Lim WonBong, Kim JiSun, Lim ChaeGwang, Kim SangWoo, Jeon SangMi, Karna Sandeep, Cho MinSung, Choi HongRan, Kim OkJoon
Department of Oral Pathology, School of Dentistry, Dental Science Research Institute, Chonnam National University, Bug-Gu, Gwangju, Korea.
Photomed Laser Surg. 2012 Aug;30(8):451-9. doi: 10.1089/pho.2011.3199. Epub 2012 Jul 9.
The aim of this study was to examine the reactive oxygen species (ROS) that are dissipated by 635 nm irradiation, and the effect of 635 nm irradiation on ROS scavenging system.
Intracellular ROS are produced in the form of superoxide anion by either nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or xanthine oxidase in response to a number of stimuli. Low-level light irradiation decreases the intracellular ROS level and has been used in clinical situations for reducing the level of oxidative stress.
Human epithelial cells were exposed to exogenous and endogenous oxidizing agents that promote the generation of harmful ROS. These were then irradiated with 635 nm LED light, 5 mW/cm(2) for 1 h, 18 J/cm(2) or by 470 nm LED light, also 5 mW/cm(2) for 1 h, 18 J/cm(2) on a 9 cm cell culture dish. After irradiation, the MTT reduction method and malondialdehyde (MDA) colorimetric assay were performed in xanthine/xanthine oxidase (XXO)- or hydrogen peroxide (H(2)O(2))-treated HaCaT cells. The superoxide anion was detected by an electron spin resonance (ESR) spectrometer using 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as the spin trap and H(2)O(2) was assayed by flow cytometry using 2',7'-dichlorodihydrofluorescein diacetate (H(2)DCF-DA).
Irradiation at 635 nm enhanced cell viability in the XXO-treated HaCaT cells. Also, irradiation had a much lesser effect on cell viability in the HaCaT cells treated with exogenous H(2)O(2) as compared with that in cells treated with N-acetyl-L-cysteine. The level of the superoxide anion increased in response to XXO treatment, and then decreased after 635 nm irradiation. Irradiation with 635 nm led to a decrease in superoxide anion and lipid peroxidation levels in the presence or absence of diethyldithiocarbamate.
These results highlight the potential role of 635 nm irradiation in protection against oxidative stress by scavenging superoxide anions. Also, a pathway that is independent of the activities of intracellular enzymatic ROS scavengers, such as superoxide dismutase, glutathione peroxidase and catalase might be involved in its mechanism of action.
本研究旨在检测635 nm照射所消耗的活性氧（ROS），以及635 nm照射对ROS清除系统的影响。
细胞内ROS由烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶或黄嘌呤氧化酶在多种刺激下以超氧阴离子的形式产生。低水平光照射可降低细胞内ROS水平，并已用于临床以降低氧化应激水平。
将人上皮细胞暴露于能促进有害ROS生成的外源性和内源性氧化剂中。然后在9 cm细胞培养皿中，用5 mW/cm²的635 nm LED光照射1 h（剂量为18 J/cm²），或用同样5 mW/cm²的470 nm LED光照射1 h（剂量为18 J/cm²）。照射后，在经黄嘌呤/黄嘌呤氧化酶（XXO）或过氧化氢（H₂O₂）处理的HaCaT细胞中进行MTT还原法和丙二醛（MDA）比色测定。使用5,5-二甲基-1-吡咯啉-N-氧化物（DMPO）作为自旋捕获剂，通过电子自旋共振（ESR）光谱仪检测超氧阴离子，使用二氯二氢荧光素二乙酸酯（H₂DCF-DA）通过流式细胞术测定H₂O₂。
635 nm照射增强了XXO处理的HaCaT细胞的活力。此外，与用N-乙酰-L-半胱氨酸处理的细胞相比，照射对用外源性H₂O₂处理的HaCaT细胞活力的影响要小得多。超氧阴离子水平在XXO处理后升高，然后在635 nm照射后降低。在有或没有二乙基二硫代氨基甲酸盐的情况下，635 nm照射导致超氧阴离子和脂质过氧化水平降低。
这些结果突出了635 nm照射通过清除超氧阴离子在抵御氧化应激方面的潜在作用。此外，其作用机制可能涉及一条独立于细胞内酶促ROS清除剂（如超氧化物歧化酶、谷胱甘肽过氧化物酶和过氧化氢酶）活性的途径。
