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阿托伐他汀对同型半胱氨酸所诱导内皮祖细胞功能损伤的保护作用

《心脏杂志》[ISSN:1009-7236/CN:61-1268/R]

期数:
2010年第3期
页码:
332-337
栏目:
基础研究
出版日期:
2010-04-06

文章信息/Info

Title:
Atorvastatin antagonizes homocysteine-induced functional injury of endothelial progenitor cells
作者:
鲍晓梅; 吴春芳; 陆国平
上海交通大学附属瑞金医院心内科，上海 200025
Author(s):
BAO Xiao-mei;  Wu Chun-fang;  Lu Guo-ping
Department of Cardiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
关键词:
阿托伐他汀; 同型半胱氨酸; 内皮祖细胞功能; 活性氧; NADPH氧化酶; 一氧化氮
Keywords:
atorvastatin;  homocysteine;  endothelial progenitor cell function;  reactive oxygen species;  NADPH oxidase;  nitric oxide
分类号:
Q256
DOI:
-
文献标识码:
A
摘要:
目的: 探讨同型半胱氨酸(homocysteine, Hcy)诱导小鼠骨髓内皮祖细胞（endothelial progenitor cells,EPCs）功能损伤的氧化应激机制及阿托伐他汀的拮抗作用。方法: 密度梯度离心法获取小鼠骨髓单个核细胞，培养7 d后收集贴壁细胞，FITC-UEA-1荧光染色鉴定EPCs。EPCs与不同浓度Hcy(0 μmol/L,50 μmol/L,500 μmol/L)共孵育24 h或分别经不同浓度的阿托伐他汀(0.1 μmol/L,1 μmol/L,10 μmol/L)预孵育0.5 h后，再加入500 μmol/L Hcy共孵育24 h。用MTT比色法，改良Boyden小室、黏附能力测定和体外血管生成试剂盒，分别观察EPCs的增殖能力、迁移能力、黏附能力和体外血管生成能力。荧光探针H2DCF-DA法检测细胞内活性氧水平，光泽精化学发光法检测NADPH氧化酶活性，硝酸还原酶法测定细胞培养液中一氧化氮(NO)含量，RT-PCR法测定eNOS基因表达。结果: Hcy诱导EPCs增殖、迁移、黏附和体外血管生成功能下降，活性氧的产生及NADPH氧化酶的活性增加， eNOS基因表达及细胞培养液中NO含量减少，与无Hcy处理组相比有明显差异（P<0.05或P<0.01）。与500 μmol/L Hcy组相比，阿托伐他汀预处理可呈剂量依赖性地拮抗Hcy诱导的上述改变（P<0.05或P<0.01）。结论: Hcy可能通过激活NADPH氧化酶，诱导EPCs活性氧的产生及降低eNOS基因表达和NO水平，导致EPCs增殖、迁移、黏附和体外血管生成功能下降。阿托伐他汀部分拮抗Hcy的作用。
Abstract:
AIM: To investigate the oxidative mechanism of homocysteine (Hcy)-induced functional injury in endothelial progenitor cells (EPCs) and the protective effects of atorvastatin. METHODS: Total mononuclear cells were isolated from the mouse bone marrow by Ficoll density gradient centrifugation and cultured in vitro. After being cultured for 7 days，the cells were identified by fluorescence microscopy. Cells were divided into six groups. Cells were treated with Hcy (0 μmol/L, 50 μmol/L, 500 μmol/L) for 24 h or pretreated with atorvastatin (0.1 μmol/L, 1 μmol/L, 10 μmol/L) for 0.5 h, then cultured with 500 μmol/L Hcy for 24 h. EPC proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPC adhesion assay was performed by replating those on fibronectin-coated dishes, and then adherent cells were counted. Reactive oxygen species (ROS) levels in cells were measured using H2DCF-DA as a fluorescence probe. Activities of NADPH oxidases were evaluated with lucigenin-enhanced chemiluminescence. NO in the supernatant was detected by nitrate reductase assay. RT-PCR was performed to determine the eNOS expression. RESULTS: Hcy impaired the proliferation, adhesion, migration activity and in vitro vasculogenesis capacity of EPCs, increased ROS accumulation, NADPH oxidase activation, and decreased the secretion of NO, and eNOS mRNA expression, compared with the control group (P<0.05 or P<0.01). Atorvastatin inhibited the effects of Hcy in a dose-dependent manner, compared with the 500 μmol/L Hcy group (P<0.05 or P<0.01). CONCLUSION: Hcy induces ROS via activating NADPH oxidase, decreases NO secretion and eNOS mRNA expression leading to EPC functional injury. Atorvastatin partially antagonizes these effects.

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备注/Memo

备注/Memo:
收稿日期：2009-06-17.作者简介：鲍晓梅,博士生mail:baoxiaomei_nt@yahoo.com.cn
更新日期/Last Update: 2010-04-09