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谭维, 傅馨莹, 杨仁义, 马露, 丁煌, 刘晓丹, 张伟.黄芪甲苷调控Nrf2/HO-1信号通路对血管内皮细胞氧化损伤的影响[J].湖南中医药大学学报,2024,44(9):1592-1600[点击复制] |
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| 黄芪甲苷调控Nrf2/HO-1信号通路对血管内皮细胞氧化损伤的影响 |
| 谭维,傅馨莹,杨仁义,马露,丁煌,刘晓丹,张伟 |
| (湖南中医药大学中西医结合学院, 湖南 长沙 410208;中西医结合心脑疾病防治湖南省重点实验室, 湖南 长沙 410208) |
| 摘要: |
| 目的 基于细胞实验研究黄芪甲苷（astragaloside Ⅳ, AST-Ⅳ）调控核转录因子红系2相关因子2/血红素加氧酶-1（nuclear factor-erythroid 2-related factor 2/heme oxygenase-1, Nrf2/HO-1）信号通路对血管内皮氧化损伤的影响。方法 采用1-棕榈酰基-2-（5'-氧-戊酰基）-sn-甘油-3-磷酸胆碱[1-palmitoyl-2-（5-oxovaleroyl）-sn-glycero-3-phosphocholine, POVPC]刺激血管内皮细胞24 h建立血管内皮细胞氧化损伤模型，随机分为模型（Model）组（35 μmol/L POVPC）、AST-Ⅳ低剂量（AST-L）组（35 μmol/L POVPC+50 μmol/L AST-Ⅳ）、AST-Ⅳ高剂量（AST-H）组（35 μmol/L POVPC+100 μmol/L AST-Ⅳ）及AST-Ⅳ高剂量+ML385（Nrf2抑制剂）（AST-H+ML385）组（35 μmol/L POVPC+100 μmol/L AST-Ⅳ+5 μmol/L ML385），以正常未处理细胞作为对照（Control）组。免疫荧光鉴定大鼠胸主动脉内皮细胞（aortic vascular endothelium cell, AVEC），CCK-8检测AST-Ⅳ细胞增殖情况，Transwell小室检测AVEC迁移情况，Matrigel基质胶检测细胞血管形成功能，鬼笔环肽染色检测细胞骨架结构，DCFH-DA荧光探针检测细胞活性氧（reactive oxygen species, ROS）水平，试剂盒法检测细胞培养上清液中超氧化物歧化酶（superoxide dismutase, SOD）水平，RT-qPCR和Western blot检测Nrf2和HO-1的 mRNA及蛋白表达情况。结果 免疫荧光鉴定所提取的细胞，可特异性表达血管性血友病因子（von willebrand factor, vWF），鉴定为AVEC。与Control组相比，Model组细胞活力、迁移能力和血管形成功能显著下降（P＜0.01），细胞骨架破坏明显，细胞内ROS水平显著上升（P＜0.01），细胞培养上清液中SOD含量显著减少（P＜0.01），细胞中Nrf2和HO-1的mRNA及蛋白表达水平下调（P＜0.01或P＜0.05）。与Model组比较，AST-L组及AST-H组细胞活力、迁移能力和血管形成功能显著升高（P＜0.01），细胞骨架破坏得到较好修复，细胞内ROS水平显著下降（P＜0.01），细胞培养上清液中SOD含量显著增加（P＜0.01），细胞中Nrf2和HO-1的mRNA及蛋白表达水平显著上调（P＜0.01），且以AST-H组效果更为显著（P＜0.01）。与AST-H组比较，AST-H+ML385组迁移能力和血管形成功能显著下降（P＜0.01），细胞骨架破坏增加，细胞内ROS水平显著上升（P＜0.01），细胞培养上清液中SOD含量显著减少（P＜0.01），细胞中Nrf2和HO-1的mRNA及蛋白表达水平显著下调（P＜0.01）。结论 AST对血管内皮氧化损伤具有保护作用，且有一定的剂量依赖性，其机制可能是通过激活Nrf2/HO-1信号通路发挥作用。 |
| 关键词: 心血管疾病 黄芪甲苷 Nrf2/HO-1信号通路 氧化损伤 血管内皮细胞 |
| DOI：10.3969/j.issn.1674-070X.2024.09.006 |
| 投稿时间：2023-11-14 |
| 基金项目:国家自然科学基金项目（82174218）；湖南省科技创新计划资助项目（2023SK2021）；湖南省科技创新领军人才项目（2023RC1066）。 |
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| Effects of astragaloside Ⅳ on oxidative damage of vascular endothelial cells by regulating Nrf2/HO-1 signaling pathway |
| TAN Wei, FU Xinying, YANG Renyi, MA Lu, DING Huang, LIU Xiaodan, ZHANG Wei |
| (School of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, Hunan 410208, China;Hunan Key Laboratory of Integrated Chinese and Western Medicine for Prevention and Treatment of Heart and Brain Diseases, Changsha, Hunan 410208, China) |
| Abstract: |
| Objective To explore the effects of astragaloside Ⅳ (AST-Ⅳ) on vascular endothelial oxidative damage by modulating the nuclear factor-erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling pathway, based on cell experiments. Methods An endothelial cell oxidative damage model was established by stimulating vascular endothelial cells with 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) for 24 hours. The cells were randomly divided into model group (35 μmol/L POVPC), low-dose AST-Ⅳ (AST-L) group (35 μmol/L POVPC+50 μmol/L AST-Ⅳ), high-dose AST-Ⅳ (AST-H) group (35 μmol/L POVPC+100 μmol/L AST-Ⅳ), and high-dose AST-Ⅳ+ ML385 (Nrf2 inhibitor) (AST-H+ML385) group (35 μmol/L POVPC+100 μmol/L AST-Ⅳ+5 μmol/L ML385). Untreated normal cells were used as the control group. Rat aortic vascular endothelium cells (AVECs) were identified by immunofluorescence. The proliferation of AST-Ⅳ cells was tested by CCK-8 assay. Transwell chambers were utilized to evaluate AVEC migration, while Matrigel matrix gel was employed to assess angiogenic function of the cells. The cytoskeletal structure was determined by phalloidin staining, reactive oxygen species (ROS) levels were measured by the DCFH-DA fluorescence probe, and superoxide dismutase (SOD) levels in cell culture supernatants were measured by kit method. The mRNA and protein expressions of Nrf2 and HO-1 were determined by RT-qPCR and Western blot. Results The expression of extracted cell surface marker von willebrand factor (vWF) was determined by immunofluorescence and was identified as AVEC. Compared with control group, the cell viability, migration ability, and angiogenic function in Model group decreased significantly (P＜0.01), with obvious cytoskeleton disruption, a significant increase in intracellular ROS level (P＜0.01), a significant decrease in SOD content in the cell culture supernatant (P＜0.01), and downregulated mRNA and protein expression levels of Nrf2 and HO-1 in cells (P＜0.01 or P＜0.05). Compared with model group, AST-L group and AST-H group showed significantly increased cell viability, migration ability, and angiogenic function (P＜0.01), with better repair of cytoskeleton disruption, a significant decrease in intracellular ROS level (P＜0.01), a significant increase in SOD content in the cell culture supernatant (P＜0.01), and significantly upregulated mRNA and protein expression levels of Nrf2 and HO-1 in cells (P＜0.01), with more pronounced effects in AST-H group (P＜0.01). Compared with AST-H group, AST-H+ML385 group showed significantly decreased migration ability and angiogenic function (P＜0.01), increased cytoskeleton disruption, a significant increase in intracellular ROS level (P＜0.01), a significant decrease in SOD content in the cell culture supernatant (P＜0.01), and significantly downregulated mRNA and protein expression levels of Nrf2 and HO-1 in cells (P＜0.01). Conclusion AST has protective effects on vascular endothelial oxidative damage, with a certain dose dependence, and its mechanism may involve the activation of Nrf2/HO-1 signaling pathway. |
| Key words: cardiovascular disease astragaloside Ⅳ Nrf2/HO-1 signaling pathway oxidative damage vascular endothelial cell |
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