食品科学 ›› 2018, Vol. 39 ›› Issue (19): 169-174.doi: 10.7506/spkx1002-6630-201819026

• 营养卫生 • 上一篇    下一篇

小米抗氧化肽的纯化及其抑制H2O2诱导损伤的胰岛素瘤细胞氧化应激作用

王 帅,刘剑利,霍雅鹏,阎 笑,田思琪,贺 音,曹向宇*   

  1. 辽宁大学生命科学院,辽宁 沈阳 110036
  • 出版日期:2018-10-15 发布日期:2018-10-24
  • 基金资助:
    国家自然科学基金面上项目(31770017);辽宁省农业领域青年科技创新人才培养资助计划项目(2015013);辽宁省博士科研启动基金项目(20170520258);辽宁省高等学校优秀人才支持计划项目(LJQ2013002);辽宁省高等学校科学研究一般项目(L2014007)

Purification of Antioxidant Peptide from Millet and Its Protective Effect against Oxidative Stress Induced by H2O2 in INS-1 Cells

WANG Shuai, LIU Jianli, HUO Yapeng, YAN Xiao, TIAN Siqi, HE Yin, CAO Xiangyu*   

  1. School of Life Science, Liaoning University, Shenyang 110036, China
  • Online:2018-10-15 Published:2018-10-24

摘要: 本研究利用Sephadex G-25和DEAE-32对小米抗氧化肽进行纯化,利用醋酸纤维素薄膜电泳法测定小米抗氧化肽的纯度,以1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl,DPPH)自由基、超氧阴离子自由基、羟自由基清除实验考察纯化的小米抗氧化肽对自由基的清除能力。在细胞水平研究小米抗氧化肽对胰岛细胞的保护作用,利用H2O2进行大鼠胰岛素瘤细胞(INS-1)氧化应激造模,采用2’,7’-二氯二氢荧光素二乙酸酯荧光探针法检测细胞内活性氧(reactive oxygenspecies,ROS)水平,3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法检测细胞活力,采用流式细胞技术检测细胞凋亡率,采用实时荧光定量聚合酶链式反应检测抗氧化酶的表达水平。结果表明:经过Sephadex G-25和DEAE-32两次层析,小米抗氧化肽达到了电泳纯;50 μg/mL小米抗氧化肽对超氧阴离子自由基、羟自由基和DPPH自由基的清除率分别为(62.71±3.86)%、(73.56±4.51)%和(82.62±5.25)%;小米抗氧化肽能显著提升H2O2损伤后细胞活力,降低细胞内ROS水平,抑制H2O2诱导损伤的INS-1细胞的凋亡(P<0.05),其可能的机制与超氧化物歧化酶1、过氧化氢酶、谷胱甘肽巯基转移酶、醌氧化还原酶1等抗氧化酶系的表达上调有关。结论:小米抗氧化肽对H2O2诱导损伤的INS-1细胞氧化应激具有一定的保护作用。

关键词: 小米抗氧化肽, 氧化应激, 胰岛细胞, 凋亡

Abstract: Antioxidant peptides were purified from millet protein hydrolysate by sequential column chromatographies on Sephadex G-25 and DEAE-32. Cellulose acetate electrophoresis was used to determine the purity of the purified peptides. The antioxidant effect was investigated by detecting radical scavenging activity against superoxide anion, hydroxyl and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. In order to investigate the protective effect of the antioxidant peptides on islet cells, an INS-1 cell model of oxidative stress induced by H2O2 was used. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide method was applied to detect the cell viability, flow cytometry was used to detect the cell apoptosis, and real-time polymerase chain reaction was employed to analyze the gene expression of antioxidant enzymes. The results showed that the peptides were purified to electrophoretic homogeneity. The percentage scavenging of superoxide anion, hydroxyl and DPPH radicals by the peptides were (62.71 ± 3.86)%, (73.56 ± 4.51)% and (82.62 ± 5.25)%, respectively. The peptides could significantly promote cell viability, reduce the level of cellular reactive oxygen species, and inhibit cell apoptosis (P < 0.05). The mechanism may be related to the up-regulated expression of antioxidant enzymes including superoxide dismutase 1, catalase enzymes, glutathione S-transferases, and quinone oxidoreductase 1. This study conclusively demonstrated that the antioxidant peptides from millet showed a significant protection against oxidative stress induced by H2O2 in INS-1 cells.

Key words: antioxidant peptides from millet, oxidative stress, islet cell, apoptosis

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