Explore The Meaning Of Artemisinin Resistance From The Point Of View Of Controlling Malaria Infection By Spleen

According to the World Health Organization(WHO),there were an estimated 229 million malaria cases worldwide in 2019,including 409,000 deaths.The majority of malaria cases are in Africa,followed by Southeast Asia and the eastern Mediterranean[1].Artemisinin and its derivatives have high antimalarial activity against Plasmodium falciparum in the erythrocyte phase.In order to avoid the potential risk of drug resistance caused by single drug,WHO proposed artemisinin-based combination therap(ACT)as a first-line treatment for simple malaria[2].The treatment was initially effective against malaria worldwide,but since 2009,Southeast Asian countries have reported that after three days of ACT treatment,the clearance time of the malaria parasite in some malaria patients has been prolonged.This has led to a series of discussions and studies on the resistance of malaria parasites to artemisinin.In 2015,WHO defined artemisinin resistance as the clearance half-life of parasites in the blood of malaria patients treated with artesunate or ACTs alone? 5h[3].Mutations in Plasmodium falciparum kelch13(Pfkelch13)genes are believed to be the main cause of resistance to artemisinin-based drugs.However,not all mutations in the K13 gene can cause resistance to artemisinin drugs.Even in patients infected with the K13 mutation,the clearance time of the malaria parasite during the antimalarial treatment is very different[4].K13 mutation may be only one of the mechanisms of artemisinin resistance.The molecular mechanisms involved still need to be further studied.The definition of artemisinin resistance is not consistent with the traditional pharmacological definition of drug resistance,which defines drug resistance as a heritable physiological characteristic in which the susceptibility of a pathogen to a drug decreases or even dissolves after repeated contact with the drug,resulting in reduced or ineffective efficacy of the drug against the pathogen.The mechanism of pathogen resistance is:(1)Bacteria produce enzymes that inactivate antimicrobial agents and inactivate antimicrobial agents;(2)Changes in target sites of antimicrobial agents;(3)Changes in the permeability of bacterial outer memberane[5].The traditional pharmacological definition of drug resistance focuses on the reduction or even disappearance of susceptibility to drugs caused by the internal changes of the microorganism.Artemisinin resistance emphasizes the retention curve of the parasite itself in the host.Although there is some overlap in the description of the two,they are very different in nature.The former attaches importance to the micropathogenicity Changes in the genotype or epigenetic nature of the organism,which almost entirely eliminate the description of the phenomenon.In vivo clearance of plasmodium is a complex process involving the insecticidal efficacy of antimalarial drugs,the regulation of host immune function(such as the recognition and processing of phagocytes),and the expulsion of foreign bodies by immune organs(such as spleen)[6].Moreover,ACT 3-day therapy has never been established as a cure.Prolongation of treatment time or adjustment of combination therapy can still achieve the efficacy of malaria treatment.Regardless of host defense mechanisms,should delay in clearance time after artemisinin-based combination therapy be defined as artemisinin "resistance"?The pathogenesis and clinical manifestations of malaria are affected by host age,immunity and genetic background,environmental conditions and parasitic genetics and other factors[7-8].In the presence or absence of artemisinin treatment,host defense mechanisms(e.g.clearance of circulating parasites via the spleen and mononuclear phagocytic system(MPS))play an important role in rapid infection control.Southeast Asia has always been the first place to develop resistance to various antimalarial drugs.After developing resistance to chloroquine,sulfadoxine,quinine and mefloquine,P.falciparum was found to develop resistance to artemisinin drugs for the first time.In Africa,which accounts for 90 percent of the world's malaria cases,few cases of artemisinin resistance have been reported.From the perspective of host control of malaria infection,it is particularly necessary to find out the reasons for the prolonged clearance time of malaria parasites.The spleen plays a key role in the control of malaria infection by eliminating malaria parasites through specific pitting.However,it is not clear whether there is any correlation between the clearance of Plasmodium by spleen and the prolonged clearance time of Plasmodium after ACTs treatment,i.e.drug resistance.Moreover,it is necessary to further study whether there are differences in the ability of spleen to clear plasmodium in different regions,people who are infected with malaria for the first time or repeatedly infected with malaria,and thus affect the clearance time of plasmodium.Objective:1.To explore the importance of the spleen's ability to clear malaria parasites in the control of malaria infection.2.To explore the main ways in which the spleen clears malaria parasites from blood circulation.3.To explore the factors affecting spleen clearance of plasmodium from blood circulation.4.To explore the nature of artemisinin drug resistance,and to provide a theoretical basis for solving the problem of artemisinin drug resistance.Method:Mice of four strains,C57BL/6,BALB/c,ICR and KM,were divided into control group and infection group.Mice of infection group were inoculated intraperitoneally with 1×107 artemisinin-sensitive erythrocytes of P.herghei K173(PbK173 iRBCs).The body weight,survival time and tail vein blood smear were recorded every day after infection in the test survival group,and the infestation rate was calculated.The peripheral blood,heart,liver and spleen of the other infection groups were collected at 1,3,5 and 8 days after infection,respectively.The peripheral blood parameters of mice were measured by automatic blood analyzer,and each organ was weighed and the organ coefficient was calculated.The spleens were divided into two parts:one was fixed with 4%paraformaldehyde fixation solution for 24 hours and then paraffin section was made for pathological analysis;Spleen cells in the remaining spleen were detected by flow cytometry.PbK173 artemisinin sensitive strain and resistance strain parallel experiments,the use of C57BL/6 mice,according to the weight were randomly divided into control group and the group of infection,the infection of mice respectively and intraperitoneal inoculation 1×107 sensitive/resistant strains iRBCs,lifetime measurement team records every day after infection in mice weight and tail vein blood smear dyeing rate,other infection group respectively in 2,5,9 days after infection to collect peripheral blood,heart,liver and spleen in mice.Peripheral blood fluid parameters,organ coefficients,pathological sections of spleen and spleen cells were detected in each group.PbK173 artemisinin-sensitive and artemisinin-resistant strains were used in parallel drug therapy experiment.C57BL/6 mice were randomly divided into control group and infected group according to body weight.The infected group was divided into model group and drug treatment group,and the drug treatment group consisted of flanadine group(6 mg/kg),DHA-L group(10 mg/kg),DHA-M(20 mg/kg)and DHA-H(40 mg/kg).In the infected group,1×107 iRBCs of sensitive/resistant strains were intraperitoneally inoculated,respectively.In the survival group,the body weight and tail vein blood smear were recorded every day after treatment and the infection rate was calculated.In the other groups,peripheral blood,heart,liver and spleen were collected on the first day after treatment and the fifth day after treatment.Peripheral blood fluid parameters,organ coefficients and pathological sections of spleen were detected in each group.Results:1.Different strains of mice had different tolerance to PbK173 artemisinin-sensitive strains,and the course of disease was different among strains.ICR mice developed disease rapidly and died quickly.Although the onset time of BALB/c mice was earlier than that of KM mice,the survival time of BALB/c mice was not significantly different from that of KM mice.The lethal infecting rate of KM mice was 65%,while the lethal infecting rate of other strains was over 80%.The growth rate of C57BL/6 mice was slower than that of other strains,and the survival period was longer than that of other strains.2.Spleen enlargement is a typical symptom of malaria infection,but the tolerance or pathological response of spleens of different strains of mice to malaria parasites varies:On the 8th day of infection with PbK173,the spleen parenchyma of mice infected with C57BL/6 and BALB/c was intact,while the spleen of mice infected with ICR and KM showed severe vacuolar pathological changes,and the spleen parenchyma was incomplete.On the fifth day,vacuolar pathological changes were found in the spleen of KM infected mice,especially in the red pulp.By the eighth day of infection,vacuolar pathology pervaded the entire spleen.3.There were differences in the function of physical intercepting plasmodium in the spleens of mice of different strains.During the period of infection with PbK173,the plasmodium in the spleens of mice infected with C57BL/6 and BALB/c were distributed in each growth stage of plasmodium.At the initial stage of infection,the plasmodium in each growth stage could be trapped in the spleen of KM infected mice.On the eighth day of infection,pathological changes occurred in the structure of the spleen,and the size of plasmodium remaining in the spleen was too large.At the initial stage of infection,plasmodium was more abundant in trophozoites in the spleen of ICR infected mice.4.In this study,the infection rate of PbK173 artemisinin-resistant strain and PbK173 artemisinin-sensitive strain showed no significant difference at the late stage of infection,but the survival period of PbK173 artemisinin-resistant strain was shorter than that of resistant strain(P<0.01),suggesting that the lethality or toxicity of PbK173 artemisinin-resistant strain was reduced.5.In C57BL/6 mice infected with PbK173 artemisinin-sensitive/artemisinin-resistant strain,the spleen coefficient of the resistant strain was always higher than that of the sensitive strain after the fifth day of infection without drug treatment(P<0.01).6.The peripheral blood smear of infected mice was observed under 100x oil microscope.The normal red blood cells were round and pale red.At the early stage of infection with Plasmodium,the body was stained blue and the nucleus was red.In the middle and late stage of infection,the erythrocyte volume increased with the growth of the worm body,and both the worm body and nucleus were blue.The curative effect of MD and DHA-H on the susceptible strain infected mice was 100%,and the blood of the two groups was observed on the first day of withdrawalThe liquid smear contains red blood cells of uneven size,mixed with some large,insect-free blue cells(damaged red blood cells).In the blood smear on day 5 of drug withdrawal,the damaged red blood cells were significantly reduced and the size of red blood cells was more uniform.This may be because the infected red blood cells remain in the blood circulation despite the inhibition of the malaria parasite by antimalarial drugs.The dysfunction of the blood system caused by the malaria parasite infection in mice does not recover immediately,but is alleviated by the body's own immunity(such as the spleen filtering and clearing the damaged red blood cells)after the treatment is stopped.This result needs further verification.7.In the MD treatment group,the cure rate of PbK173 artemisinin-sensitive or artemisinin-resistant strains was 100%.On the first day of drug withdrawal,there was no statistical difference in spleen coefficient and red blood cell count between the two strains.The blood smear shows the presence of blue cells(damaged red blood cells)without insects,and the cell size is not uniform.However,on the fifth day of withdrawal,the erythrocyte count and hemoglobin concentration in the peripheral blood of the susceptible strain infected mice were observedCompared with the resistant strain(P<0.05),the spleen of the sensitive strain continued to increase after drug withdrawal,and the damaged red blood cells were significantly reduced in the blood smear,and the size of red blood cells was more uniform.During this period,the spleen of the resistant strain infected mice did not continue to increase,and the cell size heterogeneity in the blood smear did not improve significantly.This contrast may be due to the fact that the spleen,which is capable of retaining damaged red blood cells from the circulation,is more sensitive to damaged red blood cells from the sensitive strains,so the spleens of the infected mice continued to grow after treatment.This result needs further verification.Conclusions:1.The intercepting function of spleen through the intercellular space of splenic venous sinus and the immune response of macrophages work together to remove plasmodium from the blood circulation.2.The ability of host spleens with different genetic backgrounds to clear malaria parasites is different.3.The interception of endothelial cell space in splenic venous sinus plays a major role in the clearance of plasmodium from spleen.4.The elasticity of the stress fibers in the splenic sinus and the flexibility of the erythrocytes affect the retention of the spleen.5.There may be differences in the flexibility of erythrocytes infected with different strains sensitive to artemisinin.