Mechanism Of Methylglyoxal Damage On Microvascular Endothelial Cell Function Based On Metabolomics And Possible Intervention Strategies

ObjectivesThe impairment of endothelial cell function is an important initial factor of diabetic vascular complications.Methylglyoxal(MGO)is a highly reactive metabolite produced in the process of glucose metabolism and has a damaging effect on endothelial cell function.In this study,untargeted metabolomics and transcriptomics methods were used to analyze the metabolic pathway changes after MGO intervention in microvascular endothelial cells,which further clarify the mechanism of MGO damage on microvascular endothelial cell function and explore differential metabolites that can improve the detrimental effect of MGO,in order to provide new ideas for the treatment of diabetic vascular complications.Methods Part OneThe microvascular endothelial cells were cultured in vitro and divided into control group and MGO group.The cell proliferation ability was detected by CCK8 method.The cell apoptosis and reactive oxygen species(ROS)level were detected by flow cytometry.The cell tubulization function was detected by tube formation assay.The cell migration ability was detected by scratch assay.The supernatant NO content was detected by NO reagent kit.The m RNA expression level of related genes was detected by q PCR.The expression of endothelial nitric oxide synthase(e NOS)protein was detected by Western Blot.The differential genes and involved important signaling pathways were screened by RNA-seq technology,and the differential metabolites and metabolic pathways were analyzed by non-targeted cell metabolomics technology.Part TwoWe used L-Arginine(Arg)to interfere with MGO-induced microvascular endothelial cells.The experiment was divided into control group,Arg group,MGO group and MGO+ Arg group.CCK8 method was used to detect the cell proliferation ability.Flow cytometry was used to detect cell apoptosis.Tube formation experiment was used to detect cell tube formation function.Scratch experiment was used to detect cell migration ability and NO reagent kit was used to detect the content of NO in cell supernatant.The expression of e NOS protein was detected by Western Blot.Results Part One1.A model of MGO-induced injured microvascular endothelial cells was successfully established in vitro.Compared with the control group,the activity of cells in the MGO group decreased significantly and the apoptosis level increased significantly.ROS production increased and inflammatory reaction increased significantly in the MGO group.Importantly,the ability of forming tube and migration decreased significantly in the MGO group.Furthermore,the amount of NO in the cell supernatant decreased significantly and the expression of e NOS protein decreased significantly.2.The results of the cell transcriptome analysis showed that the gene transcription level of the MGO-intervention group changed significantly.A total of 2844 differentially expressed genes were screened out in the control group and MGO group.A total of 884 genes were up-regulated and 1960 genes were down-regulated,including genes closely related to vascular formation.The KEGG pathway enrichment analysis showed that the cell-extracellular matrix receptor interaction and the local adhesion signal pathway were the two pathways with the most significant enrichment.3.The results of the cell non-targeted metabolomics detection showed that MGO mainly induced the amino acid metabolism reprogramming of microvascular endothelial cells.MGO intervention significantly changed the content of 278 cellular metabolites,including 21 metabolites up-regulated and 257 metabolites down-regulated.KEGG metabolic pathway analysis showed that the significantly different metabolic pathways in the control group and MGO group were mainly the amino acid metabolism pathway and the purine metabolism pathway.Part TwoCompared with the MGO group,Arg intervention could significantly improve the vitality of microvascular endothelial cells and reduce the apoptosis induced by MGO.Arg intervention also could significantly promote the tubulization and migration ability of endothelial cells damaged by MGO,as well as significantly increase the expression of e NOS proteinthe and content of up-regulating NO in endothelial cells.Conclusion1.MGO intervention significantly damaged the functions of proliferation,tube formation and migration of microvascular endothelial cells.2.MGO intervention affected the expression of genes related to angiogenesis,and the extracellular matrix receptor interaction and local adhesion signaling pathway were significantly enriched signaling pathways of differentially expressed genes.3.MGO intervention caused metabolic reprogramming of microvascular endothelial cells,among which amino acid and purine metabolism pathways showed significant changes,which may be a new mechanism of MGO-induced endothelial cell dysfunction.4.Differential metabolites Arg identified by metabolomics could significantly ameliorate the damaging effects of MGO on endothelial cell function.