白骨壤叶片细胞外囊泡促进糖尿病创面愈合的作用研究

Effects of Avicennia marinaleaf-derived extracellular vesicles on promoting diabetic wound healing

摘要

目的：探讨白骨壤叶片来源细胞外囊泡（Avicennia marina leaf-derived extracellular vesicles，AmLEVs）对糖尿病大鼠皮肤创面愈合的作用及其潜在机制。方法：采用差速离心联合超速离心法从白骨壤叶片中分离细胞外囊泡；使用透射电子显微镜（TEM）观察细胞外囊泡形态；使用马尔文粒径仪（DLS）检测AmLEVs粒径和Zeta电位。使用DPPH、ABTS、羟自由基检测试剂盒检测AmLEVs抗氧化能力。以人脐静脉内皮细胞（HUVECs）和人皮肤成纤维细胞（HSFs）为研究对象，使用葡萄糖浓度为25 mmol/L的高糖培养基模拟高糖环境。AmLEVs与细胞共培养后，使用DCFH-DA探针检测细胞活性氧（reactive oxygen species，ROS）水平；通过细胞计数试剂盒（CCK-8）、划痕实验、血管形成实验检测细胞活力、细胞迁移和血管形成能力；采用实时荧光定量PCR（RT-qPCR）和免疫荧光检测 VEGF mRNA和VEGF蛋白水平的表达。构建链脲佐菌素诱导的糖尿病SD大鼠全皮层创面模型，评价AmLEVs对创面愈合的促进作用。结果：成功分离出白骨壤叶片来源细胞外囊泡，粒径分布在55 nm左右、具有典型杯状形态外囊泡样纳米颗粒。体外实验表明，AmLEVs具有良好清除DPPH、ABTS、羟自由基能力；100 μg/mL AmLEVs可有效降低高糖诱导的ROS水平，从而缓解高糖环境对HUVECs增殖、迁移和血管形成的抑制作用，提高HSFs的细胞活力。与对照组相比，高糖损伤组中VEGF mRNA和蛋白水平表达下降，通过100 μg/mL AmLEVs处理后VEGF mRNA和蛋白表达水平上升。动物实验显示，与对照组大鼠相比，糖尿病组大鼠创面愈合率降低，而AmLEVs治疗后能显著提高糖尿病大鼠创面愈合率，HE和Masson染色结果显示，与糖尿病模型组相比，AmLEVs治疗后创面再上皮化增强，胶原沉积增加；免疫荧光、RT-qPCR、western blotting结果证实，与对照组相比糖尿病模型组大鼠皮肤组织中VEGF mRNA和蛋白表达下降，AmLEVs治疗后皮肤组织中VEGF mRNA和蛋白表达水平上升。结论：白骨壤叶片来源细胞外囊泡能有效促进糖尿病创面愈合，其机制可能与调节细胞氧化应激能力、促进血管生成、改善细胞迁移功能，为糖尿病创面的治疗提供潜在药物。

Abstract

Objective: To investigate the effects of Avicennia marina leaf-derived extracellular vesicles (AmLEVs) on skin wound healing in diabetic rats and explore the underlying mechanisms. Methods: Extracellular vesicles were isolated from Avicennia marina leaves by differential centrifugation combined with ultracentrifugation. The morphology of the extracellular vesicles was observed by transmission electron microscopy (TEM), and the particle size and zeta potential of AmLEVs were measured by dynamic light scattering (DLS) using a Malvern instrument. The antioxidant capacity of AmLEVs was evaluated using DPPH, ABTS, and hydroxyl radical assay kits. Human umbilical vein endothelial cells (HUVECs) and human skin fibroblasts (HSFs) were selected as experimental cells, and a high-glucose medium containing 25 mmol/L glucose was used to mimic the diabetic microenvironment. After co-culture with AmLEVs, the levels of intracellular reactive oxygen species (ROS) were detected using the DCFH-DA probe. Cell viability, cell migration, and angiogenic capacity were assessed by cell counting kit-8 (CCK-8) assay, scratch wound assay, and tube formation assay, respectively. The expression levels of VEGF mRNA and VEGF protein were determined by real-time quantitative PCR (RT-qPCR) and immunofluorescence staining. In addition, a full-thickness skin wound model was established in streptozotocin-induced diabetic Sprague-Dawley (SD) rats to evaluate the wound-healing effects of AmLEVs. Results: AmLEVs were successfully isolated, with a typical cup-shaped morphology of the nanovesicles and an average particle size of approximately 55 nm. In vitro experiments showed that AmLEVs exhibited strong scavenging activities against DPPH, ABTS, and hydroxyl radicals. Moreover, 100 μg/mL AmLEVs effectively reduced high glucose-induced ROS accumulation, thereby reversing the inhibitory effects of the hyperglycemic environment on the proliferation, migration, and tube formation of HUVECs, thus enhancing the viability of HSFs. Compared with the control group, the mRNA and protein expression levels of VEGF were decreased in the high-glucose injury group. However, these levels were increased when the high-glucose injury group was treated with 100 μg/mL AmLEVs. In vivo experiments demonstrated that the wound healing rate in the rats of diabetic group was significantly decreased compared with that in the rats of the control group. However, the wound healing rate was significantly increased when the diabetic rats were treated with AmLEVs. HE and Masson staining revealed that AmLEV treatment enhanced re-epithelialization and increased collagen deposition compared with the diabetic model group. Furthermore, immunofluorescence staining, RT-qPCR, and western blotting confirmed that VEGF mRNA and protein expressions in skin tissues were downregulated in diabetic rats but were significantly upregulated following AmLEV treatment. Conclusion: AmLEVs effectively promote diabetic wound healing. The underlying mechanisms may involve modulating cellular oxidative stress, enhancing angiogenesis, and improving cell migration, suggesting that AmLEVs can serve as a potential therapeutic agent for diabetic wound treatment.

关键词

白骨壤叶片 / 细胞外囊泡 / 糖尿病创面 / 抗氧化 / 血管生成 / 血管内皮生长因子 / 细胞迁移 / 人脐静脉内皮细胞

Key words

Avicennia marina leaves / extracellular vesicles / diabetic wound healing / antioxidation / angiogenesis / vascular endothelial growth factor / cell migration / human umbilical vein endothelial cells

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基金资助

国家自然科学基金资助项目(82360426)

广西重点研发计划资助项目(桂科AB23026049)