Li Xue-Yin, Liu Yang, Jiang Wei-Dan, Jiang Jun, Wu Pei, Zhao Juan, Kuang Sheng-Yao, Tang Ling, Tang Wu-Neng, Zhang Yong-An, Zhou Xiao-Qiu, Feng Lin
Animal Nutrition Institute, Sichuan Agricultural University, Chengdu, Sichuan, China.
Fish Nutrition and Safety Production University Key Laboratory of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan, China.
PLoS One. 2016 Jan 26;11(1):e0147408. doi: 10.1371/journal.pone.0147408. eCollection 2016.
The aim of the work was primarily to explore the protective activity pathways of lysine against oxidative damage in fish in vivo and in enterocytes in vitro. First, grass carp were fed diets containing six graded levels of lysine (7.1-19.6 g kg-1 diet) for 56 days. Second, the enterocytes were treated with different concentrations of lysine (0-300 mg/L in media) prior to (pre-treatment), along with (co-treatment) or following (post-treatment) with 6 mg/L of Cu for 24 h. The results indicated that lysine improved grass carp growth performance. Meanwhile, lysine ameliorated lipid and protein oxidation by elevating the gene expression and activity of antioxidant enzymes (superoxide dismutase (SOD), glutathioneperoxidase (GPx), glutathione-S-transferase (GST) and reductase (GR)), and nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels in fish intestine. The in vitro studies showed that co- and post-treatment with lysine conferred significant protection against Cu-induced oxidative damage in fish primary enterocytes as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) OD values, along with alkaline phosphatase (ALP) and lactate dehydrogenase activities, and the depletion of protein carbonyl (PC), malondialdehyde (MDA) and 8-hydroxydeoxyguanosine contents. Moreover, lysine co-treatment decreased the activities and mRNA level of cellular SOD, GPx, GST and GR compared with the Cu-only exposed group. Gene expression of the signalling molecule Nrf2 showed the same pattern as that of SOD activity, whereas Kelch-like ECH-associated protein 1b (Keap1b) followed the opposite trend, indicating that co-treatment with lysine induced antioxidant enzymes that protected against oxidative stress through Nrf2 pathway. In addition, post-treatment with lysine increased proteasomal activity and blocked the Cu-stimulated increase in mRNA levels of GST and associated catalase (CAT) and GST activities (P<0.01 and P<0.001). GR activity and gene expression, and glutathione (GSH) content followed an opposite trend to GST activity (P<0.05). Thus, post-treatment of lysine elevated protein and DNA repair abilities and ameliorated the cellular redox state of enterocytes. The overall results suggest that lysine plays a significant role in the protection of fish intestine in vivo and in vitro through the induction of key antioxidant protection.
这项工作的目的主要是探索赖氨酸在体内对鱼类以及在体外对肠细胞氧化损伤的保护作用途径。首先，用含有六个梯度水平赖氨酸（7.1 - 19.6 g/kg饲料）的饲料喂养草鱼56天。其次，在肠细胞用6 mg/L铜处理24小时之前（预处理）、同时（共处理）或之后（后处理），用不同浓度的赖氨酸（培养基中0 - 300 mg/L）处理肠细胞。结果表明，赖氨酸改善了草鱼的生长性能。同时，赖氨酸通过提高抗氧化酶（超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GPx）、谷胱甘肽 - S - 转移酶（GST）和还原酶（GR））的基因表达和活性以及鱼肠道中核因子红细胞2相关因子2（Nrf2）的mRNA水平，减轻了脂质和蛋白质氧化。体外研究表明，用3 - （4,5 - 二甲基噻唑 - 2 - 基） - 2,5 - 二苯基四氮唑溴盐（MTT）OD值、碱性磷酸酶（ALP）和乳酸脱氢酶活性以及蛋白质羰基（PC）、丙二醛（MDA）和8 - 羟基脱氧鸟苷含量来衡量，赖氨酸共处理和后处理对铜诱导的鱼类原代肠细胞氧化损伤具有显著的保护作用。此外，与仅暴露于铜的组相比，赖氨酸共处理降低了细胞SOD、GPx、GST和GR的活性以及mRNA水平。信号分子Nrf2的基因表达与SOD活性呈现相同模式，而类 Kelch 样 ECH 相关蛋白1b（Keap1b）则呈现相反趋势，表明赖氨酸共处理诱导了通过Nrf2途径抵御氧化应激的抗氧化酶。此外，赖氨酸后处理增加了蛋白酶体活性，并阻止了铜刺激的GST以及相关过氧化氢酶（CAT）的mRNA水平升高和GST活性增加（P<0.01和P<0.001）。GR活性和基因表达以及谷胱甘肽（GSH）含量与GST活性呈现相反趋势（P<0.05）。因此，赖氨酸后处理提高了蛋白质和DNA修复能力，并改善了肠细胞的细胞氧化还原状态。总体结果表明，赖氨酸通过诱导关键的抗氧化保护作用，在体内和体外对鱼类肠道的保护中发挥着重要作用。
