Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: attenuation by L-ascorbic acid.
Peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells: attenuation by L-ascorbic acid.
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DOI：
10.1016/s0891-5849(96)00374-7
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发表时间：
1997
影响因子：
7.4
通讯作者：
M. Sandoval;Xiao-Jing Zhang;Xiaoping Liu;E. Mannick;D. Clark;Mark J. S. Miller
中科院分区：
文献类型：
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作者：
M. Sandoval;Xiao-Jing Zhang;Xiaoping Liu;E. Mannick;D. Clark;Mark J. S. Miller
自由基一氧化氮和超氧化物反应形成过氧亚硝酸盐（ONOO−），一种有效的细胞毒性氧化剂。本研究旨在评估是否添加L-抗坏血酸（AsC）到培养基中减少过氧亚硝基诱导的人肠上皮细胞（T84）和小鼠巨噬细胞（RAW 264.7）细胞系的凋亡。在实验1中，将T84和RAW 264.7细胞分为两种方案：（1）用100-300 μM ONOO-处理并孵育4 h，以及（2）用10-100 μM ONOO-处理并孵育过夜（14 h）。在实验2中，用300 μM ONOO−和500 μM AsC处理T84和RAW 264.7细胞，并孵育4小时。在实验3中，T84和RAW 264.7细胞与500 μM AsC预孵育2小时，然后暴露于300 μM ONOO− 4小时。通过台盼蓝染料排除法评估细胞活力（坏死）。用细胞死亡检测ELISA测定法定量细胞凋亡。在4 h方案中，ONOO−在100-300 μM水平下诱导T84和RAW 264.7细胞凋亡。浓度大于300 μM的ONOO−会导致坏死。相反，将实验延长至14 h表明，ONOO−在较低浓度（50;-75 μM）下诱导细胞凋亡，浓度> 75 μM时导致坏死。AsC给药的媒体或预孵育加冲洗，减少过氧亚硝基诱导的T84和RAW 264.7细胞凋亡。这些结果表明，ONOO−可能通过促进细胞死亡而促进肠道炎症的病理生理学，而抗坏血酸可能保护肠道免受过氧亚硝酸盐诱导的损伤。版权所有© 1996 Elsevier Science Inc.
The free radicals nitric oxide and superoxide react to form peroxynitrite (ONOO−), a potent cytotoxic oxidant. This study was designed to evaluate whether addition of l-Ascorbic acid (AsC) into the culture medium decreases peroxynitrite-induced apoptosis in human intestinal epithelial (T84) and murine macrophage (RAW 264.7) cell lines. In Experiment 1, T84 and RAW 264.7 cells were divided in two protocols: (1) treated with 100–300 μM ONOO−and incubated for 4 h, and (2) treated with 10–100 μM ONOO−and incubated overnight (14 h). In Experiment 2, T84 and RAW 264.7 cells were treated with 300 μM ONOO−and 500 μM AsC and incubated for 4 h. In Experiment 3, T84 and RAW 264.7 cells were preincubated for 2 h with 500 μM AsC then exposed to 300 μM ONOO−for 4 h. Cell viability (necrosis) was assessed by trypan blue dye exclusion. Apoptosis was quantified with a cell death detection ELISA assay. In the 4 h protocol, ONOO−induced apoptosis in T84 and RAW 264.7 cells, at levels of 100–300 μM. Concentrations of ONOO−greater than 300 μM caused necrosis. In contrast, extension of the protocol to 14 h indicated that ONOO−induced apoptosis at lower concentrations (50;–75 μM), with concentrations > 75 μM resulting in necrosis. AsC administered to the media or with preincubation plus washout, decreased peroxynitrite-induced apoptosis in T84 and RAW 264.7 cells. These results indicate that ONOO−may contribute to the pathophysiology of gut inflammation by promoting cell death and ascorbic acid may protect against peroxynitrite-induced damage.Copyright © 1996 Elsevier Science Inc.