蝇蛆抗氧化肽对IPEC-J2细胞氧化应激损伤的保护作用

doi:10.16431/j.cnki.1671-7236.2023.05.011

摘要/Abstract

摘要： 【目的】以蝇蛆抗氧化肽(FTP)为研究对象,利用体外化学和细胞试验,探究FTP对DPPH和ABTS⁺·自由基清除能力以及对IPEC-J2细胞氧化应激损伤的保护作用。【方法】通过测定不同浓度(500、1 000、2 000、3 000、4 000 μg/mL)FTP对DPPH、ABTS⁺·自由基的清除率检测FTP的体外抗氧化能力;将IPEC-J2细胞用不同浓度(0、100、250、500、1 000、2 000 μg/mL)FTP培养,利用MTT法测定孵育24 h后各组细胞的存活率;利用MTT法筛选出适宜的过氧化氢(H₂O₂)浓度构建氧化应激模型;根据构建的氧化应激模型和FTP适宜的浓度梯度,将IPEC-J2细胞随机分为空白组、模型组、试验组(100、250、500、1 000 μg/mL),分别测定各组细胞存活率、活性氧(ROS)水平以及细胞内超氧化物歧化酶(SOD)、过氧化氢酶(CAT)活性和丙二醛(MDA)水平。【结果】当FTP浓度为4 000 μg/mL时DPPH和ABTS⁺·自由基的清除率分别达到了54.82%和81.90%;不同浓度FTP对IPEC-J2细胞均无生长抑制作用,细胞存活率均高于100%,呈浓度梯度性增长,且FTP浓度为2 000 μg/mL的细胞存活率是空白组的1.22倍;H₂O₂构建细胞氧化应激模型的适宜浓度为1 000 μmol/L。与空白组相比,模型组细胞存活率极显著降低,ROS和MDA水平极显著提高,SOD和CAT活性极显著降低(P＜0.01);与模型组相比,4个试验组细胞存活率均极显著增高(P＜0.01),500 μg/mL FTP组细胞存活率是模型组的2.37倍,500和1 000 μg/mL FTP组的ROS和MDA水平极显著降低(P＜0.05),SOD和CAT活性极显著或显著升高(P＜0.01;P＜0.05),且均呈剂量效应关系。【结论】FTP对IPEC-J2细胞氧化应激损伤有显著的保护作用,且呈剂量效应。

关键词: 蝇蛆抗氧化肽; 过氧化氢(H₂O₂); IPEC-J2细胞; 氧化应激

Abstract: 【Objective】 Taking the antioxidant peptide of fly maggot (FTP) as the research object,the protective effects of FTP on DPPH and ABTS⁺· free radicals scavenging ability and oxidative stress injury of IPEC-J2 cells were investigated using in vitro chemical and cell tests.【Method】 By measuring different concentrations of FTP (500,1 000,2 000,3 000 and 4 000 μg/mL) scavenging rate of DPPH,ABTS⁺· free radicals to detect the antioxidant capacity of FTP in vitro. IPEC-J2 cells were cultured with different concentrations of FTP (0,100,250,500,1 000 and 2 000 μg/mL),and the cell survival rate of each group was measured by MTT method after 24 h of incubation.The appropriate hydrogen peroxide H₂O₂ concentration was selected by MTT method to construct oxidative stress model.According to the constructed model of oxidative stress and the appropriate concentration gradient for FTP,IPEC-J2 cells were randomly divided into blank,model,and test groups (100,250,500 and 1 000 μg/mL),and the levels of cell viability,reactive oxygen species (ROS),and intracellular SOD,CAT and MDA were measured in each group.【Result】 When the concentration of FTP was 4 000 μg/mL,the scavenging rates of DPPH and ABTS⁺· radicals reached 54.82% and 81.90%,respectively.Different concentrations of FTP had no inhibitory effect on the growth of IPEC-J2 cells,and the cell survival rate was higher than 100%,showing a concentration gradient growth,and the cell survival rate of the FTP concentration of 2 000 μg/mL was 1.22 times higher than that of blank group.The suitable concentration for H₂O₂ to construct the cellular oxidative stress model was 1 000 μmol/L.Compared with blank group,the cell survival rate of model group was extremely significantly decreased,the levels of ROS and MDA were extremely significantly increased,and the activities of SOD and CAT were extremely significantly decreased (P＜0.01).Compared with model group,the cell survival rate of 4 test groups was extremely significantly increased (P＜0.01),and the cell survival rate of 500 μg/mL FTP group was 2.37 times that of model group.The levels of ROS and MDA in 500 and 1 000 μg/mL FTP groups were significantly decreased (P＜0.05),and the activities of SOD and CAT were extremely significantly or significantly increased(P＜0.01 or P＜0.05),both in a dose-effect relationship.【Conclusion】 FTP had a significant protective effect on oxidative stress injury of IPEC-J2 cells in a dose-dependent manner.

Key words: maggot antioxidant peptides; hydrogen peroxide (H₂O₂); IPEC-J2 cells; oxidative stress

中图分类号:

S811.3

李永强, 包瑞莹, 王琴, 邱平飞, 李笑春, 石慧宇, 张海文, 王学梅. 蝇蛆抗氧化肽对IPEC-J2细胞氧化应激损伤的保护作用[J]. 中国畜牧兽医, 2023, 50(5): 1836-1844.

LI Yongqiang, BAO Ruiying, WANG Qin, QIU Pingfei, LI Xiaochun, SHI Huiyu, ZHANG Haiwen, WANG Xuemei. Protective Effects of Antioxidant Peptides from Maggot on Oxidative Stress Injury of IPEC-J2 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1836-1844.

参考文献

[1] 李熙,何俊,宋宇琨,等.蝇蛆粉对黄羽肉鸡生长和屠宰性能、肉品质及血清生化指标的影响[J].饲料工业,2022,43(18):18-23. LI X,HE J,SONG Y K,et al.Effects of maggots powder on growth performance,slaughter performance,meat quality and serum biochemistry of broiler chickens[J].Feed Industry,2022,43(18):18-23.(in Chinese)
[2] SMETANA S,SCHMITT E,MATHYS A.Sustainable use of Hermetia illucens insect biomass for feed and food:Attributional and consequential life cycle assessment[J].Resources,Conservation and Recycling,2019,144:285-296.
[3] 杨东雪.白藜芦醇对过氧化氢诱导的IPEC-J2细胞凋亡的保护作用[D].长春:吉林大学,2017. YANG D X.Protective effect of resveratrol on H₂O₂-induced IPEC-J2 cells apoptosis[D].Changchun:Jilin University,2017.(in Chinese)
[4] CONTRERAS G A,STRIEDER-BARBOZA C,DE KOSTER J.Symposium review:Modulating adipose tissue lipolysis and remodeling to improve immune function during the transition period and early lactation of dairy cows[J].Journal of Dairy Science,2018,101(3):2737-2752.
[5] MULLIGAN F J,DOHERTY M L.Production diseases of the transition cow[J].The Veterinary Journal,2008,176(1):3-9.
[6] CONTRERAS G A,SORDILLO L M.Lipid mobilization and inflammatory responses during the transition period of dairy cows[J].Comparative Immunology,Microbiology and Infectious Diseases,2011,34(3):281-289.
[7] CAMPBELL J M,CRENSHAW J D,POLO J.The biological stress of early weaned piglets[J].Journal of Animal Science and Biotechnology,2013,4(2):124-127.
[8] ZHENG L,YU H,WEI H,et al.Antioxidative peptides of hydrolysate prepared from fish skin gelatin using ginger protease activate antioxidant response element-mediated gene transcription in IPEC-J2 cells[J].Journal of Functional Foods,2018,51:104-112.
[9] ZHOU Y,SHE X,CHEN Z,et al.Tartary buckwheat (Fagopyrum tataricum(L.) Gaertn) protein-derived antioxidant peptides:Mechanisms of action and structure-activity relationship in Caco-2 cell models[J].Food Science and Human Wellness,2022,11(6):1580-1590.
[10] MIRDAMADI S,MIRZAEI M,SOLEYMANZADEH N,et al.Antioxidant and cytoprotective effects of synthetic peptides identified from Kluyveromyces marxianus protein hydrolysate:Insight into the molecular mechanism[J].LWT-Food Science and Technology,2021,148:111792.
[11] ClLARKSON P M,THOMPSON H S.Antioxidants:What role do they play in physical activity and health?[J].The American Journal of Clinical Nutrition,2000,72(2 Suppl):637S-646S.
[12] GARCÍA-NEBOT M J,RECIO I,HERNÁNDEZ-LEDESMA B.Antioxidant activity and protective effects of peptide lunasin against oxidative stress in intestinal Caco-2 cells[J].Food and Chemical Toxicology,2014,65:155-161.
[13] DUARTE M E,ZHOU F X,DUTRA W M.et al.Dietary supplementation of xylanase and protease on growth performance,digesta viscosity,nutrient digestibility,immune and oxidative stress status,and gut health of newly weaned pigs[J].Animal Nutrition,2019,5(4):351-358.
[14] JAYARAMAN B,NYACHOTI C M.Husbandry practices and gut health outcomes in weaned piglets:A review[J].Animal Nutrition,2017,3(3):205-211.
[15] XIE Z J,HUANG J,XU X,et al.Antioxidant activity of peptides isolated from alfalfa leaf protein hydrolysate[J].Food Chemistry,2008,111(2):370-376.
[16] KONG B H,XIONG Y L.Antioxidant activity of zein hydrolysates in a liposome system and the possible mode of action[J].Journal of Agricultural and Food Chemistry,2006,54(16):6059-6068.
[17] GEENS M M,NIEWOLD T A.Optimizing culture conditions of a porcine epithelial cell line IPEC-J2 through a histological and physiological characterization[J].Cytotechnology,2011,63(4):415-423.
[18] ESSICK E E,WILSON R M,PIMENTEL D R,et al.Adiponectin modulates oxidative stress-induced autophagy in cardiomyocytes[J].PLoS One,2013,8(7):e68697.
[19] WANG S,ZHU S,ZHANG J,et al.Supplementation with yeast culture improves the integrity of intestinal tight junction proteins via NOD1/NF-κB P65 pathway in weaned piglets and H₂O₂-challenged IPEC-J2 cells[J].Journal of Functional Foods,2020,72:104058.
[20] 李佳艳,邬静,易金娥,等.钩吻素子对H₂O₂诱导的IPEC-J2细胞氧化应激和凋亡的影响[J].中兽医医药杂志,2019,38(3):4-7. LI J Y,WU J,YI J E,et al.Effect of koumine onoxidative stress and apoptosis of IPEC-J2 cells[J].Journal of Traditional Chinese Veterinary Medicine,2019,38(3):4-7.(in Chinese)
[21] WANG L Y,DONG L,YU Z P,et al.Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells[J].Food Research International,2016,90:33-41.
[22] HE Y,BU L,XIE H,et al.Antioxidant activities and protective effects of duck embryo peptides against H₂O₂-induced oxidative damage in HepG2 cells[J].Poultry Science,2019,98(12):7118-7128.
[23] HU Y M,LU S Z,LI Y S,et al.Protective effect of antioxidant peptides from grass carp scale gelatin on the H₂O₂-mediated oxidative injured HepG2 cells[J].Food Chemistry,2022,373:131539.
[24] WANG L,DING L,YU Z,et al.Intracellular ROS scavenging and antioxidant enzyme regulating capacities of corn gluten meal-derived antioxidant peptides in HepG2 cells[J].Food Research International,2016,90:33-41.
[25] WANG K,HAN L,TAN Y,et al.Generation of novel antioxidant peptides from silver carp muscle hydrolysate:Gastrointestinal digestion stability and transepithelial absorption property[J].Food Chemistry,2023,403:134136.
[26] ZOROV D B,JUHASZOVA M,SOLLOTT S J.Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release[J].Physiological Reviews,2014,94(3):909-950.
[27] STADTMAN E R.Protein oxidation and aging[J].Free Radical Research,2006,40(12):1250-1258.
[28] WANG Y M,LI X Y,WANG J,et al.Antioxidant peptides from protein hydrolysate of skipjack tuna milt:Purification,identification,and cytoprotection on H₂O₂ damaged human umbilical vein endothelial cells[J].Process Biochemistry,2022,113:258-269.
[29] JIHONG W,BAOGUO S,XUELIAN L,et al.Cytoprotective effects of a tripeptide from Chinese Baijiu against AAPH-induced oxidative stress in HepG2 cells via Nrf2 signaling[J].RSC Advances,2018,8(20):10898-10906.
[30] LIANG L,CAI S,GAO M,et al.Purification of antioxidant peptides of Moringa oleifera seeds and their protective effects on H₂O₂ oxidative damaged Chang liver cells[J].Journal of Functional Foods,2020,64(C):103698.
[31] SHI Y,KOVACS-NOLAN J,JIANG B,et al.Peptides derived from eggshell membrane improve antioxidant enzyme activity and glutathione synthesis against oxidative damage in Caco-2 cells[J].Journal of Functional Foods,2014,11:571-580.