Study Of Protection And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell-derived Exosomes On Aorta Of Diabetic Mice

BACKGROUND:Diabetes mellitus has become one of the global diseases threatening human health,and its related metabolic abnormalities will gradually cause vascular diseases,including microangiopathy and macroangiopathy with high morbidity and mortality.The main characterization of macroangiopathy are the formation of atherosclerotic plaques in the main arteries,coronary arteries and carotid arteries,which can cause lumen stenosis or even occlusion.The pathophysiological mechanisms of diabetic macroangiopathy are disorders of glucose and lipid metabolism,hyperactivation of platelets,injury of endothelial cells,inflammatory response and oxidative stress.So far,there is no effective treatment for diabetic macroangiopathy.Exosome is a kind of biological nanovesicle secreted by cells,which is the medium for the biological function of a variety of cells,and plays a therapeutic role in a variety of diseases.Studies have shown that stem cell-derived exosomes promote tissue regeneration with no tumor risk and low immune response.Current studies have shown that exosomes play a role in the treatment of diabetic cardiomyopathy,diabetic nephropathy and diabetic foot,but there are few studies on diabetic macrovascular disease.Metabolomics can detect small molecule metabolites(molecular weight<1000),and can describe the change of metabolites,to accurately describe the metabolic changes in different states.Since one of the pathophysiological mechanisms of diabetic macroangiopathy is the disorder of glucose and lipid metabolism,Thus,metabolomics is very important in the study of mechanism of human umbilical cord mesenchymal stem cell-derived exosomes(hUCMSCexos)to diabetic macroangiopathy.Protein is the reflect of gene,and any pathological state will cause the change of protein.Proteomics is now widely used in the search for biomarkers of disease.Differentially expressed proteins in endothelial cells and plasma were detected by proteomics to elucidate the mechanism of diabetic macroangiopathy.However,proteomics on the changes in aorta of hUCMSC-exos treated diabetic mice is rarely reported.In this study,firstly,we detected the effect on hUCMSC-exos on aortas of diabetic mice from pathological changes and other aspects.Secondly,metabolomics and proteomics were performed to explore the mechanism of hUCMSC-exos to aortas of diabetic mice.OBJECTIVE:1.To study the effect of hUCMSC-exos on aorta in diabetic mice.2.Metabolomics and proteomics were performed to explore the mechanism of hUCMSC-exos to aorta of diabetic mice.METHODS:1.Extraction of hUCMSC-exosSize exclusion chromatography was used to extract hUCMSC-exos.HUCMSCs of generation 3-7 was used.2.Animal groupingTwelve C57BLKS/J db/db mice and six db/m mice were purchase at 8 weeks.The db/db mice were divided into two groups,one group was diabetes millitus aorta(DMA,n=6).They were injected saline;another group was hUCMSC-exos treated diabetes millitus aorta(DMTA,n=6).They were injected hUCMSC-exos.db/m mice were normal control aorta(NCA,n=6).3.Animal handlingBody weight and fasting blood glucose were measured at 12,17 and 22 weeks.During the whole intervention process,the general conditions of mice,such as activity,diet and mental state were observed.After the intervention,the mice were sacrificed,the serum of the mice was collected to detect the levels of triglyceride and total cholesterol,and the aortas were fixed and frozen.4.Histopathological testHematoxylin-eosin(H&E)staining was used to observe the aorta remodeling in mice.The collagen of aortas were observed by sirius red staining.The expressions of α-SMA and OPN in aorta were observed by immunohistochemical staining.The fluorescence intensity of α-SMA and OPN were observed by immunofluorescence co-staining.The ultrastructure of aortas were observed by transmission electron microscope.5.MetabolomicsThe frozen aortas were handled by metabolites extraction,high performance liquid chromatography-mass spectrometry and bioinformatics.The differential metabolites were searched for and their functional analysis.6.ProteomicsThe frozen aortas were handled by protein extraction,liquid chromatography-secondary mass spectrometry and bioinformatics.The differential proteins were searched for and their functional analysis.7.Integreted analysis of Metabolomics and proteomicsThe pathways enriched by differential metabolites in metabolomics analysis were crossed with those enriched by differential proteins in proteomics analysis to find the common pathways between them.8.Verification of significant moleculeQuantitative Polymerase Chain Reaction(qPCR)and IHC staining were used to verify the significant molecules in the results.RESULTS:1.At 12,17 and 22 weeks,body weight and fasting blood glucose of db/db mice were significantly higher than those of db/m mice.The body weight of mice in DMTA was significantly lower than that in DMA at 17 weeks and 22 weeks,and no significant changes in FBG.TC and TG of db/db mice were significantly higher than those of db/m mice,but no significant changes in DMT A compared with DMA.2.HE staining,sirius-red staining,α-SMA and OPN immunohistochemical staining,αSMA/OPN immunofluorescence co-staining results confirmed that hUCMSC-exos could improve aortic remodeling in diabetic mice.3.Seventy-four metabolites were significantly changed in DMA VS NCA,among which glucose metabolites were significantly down-regulated and fatty acid metabolites were significantly up-regulated.After hUCMSC-exos treatment,15 metabolites were significantly changed,among which glucose metabolites were significantly up-regulated and fatty acid metabolites were significantly down-regulated.KEGG pathway analysis showed that central carbon metabolism and galactose metabolism pathways were up-regulated after hUCMSC-exos treatment.4.Ninety-nine proteins were up-regulated and 154 proteins were down-regulated in DMA VS NCA.Consistent with the results of metabolomics,the expression of enzymes related to glucose metabolism decreased and that related to fatty acid metabolism increased.Compared with the DMA group,81 proteins were significantly altered in the DMT A group,and 30 proteins were adjusted to normal levels after hUCMSCs-exos treatment nearly,including Stromal Cell-Derived Factor 2-Like Protein 1(SDF2L1),Leukemia Inhibitory Factor Receptor,LIFR),Peroxisomal Membrane Protein 16(PEX16)and E3 Ubiquitin-Protein Ligase MYCBP2(E3 Ubiquitin-protein Ligase MYCBP2,MYCBP2).KEGG pathway analysis revealed Janusassociated kinase signal transduction and transcriptional activator(JAK-STAT)pathways was significantly enriched.5.Integrated analysis between metabolomics and proteomics shows,the top 5 KEGG pathways were co-enriched,including:ABC transporters,taste transduction,purine metabolism,phospholipase D signaling pathways and Gap connections.These pathways are involved in the active transport and metabolism of various substrates,such as membrane transport,actin cytoskeletal remodeling,cell proliferation and cell survival.CONCLUSION:1.HUCMSC-exos can improve vascular remodeling in the aorta of diabetic mice.2.HUCMSC-exos protect aorta of diabetic mice by regulating energy metabolism and oxidative stress.