Bedouhène Samia, Moulti-Mati Farida, Hurtado-Nedelec Margarita, Dang Pham My-Chan, El-Benna Jamel
INSERM-U1149, CNRS-ERL8252, Centre de Recherche sur l'InflammationParis, France.
Université Paris Diderot, Sorbonne Paris Cité, Laboratoire d'Excellence Inflamex, DHU FIRE, Faculté de Médecine, Site Xavier BichatParis, France.
Am J Blood Res. 2017 Jul 25;7(4):41-48. eCollection 2017.
Reactive oxygen species (ROS) are produced by numerous biological systems and by several phagocytes such as neutrophils and macrophages. ROS include mostly superoxide anion, hydrogen peroxide, singlet oxygen and hydroxyl radical, which are involved in a variety of biological processes such as immunity, inflammation, apoptosis and cell signaling. Thus, there is a need for a sensitive and reliable method to measure ROS. The luminol-amplified chemiluminescence technique is widely used to measure ROS production by neutrophils; however, it is unclear which ROS species are detected by this technique. In this study, we show that Xanthine/Xanthine oxidase (XXO), a known superoxide-producing system, stimulated a luminol-amplified chemiluminescence in the absence of horseradish peroxidase (HRPO), while the presence of HRPO enhanced the response. Both reactions were inhibited by superoxide dismutase (SOD), but not by catalase, confirming that superoxide anion, and not hydrogen peroxide, is the species oxidizing luminol to produce chemiluminescence. Glucose/Glucose oxidase (GGO), a known hydrogen peroxide-producing system, did not induce luminol-amplified chemiluminescence in the absence of HRPO; however, addition of HRPO resulted in a chemiluminescence response, which was inhibited by catalase, but not by SOD. Myeloperoxidase (MPO), isolated from human neutrophils, was also able to enhance the superoxide- and hydrogen peroxide-dependent luminol-amplified chemiluminescence. The production of ROS by stimulated human neutrophils was detected by luminol-amplified chemiluminescence, which was only partially inhibited by SOD and catalase. Interestingly, adding HRPO to stimulated neutrophils increased the luminol-amplified chemiluminescence, which was strongly inhibited by SOD, but not by catalase. These results show that (a) luminol-amplified chemiluminescence is able to detect superoxide anion in the absence of peroxidases, but not hydrogen peroxide; (b) in the presence of peroxidases, luminol-amplified chemiluminescence is able to detect both superoxide anion and hydrogen peroxide; and
活性氧（ROS）由众多生物系统以及几种吞噬细胞（如中性粒细胞和巨噬细胞）产生。ROS主要包括超氧阴离子、过氧化氢、单线态氧和羟基自由基，它们参与多种生物过程，如免疫、炎症、细胞凋亡和细胞信号传导。因此，需要一种灵敏且可靠的方法来测量ROS。鲁米诺增强化学发光技术被广泛用于测量中性粒细胞产生的ROS；然而，尚不清楚该技术检测到的是哪种ROS种类。在本研究中，我们表明黄嘌呤/黄嘌呤氧化酶（XXO），一种已知的超氧产生系统，在没有辣根过氧化物酶（HRPO）的情况下能刺激鲁米诺增强化学发光，而HRPO的存在会增强该反应。两种反应均被超氧化物歧化酶（SOD）抑制，但不被过氧化氢酶抑制，这证实了是超氧阴离子而非过氧化氢将鲁米诺氧化以产生化学发光。葡萄糖/葡萄糖氧化酶（GGO），一种已知的过氧化氢产生系统，在没有HRPO的情况下不会诱导鲁米诺增强化学发光；然而，添加HRPO会导致化学发光反应，该反应被过氧化氢酶抑制，但不被SOD抑制。从人中性粒细胞中分离出的髓过氧化物酶（MPO）也能够增强超氧和过氧化氢依赖性的鲁米诺增强化学发光。通过鲁米诺增强化学发光检测到刺激后人中性粒细胞产生的ROS，该化学发光仅被SOD和过氧化氢酶部分抑制。有趣的是，向刺激后的中性粒细胞中添加HRPO会增加鲁米诺增强化学发光，该化学发光被SOD强烈抑制，但不被过氧化氢酶抑制。这些结果表明：（a）鲁米诺增强化学发光在没有过氧化物酶的情况下能够检测超氧阴离子，但不能检测过氧化氢；（b）在有过氧化物酶存在的情况下，鲁米诺增强化学发光能够检测超氧阴离子和过氧化氢；并且
