis inducers(ERA,RSL3,SOR)and DHA in combination with ferroptotic inhibitors(DFO,Lip-1).The labile iron pool and membrane lipid peroxide are two key indicators of ferroptosis.Since ferroptosis eventually leads to an increase in Fe2+-dependent lipid peroxide,two aspects are involved:one is the membrane lipid peroxide,the other is cause of elevated ferrous iron concentration.The lipidomics techniques were used to analyze the changes of lipids in the plasma membrane system after the cure of DHA,and to study the molecular mechanism of increased lipid peroxide.RT-PCR was used to analyze the expression levels of intracellular iron-sulfur cluster proteins and its assembly pathways,after the treatment of DHA on parasites,to study the causes of elevated Fe2+levels.Results:1.Ferroptosis inducers can induce P.falciparum death in a dose-dependent manner.The susceptibility assay in vitro was performed against.falciparum 3D7 in order to analyse the antiparasitic effect(APE)of DHA,ERA,RSL3,SOR,DFO,Lip-1.DHA,ERA,RSL3,SOR treatment inhibited the growth of parasites in a dose-dependent manner.The IC50 levels for the drugs alone were 4.37±0.82 nM,6.40±0.67 ?M,4.87±0.26 ?M and 12.40±0.82 ?M,respectively,for DHA,ERA,RSL3,SOR.After the ferroptosis inhibitor DFO and Lip-1 alone acted on parasties for 72 h,the parasites were also significantly killed in large doses with IC50 values of 17.10±0.93 ?M and 3.20±0.23 ?M,respectively.2.The combination of ferroptosis inducer and DHA showed synergistic or additive effects in malaria parasite.The above findings provide a new idea for the combination of artemisinins.To estimate the combinatory effects of DHA with ERA,RSL3,SOR isobolographic analyses were performed.From the dose-effect results between DHA/ERA,it was found that a synergistic or additive effect was observed when the APE was 40-90%.Combination of DHA with RSL3 proved a nearly additive and synergistic effect at APE 50-90%.Combination of DHA with SOR proved a nearly additive and synergistic effect at APE of 30-90%.3.Ferroptosis inhibitors reduce the antimalarial effect of DHA in malaria parasite.DFO reduce the antiparasitic effects of DHA from the dose-effect results of DHA/DFO at some dose levels.In the isobolographic analyses,the antagonistic effect of DHA/DFO was observed at APE of 10-90%.The combined treatment of DHA/DFO reduced the antimalarial effect of DHA compared to treatment with DHA alone or DFO,especially at low doses.The combination treatment of DHA/Lip-1 produced a more significant reduction in antiparasitic effects of DHA compared to the use of only DHA or Lip-1 treatment.Antagonism of DHA/Lip-1 was observed at 10-90%antimalarial effect.Although Lip-1 alone is harmful(possibly non-specific)to P.falciparum at large doses,it can block and antagonize antimalarial effect of DHA at non-toxic doses.Pretreated with DHA(4.4 nM)1 h or 2 h,non-toxic doses of Lip-1 can almost block the anti-parasitic effects of DHA.While to combination with lethal dose of DHA(20 or 40 nM)for 72 h,some doses of Lip-1 also can save about 20-50%parasites.Lip-1(2.35 ?M)interacted with different concentrations of DHA and found that Lip-1 inhibited the anti-malarial effect of DHA and the blocking effect of DHA was weaker with the increase of DHA dose,but there were still a small amount of parasites survived.4.For the two core indicators of ferroptosis,intracellular free Fe2+ and membrane lipid peroxide,DHA has similar action characteristics as ferroptosis inducer,which simultaneously increases ferrous iron and membrane lipid peroxidation in P.falciparum cells.Conversely,ferroptosis inhibitors can reduce DHA-induced increases in ferrous iron and membrane lipid peroxide accumulation in parasites.Similarly to the ferroptosis inducers treatment,DHA(40-200 nM)significantly enhances intracellular ferrous iron and membrane lipid peroxide in trophozoite stage parasitized RBC(pRBC)in a dose-dependent manner.ERA(3.5-3500 ?M),RSL3(50-250 ?M),and SOR(6-600 ?M)treatments were also effective in enhancing ferrous iron and membrane lipid peroxide in the pRBC.DHA has similar features with ferroptosis inducers in killing parasites,and the change of intracellular ferrous iron is more serious than the change of lipid peroxide.DHA simultaneously enhances ferrous iron and membrane lipid peroxide in pRBC,and increasing of intracellular ferrous iron concentration and the increase in membrane lipid peroxide are characteristic of ferroptosis.Ferroptosis inhibitors reduce DHA-induced elevation of ferrous iron and membrane lipid peroxide concentrations in parasites.DHA(20 and 40 nM)combination with the all doses of DFO/Lip-1 produced high percentage suppression in ferrous iron and membrane lipid peroxide.DFO significantly removed intracellular non-haem iron in pRBC in a concentration-dependent manner,and then reduced the lipidROS accumulation.Lip-1 significantly removed intracellular lipidROS in parasitized RBC in a concentration-dependent manner.Therefore,both DFO and Lip-1 can reduce DHA-induced elevation of ferrous iron and membrane lipid peroxide in malaria parasite.5.Lipidomics studies have shown that DHA can affect the membrane lipid metabolism in P.falciparum,Lipidomics analysis was used to perform positive and negative ion scanning on parasite samples on high-resolution mass spectrometry.A total of 270 lipids were obtained in negative ion mode,and 8 lipids changes more than one-fold change after DHA treatment The eight major lipids include phosphatidylcholine(PC),phosphatidylethanolamine(PE),phosphatidylserine(PS),and cardiolipin(CL).In the positive ion mode,a total of 191 lipids were obtained.Among them,there were 13 lipids with more than one-fold change after DHA treatment.The main change lipids included phosphatidylcholine(PC),sphingomyelin(SM),and triglyceride(TG).After DHA treatment,a total of 21 lipids were detected from the positive and negative ion modes,accounting for about 4.5%of the total number of lipids tested.Among them,four kinds of membrane lipids have obvious reduction in content,three are PC,and one is PS.The PC is often located in the outer layer of the membrane lipid layer,and PS is often positioned in the inner layer.They are all di-or tri-tetraenoic acids containing eighteen carbons and are susceptible to attack by ferrous iron6.DHA can significantly affect the expression of some iron-sulfur cluster protein genes in.falciparum.The trophozoite stage parasites were successfully extracted and RT-PCR was performed.A total of 45 genes related to iron-sulfur cluster genes in P.falciparum 3D7 were detected,including 31 iron-sulfur cluster protein genes and 14 iron-sulfur cluster assembly genes.After the treatment of DHA,44 genes were changed,and 1 genes were not significantly changed.Among the expression-changing genes,11 genes were up-regulated,22 were down-regulated,and 11 genes were low-dose down-regulated and high-dose up-regulated.DHA mainly affects the apicoplast,mitochondrial iron-sulfur cluster assembly pathway and key iron-sulfur cluster proteins in apicoplast,mitochondria,nuclear and cytoplasm,and site-specific.DHA has a certain effect on apicoplast,mitochondria,nucleus and cytoplasmic iron-sulfur cluster protein,which is dose-dependent.DHA can down-regulate the genes such as LipA and LipB in the apicoplast fatty acid synthesis pathway.DHA has a regulatory effect on mitochondrial IRP,ISD,ATF and other genes.DHA has a great influence on the nuclear and cytoplasmic iron-sulfur cluster protein genes.DHA has a regulatory effect on DRE2,DLS and other genes with uncertain sites.Therefore,DHA may cause the increase of ferrous iron in P.falciparum cells by regulating iron-sulfur cluster proteins and their assembly pathways,which provides a preliminary basis for further elucidating the deep mechanism of artemisinin regulating iron metabolism.Conclusions:In summary,these in vitro results suggest that ferroptosis is an important way to induce the cell death in malaria parasite,and also one of the main mechanisms of artemisinin in parasite killing.