Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells
Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells
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可见光可能直接诱导核DNA损伤，从而触发RGC-5细胞的死亡途径
作者：
Li, Guang-Yu;Fan, Bin;Ma, Tong-Hui
研究表明，可见光可通过线粒体途径诱导视网膜神经节细胞(RGC)-5细胞死亡。目前的研究旨在确定可见光是否也可能通过破坏核DNA直接触发死亡途径。方法将RGC-5细胞暴露于不同强度和时间的可见光下。采用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑测定法和碘化丙啶染色法监测细胞活力和死亡情况。采用质粒法、基因组DNA法和原位末端脱氧核苷酸转移酶dUTP缺口末端标记法测定光致核DNA损伤。western blot检测核酶poly（adp -核糖）聚合酶-1 （PARP-1）随后的活化情况，并采用特异性抑制剂评估PARP-1在死亡通路中的作用。用聚（adp -核糖）糖水解酶和凋亡诱导因子（AIF）抑制剂来显示它们对光诱导细胞死亡的影响。用fura-2法和钙通道阻滞剂检测钙内流。结果可见光诱导RGC-5细胞死亡具有时间依赖性和强度依赖性。光照强度增加到2,600 lx后，通过检测体外双链DNA断裂和核DNA损伤，可以清楚地观察到RGC-5细胞死亡通路的激活。核酶PARP-1在2600 lx光照下暴露2天后立即被激活，PARP-1特异性抑制剂具有显著的神经保护作用。聚（adp -核糖）糖水解酶抑制剂单宁酸和AIF抑制剂n -苯基马来酰亚胺部分保护RGC-5细胞免受光损伤。光照2天后检测到大量钙流入，钙通道阻滞剂部分保护细胞免受光损伤。结论可见光照可直接引起核DNA损伤，从而激活PARP-1。此外，2,600 lx光照损伤的RGC-5细胞可作为筛选神经保护药物的合适细胞死亡模型，因为该处理可在2天内诱导显著的细胞死亡。此外，这些结果表明，2600 lx的光照比1000 lx的光照更明显地激活了死亡途径，这是之前研究中使用的。
Purpose Visible light has been previously demonstrated to induce retinal ganglion cell (RGC)-5 cell death through the mitochondrial pathway. The present study was designed to determine whether visible light might also directly trigger the death pathway by damaging nuclear DNA. Methods RGC-5 cells were exposed to various intensities and durations of visible light exposure. Cell viability and death were monitored with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide staining. Nuclear DNA damage caused by light was determined with the plasmid assay, genome DNA assay, and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling. The subsequent activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was measured with western blot, and PARP-1’s role in the death pathway was assessed by using specific inhibitors. Poly (ADP-ribose) glycohydrolase and apoptosis-inducing factor (AIF) inhibitors were used to show their influence on light-induced cell death. Calcium influx was examined with the fura-2 assay and calcium channel blocker. Results We found that visible light induced RGC-5 cell death in a time- and intensity-dependent manner. After the light intensity was increased to 2,600 lx, activation of the death pathway in RGC-5 cells was clearly observed by detecting double-strand DNA breaks and nuclear DNA damage in vitro. Nuclear enzyme PARP-1 was promptly activated after exposure to 2,600 lx of light for 2 days, and specific inhibitors of PARP-1 had significant neuroprotective effects. The poly(ADP-ribose) glycohydrolase inhibitor tannic acid and AIF inhibitor N-phenylmaleimide partially protected RGC-5 cells from light injury. A massive calcium influx was detected after 2 days of light exposure, and a calcium channel blocker partially protected cells against light injury. Conclusions These results suggest that visible light exposure may directly cause nuclear DNA damage, which consequently activates PARP-1. In addition, RGC-5 cells damaged by 2,600 lx of light exposure can be used as an appropriate cell death model for screening neuroprotective drugs, since this treatment induced remarkable cell death within 2 days. Moreover, these results show that 2,600 lx of light exposure provides a more apparent activation of the death pathway than 1,000 lx of light exposure, which was used in a previous study.
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DOI：
10.1016/0027-5107(91)90121-4
发表时间：
1991
期刊：
Mutation research
影响因子：
--
作者：
J. G. Peak;M. Peak
通讯作者：
J. G. Peak;M. Peak
影响因子：
4.4
作者：
Van Bergen, Nicole J.;Wood, John P. M.;Crowston, Jonathan G.
通讯作者：
Crowston, Jonathan G.
DOI：
--
发表时间：
1966-10
期刊：
Investigative ophthalmology
影响因子：
--
作者：
W. Noell;Virgil S. Walker;B. Kang;Steven Berman
通讯作者：
W. Noell;Virgil S. Walker;B. Kang;Steven Berman
影响因子：
2.9
作者：
Li, Guang-Yu;Osborne, Neville N.
通讯作者：
Osborne, Neville N.
影响因子：
2.2
作者：
I. Surgucheva;Alejandra D. Weisman;J. Goldberg;A. Shnyra;A. Surguchov
通讯作者：
I. Surgucheva;Alejandra D. Weisman;J. Goldberg;A. Shnyra;A. Surguchov