| Background and purpose :The incidence of diabetes mellitus(DM)is increasing dramatically worldwide and will become the seventh leading cause of death worldwide by 2030,among which the most common vascular complication causing death is diabetic nephropathy.In the T2 D epidemiological survey of large cities,diabetic nephropathy accounted for 39.7%of diabetic microangiopathy.Autophagy,as a cellular process,plays a crucial role in cellular physiology mainly through the continuous removal of damaged organelles and long-lived proteins from lysosomes and recovery of metabolites and nutrients to meet its own needs.Studies have shown that Bcl-2 not only inhibits apoptosis by binding to pro-apoptotic proteins containing the BH3 domain,but also regulates autophagy by binding to the BH3 domain of Beclin1 to prevent Beclin1-dependent autophagy.Bcl-2 is the most classical anti-apoptotic gene,and its role in regulating autophagy has attracted more and more attention in recent years.Bcl-2 can bind to Beclin1,an autophagy related protein,through the BH3 domain to prevent Beclin1-dependent autophagy.The effect of Bcl-2/Beclin1 on autophagy in DKD has not been reported.mesenchymal stem cells(MSCs)play a key role in the field of cell therapy and regenerative medicine because of their ability to differentiate into a variety of cell types and their strong immunomodulatory activities.Studies have found that the beneficial effects of MSCs therapy may be mediated by paracrine mechanisms,in which exosomes play a role that has attracted much attention,promoting intercellular communication and regulating the function of recipient cells through the delivery of proteins,mi RNAs,etc.We hypothesize that mesenchymal stem cell exosomes(MSC-exos)may induce autophagy to reduce podocyte injury by targeting Bcl-2/Beclin1 through mi RNA delivery.In this study,a model of high sugar-induced podocyocytes injury was established in vitro.In this study,a model of high glucose induced podococyte injury was established in vitro.By observing whether BMSCs-ex Os alleviates podococyte injury through regulation of autophagy,and thus plays a protective role in the kidney,BMSCs-Exo may be mediated by mi R-143-3p/ Bcl-2/Beclin1 Mechanism of autophagy induced by signaling pathway in podocytes.Methods:1.The expressions of CD73,CD90,CD105,CD19,CD31 and HLA-DR were detected by flow cytometry in primary C57BL/6 mouse BMSCs;2.BMSC-exos was extracted by overspeed centrifugal method,and its morphological characteristics were observed by projection electron microscopy.The positive rate of BMSC-exos surface protein was determined by nano-flow cytometry,and the surface marker protein CD63 CD9 of exosomes was detected by Western blot;3.The model of podocyte injury induced by high glucose(50m M)in vitro was established;4.To verify the therapeutic effect of BMSC-exos on MPC5 injury induced by high sugar,the experiment was divided into the following 3 groups :NC group,HG group,HG+exo group;5.The membrane fusion ability of BMSC-exos after PKH67 staining and MPC5 after high glucose injury was detected;6.To explore related mi RNAs,extractions of exosomes derived from MPC5 culture supernatant,exosomes derived from MPC5 culture supernatant stimulated by high sugar and BMSC-exos were respectively extracted,and total mi RNAs were extracted using mi RVana TM mi RNA Isolation Kit.mi RNA expression profiles were obtained by Illumina Hi Sep2500 sequencer and 50 SE sequencing;7.BMSCs were verified to regulate Bcl-2/Beclin1 pathway inducing autophagy through exosomal delivery of mi R-143-3p targeting,the experiment was divided into four groups: HG group,HG+BMSC-exos-mi R-143-3p-NCgroup,H + BMSC-exos-mi R-143-3p-inhibitorgroup,HG+BMSC-exos-mi R-143-3p-mimic group;8.Dual luciferase reporter gene was used to detect the interaction between mi R-143-3p and Bcl-3 UTR region.Cellular immunofluorescence was used to detect the expression of podocin in MPC5 before and after BMSC-exos treatment,and the related protein Beclin1 LC3 was detected by Western blot Beclin1 Bcl-2 m RNA expression level was detected by PCR.Results:1.The morphology of primary BMSCs showed long spindle shape and polar arrangement,CD73 、 CD90 、 CD105 molecules were highly expressed on the surface,and CD19、CD31、CD34、CD45、CD11,and HLA-DR molecules were low expressed on the surface.2.The isolated BMSC-exos was spherical under transmission electron microscopy,and the average particle size of the NTA particle size analysis was68.42 nm.Western blot showed that the expression of the surface labeled protein CD9CD63 was significantly higher than that of the BMSCs lysate.3.The expression of podocin in MPC5 decreased significantly after high glucose induction.The expression levels of Beclin1 and LC3Ⅱ were significantly increased after BMSC-exos treatment of high-sugar-induced MPC5,suggesting that BMSC-exos up-regulated the autophagy level of high-sugar-induced MPC5.4.The expression of differential mi RNA was screened by high-throughput sequencing to identify the autophagy related mi R-143-3p.5.The exosomes stained with PKH67 dye were co-incubated with MPC5 after high glucose injury for 48 h,and the green fluorescence in the cytoplasm of podocytes was observed by laser confocal microscopy,suggesting that exosomes could be extrinsic ingested into podocytes after hypercentrifugation6.Beclin1 and LC3 expression were significantly up-regulated in HG +BMSC-exos-mi R-143-3p-NC group and HG + BMSC-exos-mi R-143-3p-mimic group,and the autophagy level of MPC5 after high glucose injury was up-regulated,while the expression level of Bcl-2 protein was decreased.There was no significant difference between HG+BMSC-exos-mi R-143-3p-inhibitor group and HG group.7.Luciferase reporter gene detected that mi R-143-3p had targeted inhibition on Bcl-2 gene.Conclusion:The results of this study suggest that BMSC-exos can significantly improve podocyte injury,and its potential mechanism may be through the regulation of the Bcl-2/Beclin1 pathway through the delivery of mi R-143-3p,up-regulating the autophagy level of MPC5 damaged by high glucose,alleviating the damage of MPC5,and thus delaying the further development of diabetic kidney disease This study provides an experimental basis for the development of exosomes for the treatment of diabetic kidney disease. |
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