The Role Of Phospholipid Metabolism In Experimental Autoimmune Myasthenia Gravis Based On Lipidomics

BackgroundMyasthenia gravis(MG)is one of the most common autoimmune diseases of the nervous system.Its immunological mechanisms is complex,recent research demonstrated that immune tolerance state is destroyed by external antigens or misidentification of certain autoantigen in MG.,And the antigen-specific CD4+T cells are reactivated.The imbalance of inflammatory and anti-inflammatory cytokines and the imbalance of CD4~+T subsets have resulted in the abnormal of antibodies producing.The pathogenesis of MG is not very clear,the abnormal changes amount and functional deficiency of regulatory T cells and effector T cells seems to be the trigger point of MG.Studies have found that the activation and differentiation of immune cells is accompanied by the reconstruction of metabolic patterns.In recent years,with the development of lipidomics technology,more and more studies have shown that lipid metabolism also plays an important role in immune cells.In addition,abnormal lipid metabolism exists in the peripheral blood of patients with autoimmune diseases.Based on this,this study used extensive targeted lipidomics to analyze the changes in lipid metabolism of CD4~+T cells in EAMG by constructing an EAMG rat model to identify changes in cell metabolism,looking for differential lipid metabolites,to explore the role of lipid metabolism in the pathogenesis of EAMG and provides new ideas for future research on targeted immunotherapy of myasthenia gravis.Methods1.Establishment the rat model of EAMG and Experimental grouping:The healthy,6-week-old female Lewis rats were selected and randomly divided into 2groups.One group of rats were induced by subcutaneous injection into with antigen-containing emulsion that consist of R97–116 peptides Rat-derived Acetylcholine Receptor(ACh R)and were boosted by the same method against after1month from first immunization to set up the model of Myasthenia gravis.At the same time Normal Control(NC)group rats were immunized with a solvent emulsion without contain ACh R antigen by the same methods with EANG group.The clinical score and body weight were measured and recorded every 1 day after immunization.2.Preparation of rat spleen cell suspension and flow cytometry cell sorting:Rats in the EAMG group with clinical score greater than or equal to 2 and the control group Rats were selected,and killed by carbon dioxide asphyxation.Spleens were taken under aseptic conditions and mononuclear cell suspension was prepared.flow cytometry sorting of spleen CD4+CD25-T cells.3.Detection of lipid metabolism:T cells samples from all the groups were analyzed for lipidomics by Ultra Performance Liquid Chromatography and Tandem mass spectrometry(UPLC-MS/MS)technology,screening the Different lipid metabolites between the EAMG group and in the NC group and based on the KEGG database to find the affected metabolic pathways.4.QRT-PCR validation experiment:q RT-PCR technology was used to detect the expression levels of key enzymes in the differential lipid metabolite pathway,and conducted further verification experiments.Results1.A total of 191 lipid metabolites were detected in this experiment.Compared with the EAMG rats in the NC group,there were 66 lipid metabolism differences,and they were all metabolically up-regulated(difference times>2),involving 3 lipid subclasses.Including glycerophospholipids(Glycerophospolipids,GP),sphingolipids(Sphingolipids,SP),fatty acyls(Fatty Acyls,FA).Glyerophospholipids(GP)accounted for 89%of the difference,of which phosphatidylcholine(PC)accounted for 59%and phosphatidylethanolamine(PE)accounted for 27%.2.KEGG pathway enrichment analysis indicates that the glycerophospholipids pathway is the most enriched.3.The qRT-PCR experiment found that the rate-limiting enzyme PCYT2 of the de novo phosphatidylethanolamine synthesis pathway of effector T cells in the EAMG group was significantly higher than that of the NC group,while the expression of the key enzyme LPCAT2 in the phosphatidylcholine remodeling pathway decreased.conclusion1.There is a lipid metabolism disorder in EAMG rat effector T cells,among which phosphatidylcholine and phosphatidylethanolamine are the most significant metabolic differences.2.Phospholipid metabolism pathway is the most affected metabolic pathway of effector T cells in EAMG rats.3.Abnormal phospholipid metabolism in effector T cells may be one of the pathogenesis of EAMG.