Oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) in cultured bovine articular chondrocytes increases production of intracellular reactive oxygen species (ROS) resulting in the activation of NF-kappaB.
Oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) in cultured bovine articular chondrocytes increases production of intracellular reactive oxygen species (ROS) resulting in the activation of NF-kappaB.
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发表时间：
2004
影响因子：
7
通讯作者：
Shunji Nishimura;M. Akagi;Koji Yoshida;S. Hayakawa;T. Sawamura;H. Munakata;C. Hamanishi
中科院分区：
文献类型：
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作者：
Shunji Nishimura;M. Akagi;Koji Yoshida;S. Hayakawa;T. Sawamura;H. Munakata;C. Hamanishi
目的研究氧化低密度脂蛋白（ox-LDL）对牛关节软骨细胞（BACs）细胞内活性氧（ROS）产生的影响，并探讨这种增加是否通过与凝集素样ox-LDL受体-1 （LOX-1）结合而发生。此外，为了确定ox-LDL与LOX-1的结合是否会导致NF-kappaB活化。设计BACs与2',7'-二氯荧光素二乙酸酯（DCFH-DA）预孵育，这种染料可以通过荧光光谱法监测DCF细胞内ROS的产生。将BACs与天然LDL和ox-LDL（10、50和100微克/毫升）在37℃下孵育5分钟，观察DCF的形成。BACs也用抗lox -1单抗（40微克/毫升）或抗坏血酸（10微克/毫升）预孵育。以50 μ g/ml ox-LDL处理指定时间的BACs核提取物，并与抗lox -1单抗或抗坏血酸预孵育，制备并通过电泳迁移转移试验（EMSA）进行分析。结果ox-LDL在与BACs孵育5分钟后诱导ROS产生显著的剂量依赖性增加（P < 0.001）。在抗lox -1单抗和抗坏血酸预孵育的BACs中，ROS的形成明显减少（P < 0.001）。与ox-LDL孵育5分钟后，转录因子NF-kappaB在BACs中明显活化，并被抗lox -1单抗和抗坏血酸减弱。结论ox-LDL与bac中LOX-1的结合增加了细胞内ROS的产生并激活了NF-kappaB。NF-kappaB被抗坏血酸激活的减少表明，至少部分的激活是ros依赖的。这些观察结果支持了高胆固醇血症是关节炎的几个危险因素之一的假设，并且脂质过氧化产物如ox-LDL参与软骨基质降解。
OBJECTIVE To examine the effect of oxidized low-density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine articular chondrocytes (BACs) and to investigate whether this increase occurs through binding to the receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, to ascertain whether the binding of ox-LDL to LOX-1 results in NF-kappaB activation. DESIGN BACs were preincubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye that allows the monitoring of intracellular ROS production for DCF by spectrofluorometry. BACs were incubated with native LDL and ox-LDL (10, 50, and 100 microg/ml) for 5 min at 37 degrees C and DCF formation was observed. BACs were also preincubated with anti-LOX-1 mAb (40 microg/ml) or ascorbic acid (10 microM). Nuclear extracts from BACs treated for the indicated periods with 50 microg/ml ox-LDL, and preincubated with anti-LOX-1 mAb or ascorbic acid, were prepared and analyzed by electrophoretic mobility shift assay (EMSA). RESULTS ox-LDL induced a significant dose-dependent increase in ROS production after 5-min incubation with BACs (P < 0.001). ROS formation was markedly reduced in BACs preincubated with anti-LOX-1 mAb and ascorbic acid (P < 0.001). Activation in BACs of the transcription factor NF-kappaB was evident after 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 mAb and ascorbic acid. CONCLUSION ox-LDL binding to LOX-1 in BACs increased the production of intracellular ROS and activated NF-kappaB. Reduction of NF-kappaB activation by ascorbic acid indicates that the activation, at least in part, is ROS-dependent. These observations support the hypothesis that hypercholesterolemia is one of several risk factors for arthritis, and that lipid peroxidation products such as ox-LDL are involved in cartilage matrix degradation.