蛋清源抗氧化肽对HEK293细胞氧化应激损伤的抑制作用及机制
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国家自然科学基金项目(31471597);; 中央高校基本科研业务费专项(451170301197);; 吉林省科学技术厅重点实验室项目(20160622030JC)


The Anti-oxidative Effects and Mechanism of Antioxidant Peptides from Egg White against Oxidative Stress Injury in Human Embryonic Kidney 293 Cells
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    摘要:

    食源性抗氧化肽以其安全性高,抗氧化能力好,逐渐成为多肽研究领域的热点。本文以蛋清卵黏蛋白胃蛋白酶水解产物为原料,以蛋清源五肽作为研究对象,首先,采用基于不同机理的体外抗氧化活性化学测定方法,对8条蛋清源五肽的抗氧化活性进行评价。DPPH自由基清除活性最强的为五肽CFDVF,浓度为1.0 mmol/L时,其自由基清除率为50.14%±2.1%;ABTS+·自由基清除能力最强的为五肽VYQFL,浓度为100μmol/L时,TEAC值为(94.66±0.37)μmol/L TE/100μmol/L;ORAC活性最强的为五肽WNWAD,TEAC值为(2.53±0.18)μmol/L TE/μmol/L。接着,利用HEK293细胞氧化损伤模型评价8条五肽的抗氧化活性,其中五肽WNWAD(Trp-Asn-Trp-Ala-Asp)的氧化应激抑制活性最强。同时,细胞毒性试验证明,五肽本身对细胞无毒副作用,也无促进增殖作用。最后,综合体外化学和细胞试验结果,筛选出WNWAD进行蛋清肽氧化应激抑制机制研究。首先采用试剂盒方法检测细胞内LDH活性和MDA含量,以及抗氧化酶CAT、T-SOD和GSH-PX的活性,结果表明H2O2可导致细胞LDH释放率上升,MDA含量增高,而WNWAD可以抑制脂质过氧化进程,维持细胞膜完整性,提高细胞内LDH活性,降低MDA含量;H2O2可以抑制细胞内抗氧化酶CAT、T-SOD和GSH-PX的活性,而经过WNWAD预处理,可以显著增强其活性;随着肽浓度的升高,3种酶的活性均有所提高,呈浓度依赖型关系,尤其在肽的浓度为1.0μmol/L时,过氧化氢酶(CAT)活力为(20.63±0.51) U/mg蛋白、总超氧化物歧化酶(T-SOD)活力为(179.39±5.73)U/mg蛋白,谷胱甘肽过氧化物酶(GSH-PX)为(33.97±5.02)个活力单位,较对应损伤组的酶活性均有显著性提高(P<0.01);最后,利用蛋白质免疫印迹技术检测细胞内抗氧化酶相关蛋白表达水平,结果表明:WNWAD预处理可在一定程度上恢复H2O2诱导损伤的HEK293细胞中抗氧化酶CAT、SOD和GSH-PX的蛋白表达水平。

    Abstract:

    In recent years, antioxidant peptides with high safety, potent antioxidant capacity, become a hot research field. This research is mainly engaged in oxidative stress inhibitory activity of egg white peptides. This study takes the pepsin hydrolysates from egg white ovomucin as raw materials, and pentapeptides from egg white as research object. The antioxidant activity of pentapeptides from egg white protein were evaluated using different chemical methods with different mechanisms. The pentapeptide CFDVF showed extraordinary DPPH scavenging activity (50.14% ± 2.1% at the concentration of 1.0 mmol/L), VYQFL exhibited the strongest ABTS free radical scavenging ability [TEAC value = (94.66 ± 0.37) μmol/L TE/100 μmol/L], WNWAD showed extraordinary oxygen radical absorption capacity [ORAC value = (2.53 ± 0.18) μmol/L TE/μmol/L]. Secondly, in order to further examine the antioxidant activity of pentapeptides from egg white, the oxidative stress model of HEK293 cells was established by MTS assay. WNWAD (Trp-Asn-Trp-Ala-Asp) exhibited the strongest oxidative stress inhibitory activity. The pentapeptides showed no stimulatory or cytotoxic effects in HEK293 cells. Finally, the WNWAD was screened out to carry out the research on mechanism of pentapeptides from egg white against oxidative stress in human embryonic kidney 293 cells. The MDA production and cellular activity of LDH, CAT, T-SOD, and GSH-PX were measured by kits. The results showed that WNWAD inhibited cell oxidative stress by increasing intracellular LDH activity and reducing intracellular MDA content. Enzyme activity results displayed that WNWAD dose-dependently increased the activity of SOD1, CAT and GPx1 in HEK293 cells. The effect of WNWAD on the protein expression of SOD1, CAT and GPx1 in HEK293 cells was analyzed with western blotting. Compared to the control, a concentration-dependent up regulation of SOD1, CAT and GPx1 protein expression by the WNWAD could be seen.

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张燕;陈志飞;赵颂宁;张婷;刘静波;.蛋清源抗氧化肽对HEK293细胞氧化应激损伤的抑制作用及机制[J].中国食品学报,2019,19(10):11-22

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  • 在线发布日期: 2019-11-21
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