Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, China.
Aquat Toxicol. 2013 Jan 15;126:169-79. doi: 10.1016/j.aquatox.2012.11.005. Epub 2012 Nov 12.
The present study explored the protective effects of glutamine (Gln), alanine (Ala), citrulline (Cit) and proline (Pro) on hydroxyl radical (·OH)-induced apoptosis in isolated carp erythrocytes. Hydroxyl radicals were generated by ferrous ion (Fe(2+))-mediated decomposition of hydrogen peroxide (H(2)O(2)) (Fenton reaction). In order to select an optimal ·OH concentration to induce apoptosis, cultures were treated with different concentrations of FeSO(4)/H(2)O(2) (0 μM/0 μM-50 μM/25 μM). The results showed that exposure to FeSO(4)/H(2)O(2) (0 μM/0 μM-40 μM/20 μM) increased apoptosis in a dose-dependent manner. Moreover, apoptosis was at its highest level at 40 μM FeSO(4)/20 μM H(2)O(2). We then examined the cytoprotective effects of Gln, Ala, Cit, Pro or the combination of Ala, Cit and Pro under conditions of apoptosis. Carp erythrocytes were treated with the substances listed above in the presence of 40 μM FeSO(4)/20 μM H(2)O(2) for 9 h. The controls were grown in Gln, Ala, Cit, Pro-free culture medium. The results showed that Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively protected against annexin binding, decrease of forward scatter and DNA fragmentation in carp erythrocytes induced by ·OH. Furthermore, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro effectively blocked ·OH-stimulated erythrocyte hemolysis, reduced the increase of superoxide anion and H(2)O(2) concentrations, inhibited the formation of malondialdehyde, protein carbonyls and met-hemoglobin, and prevented the decrease of superoxide dismutase, catalase and glutathione peroxidase activities and glutathione content in carp erythrocytes induced by ·OH. In addition, the results suggest that the combination of Ala, Cit and Pro produces a greater anti-apoptotic and anti-oxidative effect than their individual effects at the same concentrations. Taken together, the results showed that ·OH induces apoptosis and oxidative damage in carp erythrocytes. In addition to inhibiting apoptosis, Gln, Ala, Cit, Pro and the combination of Ala, Cit and Pro protected carp erythrocytes against oxidative damage induced by ·OH, which may be a major factor in the protection of erythrocytes from apoptosis.
本研究探讨了谷氨酰胺（Gln）、丙氨酸（Ala）、瓜氨酸（Cit）和脯氨酸（Pro）对羟自由基（·OH）诱导的鲤鱼红细胞凋亡的保护作用。羟自由基通过亚铁离子（Fe(2+)）介导的过氧化氢（H(2)O(2)）分解（Fenton 反应）产生。为了选择诱导凋亡的最佳·OH 浓度，将培养物用不同浓度的 FeSO(4)/H(2)O(2)（0 μM/0 μM-50 μM/25 μM）处理。结果表明，暴露于 FeSO(4)/H(2)O(2)（0 μM/0 μM-40 μM/20 μM）可剂量依赖性地增加凋亡。此外，在 40 μM FeSO(4)/20 μM H(2)O(2)下，凋亡水平最高。然后，我们在凋亡条件下研究了 Gln、Ala、Cit、Pro 或 Ala、Cit 和 Pro 混合物的细胞保护作用。用上述物质处理鲤鱼红细胞，同时存在 40 μM FeSO(4)/20 μM H(2)O(2)9 小时。对照品在无 Gln、Ala、Cit、Pro 的培养基中生长。结果表明，Gln、Ala、Cit、Pro 以及 Ala、Cit 和 Pro 的混合物有效保护鲤鱼红细胞免受 annexin 结合、前向散射减少和 DNA 片段化的影响，由·OH 诱导。此外，Gln、Ala、Cit、Pro 以及 Ala、Cit 和 Pro 的混合物有效阻断·OH 刺激的红细胞溶血，减少超氧阴离子和 H(2)O(2)浓度的增加，抑制丙二醛、蛋白质羰基和高铁血红蛋白的形成，并防止超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶活性和鲤鱼红细胞中谷胱甘肽含量的降低由·OH 诱导。此外，结果表明，Ala、Cit 和 Pro 的混合物在相同浓度下产生比其单独作用更大的抗凋亡和抗氧化作用。综上所述，结果表明·OH 诱导鲤鱼红细胞凋亡和氧化损伤。除了抑制凋亡外，Gln、Ala、Cit、Pro 以及 Ala、Cit 和 Pro 的混合物还可保护鲤鱼红细胞免受·OH 诱导的氧化损伤，这可能是红细胞免受凋亡保护的主要因素。
