许珂,缪秉陶,曹洪杰,任祥雨,李航婷,杨最素

• 1:浙江海洋大学食品与药学学院

摘要(Abstract)：

【目的】为探讨鮟鱇（Lophius litulon）鱼皮活性肽（Monkfish skin peptides,MSP）对棕榈酸（Palmitic acid,PA）诱导人肾皮质近曲小管上皮细胞（HK-2细胞）损伤保护的作用。【方法】以新鲜鮟鱇鱼皮为材料，选取加酶量、提取时间、pH、温度和料液比为因子，以1,1-二苯基-2-三硝基苯肼自由基（DPPH·）清除率为响应值，分别进行单因素实验和响应面实验，优化鮟鱇鱼皮肽的提取工艺；对得到的鮟鱇鱼皮肽经过超滤、葡聚糖凝胶过滤层析和全自动高压制备分离纯化得到MSP；利用液相二级质谱和多肽从头（de novo）测序技术对其进行结构分析，筛选抗氧化活性较强的肽段；通过构建PA诱导的HK-2细胞损伤模型，测定MSP对HK-2细胞中氧化应激指标、脂质代谢指标和炎症因子水平的影响。【结果】确定酸性蛋白酶与胃蛋白酶质量比2∶1的复配比例，加酶量3 800 U/g、pH 3、酶解时间4 h、温度50℃和料液比1 g∶10 mL为鮟鱇鱼皮活性肽的最佳酶解条件，此条件下制备的鮟鱇鱼皮肽对DPPH·的清除率为78.84%；经超滤，酶解多肽中<3 ku组分抗氧化活性最强，收集该组分进行凝胶色谱分离后，得到F1、F2和F3共3个峰，其中F1组分DPPH·清除率最高。F1组分经高效液相色谱法（HPLC）分离纯化后得到6个吸收峰，其中H2组分吸收峰强度最高，经氨基酸序列鉴定，从中筛选出3种寡肽，分别为Phe-Glu-Ala-Thr-Ser-Gln-Glu-Leu(FEATSQEL,FL-8)、Leu-Val-Ser-Cys-Ser-Ser-Leu(LVSCSSL,LL-7)和Pro-Leu-Glu-Asn-Tyr(PLENY,PY-5)。3条多肽在HK-2细胞脂质损伤模型中的修复作用各不相同：FL-8和PY-5能提高HK-2细胞模型的抗氧化能力，使细胞内谷胱甘肽过氧化物酶（GSH-Px）活性和总抗氧化能力（T-AOC）显著升高，降低细胞中活性氧的荧光强度，并显著降低丙二醛（MDA）水平，而LL-7能降低GSH-Px活性与T-AOC能力，能显著降低MDA的含量；在调节细胞的脂质代谢方面，FL-8与PY-5分别在不同程度上降低总胆固醇、甘油三酯和低密度脂蛋白-胆固醇含量，并显著增加高密度脂蛋白-胆固醇含量，而LL-7则会加剧细胞内的脂质蓄积；FL-8与PY-5显著降低炎症因子白细胞介素-18(IL-18)的水平，而LL-7则相反（α=0.05）。【结论】制备的MSP对PA诱导的HK-2具有保护作用，可为鮟鱇鱼皮活性肽的利用和缓解脂质肾毒性产品的开发提供理论基础和实验依据。

关键词(KeyWords)： 鮟鱇鱼皮;活性肽;分离纯化;HK-2细胞;保护

Abstract:

Keywords:

基金项目(Foundation): 浙江省公益技术研究项目（LGN21D060002）

作者(Author): 许珂,缪秉陶,曹洪杰,任祥雨,李航婷,杨最素

参考文献(References)：

• [1]何云.鮟鱇鱼骨的综合利用研究[D].上海:上海海洋大学, 2018.
• [2]张文,阎洁,梅光明,等.鮟鱇鱼皮营养成分分析与评价[J].浙江海洋大学学报(自然科学版), 2019, 38(4):322-327.
• [3]马华威,杨会成,付万冬,等.鮟鱇鱼皮胶原蛋白肽的抗氧化活性[J].食品科学, 2014, 35(9):80-84.
• [4]杨会成,郑斌,廖妙飞,等.鮟鱇皮胶原蛋白肽最佳制备工艺及自由基清除活性研究[J].食品工业科技, 2014, 35(20):159-164.
• [5] ZHANG W, ZHENG J W, TIAN X X, et al. Pepsin-soluble collagen from the skin of litulon:a preliminary study evaluating physicochemical, antioxidant, and wound healing properties[J]. Marine Drugs, 2019, 17(12):708.
• [6]曹洪杰,张文,缪秉陶,等.鮟鱇鱼皮酶溶胶原蛋白肽对小鼠非酒精性脂肪肝的影响及作用机制[J].食品科学技术学报, 2023, 41(3):72-84.
• [7] MARKOVA I, MIKLANKOVA D, HüTTL M, et al. The effect of lipotoxicity on renal dysfunction in a nonobese rat model of metabolic syndrome:a urinary proteomic approach[J]. Journal of Diabetes Research, 2019, 2019:8712979.
• [8] NAM G E, CHO K H, HAN K, et al. Obesity, abdominal obesity and subsequent risk of kidney cancer:a cohort study of 23.3 million East Asians[J]. British Journal of Cancer,2019, 121(3):271-277.
• [9] PARK J H, KIM J Y, KIM S H, et al. A latent class analysis of dietary behaviours associated with metabolic syndrome:a retrospective observational cross-sectional study[J]. Nutrition Journal, 2020, 19(1):116.
• [10] CASTRO B B A, FORESTO-NETO O, SARAIVA-CAMARA N O, et al. Renal lipotoxicity:Insights from experimental models[J]. Clinical and Experimental Pharmacology&Physiology, 2021, 48(12):1579-1588.
• [11] MIAO B T, ZHENG J W, ZHENG G P, et al. Using collagen peptides from the skin of monkfish(Lophius litulon)to ameliorate kidney damage in high-fat diet fed mice by regulating the Nrf2 pathway and NLRP3 signaling[J].Frontiers in Nutrition, 2022, 9:798708.
• [12]李洁.龙头鱼骨胶原蛋白肽的制备及其对糖尿病防治的分子机制研究[D].舟山:浙江海洋大学, 2022.
• [13]杨志友,马智慧,张永平,等.岩藻多糖对Aβ25-35诱导神经元凋亡和突起萎缩的抑制作用[J].广东海洋大学学报, 2021, 41(4):77-83.
• [14]张崟,郭思亚,熊伟,等.胶原蛋白酶解制备肽工艺研究进展[J].中国调味品, 2018, 43(2):89-95.
• [15] HU G H, DOU L, ZHANG J, et al. Exploring the antioxidant stability of sheep bone protein hydrolysate-identification and molecular docking[J]. LWT, 2024, 192:115682.
• [16] XIANG Z D, XUE Q, GAO P, et al. Antioxidant peptides from edible aquatic animals:Preparation method, mechanism of action, and structure-activity relationships[J]. Food Chemistry, 2023, 404:134701.
• [17] ZHONG S Y, MA C W, LIN Y C, et al. Antioxidant properties of peptide fractions from silver carp(Hypophthalmichthys molitrix)processing by-product protein hydrolysates evaluated by electron spin resonance spectrometry[J]. Food Chemistry, 2011, 126(4):1636-1642.
• [18] ZOU T B, HE T P, LI H B, et al. The structure-activity relationship of the antioxidant peptides from natural proteins[J]. Molecules, 2016, 21(1):72.
• [19] MITROFANOVA A, MERSCHER S, FORNONI A. Kidney lipid dysmetabolism and lipid droplet accumulation in chronic kidney disease[J]. Nat Rev Nephrol, 2023, 19(10):629-645.
• [20] YAN X, TAN X Y, LI Y X, et al. A stepwise targeting curcumin derivative, Ser@TPP@CUR, for acute kidney injury[J]. ACS Medicinal Chemistry Letters, 2022, 13(4):554-559.
• [21] DEL RIO D, STEWART A J, PELLEGRINI N. A review of recent studies on malondialdehyde as toxic molecule and biological marker of oxidative stress[J]. Nutrition, Metabolism and Cardiovascular Diseases, 2005, 15(4):316-328.
• [22] ZHANG J, LI M, ZHANG G N, et al. Identification of novel antioxidant peptides from snakehead(Channa argus)soup generated during gastrointestinal digestion and insights into the anti-oxidation mechanisms[J]. Food Chemistry,2021, 337:127921.
• [23] D’AGATI V D, CHAGNAC A, DE VRIES A P J, et al.Obesity-related glomerulopathy:clinical and pathologic characteristics and pathogenesis[J]. Nature Reviews Nephrology, 2016, 12(8):453-471.