Background Refractory diabetic wounds are a common occurrence in patients with diabetes and epidermis-specific macroautophagy/autophagy impairment has been implicated in their pathogenesis. Therefore, identifying and developing treatment strategies capable of normalizing epidermis-specific macroautophagy/autophagy could facilitate diabetic wound healing. The study aims to investigate the potential of bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) from hypoxic conditions as a treatment to normalize epidermis-specific autophagy for diabetic wound healing. Methods We compared the effects of bone marrow mesenchymal stem cell (BMSC)-sourced exosomes (BMSC-Exos) from hypoxic conditions to those of BMSC in normoxic conditions (noBMSC-Exos). Our studies involved morphometric assessment of the exosomes, identification of the microRNA (miRNA) responsible for the effects, evaluation of keratinocyte functions and examination of effects of the exosomes on several molecules involved in the autophagy pathway such as microtubule-associated protein 1 light chain 3 beta, beclin 1, sequestosome 1, autophagy-related 5 and autophagy-related 5. The experiments used human BMSCs from the American Type Culture Collection, an in vivo mouse model of diabetes (db/db) to assess wound healing, as well as the human keratinocyte HaCaT cell line. In the methodology, the authors utilized an array of approaches that included electron microscopy, small interfering RNA (siRNA) studies, RNA in situ hybridization, quantitative real-time reverse transcription PCR (qRT-PCR), the isolation, sequencing and differential expression of miRNAs, as well as the use of miR-4645-5p-specific knockdown with an inhibitor. Results Hypoxia affected the release of exosomes from hypoxic BMSCs (hy-BMSCs) and influenced the size and morphology of the exosomes. Moreover, hyBMSC-Exo treatment markedly improved keratinocyte function, including keratinocyte autophagy, proliferation and migration. miRNA microarray and bioinformatics analysis showed that the target genes of the differentially expressed miRNAs were mainly enriched in ‘autophagy’ and ‘process utilizing autophagic mechanism’ in the ‘biological process’ category and miR-4645-5p as a major contributor to the pro-autophagy effect of hyBMSC-Exos. Moreover, mitogen-activated protein kinase-activated protein kinase 2 (MAPKAPK2) was identified as a potential target of exosomal miR-4645-5p; this was confirmed using a dual luciferase assay. Exosomal miR-4645-5p mediates the inactivation of the MAPKAPK2-induced AKT kinase group (comprising AKT1, AKT2, and AKT3), which in turn suppresses AKT-mTORC1 signaling, thereby facilitating miR-4645-5p-mediated autophagy. Conclusions Overall, the results of this study showed that hyBMSC-Exo-mediated transfer of miR-4645-5p inactivated MAPKAPK2-induced AKT-mTORC1 signaling in keratinocytes, which activated keratinocyte autophagy, proliferation and migration, resulting in diabetic wound healing in mice. Collectively, the findings could aid in the development of a novel therapeutic strategy for diabetic wounds.

中文翻译：

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来自缺氧骨髓间充质干细胞的外泌体 miR-4645-5p 通过恢复角质形成细胞自噬促进糖尿病伤口愈合

背景 难治性糖尿病伤口在糖尿病患者中很常见，表皮特异性巨自噬/自噬损伤与其发病机制有关。因此，确定和开发能够使表皮特异性巨自噬/自噬正常化的治疗策略可以促进糖尿病伤口愈合。该研究旨在探讨缺氧条件下骨髓间充质干细胞衍生的外泌体（BMSC-exos）作为一种治疗方法的潜力，使表皮特异性自噬正常化，促进糖尿病伤口愈合。方法 我们比较了低氧条件下骨髓间充质干细胞 (BMSC) 来源的外泌体 (BMSC-Exos) 与常氧条件下 BMSC (noBMSC-Exos) 的影响。我们的研究涉及外泌体的形态测量评估、负责影响的 microRNA (miRNA) 的鉴定、角质形成细胞功能的评估以及外泌体对参与自噬途径的几种分子（例如微管相关蛋白 1 轻链 3）的影响检查beta、beclin 1、sequestosome 1、自噬相关 5 和自噬相关 5。实验使用来自美国典型培养物保藏中心的人类 BMSC，这是一种糖尿病体内小鼠模型 (db/db)，用于评估伤口愈合以及人类角质形成细胞 HaCaT 细胞系。在该方法中，作者采用了一系列方法，包括电子显微镜、小干扰 RNA (siRNA) 研究、RNA 原位杂交、定量实时逆转录 PCR (qRT-PCR)、分离、测序和差异表达miRNA，以及使用抑制剂进行 miR-4645-5p 特异性敲低。结果缺氧影响缺氧BMSCs（hy-BMSCs）释放外泌体，并影响外泌体的大小和形态。此外，hyBMSC-Exo 治疗显着改善角质形成细胞功能，包括角质形成细胞自噬、增殖和迁移。miRNA芯片和生物信息学分析表明，差异表达miRNA的靶基因主要富集在“生物过程”类别中的“自噬”和“利用自噬机制的过程”，而miR-4645-5p是促自噬的主要贡献者。 hyBMSC-Exos 的自噬作用。此外，丝裂原激活蛋白激酶激活蛋白激酶2（MAPKAPK2）被确定为外泌体miR-4645-5p的潜在靶标；使用双荧光素酶测定证实了这一点。外泌体 miR-4645-5p 介导 MAPKAPK2 诱导的 AKT 激酶组（包括 AKT1、AKT2 和 AKT3）失活，进而抑制 AKT-mTORC1 信号传导，从而促进 miR-4645-5p 介导的自噬。结论 总体而言，本研究结果表明，hyBMSC-Exo 介导的 miR-4645-5p 转移使角质形成细胞中 MAPKAPK2 诱导的 AKT-mTORC1 信号失活，从而激活角质形成细胞自噬、增殖和迁移，从而导致小鼠糖尿病伤口愈合。