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 H_(2)O_(2)-induced oxidative stress injury in HEK293 cells

目的评价纳豆肽的抗氧化能力以及对对H_(2)O_(2)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(·O^(2-))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H_(2)O_(2)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·O^(2-)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300μg/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H_(2)O_(2)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。

Objective To evaluate the antioxidant capacity of natto peptides and protective effects against H_(2)O_(2)-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(·O^(2-))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 H_(2)O_(2)-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·O 2-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 300μg/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 H2 O 2-induced oxidative stress damage in HEK293 cells,which provides theoretical basis for the application of natto antioxidant peptide in functional foods.