Curcumin and resveratrol act by different ways on NADPH oxidase activity and reactive oxygen species produced by equine neutrophils.

Abstract In neutrophils (PMNs), superoxide anion (O 2 − ), 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 − 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 − 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.