Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model
Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model
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DOI：
10.1007/s00403-007-0804-3
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发表时间：
2008-04-01
影响因子：
3
通讯作者：
Sakurai, Hirornu
中科院分区：
文献类型：
--
作者：
Hakozaki, Tomohiro;Date, Akira;Sakurai, Hirornu
活性氧（ROS）在紫外线引起的皮肤损伤或光老化过程中起着重要作用。虽然已经开发了许多酶和化学方法来评估ROS，但评估生命系统中ROS生成的方法相当有限。在这里，我们提出了一个独特的系统来可视化uvb诱导的ROS，并研究ROS的生物学影响。简而言之，人体皮肤等效模型（HSEM）暴露于UVB。利用化学发光探针（CLA）和超低光成像装置对HSEM发出的发光进行了可视化和半量化。通过半定量发射化学发光（CL）强度、MTT测定和8-羟基-2′-脱氧鸟苷（8-OHdG）染色评价抗氧化化合物如抗坏血酸、β -胡萝卜素、超氧化物歧化酶（SOD）和酵母发酵滤液（YFF）对HSEM的影响。利用灵敏的二维超低光显像仪成功地实现了HSEM中ROS生成的时间和空间依赖动力学的可视化。β -胡萝卜素和SOD处理有效抑制了CL强度，表明UVB照射下HSEM中产生了O-1(2)和O-2（-）。经测试的抗氧化化合物还能减轻uvb诱导的CL，并在8-OHdG形成和细胞死亡方面改善诱发的皮肤损伤。综上所述，该模型不仅可用于实时可视化uvb诱导的ROS生成，还可用于评估局部应用抗氧化化合物抑制ROS生成和减弱序列化学和生物反应的功效。
Reactive oxygen species (ROS) play important roles in the process of ultraviolet-induced skin damage or photoaging. Although many enzymatic and chemical methods have been developed for evaluating ROS, evaluation methods for ROS generation in living systems are quite limited. Here we propose a unique system to visualize UVB-induced ROS and investigate the biological impact of ROS. In brief, a human skin equivalent model (HSEM) was exposed to UVB. Emitted luminescence from the HSEM was visualized and semi-quantified by using a chemiluminescent probe (CLA) and an ultra low-light imaging apparatus. The effects of anti-oxidative compounds such as ascorbate, beta-carotene, superoxide dismutase (SOD), and yeast ferment filtrate (YFF) on the HSEM were evaluated by semi-quantification of emitted chemiluminescence (CL) intensities, MTT assay and 8-hydroxy-2'-deoxyguanosine (8-OHdG) staining. Visualization of time- and space-dependent dynamics of ROS generation in the HSEM was successfully achieved by utilizing a sensitive two-dimensional ultra-low light luminograph. Treatments with beta-carotene and SOD effectively suppressed CL intensity, indicating the generation of O-1(2) and O-2(-) in the HSEM under UVB exposure. Tested anti-oxidative compounds also attenuated UVB-induced CL and ameliorated the induced skin damages in terms of 8-OHdG formation and cell death. As a conclusion, this model is useful for not only visualizing the production of UVB-induced ROS in real-time but also evaluating the efficacy of topically applied anti-oxidative compounds to suppress ROS generation and attenuate sequential chemical and biological responses.