The refractory diabetic wound has remained a worldwide challenge as one of the major health problems. The impaired angiogenesis phase during diabetic wound healing partly contributes to the pathological process. MicroRNA (miRNA) is an essential regulator of gene expression in crucial biological processes and is a promising nucleic acid drug in therapeutic fields of the diabetic wound. However, miRNA therapies have limitations due to lacking an effective delivery system. In the present study, we found a significant reduction of miR-31-5p expression in the full-thickness wounds of diabetic mice compared to normal mice. Further, miR-31-5p has been proven to promote the proliferation, migration, and angiogenesis of endothelial cells. Thus, we conceived the idea of exogenously supplementing miR-31-5p mimics to treat the diabetic wound. We used milk-derived exosomes as a novel system for miR-31-5p delivery and successfully encapsulated miR-31-5p mimics into milk exosomes through electroporation. Then, we proved that the miR-31-5p loaded in exosomes achieved higher cell uptake and was able to resist degradation. Moreover, our miRNA-exosomal formulation demonstrated dramatically improved endothelial cell functions in vitro, together with the promotion of angiogenesis and enhanced diabetic wound healing in vivo. Collectively, our data showed the feasibility of milk exosomes as a scalable, biocompatible, and cost-effective delivery system to enhance the bioavailability and efficacy of miRNAs.

Oxidative damage is a critical cause of diabetic wounds. Exosomes from various stem cells could promote wound repair. Here, we investigated the potential mechanism by which exosomes from adipose-derived stem cells (ADSC-EXOs) promote diabetic wound healing through the modulation of oxidative stress. We found that ADSC-EXOs could promote proliferation, migration, and angiogenesis in keratinocytes, fibroblasts, and endothelial cells. Furthermore, ADSC-EXOs reduced the reactive oxygen species (ROS) levels in these cells and protected them against hypoxic and oxidative stress damage. Finally, the local injection of ADSC-EXOs at wound sites significantly increased collagen deposition and neovascularization while reducing ROS levels and cell death; thus, it led to accelerated diabetic wound closure. The mechanism underlying ADSC-EXO functions involved heat-shock protein 90 (HSP90) expressed on the cell surface; these functions could be inhibited by an anti-HSP90 antibody. Exosomal HSP90 could bind to the low-density lipoprotein receptor-related protein 1 (LRP1) receptor on the recipient cell membrane, leading to activation of the downstream AKT signaling pathway. Knockdown of LRP1 and inhibition of the AKT signaling pathway by LY294002 in fibroblasts was sufficient to impair the beneficial effect of ADSC-EXOs. In summary, ADSC-EXOs significantly accelerated diabetic wound closure through an exosomal HSP90/LRP1/AKT signaling pathway.

Diabetic peripheral neuropathy (DPN) is one of the most common chronic complications in diabetes. Previous studies have shown that chronic neuroinflammation was associated with DPN. However, further research is needed to investigate the exact immune molecular mechanism underlying the pathogenesis of DPN. Expression profiles were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were screened by R software. After functional enrichment analysis of DEGs, a protein–protein interaction (PPI) network analysis was performed. The CIBERSORT algorithm was used to evaluate the infiltration of immune cells in DPN. Next, the least absolute shrinkage and selection operator (LASSO) logistic regression and support vector machine-recursive feature elimination (SVM-RFE) algorithms were applied to identify potential DPN diagnostic markers. Finally, the results were further validated by qRT-PCR. A total of 1308 DEGs were screened in this study. Enrichment analysis identified that DEGs were significantly enriched in immune-related biological functions and pathways. Immune cell infiltration analysis found that M1 and M2 macrophages, monocytes, resting mast cells, resting CD4 memory T cells and follicular helper T cells were involved in the development of DPN. LTBP2 and GPNMB were identified as diagnostic markers of DPN. qRT-PCR results showed that 15 mRNAs, including LTBP2 and GPNMB, were differentially expressed, consistent with the microarray results. In conclusion, LTBP2 and GPNMB can be used as novel candidate molecular diagnostic markers for DPN. Furthermore, the infiltration of immune cells plays an important role in the progression of DPN.

Adipose-derived stem cells (ADSCs) can differentiate into Schwann cells (SCs) at the site of nerve injury, where Schwann cell-derived exosomes (SC-Exos) are suspected to exert an induction effect. Our study aimed to induce the differentiation of ADSCs in vitro using SC-Exos and to investigate the mechanisms involved through miRNA sequencing. Subcutaneous fat was used to extract ADSCs. Exosomes were extracted from Schwann cell lines (RSC96) using ultracentrifugation and were able to be taken up by human ADSCs. After 8 days of induction of ADSCs by SC-Exos, phenotypic characteristics were observed by examining the expression of SC markers (S100ß, NGFR, MPZ, GFAP) through RT-qPCR, Western blot and immunofluorescence. The RNA and protein expression levels of S100ß, NGFR, MPZ, and GFAP were found to be significantly higher in the SC-Exo induction group than in the uninduced group, which was also consistent with the immunofluorescence results. Additionally, miRNA sequencing was performed on exosome-induced ADSCs, followed by bioinformatic analysis and validation of the results. According to the sequencing results, there were a total of 94 differentially expressed miRNAs. Bioinformatics analysis indicated that 3506 Gene Ontology terms and 98 Kyoto Encyclopedia of Genes and Genomes pathways were significantly enriched. Ten miRNAs, 5 target mRNAs and elevated expression of the PIK3CD/Akt pathway were validated by RT-qPCR or Western blot, which is consistent with the sequencing results. Our study demonstrates that the utility of SC-Exos is effective in inducing the differentiation of ADSCs into SCs, in which these validated differentially expressed miRNAs exert a vital effect. This work provides a new paradigm via rationally applying Schwann cell-derived exosomes as a promising therapeutic option for repairing peripheral nerve injury.

Adipose-derived stem cells (ADSCs) have been widely applied in translational and regenerative medicine. During aging, there is a recognized functional decline in ADSCs, which compromises their therapeutic effectiveness. Currently, the mechanisms of aging-induced stem cell dysfunction remain unclear, hence there is a need to elucidate these mechanisms and propose strategies for reversing this functional impairment. In this study, we found that ADSCs isolated from old donors (O-ADSCs) presented inferior phenotypes and decreased miR-145-5p levels compared to those from young donors (Y-ADSCs). To interrogate the role of miR-145-5p in ADSCs, gain- and loss-of-function assays were performed. The results indicated that miR-145-5p overexpression in O-ADSCs promoted cellular proliferation and migration, while reducing cell senescence. Further study demonstrated that miR-145-5p could regulate ADSCs function by targeting bone morphogenetic protein binding endothelial cell precursor-derived regulator (BMPER), which is a crucial modulator in angiogenesis. Moreover, in vivo experiments showed that miR-145-5p-overexpressing O-ADSCs accelerated wound healing by promoting wound re-epithelialization and angiogenesis. Collectively, this study indicates that miR-145-5p works as a positive regulator for optimizing O-ADSCs function, and may be a novel therapeutic target for restoring aging-associated impairments in stem cell function.