The Role And Mechanism Of Mesenchymal Stem Cell-Derived Small Extracellular Vesicles In Alleviating Type 2 Diabetes By Inhibiting The Apoptosis And Dedifferentiation OfΒCell

Objective:Characterized by the islet β cell dysfunction and insulin resistance,the global incidence rate of type 2 diabetes is high.Long-term hyperglycaemia can cause the injury of many tissues and organs,resulting in the occurrence of serious diabetic complications.Current treatment methods include oral hypoglycaemic drugs and insulin injection,which can only temporarily reduce blood glucose but cannot completely solve the cause of disease with poor efficacy and side effects.In recent years,mesenchymal stem cell-derived small extracellular vesicles(MSC-sEVs)have been reported to exert important roles in tissue regeneration and considered as a novel cell-free strategy.The purpose of this study is to explore the therapeutic role and specific mechanism of MSC-sEVs in islet β cell injury,and provide a novel method for type 2 diabetes therapy.Methods:The primary MSC was isolated by the adherent method of umbilical cord tissue.After the adipogenic and osteogenic induction,Oil-Red-O and Alizarin-Red-S staining were performed to identify the multidirectional differentiation ability of MSC.The expression of surface antigen of MSC was detected by flow cytometry.MSC-sEVs were extracted from the conditioned culture supernatant of MSC by ultracentrifugation.The morphology and structure of MSC-sEVs were detected by transmission electron microscopy.The particle size and concentration of MSC-sEVs were detected by nanoparticle tracking analysis.The protein marker expression of MSC-sEVs was detected by Western blot.The db/db mice were used as the animal model of type 2 diabetes.MSC-sEVs and PBS were injected into the tail vein of db/db mice respectively,and the db/m mice treated with PBS were used as the control group.The distribution of MSC-sEVs in vivo was observed by the small animal imaging instrument.The therapeutic effect of MSC-sEVs in type 2 diabetes was evaluated by the measurement of fasting blood glucose,body weight,serum insulin level,intraperitoneal glucose tolerance and intraperitoneal insulin tolerance.The islet structure was observed by hematoxylin and eosin(H&E)staining.The number of β cells in islet was detected by immunofluorescence staining.Western blot and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling(TUNEL)staining were adopted to determine the apoptosis level of islet βcell.Immunofluorescence staining and Western blot were adopted to detect the expression of dedifferentiation-related indicators in islet β cell.In vitro,rat insulinoma(INS-1)cells were stimulated by 33.3mmol/L glucose medium,and then treated with MSC-sEVs.Immunofluorescence staining and Western blot were adopted to determine the apoptosis and dedifferentiation of INS-1 cells.In order to further investigate whether MSC-sEVs alleviate islet β cell injury by regulating ecotropic viral integration site 1(EVI1),the expression of EVI1 after MSC-sEVs treatment was analyzed by immunofluorescence staining,Western blot and real-time quantitative reverse transcription polymerase chain reaction(q RT-PCR).Subsequently,the apoptosis and dedifferentiation of INS-1 cells transfected with the small interfering RNA(siRNA)of EVI1 were detected by immunofluorescence staining and Western blot.MiRNA sequencing and bioinformatics software were used to analyze the enriched miRNAs in MSC-sEVs and screen the specific miRNA to target EVI1.The biological function of miRNA in high glucose-induced islet β cell damage was evaluated by detecting the apoptosis and dedifferentiation of INS-1 cells after miRNA mimics transfection.Results:MSC isolated from umbilical cord tissue displayed the ability of adipogenic and osteogenic differentiation,positively expressed CD29,CD44 and CD166,and negatively expressed CD14,CD45 and human leukocyte antigens-DR(HLA-DR).MSC-sEVs isolated by ultracentrifugation exhibited cup-shaped vesicles under the transmission electron microscope with the particle size of 171.3nm.MSC-sEVs were positive for the expression of protein markers such as CD9,CD63,heat shock protein 70(HSP70),tumor susceptibility gene 101(TSG101)and Alix,and negative for the expression of Calnexin.MSC-sEVs were observed to locate in pancreatic tissue after tail vein injection.The db/db mice treated with MSC-sEVs exhibited the reduced fasting blood glucose,decreased body weight,enhanced serum insulin level,improved glucose tolerance and insulin tolerance,and recovered islet βcell function.In addition,MSC-sEVs could increase the number of islets in pancreatic tissue,improve the structure of islets,increase the number of β cells in islet tissue,reduce TUNEL positive cells and inhibit islet β cell apoptosis.Islet β cells after MSC-sEVs treatment showed the increased expression of mature cell phenotypic markers such as pancreatic and duodenal homeobox 1(PDX1)and forkhead box O1(FOXO1),and the decreased expression of primitive cell phenotypic markers including aldehyde dehydrogenase 1 family member A3(ALDH1A3),octamer-binding transcription factor 4(OCT4)and Nanog homeobox(NANOG),indicating that MSC-sEVs could alleviate the dedifferentiation of islet β cells.In vitro,MSC-sEVs could reverse the high glucose-induced upregulation of apoptosis and dedifferentiation in INS-1 cells.The mechanism study demonstrated that MSC-sEVs inhibited the activation of EVI1,and the knockdown of EVI1 could significantly alleviate the apoptosis and dedifferentiation of INS-1 cells under high glucose condition.The miRNA sequencing results showed that miR-4436 was highly expressed in MSC-sEVs.Transfection with miR-4436 mimics could reduce the EVI1 expression and improve the apoptosis and dedifferentiation of INS-1 cells.Conclusions:MSC-sEVs ameliorate the apoptosis and dedifferentiation of islet β cells and recover their function by miR-4436-mediated EVI1 inhibition,leading to the alleviation of type 2diabetes.