The Mechanism And Effect Of Mesenchymal Stem Cell Derived Exosomes On Improving Diabetic Microangiopathy

Background:Type 2 diabetes mellitus(T2DM)is caused by insufficient insulin secretion or insulin resistance in human body due to a variety of causes,and its chronic complications mainly include macrovascular,microvascular and nerve lesions.Among them,diabetic microangiopathy is the main predisposing factor of diabetic foot,gangrene,amputation and disability.The main pathophysiological basis is the damage of endothelial cell structure and function,which leads to increased vascular permeability,lipid deposition and atherosclerosis.Therefore,endothelial damage and increased vascular permeability are a key pathological basis in type 2 diabetic vascular disease.Recent reports indicate that stem cells inhibit cell apoptosis,promote endothelial cell migration and proliferation,and participate in the entire process of angiogenesis.Paracrine signaling is considered to be one of the main potential mechanisms behind the therapeutic effects of stem cells.Exosomes are especially important paracrine factors for intercellular communication.The small diameter of exosome and its no nuclear structure have the advantages,which avoids some adverse events caused by direct transplantation of stem cells.Exosomes contain abundant proteins and miRNAs that can improve endothelial cell injury,but there are few reports on the efficacy and mechanism of exosomes in improving microvascular disease.Objective:To investigate the therapeutic effect and potential mechanism of mesenchymal stem cell-derived exosomes on microangiopathy in T2DM.Methods:1.Mesenchymal stem cell-derived exosomes were extracted by hypervelocity centrifugation method,and exosome morphology was observed by transmission electron microscopy,exosome diameter distribution was detected by particle size analysis,and exosome specific proteins were identified by Western blot.2.DB/DB mouse were randomly divided into exosomes group and PBS group.Exosomes and PBS were injected into the tail vein,respectively,and the calf muscle tissue was taken 28 days later.0.5%Evans blue fluorescence assessment microvascular permeability.The expression of Ki67,Bax and Bcl-2 in muscle tissue was observed by immunohistochemistry.The expression of CD31 was detected by immunofluorescence.The morphology and function of microvessels in muscle tissue of lower limbs was evaluated by transmission electron microscopy.3.TMT proteomics was used to detect the changes of differential protein expression in lower limb muscle tissues of the PBS group and the exosome group,and data analysis was performed to screen key signal molecules and their involved biological pathways.Key signal molecules CD105 were verified by Western blot.The expression of TGF-βin exosomes were evaluated by Western blot.Results:1.Electron microscopy showed that hUCMSCs exosomes presented a uniform vesicle structure,and NT A showed that its diameter was about 160 nm.Western blot showed positive expression of specific proteins CD9,CD81 and TSG101 on exosomes.2.There is no significant change in blood glucose and body weight before and after the exosome treatment.The exosome group can significantly reduce the exudation of Evans blue.Compared with the PBS group,The expression of Bax in the exosome group was significantly reduced,while the expression of Ki67 and Bcl-2 increased.Meanwhile,CD31 immunofluorescence showed that the red fluorescence of exosome treatment was significantly increased,which was higher than that of PBS group.Transmission electron microscopy showed smooth capillary lumen and smooth and complete surface of endothelial cells in the exosome group,while narrow capillary lumen and fingerlike protrusion of endothelial cells in the PBS group.3.Quantitative analysis of TMT proteomics showed that there were 82 differential proteins,including 49 down-regulated proteins and 33 up-regulated proteins.Go enrichment analysis showed that the differential proteins were involved in molecular function,biological process,cell components,among which CD 105 was one of the up-regulated proteins.Through literature search,CD 105 was found to be related to endothelial cell proliferation.Therefore,this study verified the changes of CD 105 in the exosome group,and it was used as the mechanism study of this study.4.Western blot analysis showed that the expression of CD105 protein in lower limb muscle tissue of exosome group was significantly increased compared with that of PBS group.Based on the fact that CD105 is a component of the TGF-β receptor complex and exosomes are rich in growth factors and cytokines,this study further examined the expression of TGF-β in exosomes,and the results showed that exosomes had high expression of TGF-β.Conclusion:By improving the integrity of microvascular endothelial cells,hUCMSCs exosomes can improve the permeability of microvessels in diabetic lower muscle tissue,further promote the proliferation of lower limb muscle cells and inhibit the apoptosis of tissue cells.The mechanism may be associated with exosomes rich in TGF-β,which is likely to promote endothelial cell proliferation and improve permeability through binding to the endothelial CD105/TβR-Ⅱ receptor complex,while promoting angiogenesis and protecting skeletal muscle cells from apoptosis.