Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils
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DOI：
10.1016/j.cbi.2013.09.011
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发表时间：
2013-11-25
影响因子：
5.1
通讯作者：
Serteyn, Didier
中科院分区：
文献类型：
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作者：
Derochette, Sandrine;Franck, Thierry;Serteyn, Didier
在中性粒细胞（PMN）中，超氧阴离子（O-2中心点（-）），即杀死病原体的第一个活性氧（ROS），由NADPH氧化酶产生，NADPH氧化酶是一种酶复合物，由胞质亚基移位至膜黄细胞色素B形成（558）。在马中，PMN的过度活化通常与致命的病理学相关，并且通过作用于NADPH氧化酶来调节其ROS产生是管理炎症的主要目标。我们开发了一种无细胞测定法来测量体外组装的马NADPH氧化酶的活性，以测试天然或合成化合物对酶活性或组装的影响。无细胞测定用氯化二苯碘铵和Gp 91 ds-tat（主要描述为人NADPH氧化酶的两种抑制剂）进行验证。姜黄素和白藜芦醇在终浓度范围为10（-4）至10（-6）M的抗氧化作用通过化学发光（CL）和无细胞测定法在全细胞上进行了研究，其中分子在酶组装之前或之后加入。化学发光实验表明姜黄素能有效抑制O-2中心点（-）的产生，并能进入中性粒细胞或与细胞膜相互作用。无细胞实验表明，姜黄素在10（-5）M时仍能作用于NADPH氧化酶的重组，而白藜芦醇在CL中是从细胞外作用，在无细胞实验中是在复合物组装后作用，因此白藜芦醇不是NADPH氧化酶活性的抑制剂，而是O-2中心点（-）清除剂。通过直接作用于NADPH氧化酶，姜黄素应该是治疗涉及过量ROS产生的急性或炎性疾病的良好候选物。(C)2013爱思唯尔爱尔兰有限公司保留所有权利。
In neutrophils (PMNs), superoxide anion (O-2 center dot(-)), the first reactive oxygen species (ROS) produced to kill pathogenic agents, is generated by NADPH oxidase, an enzymatic complex formed by the translocation of cytosolic subunits to the membrane flavocytochrome b(558). In horses, excessive activation of PMNs is often associated with deadly pathologies and the modulation of their ROS production by acting on NADPH oxidase is a prime target to manage inflammation. We developed a cell-free assay to measure the activity of equine NADPH oxidase assembled in vitro, in order to test the effects of natural or synthetic compounds on the enzyme activity or assembly. The cell-free assay was validated with diphenyleneiodonium chloride and Gp91ds-tat, two inhibitors largely described for human NADPH oxidase. The anti-oxidant effects of curcumin and resveratrol at final concentration ranging from 10(-4) to 10(-6) M were studied on whole cells by chemiluminescence (CL) and by cell-free assay, in which the molecule was added before or after the enzyme assembly. The CL assay demonstrated that curcumin efficiently inhibited the O-2 center dot(-) production and easily entered into PMNs or interacted with their membrane. Cell-free assay showed that curcumin acted on the reconstitution of NADPH oxidase even at 10(-5) M, while resveratrol appeared to be an O-2 center dot(-) scavenger rather than an inhibitor of NADPH oxidase activity, since it acted from outside the cell in CL and after the complex assembly in cell-free assay. By acting directly on NADPH oxidase, curcumin should be a good candidate for the treatment of acute or inflammatory diseases involving an excessive ROS production. (C) 2013 Elsevier Ireland Ltd. All rights reserved.