Homocysteine reduces protein S-nitrosylation in endothelium
Homocysteine reduces protein S-nitrosylation in endothelium
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DOI：
10.3892/ijmm.2014.1920
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发表时间：
2014-11-01
影响因子：
5.4
通讯作者：
Liu, Enqi
中科院分区：
文献类型：
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作者：
Chen, Yulong;Zhao, Sihai;Liu, Enqi
高同型半胱氨酸血症(HHcy)是心血管疾病的危险因素。蛋白质的S亚硝化参与了心血管功能的调节。然而，同型半胱氨酸(Hcy)是否通过抑制血管内皮细胞内的蛋白S亚硝化而损害血管功能仍有待确定。实验是在人脐静脉内皮细胞(HUVECs)中进行的。雄性SD大鼠，在给予或不给予L蛋氨酸的情况下，用于体内验证结果。用亚硝半胱氨酸免疫荧光法对S亚硝化产物进行分析，并用生物素切换法进一步证实。2‘，7’-二氯二乙酸二氯荧光素(DCFH-DA)染色检测ROS水平。硝酸还原法测一氧化氮(NO)水平。蛋白表达采用免疫印迹分析。用凝胶迁移率改变分析法(EMSA)检测核因子-kappaB的活性。用双抗体夹心法测定血浆同型半胱氨酸水平。结果表明，海参能显著降低人脐静脉内皮细胞和血管内皮细胞的S亚硝化蛋白水平。这种蛋白S亚硝化水平的降低伴随着HUVECs内ROS的升高、Akt和内皮型一氧化氮合酶(ENOS)磷酸化水平的降低以及一氧化氮水平的降低。此外，还发现同型半胱氨酸通过抑制胞浆中核因子-kappaB的S亚硝化而增加血管细胞黏附分子-1的蛋白表达(P65)。提示同型半胱氨酸通过抑制血管内皮细胞蛋白S亚硝化而损害血管内皮细胞功能。
Hyperhomocysteinemia (HHcy) is a risk factor for cardiovascular disease. The S-nitrosylation of proteins is involved in the regulation of cardiovascular functions. However, whether homocysteine (Hcy) impairs vascular functions through the inhibition of protein S-nitrosylation in the endothelium remains to be determined. The experiments were performed in human umbilical vein endothelial cells (HUVECs). Male Sprague-Dawley rats, with or without administration of L-methionine, were used for the in vivo validation of findings. S-nitrosylation was analyzed using immunofluorescence for nitrosocysteine, and further confirmed by the biotin switch method. The levers of reactive oxygen species (ROS) were detected by 2',7'-dichlorofluorescein diacetate (DCFH-DA) staining. The levels of nitric oxide (NO) were determined by the nitrate reduction method. Protein expression was analysed by western blot analysis. The activity of nuclear factor kappa B (NF-kappa B) was evaluated by an electrophoretic mobility shift assay (EMSA). The levels of plasma Hcy were measured by ELISA. The results showed that Hey significantly reduced the levels of protein S-nitrosylation in HUVECs and endothelial S-nitrosylation of aorta. This reduction of protein S-nitrosylation was accompanied by increasing ROS, decreasing phosphorylation levels of A kt and endothelial nitric oxide synthase (eNOS), and reduced levels of nitric oxide in HUVECs. In addition, it was found that Hcy increased the protein expression of vascular cell adhesion molecule-1 by attenuating the cytoplasm S-nitrosylation of NF-kappa B (p65). These data suggested that Hcy impairs endothelial functions by inhibiting endothelial protein S-nitrosylation.