| ObjectiveMalaria is an insect-borne infectious disease whose pathogen is Plasmodium and can be transmitted by the bite of female Anopheles mosquitoes.Infection of malaria can cause periodic symptoms such as high fever,sweating,dizziness and headache.Repeated attacks may lead to splenomegaly or anemia.This ancient disease still poses a great threat to human health nowadays.Malaria is endemic worldwide,especially in Southeast Asia and Africa,causing hundreds of millions of cases and hundreds of thousands of deaths each year.More efforts 4 are needed to fight against malaria.The emergence and spread of resistance of Plasmodium to various antimalarials a huge obstacle on the way to eliminating malaria.Artemisinin Combination Therapy(ACTs)is the first-line anti-malarial program recommended by the World Health Organization,which usually composed of an artemisinin drug,such as artemisinin or its derivatives,dihydroartemisinin,artesunate and artemether,a drug with a longer half-life and a slower action,such as Piperquineo It can not only improve the curative effect,but also protect each other from the rapid emergence of drug resistance.Artemisinin is a treasure of traditional Chinese medicine.Treatment of Malaria with Artequick(ATQ),whose compositions are artemisinin and pIperaquine,is a successful practice of traditional Chinese medicine in the prevention and treatment of major diseases.However,in recent years,there has been a decline in the susceptibility of Plasmodium to ACTs in some parts of Southeast Asia,which makes it more urgent and important to study the resistance mechanism of artemisinin and its components.In order to study the molecular mechanism of resistance to artemisinin in Plasmodium,the resistance of Plasmodium berghei to artemisinin and piperaquine was cultured in a murine model,and the differences of drug resistance and resistance degree were observed.The genes of artemisinin and piperaquine resistant strains were sequenced to explore the relationship between gene mutation and drug resistance,all of them are the basis for studying the molecular mechanism of drug resistance.Methods1.The murine model was established in Kunming mice.The resistance of Plasmodium berghei K173 strains to artemisinin and piperaquine was cultured by drug incremental method.During the process of resistance cultivation,the resistance level was determined every five generations.The resistance level was determined by Peters 4-D inhibition experiment.According to the level of resistance measured and the monitoring of infection rate during cultivation,The dosage should be adjusted appropriatelly.but the dosage should not be lower than that used in the algebraic resistance cultivation of the previous resistance level determination.The cultivation of resistance was analyzed.2.During the process of resistance cultivation,at the end of each generation of resistance cultivation,eyeballs were taken for blood collection.DNA extraction was done and the Kelchl3 fragment of the Plasmodium berghei resistant strain was analyzed by polymerase chain reaction(PCR),following by gene sequencing.3.Forty Kunming mice were randomLy divided into four groups and were infected with different Plasmodi'um berghei K173 strains,named as K173 group;A30 group;,A50 group and P50 group respectively.Thin blood smear from the tail blood of mice was smeared every day to monitor the increase of infection rate.When the infection rate reached 1.5%,Eyeballs were taken to collect blood.The PI3K content in serum was measured by Mouse Phosphotylinosital 3 kinase(PI3K)ELISA Kit.In addition,Plasmodium was collected for lipid extraction and PI(3)P Mass ELISA Kit was used to determine the content of PI(3)P,and the differences among the groups were compared.Results1.Plasmodium berghei showed resistance to artemisinin and piperaquine after 50 generations of resistance cultivation.The resistance of Plasmodium to artemisinin was cultivated from 21 to 50 generations,and the dosage increased from 574.20mg/kg to 883.96mg/kg.Resistance index increased from 21 generations to 30 generations,but then declined again.During 40 to 50 generations,the resistance index fluctuated about 5.At the end of resistance cultivation,the resistance index was 4.97.The dosage of piperaquine increased from 47.67mg/kg in the 21st generation to 76.82mg/kg in the 50th generation,and the resistance index continued to rise,reaching 148.82 in the 50 generation.2.Amplification and sequencing of K13 fragments of both artemisinin sensitive and resistant strains of Plasmodium berghei were performed.The sequencing results were compared with the sequence of K13 fragment(CXJ03505)of Plasmodium berghei K173 strain in ENA.More than 30 mutations were found in resistant strains while no genetic mutation occurred in sensitive strain.There are 9 missense mutations,they are A128T?A189G?A311G?G361A?A368G?G398A?A464G?A531T and C1643T,and the corresponding amino acid changes were as follows:Y43F?I63M?N104S?A121T?N123S?S133N?N155S?E177D and S548L,the others were silent mutations.3.The content of PI3K from mouse serum in K173 group,A30 group,A50 group and P50 group were 82.67+15.09 pmol/L,84.33+19.39 pmol/L,109.27+10.13 pmol/L 99.54+10.98 pmol/L respectively.The PI3K content in A50 group was higher than that in K173 group or A30 group(P<0.05).The PI(3)P content of Plasmodium berghei in resistant strains were higher than that in sensitive strain.The difference was not statistically significant.Conclusions1.During the resistance cultivation,the resistance of Plasmodium berghei K173 strain to artemisinin fluctuated and growed slowly while resistance to piperaquine was quickly obtained.Eventually,strains resistant to artemisinin and piperaquine were obtained.2.Under sustained drug pressure,several gene mutations in the K13 fragment of Plasmodium berghei,nine of them were missense.Artemisinin and piperaquine caused the same mutations.3.Plasmodium berghei resistant strains containing K13 gene mutations caused the increase of host serum PI3K level and PbPI(3)P,which may be related to the mechanism of artemisinin resistance. |
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