Adipose Mesenchymal Stem Cell-derived Exosomes Promote Diabetic Mice Skin Wound Healing By Regulating Epidermal Cell Autophagy

Objective: Refractory diabetic skin wounds is one of the most common complications of diabetes.The existing clinical treatment options still cannot effectively solve the problem of refractory diabetic skin wounds.Many studies have shown that adiposederived mesenchymal stem cells(ADSC-Exos)have the function of promoting tissue repair and regeneration and can avoid the immunologic rejection and tumorigenesis risk caused by stem cell transplantation.Therefore,ADSC-Exos have great prospects in the field of tissue repair and regeneration medicine.Cells degrade and reuse the accumulated protein and damaged organelles through the lysosomal system,a metabolic pathway known as autophagy by researchers.Autophagy is also one of the mechanisms by which cells maintain their homeostasis and survival.Studies confirmed that autophagy was involved in the process of skin wound healing.At the same time,more and more researchers have noticed that exosomes in biogenesis are closely related to the level of molecular signals and intracellular autophagy.Therefore,the purpose of this study was to investigate whether ADSC-Exos promotes the repair of diabetic skin wounds by regulating autophagy,to provide a new method and theoretical basis for the treatment of diabetic skin wounds.Methods: Identification of ADSC-Exos was completed by extracting ADSC-Exos by ultra-fast filtration,evaluating its particle size by nanoparticle tracking analysis(NTA),analyzing its morphology by transmission electron microscopy(TEM),and detecting the expressions of its marker proteins Alix,CD9,and CD63 by Western Blot.The ADSC-Exos was labeled with PKH26 and co-incubated with human immortalized epidermal cell line(Ha Ca T)for observation of ADSC-Exos uptake by the cells under a laser confocal microscope.The in vitro high glucose model was constructed using Ha Ca T cells.Western Blot was used to detect the expression of autophagy marker proteins MAP1LC3 B and SQSTM1,and the transfection of autophagy double-labeled adenovirus to monitor the changes of autophagy flow after the action of high glucose and ADSC-Exos.The effects of ADSC-Exos on the proliferation and migration of Ha Ca T cells after high glucose treatment were observed using CCK-8,live cell workstation and scratch test.At the same time,the cells were treated with the autophagy inhibitor Bafilomycin A1(Baf A1)in combination to evaluate the effects of autophagy on cell function.The diabetic mouse model was constructed by injection of streptozotocin(STZ),and the skin wound injury model was constructed by performing full-cortex resection on the back of the mouse.The wound healing rate was calculated by collecting wound images at different time points and the wound tissue was collected for HE staining to analyze the effect of ADSC-Exos on diabetic skin wound healing.The expressions of LC3 and Beclin-1 were detected by immunohistochemistry to evaluate the effects of diabetes and ADSC-Exos on the autophagy level during skin wound healing.Results: Alizarin red,oil red O and Alcian blue staining demonstrated the multidirectional differentiation potential of ADSCs.Flow cytometry analysis revealed surface markers of ADSCs.NTA,TEM and Western Blot results indicated that ADSCExos was successfully isolated.The results of PKH26 staining showed that ADSCExos were successfully ingested by Ha Ca T cells.In vitro experiments showed that high glucose inhibited the autophagic flow of Ha Ca T cells,and ADSC-Exos could activate the autophagic flow of Ha Ca T cells inhibited by high glucose to a certain extent.At the same time,CCK-8 and Living Cell Workstation monitored cell proliferation and cell scratching to assess cell migration,all of which indicated that ADSC-Exos could improve the cell function of Ha Ca T inhibited by high glucose.The combination of Baf A1,an autophagy inhibitor,showed that Baf A1 could attenuate the effects of ADSC-Exo by inhibiting autophagy in a certain range.In vivo experiments confirmed that ADSC-Exos could promote the healing of diabetic skin wounds,and tissue section IHC confirmed that ADSC-Exos could up-regulate the autophagy level of diabetic wounds.Finally,the sequencing results showed that the increased expression of autophagy-related genes(NAMPT,CD46,VAMP7,VAMP3 and EIF2S1)may be the underlying mechanism for the action of ADSC-Exos.Conclusion: High glucose inhibits the proliferation,migration,and autophagy flow of epidermal cells,while ADSC-Exos promote the proliferation and migration of epidermal cells by up-regulating autophagy,thereby improving the healing of diabetic mice skin wounds.