Rauen U, Elling B, Gizewski E R, Korth H G, Sustmann R, de Groot H
Institut für Physiologische Chemie, Universitätsklinikum, Essen, Germany.
Free Radic Biol Med. 1997;22(1-2):17-24. doi: 10.1016/s0891-5849(96)00273-0.
We have previously demonstrated an energy-dependent injury to cultured liver endothelial cells during cold incubation in University of Wisconsin (UW) solution. In the present study, we report experimental evidence for the involvement of reactive oxygen species in this injury: LDH release during 48 h of cold incubation in UW solution was decreased from 40-55% under aerobic conditions to less than 20% under hypoxic conditions or by the presence of KCN (1 mM). Similar protection was achieved by the addition of the spin trap 5,5-dimethyl-1-pyrroline N-oxide, the hydroxyl radical scavenger dimethyl sulfoxide, or the flavonoid silibinin to UW solution under aerobic conditions. Preincubating the cells with the iron chelator deferoxamine even decreased the injury to less than 5%. The residual injury (as observed after longer incubation times) under hypoxic conditions or in cells preincubated with deferoxamine was no longer energy dependent. The amount of thiobarbituric acid-reactive substances markedly increased during cold incubation of the cells in UW solution. This increase was not observed in UW solution to which KCN had been added, i.e., under the conditions of energy depletion. These results suggest that an iron-dependent generation of reactive oxygen species with subsequent lipid peroxidation is involved in the pathogenesis of the injury to cultured liver endothelial cells in cold UW solution.
我们之前已经证明，在威斯康星大学（UW）溶液中进行冷孵育期间，培养的肝内皮细胞会发生能量依赖性损伤。在本研究中，我们报告了活性氧参与这种损伤的实验证据：在UW溶液中进行48小时冷孵育期间，乳酸脱氢酶（LDH）释放量在有氧条件下从40%-55%降至缺氧条件下或存在KCN（1 mM）时的20%以下。在有氧条件下，向UW溶液中添加自旋捕获剂5,5-二甲基-1-吡咯啉N-氧化物、羟基自由基清除剂二甲基亚砜或类黄酮水飞蓟宾也能实现类似的保护作用。用铁螯合剂去铁胺对细胞进行预孵育甚至可将损伤降低至5%以下。缺氧条件下或用去铁胺预孵育的细胞中（长时间孵育后观察到的）残余损伤不再依赖能量。在UW溶液中对细胞进行冷孵育期间，硫代巴比妥酸反应性物质的量显著增加。在添加了KCN的UW溶液中，即在能量耗竭的条件下，未观察到这种增加。这些结果表明，铁依赖性产生活性氧并随后发生脂质过氧化参与了冷UW溶液中培养的肝内皮细胞损伤的发病机制。
