纳豆抗氧化肽对H2O2诱导HEK293细胞氧化应激损伤的保护作用

Protective effects of natto antioxidant peptide against H2O2-induced oxidative stress injury in HEK293 cells

中文关键词:  纳豆抗氧化肽  超滤  HEK293细胞  氧化损伤  保护作用

英文关键词:natto antioxidant peptide  ultrafiltration  HEK293 cells  oxidative damage  protective effect

中文摘要:

      目的 评价纳豆肽的抗氧化能力以及对对H2O2诱导HEK293细胞氧化应激损伤的保护作用。方法 首先以1,1-二苯基-2-三硝基苯(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基(DPPH·)、2,2’-联氮-二(3-乙基-苯并噻唑啉-6-磺酸)二铵盐[2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt, ABTS]阳离子自由基(ABTS+·)、羟自由基(·OH)和超氧阴离子自由基(·O2-)清除能力以及总还原能力为指标, 测定酶解得到纳豆肽粗品的抗氧化能力。利用超滤技术对纳豆肽进行分离, 测定分离后各组分在不同浓度下对DPPH·及ABTS+·清除活性。将活性最强的两个组分作为纳豆抗氧化肽, 测定其对H2O2诱导氧化损伤HEK293细胞抗氧化酶含量的影响, 评价其对氧化损伤HEK293细胞的保护效果。结果 纳豆肽粗品在8.00 mg/mL时具有良好的DPPH·、ABTS+·、·OH和·O2-清除能力以及总还原能力。超滤后得到了相对分子质量分别大于30、10~30、3~10、小于3 kDa的4个纳豆肽组分, 其对DPPH·及ABTS+·清除活性均随着质量浓度的增加呈现上升趋势, 特别是在8 mg/mL时, 相对分子质量为3~10 kDa和小于3 kDa组分表现出最强的抗氧化活性, 后续的细胞试验表明, 这两个组分能够显著提高氧化应激下HEK293细胞的存活率。在300 μg/mL的剂量范围内, 小于3 kDa组分的纳豆抗氧化肽可使细胞存活率恢复至未损伤时的水平, 而3~10 kDa组分也使损伤细胞的存活率提高了40.8%。同时, 纳豆抗氧化肽均能提高氧化损伤HEK293细胞中的超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化酶(glutathione peroxidase, GPX)和过氧化氢酶(catalase, CAT)等抗氧化酶的含量, <3 kDa组分和3~10 kDa组分的纳豆抗氧化肽分别使SOD含量提高了49.52%和50.80%, GPX含量提高了49.52%和50.81%, CAT含量提高了93.64%和91.97%。结论 纳豆肽具有抗氧化潜力, 纳豆抗氧化肽可以有效地缓解H2O2诱导的HEK293细胞的氧化应激损伤, 为纳豆抗氧化肽在功能食品中的应用提供理论依据。

英文摘要:

      Objective To evaluate the antioxidant capacity of natto peptides and protective effects against H2O2-induced oxidative stress injury in HEK293 cells. Methods Firstly, 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (DPPH·), 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) ammonium salt (ABTS) cation radical (ABTS+·), hydroxyl radical (·OH) and superoxide anion radical (·O2-) scavenging capacity and total reducing capacity were used to measure the antioxidant capacity of crude natto peptides. Natto peptides were separated by ultrafiltration technology, and the DPPH· and ABTS+· scavenging activities of each fraction at different concentrations were determined. The 2 most active components were used as natto antioxidant peptides, and their effects on the content of antioxidant enzymes in HEK293 cells with H2O2-induced oxidative damage were determined to evaluate the protective effect on HEK293 cells with oxidative damage. Results Crude natto peptide showed good DPPH·, ABTS+·, ·OH and ·O2- scavenging abilities and total reducing ability at 8.00 mg/mL. After ultrafiltration, 4 fractions of natto peptides with relative molecular masses of more than 30 kDa, 10?30 kDa, 3?10 kDa and less than 3 kDa were obtained. Their DPPH· and ABTS+· scavenging activities showed an upward trend with the increase of mass concentration, especially at 8 mg/mL, the fraction with relative molecular mass of 3?10 kDa and less than 3 kDa showed the strongest antioxidant activity, and subsequent cell experiments showed that these 2 fractions could significantly improve the survival rates of HEK293 cells under oxidative stress. In the dose of 300 μg/mL, the less than 3 kDa fraction of natto antioxidant peptide restored the cell viability to the uninjured level, while the 3?10 kDa fraction also increased the survival rate of the injured cells by 40.8%. At the same time, natto antioxidant peptide could increase the content of superoxide dismutase (SOD), glutathione peroxidase (GPX) and catalase (CAT) in HEK293 cells with oxidative damage, the less than 3 kDa and 3?10 kDa natto antioxidant peptide could increase the content of SOD by 49.52% and 50.80%, respectively, increase the content of GPX by 49.52% and 50.81%, and increase the content of CAT by 93.64% and 91.97%, respectively. Conclusion Natto peptide has antioxidant potential. Natto antioxidant peptide can effectively alleviate H2O2-induced oxidative stress damage in HEK293 cells, which provides theoretical basis for the application of natto antioxidant peptide in functional foods.