黄芪甲苷对缺氧/复氧损伤人脐静脉内皮细胞与中性粒细胞黏附能力及细胞核转录因子κB表达的影响★
杨富国1，刘革新1，董果雄2，郭云良3
课题背景：黄芪是用于防治心脑血管疾病的传统中药。黄芪甲苷是从黄芪中提取的有效成分，其分子结构已明确，但有关黄芪甲苷对内皮细胞缺氧复氧损伤的影响报道较少。
同行评价：该项实验建立了人脐静脉内皮细胞缺氧复氧模型模拟器官组织缺血－再灌注所诱导的血管改变，从分子水平探讨黄芪甲苷对血管内皮细胞缺氧／复氧的保护作用，为缺血再灌注损伤的治疗提供用药依据，实验具有一定的创新性。
相关链接：氧化应激可激活血管内皮细胞核转录因子κB信号转导通路，活化的核转录因子κB通路又可导致细胞间黏附分子1过度表达，后者进而介导中性粒细胞的黏附和聚集，引发器官、组织及细胞的炎性损伤反应。黄芪甲苷通过清除氧自由基，抑制核转录因子κB发生核易位，减少细胞间黏附分子1的表达，从而减轻血管内皮细胞与中性粒细胞黏附，对缺氧/复氧损伤血管内皮细胞具有保护作用。
摘要
目的： 采用人脐静脉内皮细胞缺氧复氧模型模拟器官组织缺血-再灌注所诱导的血管改变，观察黄芪甲苷对缺氧/复氧损伤血管内皮细胞与中性粒细胞黏附及细胞核转录因子κB表达的干预作用。
方法：实验于2005-09/2006-05在青岛大学医学院附属医院脑血管病研究所细胞生物工程实验室进行。①实验材料：新生儿脐带（由青岛大学医学院附属医院妇产科提供，产妇及其家属知情同意，实验符合赫尔辛基宣言中的伦理学标准）。②实验过程及分组：人脐静脉内皮细胞原代培养，传至3代后，随机分为3组。对照组：常规培养；缺氧/复氧组：先缺氧处理1 h，再复氧1 h；药物干预组：在培养液中加入终浓度分别为20，40，80 mg/L的黄芪甲苷预处理，12 h后进行缺氧/复氧处理。③实验评估：分别用硫代巴比妥法和酶联免疫吸附试验测定细胞培养液中丙二醛的含量和细胞间黏附分子1浓度；免疫化学法测人脐静脉内皮细胞核转录因子κB的表达；虎红染色法检测人脐静脉内皮细胞与中性粒细胞黏附。
结果：①与对照组比较，缺氧/复氧组细胞培养液中丙二醛含量增多（P < 0.01）；随着黄芪甲苷预处理剂量的增加，丙二醛含量降低（P < 0.01），呈剂量依赖性。②与对照组比较，缺氧复氧组人脐静脉内皮细胞核转录因子κΒ阳性细胞率、细胞培养液中细胞间黏附分子1浓度及中性粒细胞黏附率升高（P均< 0.01）；随着黄芪甲苷预处理剂量的增加，核转录因子 κB表达阳性细胞率、中性粒细胞黏附率下降（P < 0.05或P < 0.01），细胞间黏附分子1浓度减少（P < 0.01），呈剂量依赖性。
结论：黄芪甲苷通过抑制缺氧复氧引起的血管内皮细胞核转录因子κB表达，减轻血管内皮细胞与中性粒细胞黏附，对缺氧/复氧损伤血管内皮细胞具有保护作用，且呈剂量依赖性。
关键词：黄芪甲苷；内皮细胞；缺氧/复氧；NF-κB；胞间粘附分子1；组织构建
杨富国，刘革新，董果雄，郭云良. 黄芪甲苷对缺氧/复氧损伤人脐静脉内皮细胞与中性粒细胞黏附能力及细胞核转录因子κB表达的影响[J].中国组织工程研究与临床康复，2008，12(15):2843-2846
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2843(ps).pdf]
1青岛大学医学院护理学院，山东省青岛市 266021；青岛大学医学院附属医院，2心内科， 3脑血管病研究所，山东省青岛市 266003
杨富国★，男，1970年生，山东省青岛市人，汉族，青岛大学医学院附属医院心内科在读硕士，讲师，主要从事冠心病的基础和临床研究。
yfuguo@163.com
中图分类号:R329.4
文献标识码:A
文章编号:1673-8225
(2008)15-02843-04
收稿日期：2008-01-05 修回日期：2008-02-02
(08-50-1-97/W·Q)
Abstract
AIM: To investigate the intervention effect of astragaloside on adhesion of polymorphonuclear neutrophils (PMN) and vascular endothelial cells and on expression of nuclear factor-κB (NF-κB) in human umbilical vein endothelial cells (hUVECs) injured by hypoxia/reoxygenation due to ischemia/reperfusion.
METHODS: The experiment was performed at the Cell Bioengineering Laboratory of Institute of Cerebrovascular Diseases, Affiliated Hospital of Medical College, Qingdao University from September 2005 to May 2006. ①Neonatal umbilical cords were offered by Department of Gynecology and Obstetrics in Affiliated Hospital of Medical College of Qingdao University, with informed consent of puerperant and their families. The experiment was accorded with ethical standard of Helsinki declaration. ②The hUVECs were cultured to the future passage. After the third passage, hUVECs were randomly divided into three groups. Cells in a control group were cultured under normal conditions; Cells in a hypoxia/reoxygenation group were cultured under in closed container with hypoxia for 1 hour, and then under normal conditions for 1 hour; The hUVECs in a astragaloside group were pretreatment by different doses of astragaloside (20, 40, 80 mg/L). After 12 hours, hUVECs suffered from hypoxia/reoxygenation. ③The concentration of intercellular adhesion molecule (ICAM)-1 and content of malondialdehyde (MDA) in supernatant were detected by enzyme linked immunosorbent assay (ELISA) and thiobarbituric acid method. Expression of NF-κB of hUVECs was analyzed by immunohistochemistry. PMN adhesion to hUVECs was measured by rose Bengal staining.
RESULTS: ①Compared to the control group, the content of MDA remarkably increased in hypoxia/reoxygenation group (P < 0.01). With the increased dose of astragaloside, MDA content decreased (P < 0.01) with a dose-dependent effect. ②Compared to the control group, the percentage of NF-κB positive endothelial cells, concentration of ICAM-1 and adhesion rate of hUVECs to PMN increased in the hypoxia/reoxygenation group (P < 0.01). With the increased dose of astragaloside, the percentage of NF-κB positive endothelial cells and adhesion rate of hUVECs to PMN decreased (P < 0.05 or P < 0.01), and concentration of ICAM-1 was reduced (P < 0.01) with a dose-dependent effect.
CONCLUSION: Astragaloside can protect vascular endothelial cells from the injury caused by hypoxia/reoxygenation by inhibiting expression of NF-κB induced by hypoxia/reoxygenation in vascular endotheliial cells and by reducing the adhesion of vascular endotheliial cells to PMN with a dose-dependent effect.
Yang FG, Liu GX, Dong GX, Guo YL. Effect of astragaloside on adhesion of human umbilical vein endothelial cells and polymorphonuclear neutrophils and on expression of nuclear factor kappa B under hypoxia/reoxygenation condition.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(15):2843-2846(China)
[www.zglckf.com/zglckf/ejournal/upfiles/08-15/15k-2843(ps).pdf]
1Nursing School, Medical College, Qingdao University, Qingdao 266021, Shandong Province, China; 2Department of Cardiology, 3Institute of Cerebrovascular Diseases, Hospital Affiliated to Medical College of Qingdao University, Qingdao 266003, Shandong Province, China
Yang Fu-guo★ Studying for master's degree, Lecturer, Nursing School, Medical College, Qingdao University, Qingdao 266021, Shandong Province, China
yfuguo@163.com
Received: 2008-01-05
Accepted: 2008-02-02
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