Background Persistent hyperglycaemia in diabetes causes functional abnormalities of human dermal fibroblasts (HDFs), partially leading to delayed skin wound healing. Extracellular vesicles (EVs) containing multiple pro-healing microRNAs (miRNAs) have been shown to exert therapeutic effects on diabetic wound healing. The present study aimed to observe the effects of EVs derived from placental mesenchymal stem cells (P-MSC-EVs) on diabetic wound healing and high glucose (HG)-induced senescent fibroblasts and to explore the underlying mechanisms. Methods P-MSC-EVs were isolated by differential ultracentrifugation and locally injected into the full-thickness skin wounds of diabetic mice, to observe the beneficial effects on wound healing in vivo by measuring wound closure rates and histological analysis. Next, a series of assays were conducted to evaluate the effects of low (2.28 x 1010 particles/ml) and high (4.56 x 1010 particles/ml) concentrations of P-MSC-EVs on the senescence, proliferation, migration, and apoptosis of HG-induced senescent HDFs in vitro. Then, miRNA microarrays and real-time quantitative PCR (RT–qPCR) were carried out to detect the differentially expressed miRNAs in HDFs after EVs treatment. Specific RNA inhibitors, miRNA mimics, and small interfering RNA (siRNA) were used to evaluate the role of a candidate miRNA and its target genes in P-MSC-EV-induced improvements in the function of HG-induced senescent HDFs. Results Local injection of P-MSC-EVs into diabetic wounds accelerated wound closure and reduced scar widths, with better-organized collagen deposition and decreased p16INK4a expression. In vitro, P-MSC-EVs enhanced the antisenescence, proliferation, migration, and antiapoptotic abilities of HG-induced senescent fibroblasts in a dose-dependent manner. MiR-145-5p was found to be highly enriched in P-MSC-EVs. MiR-145-5p inhibitors effectively attenuated the P-MSC-EV-induced functional improvements of senescent fibroblasts. MiR-145-5p mimics simulated the effects of P-MSC-EVs on functional improvements of fibroblasts by suppressing the expression of cyclin-dependent kinase inhibitor 1A and activating the extracellular signal regulated kinase (Erk)/protein kinase B (Akt) signaling pathway. Furthermore, local application of miR-145-5p agomir mimicked the effects of P-MSC-EVs on wound healing. Conclusions These results suggest that P-MSC-EVs accelerate diabetic wound healing by improving the function of senescent fibroblasts through the transfer of miR-145-5p, which targets cyclin-dependent kinase inhibitor 1A to activate the Erk/Akt signaling pathway. P-MSC-EVs are promising therapeutic candidates for diabetic wound treatment.

中文翻译：

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P-MSC 衍生的细胞外囊泡通过 miR-145-5p/CDKN1A 介导的高糖诱导的衰老成纤维细胞功能改善促进糖尿病伤口愈合

背景 糖尿病患者持续高血糖会导致人真皮成纤维细胞（HDF）功能异常，部分导致皮肤伤口愈合延迟。含有多种促愈合 microRNA (miRNA) 的细胞外囊泡 (EV) 已被证明对糖尿病伤口愈合具有治疗作用。本研究旨在观察胎盘间充质干细胞来源的EV（P-MSC-EV）对糖尿病伤口愈合和高糖（HG）诱导的成纤维细胞衰老的影响，并探讨其潜在机制。方法采用差速超速离心法分离P-MSC-EVs，局部注射至糖尿病小鼠全层皮肤创面，通过测量创面闭合率和组织学分析，观察其对体内创面愈合的有益作用。接下来，进行了一系列测定，以评估低浓度（2.28 x 1010 颗粒/ml）和高浓度（4.56 x 1010 颗粒/ml）的 P-MSC-EV 对衰老、增殖、迁移和凋亡的影响。 HG 体外诱导 HDF 衰老。然后，采用miRNA微阵列和实时定量PCR（RT-qPCR）来检测EV处理后HDF中差异表达的miRNA。使用特异性 RNA 抑制剂、miRNA 模拟物和小干扰 RNA (siRNA) 来评估候选 miRNA 及其靶基因在 P-MSC-EV 诱导的 HG 诱导的衰老 HDF 功能改善中的作用。结果将 P-MSC-EV 局部注射到糖尿病伤口中可加速伤口闭合并减少疤痕宽度，同时胶原蛋白沉积组织更好并减少 p16INK4a 表达。在体外，P-MSC-EVs以剂量依赖性方式增强HG诱导的衰老成纤维细胞的抗衰老、增殖、迁移和抗凋亡能力。发现 MiR-145-5p 在 P-MSC-EV 中高度富集。MiR-145-5p 抑制剂有效减弱 P-MSC-EV 诱导的衰老成纤维细胞功能改善。MiR-145-5p通过抑制细胞周期蛋白依赖性激酶抑制剂1A的表达并激活细胞外信号调节激酶（Erk）/蛋白激酶B（Akt）信号通路来模拟P-MSC-EV对成纤维细胞功能改善的影响。此外，局部应用 miR-145-5p agomir 模拟了 P-MSC-EV 对伤口愈合的影响。结论 这些结果表明，P-MSC-EV 通过 miR-145-5p 的转移改善衰老成纤维细胞的功能，从而加速糖尿病伤口愈合，miR-145-5p 靶向细胞周期蛋白依赖性激酶抑制剂 1A，激活 Erk/Akt 信号通路。P-MSC-EV 是糖尿病伤口治疗的有前途的候选药物。