Background: After surgery, wound recovery in diabetic patients may be disrupted due to delayed inflammation, which can lead to undesired consequences, and there is currently a lack of effective measures to address this issue. Mesenchymal stem cell (MSC)-derived exosomes (Exo) have been proven to be appropriate candidates for diabetic wound healing through the anti-inflammatory effects. In this study, we investigated whether melatonin (MT)pretreated MSCs-derived exosomes (MT-Exo) could exert superior effects on diabetic wound healing, and we attempted to elucidate the underlying mechanism. Methods: For the evaluation of the anti-inflammatory effect of MT-Exo, in vitro and in vivo studies were performed. For in vitro research, we detected the secreted levels of inflammation-related factors, such as IL-1β, TNF-α and IL-10 via ELISA and the relative gene expression of the IL-1β, TNF-α, IL-10, Arg-1 and iNOS via qRT-PCR and investigated the expression of PTEN, AKT and p-AKT by Western blotting. For in vivo study, we established air pouch model and streptozotocin (STZ)-treated diabetic wound model, and evaluated the effect of MT-Exo by flow cytometry, optical imaging, H&E staining, Masson trichrome staining, immunohistochemical staining, immunofluorescence, and qRT-PCR (α-SMA, collagen I and III). Results: MT-Exo significantly suppressed the pro-inflammatory factors IL-1β and TNF-α and reduced the relative gene expression of IL-1β, TNF-α and iNOS, while promoting the anti-inflammatory factor IL-10 along with increasing the relative expression of IL-10 and Arg-1, compared with that of the PBS, LPS and the Exo groups in vitro. This effect was mediated by the increased ratio of M2 polarization to M1 polarization through upregulating the expression of PTEN and inhibiting the phosphorylation of AKT. Similarly, MT-Exo significantly promoted the healing of diabetic wounds by inhibiting inflammation, thereby further facilitating angiogenesis and collagen synthesis in vivo.
Background Mesenchymal stem cell (MSC)-derived exosomes emerge as promising candidates for treating delayed wound healing in diabetes due to the promotion of angiogenesis. Preconditioned MSC with chemical or biological factors could possibly enhance the biological activities of MSC-derived exosomes. The purpose of this research focused on whether exosomes derived from the bone marrow MSC (BMSC) pretreated with atorvastatin (ATV), could exhibit better pro-angiogenic ability in diabetic wound healing or not and its underlying molecular mechanism. Methods We isolated exosomes from non-pretreated BMSC (Exos) and ATV pretreated BMSC (ATV-Exos) and evaluated their characterization by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and Western blotting. In vivo, we made full-thickness skin defects in streptozotocin (STZ)-induced diabetic rats and the defects received multiple-point injection with PBS, Exos, or ATV-Exos. Two weeks later, histological analysis was conducted to evaluate the impact of different treatments on wound healing and the neovascularization was measured by micro-CT. In vitro, cell proliferation, migration, tube formation, and vascular endothelial growth factor (VEGF) secretion were measured in human umbilical vein endothelial cells (HUVEC). The role of miRNAs and AKT/eNOS signaling pathway in the promoted angiogenesis of ATV-Exos were assessed with their inhibitors. Results No significant difference in morphology, structure, and concentration was observed between ATV-Exos and Exos. In STZ-induced diabetic rats, ATV-Exos exhibited excellent abilities in facilitating the wound regeneration by promoting the formation of blood vessels compared with Exos without influencing liver and kidney function. Meanwhile, ATV-Exos promoted the proliferation, migration, tube formation, and VEGF level of endothelial cells in vitro. And AKT/eNOS pathway was activated by ATV-Exos and the pro-angiogenic effects of ATV-Exo were attenuated after the pathway being blocked. MiR-221-3p was upregulated by ATV-Exos stimulation, and miR-221-3p inhibitor suppressed the pro-angiogenesis effect of ATV-Exos. Conclusions Exosomes originated from ATV-pretreated MSCs might serve as a potential strategy for the treatment of diabetic skin defects through enhancing the biological function of endothelial cells via AKT/eNOS pathway by upregulating the miR-221-3p.
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