远东拟沙丁鱼抗氧化肽对Caco-2细胞氧化应激损伤的影响

Effect of Antioxidant Peptide from Sardinops sagax on Oxidative Stress Injury on Caco-2 Cell

对远东拟沙丁鱼（Sardinopssagax）蛋白进行蛋白酶解和膜分离，得到分子质量100~3000u（F1）、3000~10000u（F2）和大于10000u（F3）的多肽组分。在生化水平上测定各组分对1,1-二苯基-2-三硝基苯肼（DPPH）、2,2-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐（ABTS）、羟基自由基和过氧自由基清除能力；在细胞水平上，以H2O2对Caco-2细胞的毒性及细胞内抗氧化体系的影响为检测指标，筛选最适浓度的H2O2建立细胞氧化应激模型；在建立该模型的基础上，进一步检测蛋白多肽F1对氧化应激损伤细胞的修复功能。结果表明：组分F1对DPPH、ABTS、羟基自由基和过氧自由基清除能力最强，清除率分别为56.03±0.14、86.26±0.32、38.34±1.70、54.85±0.75；100μmol/L的H2O2对细胞毒性作用较小，并可降低细胞中谷胱甘肽过氧化物酶（GSH-Px）、总抗氧化物歧化酶（T-SOD）、过氧化物酶（POD）的活性和谷胱甘肽（GSH）的含量，增加丙二醛（MDA）的含量，细胞氧化应激模型选择H2O2浓度为100μmol/L；在该模型条件下，F1对受损的Caco-2细胞中抗氧化体系中的GSH-Px、T-SOD、POD的活性和MDA、GSH的含量有一定恢复作用，可调节细胞中失衡的抗氧化体系。远东拟沙丁鱼多肽FI对细胞氧化应激受损有一定保护作用。

Polypeptides with different molecular weight F1 (100 – 3 000 u), F2 (3 000 –10 000 u) and F3(> 10 000 u) were prepared by the proteolysis and membrane separation from Sardinops sagax. At thebiochemical level, the antioxidant activity of these fractions was determined by its capacities ofscavenging DPPH, ABTS, OH and O2 free radicals. At the cellular level, using cell toxic and the changeof intracellular antioxidant system by the H2O2 as testing indexes, the cell model of oxidative stress withthe optimum concentration of H2O2 was established. On the basis of the model, whether F1 can repairthe Caco-2 cells oxidative damage or not was detected. The results showed that F1 had the highest activity at the biochemical level. The DPPH, ABTS, OH, and O2 free radicals capacity were(56.03±0.14)%, (86.26±0.32)%, (38.34±1.70)%, and (54.85±0.75)%, respectively.The toxic Effects of100 μmol/L H2O2 on Caco-2 cell was low and the antioxidant enzyme system of GSH-Px, T-SOD, andPOD activity and the content of GSH decreased but the content of MDA increased . It is indicated thatthe Caco-2 cells model of oxidative stress which was established by 100 μmol/L H2O2 is the best. Underthe condition of the model, F1 can repair the antioxidant enzyme system of GSH-Px, T-SOD, and PODactivity and the content of MDA and GSH in damaged Caco-2 cells. The final result showed that F1could adjust cell imbalance in antioxidant enzyme system and repair oxidative stress damage.