黑线鳕鱼皮胶原蛋白胰蛋白酶酶解多肽对UVB诱导HaCaT光损伤的抑制作用

目的:研究黑线鳕鱼(Melanogrammus aeglefinus)皮胶原蛋白胰蛋白酶酶解多肽的抗氧化、抑制光损伤作用,提高鱼皮的再利用价值。方法:利用单因素试验和响应面试验设计,探讨黑线鳕鱼皮胶原蛋白的最佳酶解工艺。将制得的酶解多肽通过基质辅助激光解离飞行时间串联质谱仪(matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry,MALDI-TOF MS/MS)测定多肽序列。利用Discovery Studio中CDOCKER模块将所得的优势肽与抗氧化相关的Kelch样环氧氯丙烷相关蛋白-1(Keap1)进行分子对接。用不同质量浓度的多肽预处理人表皮角质形成细胞(Ha Ca T)后,使用中波紫外线诱导HaCaT细胞构建光损伤模型,通过超氧化歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)活性、丙二醛(malondialdehyde,MDA)含量及细胞凋亡与周期来研究酶解多肽抑制光损伤的能力。结果:黑线鳕鱼皮胶原蛋白在胰蛋白酶质量分数1.23%、酶解温度52.95℃、酶解时间1.75 h条件下,水解度为78.33%。MALDI-TOF MS/MS测定出酶解液中的优势肽序列为V-IFFVTMGTP-R、L-SIVPV-R和L-MHT-T。通过分子对接发现V-IFFVTMGTP-R与Keap1结合最稳定,从而预测其具有抗氧化的作用。用250 mg/m L酶解液处理光损伤的HaCaT细胞,其SOD、GSHPx活性较模型组极显著提高(P0.01),MDA含量极显著降低(P0.01)。结论:黑线鳕鱼皮胶原蛋白胰蛋白酶酶解多肽具有抗氧化活性,对光损伤具有一定的抑制作用。

Inhibitory Effects of Peptides Derived from Tryptic Hydrolysate of Skin Collagen from Melanogrammus aeglefinus on UVB-Induced Photodamage in HaCaT Cells

Objective: This study aimed to examine the antioxidant activity of peptides produced by tryptic hydrolysis of skin collagen from Melanogrammus aeglefinus and its inhibitory effect against photodamage in an effort to improve the reuse potential of M. aeglefinus skin. Methods: One-factor-at-a-time method and response surface methodology were used in conjunction to determine the optimum hydrolysis process for skin collagen from M. aeglefinus. The obtained peptides were sequenced by matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF MS/MS). The dominant peptides were docked with Kelch-like ECH-associated protein 1 (Keap1) by using the CDOCKER module in Discovery Studio. At the same time, HaCaT cells were pretreated with different concentrations of the collagen hydrolysate and then induced by ultraviolet B (UVB) to establish a cellular model of photodamage. The activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) and the cell apoptosis and cycle were examined to test the ability of the peptides to inhibit photodamage. Results: Optimal hydrolysis conditions were determined as follows: hydrolysis time 1.75 h, enzyme concentration 1.23%, and temperature 52.95 ℃. The corresponding degree of hydrolysis was 78.33%. The dominant peptide sequences in the hydrolysate were identified as V-IFFVTMGTP-R, L-SIVPV-R and L-MHT-T. Molecular docking showed that the binding of V-IFFVTMGTPR to Keap1 was the most stable, thereby predicting that the peptide has antioxidant effect. The SOD and GSH-Px activities of HaCaT cells treated with the hydrolysate at 250 mg/mL were significantly higher than those of the model group (P < 0.01), accompanied by a significant decrease in MDA content (P < 0.01). Conclusion: The tryptic hydrolysis of collagen from M. aeglefinus skin has antioxidant activity, and an inhibitory effect on photodamage.