Proteomics And Metabolomics Analysis On Polarization Of Mouse Peritoneal Macrophages Induced By Excretory-secretory Products Of Fasciola Gigantica

Background : Helminthic parasites could possibly disturb the immune microenvironment,reduce or even silence the immune responses in the hosts through releasing excretory-secretory product(ESP),in order to escape from host immune attack during their migration and development.The polarization of host macrophage by helminthic ESP has been proven,while there is still a lack of research on the mechanism of Fg ESP regulation of macrophage polarization.This thesis focuses on whether Fg ESP can regulate macrophage polarization and its regulatory mechanism.Methods: After intraperitoneal injection of Fg ESP in mice,the phenotype of mouse peritoneal macrophages was identified from genes,proteins and biochemical indicators.After stimulating Fg ESP-induced mouse peritoneal macrophages with LPS and IFN-γ,the subtypes were identified using genes and biochemical indicators to obtain macrophages of different subtypes.Proteomics TMT technology and bioinformatics analysis technology were used to screen differentially expressed proteins of Fg ESP-induced macrophages and LPS and IFN-γ-induced Fg ESP-induced mouse peritoneal macrophages.GO annotation and enrichment,KEGG annotation and enrichment,and interaction network analysis of differentially expressed proteins from different group were carried out.Non-targeted metabolomics HILIC and RPLC UHPLC-Q-TOF technology and multivariate analysis methods were used to process the data to obtain sample classification information and difference variables.Screen potential biomarkers for Fg ESP-induced polarization of peritoneal macrophages,and perform heat map analysis,metabolic pathways,and metabolite interaction network analysis for potential biomarkers.Proteomics and metabolomics correlation analysis were used to integrate differentially expressed proteins and potential biomarkers,and to explore the related regulatory mechanisms and signaling pathways of the interaction between Fg ESP and macrophages.Results: After intraperitoneal injection of Fg ESP and Fh ESP,mice peritoneal macrophages were recruited,and the cells highly expressed Arg-1,RELM,and Ym-1,indicating that the peritoneal macrophages recruited by Fg ESP and Fh ESP were mainly M2 type.Fg ESP recruited macrophages were stimulated by LPS and IFN-γ for48 hours in vitro,the cells highly expressed i NOS,and the level of NO secretion in the cell supernatant increased,which suggests that M2 type is polarized to M1 type.Using TMT proteomics technology,in the M1/M0 comparison group,a total of 1151 differentially expressed proteins were identified(up 537,down 614).In the M2/M0 comparison group,a total of 1465 differentially expressed proteins were identified(up 803,down 662).A total of702 differentially expressed proteins were identified in M2/M1(up 407 and down 295).By analyzing GO and KEGG pathways of differential proteins,the cell adhesion molecule and purine metabolism pathway in the M1/M0 comparison group,ribosome pathway and Parkinson’s disease pathway in the M2/M0 group,cell adhesion molecular and lysosome pathway in the M2/M1 group have shown significant changes.Using metabolomics technology,differential metabolites were screened in all three comparative groups.Through GO and KEGG pathway analysis and metabolite interaction network analysis of differential metabolites,it was found that the changes of metabolites in the M1/M0 comparison group mainly involved glycerophospholipid metabolism,fructose and mannose metabolism and phenylalanine Biosynthesis of acid,phenylalanine metabolism,tyrosine and tryptophan,arachidonic acid metabolism,lysine degradation.The changes of metabolites in the M2/M0 comparison group mainly involved glycerophospholipid metabolism,linoleic acid metabolism,phenylpropane metabolism,phenylalanine,tyrosine and tryptophan biosynthesis,ascorbic acid and aldonic acid metabolism,arachidene Acid metabolism,lysine degradation.The changes of metabolites in the M2/M1 comparison group mainly involved glycerophospholipid metabolism,alanine,aspartic acid and glutamic acid metabolism,and niacin and nicotinamide metabolism.Through proteomics and metabolomics correlation analysis,it was found that M0 macrophages and M1 macrophages induced by LPS and IFN-γwere undergoing Huntington’s disease,lysosome,carbon metabolism,necrosis and other pathways have changed.Carbon metabolism,oxidative phosphorylation,Huntington’s disease,lysosomes,Parkinson’s disease,necrosis and purine metabolism play important roles in the process of Fg ESP-induced polarization of mouse peritoneal macrophages.In M2/M1 group,Parkinson’s disease,amino acid biosynthesis,regulation of actin cytoskeleton,purine metabolism,retrograde endocannabinoid signal transduction,pyrimidine metabolism,glycolysis/gluconeogenesis,glycerophospholipid metabolism and other pathways displayed significant changes.Conclusions: According to the analysis of experimental results,it is believed that Fg ESP can recruit M2 macrophages in vivo.Differentially expressed proteins and potential metabolites play an important role in Fg ESP-mediated macrophage polarization,and the pathways involved are mainly oxidative phosphorylation,lysosomes,carbon metabolism,necrosis,and purine metabolism.These conclusions will provide a significant reference for further in-depth study and explanation of how Fg ESP regulates the polarization and function of macrophages to achieve immune escape.