Ophthalmological Research Foundation, Oviedo, Asturias, Spain.
Acta Ophthalmol. 2019 Feb;97(1):e103-e115. doi: 10.1111/aos.13812. Epub 2018 Sep 9.
To ascertain whether red light, known to enhance mitochondrial function, can blunt a blue light insult to ARPE19 cells in culture.
Semi-confluent ARPE19 cells cultured in 10% FBS were subjected to various regimes of treatment with blue (465-475 nm, 800 lux, 26 W/m ) and red (625-635 nm, 950 lux, 6.5 W/m ) light, as well as with toxins that inactivate specific enzymes associated with mitochondrial oxidative phosphorylation. Cultures were then analysed for cell viability (MTT assay), mitochondrial status (JC-1), ROS formation, immunocytochemistry and the activation of specific proteins by electrophoresis/Western blotting. In addition, ARPE19 cells were cultured in polycarbonate membrane inserts in culture medium containing 1% FBS. Such cultures were exposed to cycles of red, blue or a combination of red and blue light for up to 6 weeks. Culture medium was changed and the trans-epithelium membrane resistance (TER) of the inserts-containing cells was measured twice weekly.
ARPE19 cells in culture are affected negatively when exposed to blue light. This is indicated by a loss of viability, a depolarization of their mitochondria and a stimulation of ROS. Moreover, blue light causes an up-regulation of HO-1 and phospho-p-38-MAPK and a cleavage of apoptosis inhibitory factor, proteins which are all known to be activated during cell death. All of these negative effects of blue light are significantly blunted by the red light administered after the blue light insult in each case. ARPE19 cell loss of viability and mitochondrial potential caused by toxins that inhibit specific mitochondrial enzyme complexes was additive to an insult delivered by blue light in each case. After a time, ARPE19 cells in culture express the tight junction protein ZO-1, which is affected by blue light. The development of tight junctions between ARPE19 cells grown in inserts reached a steady peak of resistance after about 40 days and then increased very slightly over the next 40 days when still in darkness. However, maximum resistance was significantly attenuated, when cultures were treated with cycles of blue light after the initial 40 days in the dark and counteracted significantly when the blue light cycle insult was combined with red light.
Blue light affects mitochondrial function and also the development tight junctions between ARPE19 cells, which results in a loss of cell viability. Importantly, red light delivered after a blue light insult is significantly blunted. These findings argue for the therapeutic use of red light as a noninvasive procedure to attenuate insults caused by blue light and other insults to retinal pigment epithelial cell mitochondria that are likely to occur in age-related macular degeneration.
确定已知能增强线粒体功能的红光是否能减轻蓝光对培养的 ARPE19 细胞的损伤。
半汇合的 ARPE19 细胞在 10%胎牛血清中培养，用不同的方案处理蓝光（465-475nm，800lux，26W/m）和红光（625-635nm，950lux，6.5W/m），以及用失活与线粒体氧化磷酸化相关的特定酶的毒素处理。然后通过 MTT 测定法分析细胞活力、线粒体状态（JC-1）、ROS 形成、免疫细胞化学和电泳/Western 印迹分析特定蛋白质的激活。此外，将 ARPE19 细胞在含有 1%胎牛血清的培养基中培养在聚碳酸酯膜插入物中。这种培养物接受红光、蓝光或红光和蓝光的组合的循环照射，最长可达 6 周。每周更换培养基两次，并测量含有细胞的插入物的跨上皮膜电阻（TER）。
暴露在蓝光下的培养 ARPE19 细胞受到负面影响。这表现为活力丧失、线粒体去极化和 ROS 刺激。此外，蓝光引起 HO-1 和磷酸化 p-38-MAPK 的上调，并切割凋亡抑制因子，这些蛋白质在细胞死亡过程中均被激活。所有这些蓝光的负面效应都在每次蓝光损伤后用红光治疗明显减轻。在每种情况下，抑制特定线粒体酶复合物的毒素引起的 ARPE19 细胞活力丧失和线粒体电位丧失都与蓝光损伤相加。一段时间后，培养的 ARPE19 细胞表达紧密连接蛋白 ZO-1，其受蓝光影响。插入物中生长的 ARPE19 细胞之间的紧密连接的发展在大约 40 天后达到稳定的峰值电阻，然后在接下来的 40 天内当仍处于黑暗中时略有增加。然而，当在最初的 40 天黑暗后用蓝光周期处理培养物时，最大电阻明显减弱，并且当蓝光周期损伤与红光结合时，明显减弱。
蓝光影响线粒体功能，也影响 ARPE19 细胞之间的紧密连接的发育，导致细胞活力丧失。重要的是，蓝光损伤后给予的红光明显减弱。这些发现为使用红光作为非侵入性程序来减轻与年龄相关的黄斑变性中可能发生的蓝光和其他对视网膜色素上皮细胞线粒体的损伤提供了治疗依据。
