Li Huatao, Zhou Xiaoqiu, Wu Min, Deng Mengling, Wang Chao, Hou Jingjing, Mou Pengju
College of Life Sciences, Neijiang Normal University, Neijiang 641000, China.
Conservation and Utilization of Fishes Resources in the Upper Reaches of the Yangtze River Key Laboratory of Sichuan Province, Neijiang Normal University, Neijiang 641000, China.
Anim Nutr. 2016 Dec;2(4):376-382. doi: 10.1016/j.aninu.2016.08.004. Epub 2016 Aug 12.
Erythrocytes play an essential role in transporting O and CO for respiration in fish. However, erythrocytes continuously suffer from reactive oxygen species (ROS) -induced oxidative stress and apoptosis. Thus, it is essential to expand our knowledge of how to protect erythrocytes against ROS-induced oxidative stress and apoptosis in fish. In this study, we explored the cytotoxicity and the effects of butylated hydroxyanisole (BHA), ethyl ether extracts, ethyl acetate extracts, acetone extracts (AE), ethanol extracts, and aqueous extracts of (EAm) on hydroxyl radical (•OH)-induced apoptosis in carp erythrocytes. The rat hepatocytes and carp erythrocytes were incubated with different concentrations of BHA or EAm(0.125 to 1 mg/mL). The toxicity in rat hepatocytes and carp erythrocytes was then measured using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and a haemolysis assay, respectively. The carp erythrocytes were treated with BHA or EAm in the presence of 40 μmol/L FeSO and 20 μmol/L HO at 37 °C, except for the control group. Oxidative stress and apoptosis parameters in the carp erythrocytes were then evaluated using the commercial kit. The results indicated that at high concentrations, BHA and EAm could induce toxicity in rat hepatocytes and fish erythrocytes. However, BHA was more toxic than EAm at the same concentrations. Moreover, the toxicity order of BHA and EAm in the fish erythrocytes approximately agreed with that for the rat hepatocytes. Butylated hydroxyanisole and EAm suppressed the •OH-induced phosphatidylserine exposure and DNA fragmentation (the biomarkers of apoptosis) by decreasing the generation of ROS, inhibiting the oxidation of cellular components, and restoring the activities of antioxidants in carp erythrocytes. Of all of the examined EAm, the AE showed the strongest effects. The effects of AE on superoxide anion, HO, met-haemoglobin and reduced glutathione levels, as well as glutathione reductase activity and apoptosis were equivalent to or stronger than those of BHA. These results revealed that the AE of could be used as a potential natural antioxidant or apoptosis inhibitor in fish erythrocytes.
红细胞在鱼类呼吸过程中运输氧气和二氧化碳方面发挥着重要作用。然而，红细胞不断遭受活性氧（ROS）诱导的氧化应激和凋亡。因此，有必要拓展我们对如何保护鱼类红细胞免受ROS诱导的氧化应激和凋亡的认识。在本研究中，我们探究了丁基羟基茴香醚（BHA）、乙醚提取物、乙酸乙酯提取物、丙酮提取物（AE）、乙醇提取物和水提取物对鲤鱼红细胞中羟基自由基（•OH）诱导的凋亡的细胞毒性及影响。将大鼠肝细胞和鲤鱼红细胞与不同浓度的BHA或水提取物（EAm）（0.125至1毫克/毫升）孵育。然后分别使用3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴盐（MTT）法和溶血试验测定大鼠肝细胞和鲤鱼红细胞中的毒性。除对照组外，在37℃下将鲤鱼红细胞与40微摩尔/升硫酸亚铁和20微摩尔/升过氧化氢一起用BHA或EAm处理。然后使用商业试剂盒评估鲤鱼红细胞中的氧化应激和凋亡参数。结果表明，在高浓度下，BHA和EAm可诱导大鼠肝细胞和鱼类红细胞产生毒性。然而，在相同浓度下，BHA的毒性比EAm更大。此外，BHA和EAm在鱼类红细胞中的毒性顺序与大鼠肝细胞中的大致一致。丁基羟基茴香醚和EAm通过减少ROS的产生、抑制细胞成分的氧化以及恢复鲤鱼红细胞中抗氧化剂的活性，抑制了•OH诱导的磷脂酰丝氨酸暴露和DNA片段化（凋亡的生物标志物）。在所有检测的EAm中，AE显示出最强的效果。AE对超氧阴离子、过氧化氢、高铁血红蛋白和还原型谷胱甘肽水平以及谷胱甘肽还原酶活性和凋亡的影响与BHA相当或更强。这些结果表明，的AE可作为鱼类红细胞中潜在的天然抗氧化剂或凋亡抑制剂。
