Center for Oxygen, R&D (CORD), Institute of Chemistry, B6a, University of Liège, Allée de la Chimie 3, B-4000 Liège, Belgium.
Chem Biol Interact. 2013 Nov 25;206(2):186-93. doi: 10.1016/j.cbi.2013.09.011. Epub 2013 Sep 20.
In neutrophils (PMNs), superoxide anion (O2*-), 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 b558. 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 O2(-) 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 O2*- 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.
在中性粒细胞（PMN）中，超氧阴离子（O2*-）是第一种产生的活性氧（ROS），用于杀死病原体，由 NADPH 氧化酶产生，这是一种由细胞质亚基向膜黄素细胞色素 b558 易位形成的酶复合物。在马中，PMN 的过度激活通常与致命的病理有关，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，以便测试天然或合成化合物对酶活性或组装的影响。该无细胞测定法已通过二苯乙烯碘化物和 Gp91ds-tat 进行了验证，这两种抑制剂在人类 NADPH 氧化酶中已有大量描述。姜黄素和白藜芦醇的抗氧化作用在终浓度为 10(-4) 至 10(-6) M 的范围内通过化学发光（CL）和无细胞测定法在全细胞中进行了研究，其中在酶组装之前或之后添加分子。CL 测定法表明姜黄素有效地抑制了 O2(-)的产生，并且很容易进入 PMN 或与其膜相互作用。无细胞测定法表明，即使在 10(-5) M 时，姜黄素也作用于 NADPH 氧化酶的重组，而白藜芦醇似乎是 O2*-清除剂而不是 NADPH 氧化酶活性的抑制剂，因为它在 CL 中从细胞外部起作用，并且在无细胞测定法中在复合物组装之后起作用。通过直接作用于 NADPH 氧化酶，姜黄素应该是治疗涉及过度 ROS 产生的急性或炎症性疾病的良好候选药物。
