蛋清源活性肽对过氧化氢诱导的HEK293细胞抗氧化酶活力及白细胞介素8分泌的影响

Effects of Egg White-Derived Bioactive Peptide on Antioxidative Enzyme Activities and IL-8 Secretion in HEK293 Cells under Oxidative Stress Induced by Hydrogen Peroxide

食源性抗氧化肽由于抗氧化能力较好、安全性高,逐渐成为多肽研究领域的热点。本实验以H_2O_2诱导HEK293细胞损伤建立氧化损伤模型,研究了蛋清源活性肽WNWAD(Trp-Asn-Trp-Ala-Asp)及其结构改造肽WNW(Trp-Asn-Trp)、WAD(Trp-Ala-Asp)、WN(Trp-Asn)对H_2O_2诱导损伤的HEK293细胞的存活率、活性氧(reactive oxygen species,ROS)水平、抗氧化酶活力及细胞因子白介素8(interleukin-8,IL-8)分泌的影响。结果表明:蛋清源活性肽对HEK293细胞存活率的影响极显著(P<0.01),不同浓度蛋清源活性肽WNWAD、WNW、WAD、WN作用于氧化损伤的HEK293细胞后,其存活率明显提高,呈现浓度依赖性;其中1.0μmol/L蛋清源活性肽WNWAD、WNW、WAD、WN处理细胞后,存活率从损伤组的(48.0±2.4)%分别提高到(99.7±1.8)%、(69.4±3.2)%、(78.9±5.1)%、(72.1±3.6)%,与损伤组有极显著性差异(P<0.01);H_2O_2诱导HEK293细胞内ROS的产生,经过活性肽预处理后细胞内ROS的水平极显著降低(P<0.01);随着蛋清源活性肽WNWAD、WNW、WAD、WN浓度的升高,总超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶3种酶的活力均有所提高,呈现浓度依赖型关系。最后利用酶联免疫吸附测定试剂盒测定了HEK293细胞中IL-8的分泌量,经过H_2O_2处理的HEK293细胞上清液中IL-8含量极显著升高(P<0.01),加入WNWAD、WNW、WN、WAD后,细胞中的IL-8含量均有所降低,其中WNWAD和WN影响显著(P<0.05)。以上结果显示蛋清源活性肽对降低HEK293细胞氧化应激水平和提高细胞抗氧化能力有积极的影响。

In recent years,antioxidant peptides with high safety and potent antioxidant capacity have become a hot research field.The aim of this study was to investigate the effects of egg white-derived bioactive peptides WNWAD(Trp-Asn-Trp-Ala-Asp),WNW(Trp-Asn-Trp),WAD(Trp-Ala-Asp)and WN(Trp-Asn)on the survival rate,reactive oxygen species(ROS)content,antioxidative enzyme activities and interleukin-8(IL-8)level in HEK293 cells under oxidative stress induced by H2O2.MTS assay results showed that all four peptides significantly dose-dependently inhibited H2O2-induced cellular oxidative stress in HEK-293 cells and increased the survival rate increased from(48.0±2.4)%to(99.7±1.8)%,(69.4±3.2)%,(78.9±5.1)%,and(72.1±3.6)%at 1.0μmol/L concentration,respectively.These changes were statistically significant(P<0.01).2’,7’-Dichlorofluorescin diacetate(DCFH-DA)fluorescent probe assay proved that all these peptides inhibited oxidative stress by reducing intracellular ROS accumulation in HEK293 cells.They dose-dependently increased the activity of total superoxide dismutase(T-SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)in HEK293 cells.In addition,the results of enzyme-linked immune sorbent assay(ELISA)showed that WNWAD and WN up-regulated IL-8 secretion(P<0.05).All the above results showed that the egg white-derived peptides play a positive role in protecting HEK293 cells against H2O2-induced oxidative stress and improving antioxidant capacity in HEK293 cells.