{"title":"P-MSC-derived extracellular vesicles facilitate diabetic wound healing via miR-145-5p/ CDKN1A-mediated functional improvements of high glucose-induced senescent fibroblasts.","authors":"Jianlong Su, Qian Wei, Kui Ma, Yaxi Wang, Wenzhi Hu, Hao Meng, Qiankun Li, Yuehou Zhang, Wenhua Zhang, Haihong Li, Xiaobing Fu, Cuiping Zhang","doi":"10.1093/burnst/tkad010","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>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.</p><p><strong>Methods: </strong>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 <i>in vivo</i> by measuring wound closure rates and histological analysis. Next, a series of assays were conducted to evaluate the effects of low (2.28 x 10<sup>10</sup> particles/ml) and high (4.56 x 10<sup>10</sup> particles/ml) concentrations of P-MSC-EVs on the senescence, proliferation, migration, and apoptosis of HG-induced senescent HDFs <i>in vitro</i>. 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.</p><p><strong>Results: </strong>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. <i>In vitro</i>, 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.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":9553,"journal":{"name":"Burns & Trauma","volume":null,"pages":null},"PeriodicalIF":6.3000,"publicationDate":"2023-10-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10583213/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Burns & Trauma","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1093/burnst/tkad010","RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2023/1/1 0:00:00","PubModel":"eCollection","JCR":"Q1","JCRName":"DERMATOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
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.
背景:糖尿病患者持续的高血糖会导致人类真皮成纤维细胞(HDFs)功能异常,部分导致皮肤伤口愈合延迟。含有多种促进愈合的微小RNA(miRNA)的细胞外小泡(EVs)已被证明对糖尿病伤口愈合具有治疗作用。本研究旨在观察来自胎盘间充质干细胞(P-MSC-EVs)的EVs对糖尿病伤口愈合和高糖(HG)诱导的衰老成纤维细胞的影响,并探讨其潜在机制。方法:采用差速超速离心法分离P-MSC-EV,并将其局部注射到糖尿病小鼠全层皮肤伤口中,通过测量伤口闭合率和组织学分析,观察其对体内伤口愈合的有益作用。接下来,进行了一系列测定,以评估低(2.28 x 1010颗粒/ml)和高(4.56 x 1010颗粒g/ml)浓度的P-MSC-EVs对体外HG诱导的衰老HDFs的衰老、增殖、迁移和凋亡的影响。然后,进行miRNA微阵列和实时定量PCR(RT-qPCR)来检测EVs治疗后HDFs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,具有更好组织的胶原沉积和降低的p16INK4a表达。在体外,P-MSC-EVs以剂量依赖的方式增强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抑制剂有效地减弱了P-MSC-EV诱导的衰老成纤维细胞的功能改善。MiR-145-5p模拟物通过抑制细胞周期蛋白依赖性激酶抑制剂1A的表达和激活细胞外信号调节激酶(Erk)/蛋白激酶B(Akt)信号通路,模拟P-MSC-EVs对成纤维细胞功能改善的影响。此外,miR-145-5p agomir的局部应用模拟了P-MSC-EVs对伤口愈合的影响。结论:这些结果表明,P-MSC-EVs通过转移miR-145-5p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景的候选治疗药物。
期刊介绍:
The first open access journal in the field of burns and trauma injury in the Asia-Pacific region, Burns & Trauma publishes the latest developments in basic, clinical and translational research in the field. With a special focus on prevention, clinical treatment and basic research, the journal welcomes submissions in various aspects of biomaterials, tissue engineering, stem cells, critical care, immunobiology, skin transplantation, and the prevention and regeneration of burns and trauma injuries. With an expert Editorial Board and a team of dedicated scientific editors, the journal enjoys a large readership and is supported by Southwest Hospital, which covers authors'' article processing charges.