Sandwich-like nanofiber membrane loaded with salidroside regulates macrophage polarization and promotes angiogenesis in diabetic wounds

doi:10.12307/2026.363

Abstract

Abstract: BACKGROUND: Salidroside, with its anti-inflammatory, antioxidant, and pro-angiogenic properties, has shown potential in the treatment of various diseases. However, its application in diabetic wound healing remains to be further explored.
OBJECTIVE: To explore the effectiveness of a three-layer nanofiber membrane containing salidroside on repairing diabetic skin wounds.
METHODS: (1) Electrospun polycaprolactone-polyethylene glycol-polyvinyl alcohol and polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol membranes were prepared and characterized for their morphology, water contact angle, tensile elastic modulus, and sustained drug release properties. Human umbilical vein endothelial cells were co-cultured with the two membranes, and their biocompatibility was analyzed by cell adhesion and live-dead staining. (2) Logarithmic-phase mouse mononuclear phagocyte leukemia cell line (RAW264.7) cells were treated with 1 μg/mL lipopolysaccharide. After 24 hours, the cell supernatant was collected and mixed with DMEM high-glucose medium supplemented with 10% fetal bovine serum as conditioned medium. Human umbilical vein endothelial cells were cultured in three groups: a control group without any materials, and the other two groups co-cultured with polycaprolactone-polyethylene glycol-polyvinyl alcohol electrospun membranes and polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membranes, respectively. Conditioned medium was added to induce an inflammatory response, and cell proliferation, migration, and tube formation were measured. (3) RAW264.7 cells were treated with 1 μg/mL lipopolysaccharide (inducing inflammatory response) for 24 hours and then divided into three groups: a control group without any materials, and the other two groups co-cultured with polycaprolactone-polyethylene glycol-polyvinyl alcohol membranes and polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol membranes, respectively. Intracellular nitric oxide levels and CD206 and interleukin-1β mRNA expressions were measured. (4) Twenty-four C57 mice were used to establish a diabetic model by high-fat and high-glucose feeding combined with intraperitoneal injection of streptozotocin. Three weeks after modeling, a circular full-thickness skin defect with a diameter of 8 mm was made on the back of the mice. The mice were randomly divided into three intervention groups: the blank group (n=8) was not implanted with any material; the control group (n=8) and the experimental group (n=8) were implanted with polycaprolactone-polyethylene glycol-polyvinyl alcohol electrospun membrane and polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membrane, respectively. The wound healing was observed, and hematoxylin-eosin and Masson staining and immunohistochemical staining of CD206 and vascular endothelial growth factor were performed on the wound skin tissue at the set time points.
RESULTS AND CONCLUSION: (1) Scanning electron microscopy revealed that the polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membrane exhibited a three-layer structure, with randomly arranged fibers in each layer interconnected to form a porous structure. The water contact angle of the polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membrane was smaller than that of the polycaprolactone-polyethylene glycol-polyvinyl alcohol electrospun membrane (P < 0.05), and there was no significant difference in the tensile elastic modulus between the two groups of membranes. The polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membrane exhibited excellent drug release properties. Both membranes exhibited excellent biocompatibility and effectively supported cell growth and survival. (2) Under inflammatory response, compared with the control group, polycaprolactone-polyethylene glycol-polyvinyl alcohol electrospinning membrane, polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospinning membrane could promote the proliferation, migration and tube formation ability of human umbilical vein endothelial cells. (3) Under inflammatory conditions, compared with the control group, polycaprolactone-polyethylene glycol-polyvinyl alcohol membranes, polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membranes reduced intracellular nitric oxide levels and interleukin-1β mRNA expression, and increased CD206 mRNA expression, indicating that polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membranes could effectively promote macrophage polarization toward the M2 phenotype and exert an anti-inflammatory effect. (4) Animal experiments showed that polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membranes promoted diabetic wound healing compared with control and polycaprolactone-polyethylene glycol-polyvinyl alcohol electrospun membranes. Hematoxylin-eosin and Masson staining revealed more significant angiogenesis and complete collagen fiber maturation in the skin samples of the experimental group. Immunohistochemical staining results showed that polycaprolactone-polyethylene glycol/salidroside-polyvinyl alcohol electrospun membrane significantly promoted the macrophage polarization and angiogenesis toward M2 by upregulating the expression of CD206 and vascular endothelial growth factor.

Key words: diabetes, skin wound, salidroside, electrospun membrane, three-layer structure, polycaprolactone, polyethylene glycol, polyvinyl alcohol, nanodressing

CLC Number:

Zheng Hao, Zhou Tianqi, Pan Jiazhaо, He Jialin, Zou Zihao, Teng Jianxiang, Xie Mengli, Yang Long, Tian Xiaobin. Sandwich-like nanofiber membrane loaded with salidroside regulates macrophage polarization and promotes angiogenesis in diabetic wounds[J]. Chinese Journal of Tissue Engineering Research, 2026, 30(20): 5152-5166.

Figures/Tables 9

2.8 静电纺丝膜加速糖尿病小鼠皮肤创面愈合 2.8.1 实验动物数量分析 24只小鼠全部进入结果分析。 2.8.2 各组糖尿病小鼠皮肤创面愈合情况各组小鼠术后不同时间点的创面大体观，见图8A，显示随着时间的延长，各组创面面积逐渐减小。各组小鼠术后不同时间点的创面愈合率见图8B，C。术后第4天，实验组创面愈合率高于空白组(P < 0.01)；术后第7，14天，实验组创面愈合率高于空白组、对照组(P < 0.05，P < 0.001，P < 0.000 1)，说明PCL-PEG/Sal-PVA静电纺丝膜加速了创面愈合。 2.8.3 各组糖尿病小鼠皮肤创面组织形态学观察结果术后第7，14天，各组糖尿病小鼠皮肤创面苏木精-伊红与Masson图像，见图9A，B。苏木精-伊红染色显示，相同时间下，实验组创面区域可见明显的新生血管生成，表现为血管腔隙增多、管壁薄且排列紧密，提示PCL-PEG/Sal-PVA静电纺丝膜显著促进了血管生成；空白组和对照组创面区域血管数量较少且多集中在创面边缘，表明该2组的血管生成能力较弱。Masson染色胶原沉积和重塑是糖尿病创面愈合的重要指标。Masson染色显示，3组胶原密度均有所增加，其中实验组胶原密度最高，其次是对照组，最后是空白组。苏木精-伊红染色定量分析结果显示，实验组术后第7，14天的平均血管数量多于空白组、对照组(P < 0.05，P < 0.01)，创面边缘距离小于空白组、对照组(P < 0.01，P < 0.001)，见图9C，D。Masson染色定量分析结果显示，实验组术后第7，14天的胶原沉积密度高于空白组、对照组(P < 0.001，P < 0.000 1)，见图9E。以上结果进一步验证了PCL-PEG/Sal-PVA静电纺丝膜促进创面愈合及血管生成的积极作用。免疫组化染色结果显示，实验组创面组织中观察到明显的CD206阳性染色信号，主要分布在肉芽组织和新生血管周围，表明M2型巨噬细胞数量较多；相比之下，空白组、对照组创面组织中CD206阳性信号较弱，表明M2型巨噬细胞数量较少，见图9F。此外，实验组创面组织中血管内皮生长因子阳性信号显著增强，尤其是在新生血管周围的内皮细胞中呈现强阳性染色，提示促血管生成能力较高；空白组、对照组创面组织中血管内皮生长因子阳性信号较弱，提示促血管生成能力较低，见图9F。免疫组化染色定量分析结果显示，实验组CD206、血管内皮生长因子表达高于空白组、对照组(P < 0.05，P < 0.01)，见图9G，H。表明PCL-PEG/Sal-PVA静电纺丝膜通过上调CD206和血管内皮生长因子表达显著促进了M2型巨噬细胞的极化和血管生成。 "

References

[1] PRIMADHI RA, SEPTRINA R, HAPSARI P, et al. Amputation in diabetic foot ulcer: A treatment dilemma. World J Orthop. 2023;14(5):312-318.
[2] GHOSH R, SINGH P, PANDIT AH, et al. Emerging Technological Advancement for Chronic Wound Treatment and Their Role in Accelerating Wound Healing. ACS Appl Bio Mater. 2024;7(11):7101-7132.
[3] 谢梦恬,马雨龙,孙一榕,等.生物活性水凝胶通过调控细胞行为促进皮肤慢性创面愈合[J].材料导报,2025,39(5):35-48.
[4] HUANG C, TENG J, LIU W, et al. Modulation of macrophages by a phillyrin-loaded thermosensitive hydrogel promotes skin wound healing in mice. Cytokine. 2024;177:156556.
[5] LOUISELLE AE, NIEMIEC SM, ZGHEIB C, et al. Macrophage polarization and diabetic wound healing. Transl Res. 2021;236:109-116.
[6] LI G, PU Z, GUO S, et al. Durable and biocompatible low adhesion wound dressing material based on interfacial behaviors for wound management. Colloids Surf B Biointerfaces. 2025;247: 114413.
[7] MISHRA A, KUSHARE A, GUPTA MN, et al. Advanced Dressings for Chronic Wound Management. ACS Appl Bio Mater. 2024;7(5): 2660-2676.
[8] LI K, ZHU Z, ZHAI Y, et al. Recent Advances in Electrospun Nanofiber-Based Strategies for Diabetic Wound Healing Application. Pharmaceutics. 2023;15(9):2285.
[9] XU R, FANG Y, ZHANG Z, et al. Recent Advances in Biodegradable and Biocompatible Synthetic Polymers Used in Skin Wound Healing. Materials (Basel). 2023;16(15):5459.
[10] XIAO L, LI L, HUANG J, et al. Salidroside attenuates lipopolysaccharide-induced neuroinflammation and cognitive impairment in septic encephalopathy mice. Int Immnopharmacol. 2023;117:109975.
[11] XU XW, ZHANG J, GUO ZW, et al. A narrative review of research progress on the relationship between hypoxia-inducible factor-2α and wound angiogenesis. Ann Palliat Med. 2021;10(4):4882-4888.
[12] WANG Y, HAN J, LUO L, et al. Salidroside facilitates therapeutic angiogenesis in diabetic hindlimb ischemia by inhibiting ferroptosis. Biomed Pharmacother. 2023;159:114245.
[13] ZHANG J, KASIM V, XIE YD, et al. Inhibition of PHD3 by salidroside promotes neovascularization through cell-cell communications mediated by muscle-secreted angiogenic factors. Sci Rep. 2017;7: 43935.
[14] MISHRA KP. Salidroside exerts anti‒inflammatory effect by reducing nuclear factor κB and nitric oxide production in macrophages. MOJ Immunology. 2018;6(1):1-6.
[15] XIE Y, CHI M, YANG X, et al. FeMOFs/CO loading reduces NETosis and macrophage inflammatory response in PLA based cardiovascular stent materials. Regen Biomater. 2025;12:rbae140.
[16] LIU J, QU M, WANG C, et al. A Dual-Cross-Linked Hydrogel Patch for Promoting Diabetic Wound Healing. Small. 2022;18(17):e2106172.
[17] RUDER K. Diabetic Foot Infections and Amputations Are All Too Common-Here’s What Could Move the Needle. JAMA. 2024;331(12): 998-1000.
[18] WANG F, ZHANG X, ZHANG J, et al. Recent advances in the adjunctive management of diabetic foot ulcer: Focus on noninvasive technologies. Med Res Rev. 2024;44(4):1501-1544.
[19] LEE CH, CHEN DY, HSIEH MJ, et al. Nanofibrous insulin/vildagliptin core-shell PLGA scaffold promotes diabetic wound healing. Front Bioeng Biotechnol. 2023;11:1075720.
[20] LI Y, WU T, ZHANG G, et al. A native sericin wound dressing spun directly from silkworms enhances wound healing. Colloids Surf B Biointerfaces. 2023;225:113228.
[21] BRUN NR, LENZ M, WEHRLI B, et al. Comparative effects of zinc oxide nanoparticles and dissolved zinc on zebrafish embryos and eleuthero-embryos: importance of zinc ions. Sci Total Environ. 2014;476-477: 657-666.
[22] XIE S, ZHU J, YANG D, et al. Low Concentrations of Zinc Oxide Nanoparticles Cause Severe Cytotoxicity Through Increased Intracellular Reactive Oxygen Species. J Biomed Nanotechnol. 2021;17(12): 2420-2432.
[23] 樊红艳,何树梅,杨建伟,等.藏药红景天苷对斑马鱼发育及急性毒性的评价[J].中国民族民间医药,2021,30(19):18-21.
[24] ALI MM, AL-MOKADDEM AK, ABDEL-SATTAR E, et al. Enhanced wound healing potential of arabincoside B isolated from Caralluma Arabica in rat model; a possible dressing in veterinary practice. BMC Vet Res. 2024;20(1):282.
[25] HUANG Y, SONG M, LI X, et al. Temperature-responsive self-contraction nano fiber/hydrogel composite dressing facilitates the healing of diabetic-infected wounds. Mater Today Bio. 2024;28:101214.
[26] SONG B, HUANG G, XIONG Y, et al. Inhibitory effects of salidroside on nitric oxide and prostaglandin E₂ production in lipopolysaccharide-stimulated RAW 264.7 macrophages. J Med Food. 2013;16(11): 997-1003.
[27] SUN P, SONG SZ, JIANG S, et al. Salidroside Regulates Inflammatory Response in Raw 264.7 Macrophages via TLR4/TAK1 and Ameliorates Inflammation in Alcohol Binge Drinking-Induced Liver Injury. Molecules. 2016;21(11):1490.
[28] LAN KC, CHAO SC, WU HY, et al. Salidroside ameliorates sepsis-induced acute lung injury and mortality via downregulating NF-κB and HMGB1 pathways through the upregulation of SIRT1. Sci Rep. 2017;7(1):12026.
[29] JIA X, ZHANG K, FENG S, et al. Total glycosides of Rhodiola rosea L. attenuate LPS-induced acute lung injury by inhibiting TLR4/NF-κB pathway. Biomed Pharmacother. 2023;158:114186.
[30] ZHANG Y, ZHAO Q. Salidroside attenuates interleukin-1β-induced inflammation in human osteoarthritis chondrocytes. J Cell Biochem. 2019;120(2):1203-1209.
[31] WANG XL, SUN RX, LI DX, et al. Salidroside Regulates Mitochondrial Homeostasis After Polarization of RAW264.7 Macrophages. J Cardiovasc Pharmacol. 2023;81(1):85-92.
[32] YANG BY, HU CH, HUANG WC, et al. Effects of Bilayer Nanofibrous Scaffolds Containing Curcumin/Lithospermi Radix Extract on Wound Healing in Streptozotocin-Induced Diabetic Rats. Polymers (Basel). 2019;11(11):1745.
[33] WOJDASIEWICZ P, BRODACKI S, CIEŚLICKA E, et al. Salidroside: A Promising Agent in Bone Metabolism Modulation. Nutrients. 2024; 16(15):2387.
[34] 马宇滢. 红景天苷和丹酚酸B对人内皮祖细胞增殖、迁移和凋亡的作用[D].上海:复旦大学,2007.
[35] LI R, LIU K, HUANG X, et al. Bioactive Materials Promote Wound Healing through Modulation of Cell Behaviors. Adv Sci (Weinh). 2022; 9(10):e2105152.