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

参考文献

[1] Armstrong D G, Tan T W, Boulton A J M, et al. Diabetic foot ulcers：a review[J]. The Journal of the American Medical Association, 2023, 330(1): 62.

[2] Jeffcoate W, Boyko E J, Game F, et al. Causes，prevention，and management of diabetes-related foot ulcers[J]. The Lancet Diabetes & Endocrinology, 2024, 12(7): 472-482.

[3] Xu Z, Fan J L, Tian W G, et al. Cellulose-based pH-responsive Janus dressing with unidirectional moisture drainage for exudate management and diabetic wounds healing[J]. Advanced Functional Materials, 2024, 34(3): 2307449.

[4] Huang K, Mi B B, Xiong Y, et al. Angiogenesis during diabetic wound repair：from mechanism to therapy opportunity[J]. Burns & Trauma, 2025, 13: tkae052.

[5] Lu Y F, Li H S, Wang J, et al. Engineering bacteria-activated multifunctionalized hydrogel for promoting diabetic wound healing[J]. Advanced Functional Materials, 2021, 31(48): 2105749.

[6] 莫瑞卿, 杨盛慧, 苏宏杰, 等. 创面巨噬细胞抗炎极化与MANF生成在胫骨横向骨搬移治疗糖尿病足中的作用[J]. 广西医科大学学报, 2024, 41(11): 1573-1580.

[7] Mo R Q, Yang S H, Su H J, et al. The role of wound macrophage anti-inflammatory polarization and MANF production in the treatment of diabetic foot with tibial cortex transverse transport[J]. Journal of Guangxi Medical University, 2024, 41(11): 1573-1580.

[8] Hao S Y, Yang H Y, Hu J J, et al. Bioactive compounds and biological functions of medicinal plant-derived extracellular vesicles[J]. Pharmacological Research, 2024, 200: 107062.

[9] Dad H A, Gu T W, Zhu A Q, et al. Plant exosome-like nanovesicles：emerging therapeutics and drug delivery nanoplatforms[J]. Molecular Therapy, 2021, 29(1): 13-31.

[10] Hajialiasgary Najafabadi A， Soheilifar M H， Masoudi-Khoram N . Exosomes in skin photoaging：biological functions and therapeutic opportunity[J]. Cell Communication and Signaling, 2024, 22(1): 32.

[11] Cao M, Diao N N, Cai X L, et al. Plant exosome nanovesicles（PENs）：green delivery platforms[J]. Materials Horizons, 2023, 10(10): 3879-3894.

[12] Ye H L, Tang J Z, Liang W T, et al. Dissolving microneedles loaded with Acanthopanax senticosus-derived vesicles and PEI for antibacterial activity，cfDNA scavenging，anti-inflammation，and angiogenesis in diabetic wounds[J]. Chemical Engineering Journal, 2025, 526: 171412.

[13] Zhao Q, Hu Q X, Li J P, et al. Morinda officinalis-derived extracellular vesicle-like particles promote wound healing via angiogenesis[J]. ACS Applied Materials & Interfaces, 2025, 17(21): 30454-30464.

[14] 胡晓咏, 杨朝颖, 宋乾华, 等. 丹参衍生的外泌体样纳米颗粒通过PI3K/Akt/eNOS信号通路减轻血管内皮细胞氧化损伤[J]. 中国病理生理杂志, 2025, 41(10): 1892-1899.

[15] Hu X Y, Yang C Y, Song Q H, et al. Salvia miltiorrhiza-derived exosome-like nanoparticles alleviate oxidative damage of vascular endothelial cells through the PI3K/Akt/eNOS signaling pathway [J]. Chinese Journal of Pathophysiology, 2025, 41(10): 1892-1899.

[16] Kim M, Park J H . Isolation of Aloe saponaria-derived extracellular vesicles and investigation of their potential for chronic wound healing[J]. Pharmaceutics, 2022, 14(9): 1905.

[17] Yang J C, Wang Y H, Fu Y Q, et al. Plant extracellular vesicles：a promising bionic medicine platform for disease treatment and drug delivery[J]. Interdisciplinary Medicine, 2025, 3(3): e20240101.

[18] Lakshme P S T， Roy A， Sivaperumal P， et al. Exploration of antioxidant effects of crude extract of mangrove plant - Avicennia marina [J]. Journal of Pharmaceutical Research International, 2021: 321-329.

[19] Weng J L, Chen Y Z, Zeng Y H, et al. A novel hydrogel loaded with plant exosomes and stem cell exosomes as a new strategy for treating diabetic wounds[J]. Materials Today Bio, 2025, 32: 101810.

[20] Zhang J J, Li W Q, Liu Y N, et al. OTUD1 delays wound healing by regulating endothelial function and angiogenesis in diabetic mice[J]. Journal of Advanced Research, 2026, 80: 1063-1081.

[21] Li Y L, He R Y, Huang Y, et al. A 3R（remove-remodel-repair）-integrated self-assembled Chlorella-gelatin-PEG hydrogel for diabetic wound healing[J]. Materials Today Bio, 2025, 33: 101935.

[22] 何丰来, 肖聪, 龙能吉, 等. 银杏叶提取物注射液对糖尿病足溃疡模型大鼠创面恢复的作用及机制研究[J]. 广西医科大学学报, 2022, 39(1): 67-73.

[23] He F L, Xiao C, Long N J. Effect of Ginkgo biloba injection on the recovery of diabetic foot ulcer model rats′ wounds and its mechanism [J]. Journal of Guangxi Medical University, 2022, 39(1): 67-73.

[24] Chi J, Wang X B, Song Y R, et al. Emerging plant-derived exosome-like particles reveal key therapeutic benefits. A comprehensive review of evidence[J]. International Journal of Nanomedicine, 2025, 20: 12393-12411.

[25] Cerri F, De Santes B, Spena F, et al. Phytochemical profiling，antioxidant activity，and in vitro cytotoxic potential of mangrove Avicennia marina [J]. Pharmaceuticals, 2025, 18(9): 1308.

[26] Nastić N, Barathikannan K, Hai A, et al. Comprehensive two-step sequential extraction and pharmacological profiling of Emirati plant leaves：a multi-target natural product discovery approach[J]. Journal of Agriculture and Food Research, 2026, 25: 102551.

[27] Du X L, Edelstein D, Rossetti L, et al. Hyperglycemia-induced mitochondrial superoxide overproduction activates the hexosamine pathway and induces plasminogen activator inhibitor-1 expression by increasing Sp1 glycosylation[J]. Proceedings of the National Academy of Sciences of the United States of America, 2000, 97(22): 12222-12226.

[28] Lu X X, Qin L, Guo M, et al. A novel alginate from Sargassum seaweed promotes diabetic wound healing by regulating oxidative stress and angiogenesis[J]. Carbohydrate Polymers, 2022, 289: 119437.

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)