| Objective:The purpose of this study is to establish a targeted metabolomics platform of endogenous amino acids with stable performance,compatible with serum and multiple tissues samples including liver,spleen,kidney and intestine,to investigate the pattern of amino acids metabolism in Artemisinin-sensitive/-resistant Plasmodium infected mouse models.By screening the differential amino acids and their metabolic pathways closely related to Artemisinin-resistant malaria,the consistency and specificity of metabolism perturbed under the two malaria models were explored to provide a scientific basis for studying the metabolic mechanisms of Artemisinin-resistant Plasmodium invasion in the host.The emergence of Artemisinin-resistance has exacerbated the pressure for malaria elimination.In a pre-nontargeted metabolomics analysis,it was found that chrysosplenetin（CHR）could bidirectionally regulate the metabolic phenotypic differences in hosts caused by Artemisinin-sensitive/-resistant Plasmodium in concert with artemisinin（ART）.In this thesis,we further constructed a mass spectrometry-targeted metabolomics platform to validate and confirm the total 13 target metabolites screened in the previous stage.The amino acids associated with the regulatory effects of CHR were clarified and the metabolic pathways of potentially acting amino acids were analyzed,so as to clarify the potential amino acids and their roles in the regulation of host-specific perturbations caused by Artemisinin-resistant Plasmodium by CHR and to explore the metabolic regulatory mechanism of CHR as a small molecule inhibitor for ART-resistance.Methods:1.Construction of targeted metabonomics platform for endogenous amino acids.Based on HPLC-MS/MS technology,a targeted metabonomics platform for the simultaneous determination of 13 metabolites was constructed.The mass spectrometry conditions of each target amino acid were first optimized respectively.The optimal liquid phase conditions for the simultaneous detection of all amino acids were mapped,including the chromatographic column and mobile phase composition.Examination of the compatibility with serum and tissue samples was also considered.Finally,the platform was evaluated for selectivity,the linear range,limit of detection and limit of quantification of the standard curve,intra-day and inter-day precision,matrix effect and recovery and sample stability.2.The study on differences in amino acids metabolism in Artemisinin-sensitive/-resistant Plasmodium infected mice based on targeted metabolomicsAfter constructing the Artemisinin-sensitive/-resistant infected mouse models,serum,liver,spleen,kidney and intestinal tissue samples were collected respectively.Based on the constructed targeted metabolomics platform for endogenous amino acids,data were collected and statistically analyzed to screen for differential amino acids and make relevant pathway enrichment analysis.The global metabolic pathways were mapped to explore amino acids metabolic perturbations in the two malaria models based on the KEGG database.3.The regulation of CHR in amino acids metabolism of Artemisinin-sensitive/-resistant Plasmodium infected mice.On the construction of the Artemisinin-sensitive/-resistant Plasmodium infection mouse models,mice were randomly divided into blank control group,ART monotherapy group and ART-CHR（1:2）combination group.After 7 days of continuous gavage administration,the samples of serum,liver,spleen,kidney and intestine tissue were collected respectively.The same method was used for data collection.The obtained data were then analyzed in depth to investigate the differences in the regulation of host amino acid metabolism of Artemisininsensitive/-resistant Plasmodium infected mice before and after the combination of ART and CHR.Results:1.The thesis constructed a LC-MS/MS targeted metabolomics platform for the simultaneous determination of 13 endogenous metabolites.The selectivity,the linear range,limit of detection and limit of quantification of the standard curve,intra-day and inter-day precision,matrix effect and recovery and sample stability of the platform were all in accordance with the methodological requirements.2.Analysis of biological samples from Artemisinin-sensitive/-resistant Plasmodium infected mice was completed using the targeted metabolomics platform of endogenous amino acids constructed.The two malaria mice exhibited different amino acid metabolic profiles.A total of 11 metabolites were perturbed in mice infected with Artemisinin-sensitive Plasmodium,altering the levels of glycine（Gly）,serine（Ser）,lysine（Lys）,phenylalanine（Phe）,glutamine（Gln）,tryptophan（Trp）and trimethylamine N-Oxide（TMAO）in serum.Significant alterations in the levels of Ser,valine（Val）,taurine（Tau）,Phe,alanine（Ala）,Gln,Trp and TMAO were found in liver.Val,Phe,Tau and Trp levels were changed in the spleen and Val,Gln,Tau,Phe,isoleucine（Ile）,Trp and TMAO levels in the kidney,while Trp,Lys,Tau and TMAO were disturbed in intestine.A total of 9 amino acids were perturbed in Artemisininresistant Plasmodium infected mice.It disturbed the levels of Ile,Lys and TMAO in serum,Ser,Gln,Gly,Tau and TMAO in liver,Val,Ala and Tau in spleen,Val,Tau and TMAO in kidney,Tau and TMAO in intestine.The metabolic patterns of the two malaria-infected mice were also different significantly,with greater differences in amino acids in serum and liver,but not in intestine.3.Using the targeted metabolomics platform,the analysis of biological samples from Artemisinin-sensitive/-resistant malaria mice after receiving ART alone and ART-CHR（1:2）co-used respectively was completed.Artemisinin-sensitive malaria mice treated with ART showed down-regulation of Trp level in serum,up-regulation of proline（Pro）in liver,upregulation of Phe in kidney,and altered levels of Gln and Ile in intestine.Treatment with ARTCHR（1:2）resulted in increased Gly and decreased Trp in serum,and there were differences in the levels of Gly,Pro and threonine（Thr）compared to ART administration.Phe and Thr levels were different in liver compared to ART,while the levels of Ser,Gly,Val and Trp in spleen were different.In kidney,TMAO,Val,Phe and Ile were altered,in addition Ser,Val and Tau levels were higer than ART.Gly,Tau,Gln and Ile levels were alterd in intestine,but there were no metabolic difference between the two administrations.After ART treatment of Artemisinin-resistant malaria mice,Tau was down-regulated in serum,Pro,Phe,Gly,Gln and Trp levels were altered in liver,TMAO was down-regulated in spleen,Pro,Val,Thr and Ile levels were altered in kidney,and Ile,Phe,Trp and Tau were altered in intestine.After receiving ART-CHR（1:2）treatment,Gln level was up-regulated in serum,but Trp,Ala,Val,Tau and Phe levels were altered compared to the ART administration.In liver,Phe and Tau levels were altered,but Gln and Trp were higher and Ala levels were lower than in the ART administration.In spleen,there were significant differences in amino acid metabolic profile with altered levels of Ala,Tau,Lys and Trp.However,the trends of Pro,Thr and Ile changes in the kidney were consistent with ART,and there were also significant differences in Phe and Tau.And the trends of Ile,Phe and Trp in intestine were also consistent with ART,but the trend was weaker than ART regulation.Ser,Phe and Pro were also significantly regulated.In addition,TMAO,Tau and Thr differed between the two administration groups.Conclusion:1.The targeted metabolomics platform for endogenous amino acids was successfully constructed and performed well for the determination of 13 metabolites in serum and multiple tissues including liver,spleen,kidney and intestinal tissues of mice infected Plasmodium,ensuring the next step of rapid and accurate targeted amino acid analysis of bulk biological samples;2.An endogenous amino acid-based targeted metabolomics platform was used to determine the amino acids in biological samples from Artemisinin-sensitive/-resistant malaria mice.Significant differences in metabolic profiles were exhibited in serum,liver,spleen,kidney and intestinal tissue samples,and differential amino acids were searched for in the two models,where were mainly related to the proliferation,growth and utilization of Plasmodium,immune escape,the influence of intestinal flora and homeostasis.Gly,Ile and Ala specifically regulated in Artemisinin-resistant malaria may be its metabolic characteristics,laying the foundation for explaining the mechanism of metabolic differences following resistance mutations;3.Artemisinin-sensitive/-resistant malaria mice treated with ART and ART-CHR（1:2）,respectively,exhibited certain metabolic phenotype differences in serum and various tissue samples.The co-used of CHR and ART improved the antimalarial effect of ART alone,which was most significant in the spleen,kidney and intestine of Artemisinin-resistant malaria mice,inhibited the utilization of amino acids of Plasmodium,enhanced the immune ability of mice as well as improved the intestinal flora enironment.These features help to explain the metabolic role of CHR in modulating the amino acids metabolism of Artemisinin-resistant Plasmodium perturbing hosts and provide metabolic data to support the research and development of CHR as a small molecule inhibitor of ART-resistant. |
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