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龙柳芽,胡清睿,熊武,邹晓玲,薛冰倩,罗标,张熙.抗氧化视角下黄芪甲苷修复高糖损伤内皮祖细胞体外成管功能的实验研究[J].湖南中医药大学学报,,():[点击复制] |
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| 抗氧化视角下黄芪甲苷修复高糖损伤内皮祖细胞体外成管功能的实验研究 |
| 龙柳芽,胡清睿,熊武,邹晓玲,薛冰倩,罗标,张熙 |
| (湖南中医药大学中西医结合学院, 湖南 长沙 410208;湖南中医药大学第一附属医院, 湖南 长沙 410007) |
| 摘要: |
| 目的 探讨黄芪甲苷(AS-Ⅳ)对高糖诱导损伤内皮祖细胞(EPCs)氧化应激状态及体外血管生成能力的影响。方法 自人脐带血中分离、培养EPCs，采用双荧光染色法进行鉴定。将鉴定成功的 EPCs 置于 30 mmol/L 的高糖 DMEM 培养基中处理 120 h，建立高糖诱导损伤的 EPCs 模型。将高糖损伤的 EPCs 随机分为高糖损伤 EPCs 组、AS-Ⅳ组和 AS-Ⅳ+ML385 组，并设置正常 EPCs组。AS-Ⅳ组用 100 mg/L AS-Ⅳ干预，AS-Ⅳ+ML385 组同时用 100 mg/L AS-Ⅳ和 19 μmol/L ML385 干预，正常 EPCs 组和高糖损伤 EPCs 组加入等体积 PBS，以上各组干预时间均为 48 h。采用荧光探针法检测细胞内活性氧(ROS)水平；采用流式细胞术检测细胞凋亡率和焦亡比例；采用 ELISA 检测细胞培养上清液中血管生成相关因子[包括血管内皮生长因子 A(VEGF-A)、血管内皮生长因子 B(VEGF-B)、血管内皮生长因子 C(VEGF-C)、成纤维细胞生长因子(FGF)及血管生成素 1(Ang-1)]水平，并通过基质胶成管实验分析细胞交叉点数、血管总长度和血管分支长度，以评价 EPCs 体外血管生成能力。结果 与正常 EPCs 组比较，高糖损伤 EPCs 组细胞内 ROS 水平、细胞早期凋亡率、细胞晚期凋亡率、细胞总凋亡率及细胞焦亡比例均升高(P<0.01)，细胞培养上清液中 VEGF-A、VEGF-B、VEGF-C、FGF 及 Ang-1 水平均降低(P<0.01)，细胞交叉点数、血管总长度及血管分支长度均减少(P<0.01)。与高糖损伤EPCs 组比较，AS-Ⅳ组与 AS-Ⅳ+ML385 组细胞内 ROS 水平、细胞晚期凋亡率、细胞总凋亡率、细胞焦亡比例均降低(P<0.01)，细胞培养上清液中 VEGF-A、VEGF-B、VEGF-C、FGF 及 Ang-1 水平均升高(P<0.01)，细胞交叉点数、血管总长度及血管分支长度均增加(P<0.01)；AS-Ⅳ组细胞早期凋亡率降低(P<0.01)。与 AS-Ⅳ组比较，AS-Ⅳ+ML385 组细胞内 ROS 水平、细胞早期凋亡率、细胞晚期凋亡率、细胞总凋亡率及细胞焦亡比例均升高(P<0.01)，细胞培养上清液中 VEGF-A、VEGF-B、VEGF-C、FGF 及 Ang-1 水平均降低(P约0.05，P<0.01)，细胞交叉点数、血管总长度及血管分支长度均减少(P<0.01)。结论 黄芪甲苷可改善高糖环境下 EPCs 的功能损伤，抑制凋亡与焦亡，改善血管生成相关因子分泌，并增强其体外血管生成能力，其机制可能与激活 Nrf2 信号通路、发挥抗氧化作用有关。 |
| 关键词: 黄芪甲苷 内皮祖细胞 高糖损伤 氧化应激 Nrf2信号通路 血管生成 |
| DOI： |
| 投稿时间：2026-06-17 |
| 基金项目:湖南省自然科学基金高校联合基金重点项目(2025JJ90013)；长沙市自然科学基金项目(KQ2502258)；湖南中医药大学研究生科研创新项目(2025CX073)。 |
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| Experimental study on repair of high-glucose-induced injury to the in vitro angiogenic capacity of endothelial progenitor cells by astragaloside Ⅳ from the perspective of antioxidation |
| LONG Liuya,HU Qingrui,XIONG Wu,ZOU Xiaoling,XUE Bingqian,LUO Biao,ZHANG Xi |
| (School of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China) |
| Abstract: |
| Objective To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on oxidative stress status and in vitro angiogenic capacity of endothelial progenitor cells (EPCs) with high-glucose-induced injury. Methods EPCs were isolated and cultured from human umbilical cord blood and identified by dual fluorescence staining. The successfully identified EPCs were exposed to high-glucose DMEM (30 mmol/L) continuously for 120 h to establish a high-glucose-induced injury EPCs model. The injured EPCs were randomly divided into high-glucose-induced injury EPCs (HG-EPCs), AS-Ⅳ, and AS-Ⅳ+ML385 groups, with a normal EPCs (N-EPCs) group also included. The AS-Ⅳ group was treated with 100 mg/L AS-Ⅳ, while the AS-Ⅳ+ML385 group received 100 mg/L AS-Ⅳ combined with 19 μmol/L ML385. Equal volumes of PBS were administered to the N-EPCs and HG-EPCs groups. All groups were treated continuously for 48 h. Intracellular reactive oxygen species (ROS) levels were detected using fluorescent probe assay. Flow cytometry was applied to determine apoptosis rate and pyroptosis proportion. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of angiogenesis-related factors, including vascular endothelial growth factor A (VEGF-A), vascular endothelial growth factor B (VEGF-B), vascular endothelial growth factor C (VEGF-C), fibroblast growth factor (FGF), and angiopoietin-1 (Ang-1), in cell culture supernatants. Matrigel tube formation assay was performed to evaluate the in vitro angiogenic capacity of EPCs by analyzing the number of cell junctions, total vessel length, and branch length. Results Compared with the N-EPCs group, the HGEPCs group exhibited significantly higher intracellular ROS levels, early apoptosis rate, late apoptosis rate, total apoptosis rate, and pyroptosis proportion (P<0.01), while the levels of VEGF-A, VEGF-B, VEGF-C, FGF, and Ang-1 in the supernatants were markedly lower (P<0.01), accompanied by a reduced number of junctions, total vessel length, and branch length (P<0.01). Compared with the HG-EPCs group, both AS-Ⅳ and AS-Ⅳ+ML385 groups showed decreased ROS levels, late apoptosis rate, total apoptosis rate, and pyroptosis proportion (P<0.01), while the levels of VEGF-A, VEGF-B, VEGF-C, FGF, and Ang-1 in the supernatants markedly increased (P<0.01), along with an increased number of junctions, total vessel length, and branch length (P<0.01). The early apoptosis rate was significantly lower in the AS-Ⅳ group (P<0.01). Compared with the AS-Ⅳ group, the AS-Ⅳ+ML385 group displayed increased ROS levels, early apoptosis rate, late apoptosis rate, total apoptosis rate, and pyroptosis proportion (P<0.01), reduced levels of angiogenesis-related factors including VEGF-A, VEGF-B, VEGF-C, FGF, and Ang-1 in the supernatants (P<0.05, P<0.01), and a decreased number of junctions, total vessel length, and branch length (P<0.01). Conclusion Astragaloside Ⅳ can alleviate high-glucoseinduced functional damage in EPCs, inhibit apoptosis and pyroptosis, promote the secretion of angiogenesis-related factors, and enhance their in vitro angiogenic capacity. Its underlying mechanism may be related to the activation of the Nrf2 signaling pathway and the resulting antioxidant effects. |
| Key words: astragaloside Ⅳ endothelial progenitor cells high-glucose-induced injury oxidative stress Nrf2 signaling pathway angiogenesis |
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