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目的:抗氧化性丝素蛋白(SF)胰酶水解工艺的优化。研究水解物对过氧化氢(H2O2)造成人肝癌Hep G2细胞损伤的改善作用,对其机制进行初步探讨。方法:以超氧自由基清除率为指标,设计正交试验考察丝素蛋白胰酶水解过程中反应时间、p H、温度、底物浓度、酶浓度对水解工艺的影响。测定水解产物的分子量分布及其对羟基自由基清除率和H2O2诱导的红细胞氧化溶血抑制率。用不同浓度H2O2处理细胞24 h,测定细胞活性,选择合适浓度的H2O2损伤细胞。然后再将细胞分为空白组(不加药物处理),H2O2损伤组,H2O2损伤+5 mmol·L-1N-乙酰半胱胺酸(NAC)治疗组,H2O2损伤+10,20,30,50 g·L-1SF治疗组。观察细胞活性,丙二醛(MDA)含量,肿瘤坏死因子-α(TNF-α)含量,超氧化物歧化酶(SOD)活性,过氧化氢酶(CAT)活性和总抗氧化能力(T-AOC)的变化。结果:最佳水解工艺是酶浓度6%,反应时间160 min,底物质量浓度20 g·L-1,p H 8,温度38℃,此水解条件下所得水解液对超氧自由基清除率为72.73%,水解物分子量在10 k Da以下,对羟基自由基清除率和红细胞氧化溶血抑制率均提高。H2O2损伤细胞后细胞活性降低并且细胞中MDA含量增加(P<0.05)。与H2O2损伤组比较SF治疗组中细胞活性升高,MDA的含量也降低(P<0.05)。H2O2损伤细胞后,TNF-α含量上升,SOD,CAT的活性和T-AOC能力均下降(P<0.05),SF治疗能降低TNF-α含量,提高SOD,CAT分泌量和增强T-AOC能力(P<0.05)。结论:抗氧化性丝素蛋白水解物对过氧化氢致Hep G2细胞损伤有改善作用,机制是通过降低TNF-α含量,提高细胞活力和增加细胞中抗氧化酶活性。
Objective: To optimize the enzymatic hydrolysis process of antioxidant silk fibroin (SF). To study the effect of hydrolyzate on the injury of Hep G2 cells induced by hydrogen peroxide (H2O2), and to explore its mechanism. Methods: The effects of reaction time, p H, temperature, substrate concentration and enzyme concentration on the hydrolysis process of silk fibroin trypsin were studied using orthogonal test to study the scavenging rate of superoxide radical. Determination of the molecular weight distribution of the hydrolyzate and its clearance of hydroxyl radicals and H2O2-induced erythrocyte oxidation hemolysis inhibition rate. Cells were treated with different concentrations of H2O2 for 24 h, cell viability was measured, and H2O2 injury cells were selected. Then the cells were divided into blank group (no drug treatment), H2O2 injury group, H2O2 injury +5 mmol·L-1 N-acetyl cysteine (NAC) treatment group, H2O2 injury +10,20,30,50 g · L-1SF treatment group. Cell viability, malondialdehyde (MDA) content, tumor necrosis factor-α (TNF-α) content, superoxide dismutase (SOD) activity, catalase (CAT) activity and total antioxidant capacity (T- AOC) changes. Results: The optimal hydrolysis conditions were as follows: enzyme concentration 6%, reaction time 160 min, substrate concentration 20 g · L-1, p H 8, temperature 38 ℃. The hydrolysis rate of the obtained hydrolyzate to superoxide radical scavenging rate Was 72.73%. The molecular weight of hydrolyzate was less than 10 kDa, the hydroxyl radical scavenging rate and erythrocyte oxidation and hemolysis inhibition rate increased. H2O2 injury cells decreased cell viability and MDA content increased (P <0.05). Compared with H2O2 injury group, the activity of cell increased and the content of MDA decreased in SF group (P <0.05). The content of TNF-α, the activity of SOD and CAT and the ability of T-AOC decreased after H2O2 injury (P <0.05). SF treatment could decrease the content of TNF-α, increase the secretion of SOD and CAT and enhance the ability of T-AOC (P <0.05). CONCLUSION: Antioxidant silk fibroin hydrolyzate can improve the injury of Hep G2 cells induced by H2O2 by decreasing the content of TNF-α, increasing cell viability and increasing antioxidant enzyme activity in cells.