Dong Feng, Zhang Xiaochun, Li Shi-Yan, Zhang Zhaojie, Ren Qun, Culver Bruce, Ren Jun
Division of Pharmaceutical Sciences and Center for Cardiovascular Research and Alternative Medicine, University of Wyoming, Laramie, WY 82071, USA.
Cardiovasc Toxicol. 2005;5(1):9-20. doi: 10.1385/ct:5:1:009.
Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases, although the mechanism leading to vascular dysfunction is not clear. The aim of this study was to examine the effect of homocysteine (Hcy) on oxi-dative stress and apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were challenged for 24 h with Hcy (10 microM-3 mM) in the presence of various stress signaling inhibitors, including the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin (100 microM), the p38 mito-gen-activated protein kinase inhibitor SB203580 (2.5 microM), the extracellular signal-regulated kinase inhibitor U0126 (2.5 microM), the stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) inhibitor JNK inhibitor II (10 microM), and antioxidants alpha-tocopherol (5 microg/mL) and N-acetyl cysteine (NAC, 2 mM). Reactive oxygen species (ROS) were detected using 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate. Apoptosis was evaluated by 4',6'-diamidino-2'-phenylindoladihydrochloride staining, annexin-V phosphatidyl- serine/propidium iodide, and caspase-3 assay. NADPH oxidase and SAPK/JNK signal were evaluated with immunoblotting. Hcy significantly enhanced ROS generation and apoptosis after 24-h incubation. Apocynin prevented Hcy-induced ROS generation but only partially restored Hcy-induced apoptosis. JNK inhibitor II, alpha-tocopherol, and NAC partially reduced Hcy-induced apoptosis, although SB203580 and U0126 had no effect. Immunoblotting analysis confirmed upregulation of NADPH oxidase and SAPK/JNK signaling. Collectively, our results suggested that Hcy may induce oxidative stress and apopto-sis through an NADPH oxidase and/or JNK-dependent mechanism(s).
高同型半胱氨酸血症是心血管疾病的一个独立危险因素，尽管导致血管功能障碍的机制尚不清楚。本研究的目的是检测同型半胱氨酸（Hcy）对人脐静脉内皮细胞（HUVECs）氧化应激和细胞凋亡的影响。在存在各种应激信号抑制剂的情况下，用Hcy（10微摩尔/升 - 3毫摩尔/升）对HUVECs进行24小时刺激，这些抑制剂包括烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶抑制剂夹竹桃麻素（100微摩尔/升）、p38丝裂原活化蛋白激酶抑制剂SB203580（2.5微摩尔/升）、细胞外信号调节激酶抑制剂U0126（2.5微摩尔/升）、应激激活蛋白激酶（SAPK）/c-Jun氨基末端激酶（JNK）抑制剂JNK抑制剂II（10微摩尔/升），以及抗氧化剂α-生育酚（5微克/毫升）和N-乙酰半胱氨酸（NAC，2毫摩尔/升）。使用5-(6)-氯甲基-2',7'-二氯二氢荧光素二乙酸酯检测活性氧（ROS）。通过4',6'-二脒基-2'-苯基吲哚二盐酸盐染色、膜联蛋白-V磷脂酰丝氨酸/碘化丙啶和半胱天冬酶-3测定评估细胞凋亡。用免疫印迹法评估NADPH氧化酶和SAPK/JNK信号。孵育24小时后，Hcy显著增强了ROS的产生和细胞凋亡。夹竹桃麻素可阻止Hcy诱导的ROS产生，但仅部分恢复Hcy诱导的细胞凋亡。JNK抑制剂II、α-生育酚和NAC可部分降低Hcy诱导的细胞凋亡，尽管SB203580和U0126没有效果。免疫印迹分析证实了NADPH氧化酶和SAPK/JNK信号的上调。总体而言，我们的结果表明，Hcy可能通过NADPH氧化酶和/或JNK依赖性机制诱导氧化应激和细胞凋亡。
