蛋清源活性肽对过氧化氢诱导的HEK293细胞抗氧化酶活力及白细胞介素8分泌的影响
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摘要
食源性抗氧化肽由于抗氧化能力较好、安全性高,逐渐成为多肽研究领域的热点。本实验以H_2O_2诱导HEK293细胞损伤建立氧化损伤模型,研究了蛋清源活性肽WNWAD(Trp-Asn-Trp-Ala-Asp)及其结构改造肽WNW(Trp-Asn-Trp)、WAD(Trp-Ala-Asp)、WN(Trp-Asn)对H_2O_2诱导损伤的HEK293细胞的存活率、活性氧(reactive oxygen species,ROS)水平、抗氧化酶活力及细胞因子白介素8(interleukin-8,IL-8)分泌的影响。结果表明:蛋清源活性肽对HEK293细胞存活率的影响极显著(P<0.01),不同浓度蛋清源活性肽WNWAD、WNW、WAD、WN作用于氧化损伤的HEK293细胞后,其存活率明显提高,呈现浓度依赖性;其中1.0μmol/L蛋清源活性肽WNWAD、WNW、WAD、WN处理细胞后,存活率从损伤组的(48.0±2.4)%分别提高到(99.7±1.8)%、(69.4±3.2)%、(78.9±5.1)%、(72.1±3.6)%,与损伤组有极显著性差异(P<0.01);H_2O_2诱导HEK293细胞内ROS的产生,经过活性肽预处理后细胞内ROS的水平极显著降低(P<0.01);随着蛋清源活性肽WNWAD、WNW、WAD、WN浓度的升高,总超氧化物歧化酶、过氧化氢酶和谷胱甘肽过氧化物酶3种酶的活力均有所提高,呈现浓度依赖型关系。最后利用酶联免疫吸附测定试剂盒测定了HEK293细胞中IL-8的分泌量,经过H_2O_2处理的HEK293细胞上清液中IL-8含量极显著升高(P<0.01),加入WNWAD、WNW、WN、WAD后,细胞中的IL-8含量均有所降低,其中WNWAD和WN影响显著(P<0.05)。以上结果显示蛋清源活性肽对降低HEK293细胞氧化应激水平和提高细胞抗氧化能力有积极的影响。
In recent years, antioxidant peptides with high safety and potent antioxidant capacity have become a hot research?eld. The aim of this study was to investigate the effects of egg white-derived bioactive peptides WNWAD(Trp-Asn-TrpAla-Asp), WNW(Trp-Asn-Trp), WAD(Trp-Ala-Asp) and WN(Trp-Asn) on the survival rate, reactive oxygen species(ROS) content, antioxidative enzyme activities and interleukin-8(IL-8) level in HEK293 cells under oxidative stress induced by H_2O_2. MTS assay results showed that all four peptides signi?cantly dose-dependently inhibited H_2O_2-induced cellular oxidative stress in HEK-293 cells and increased the survival rate increased from(48.0 ± 2.4)% to(99.7 ± 1.8)%,(69.4 ± 3.2)%,(78.9 ± 5.1)%, and(72.1 ± 3.6)% at 1.0 μmol/L concentration, respectively. These changes were statistically signi?cant(P < 0.01). 2',7'-Dichloro?uorescin diacetate(DCFH-DA) ?uorescent probe assay proved that all these peptides inhibited oxidative stress by reducing intracellular ROS accumulation in HEK293 cells. They dose-dependently increased the activity of total superoxide dismutase(T-SOD), catalase(CAT) and glutathione peroxidase(GSH-Px) in HEK293 cells.In addition, the results of enzyme-linked immune sorbent assay(ELISA) showed that WNWAD and WN up-regulated IL-8 secretion(P < 0.05). All the above results showed that the egg white-derived peptides play a positive role in protecting HEK293 cells against H_2O_2-induced oxidative stress and improving antioxidant capacity in HEK293 cells.
In recent years, antioxidant peptides with high safety and potent antioxidant capacity have become a hot research?eld. The aim of this study was to investigate the effects of egg white-derived bioactive peptides WNWAD(Trp-Asn-TrpAla-Asp), WNW(Trp-Asn-Trp), WAD(Trp-Ala-Asp) and WN(Trp-Asn) on the survival rate, reactive oxygen species(ROS) content, antioxidative enzyme activities and interleukin-8(IL-8) level in HEK293 cells under oxidative stress induced by H_2O_2. MTS assay results showed that all four peptides signi?cantly dose-dependently inhibited H_2O_2-induced cellular oxidative stress in HEK-293 cells and increased the survival rate increased from(48.0 ± 2.4)% to(99.7 ± 1.8)%,(69.4 ± 3.2)%,(78.9 ± 5.1)%, and(72.1 ± 3.6)% at 1.0 μmol/L concentration, respectively. These changes were statistically signi?cant(P < 0.01). 2',7'-Dichloro?uorescin diacetate(DCFH-DA) ?uorescent probe assay proved that all these peptides inhibited oxidative stress by reducing intracellular ROS accumulation in HEK293 cells. They dose-dependently increased the activity of total superoxide dismutase(T-SOD), catalase(CAT) and glutathione peroxidase(GSH-Px) in HEK293 cells.In addition, the results of enzyme-linked immune sorbent assay(ELISA) showed that WNWAD and WN up-regulated IL-8 secretion(P < 0.05). All the above results showed that the egg white-derived peptides play a positive role in protecting HEK293 cells against H_2O_2-induced oxidative stress and improving antioxidant capacity in HEK293 cells.
引文
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[19]TANZADEHPANAH H,ASOODEH A,CHAMANI J.An antioxidant peptide derived from ostrich(Struthio camelus)egg white protein hydrolysates[J].Food Research International,2012,49(1):105-111.DOI:10.1016/j.foodres.2012.08.022.
[20]REN Y,WU H,LI X F,et al.A two-step,one-pot enzymatic method for preparation of duck egg white protein hydrolysates with high antioxidant activity[J].Applied Biochemistry and Biotechnology,2014,172(3):1227-1240.DOI:10.1007/s12010-013-0578-3.
[21]DAVALOS A,MIGUEL M,BARTOLOME B,et al.Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J].Journal of Food Protection,2004,67(9):1939-1944.DOI:10.4315/0362-028x-67.9.1939.
[22]FUJITA H,SASAKI R,YOSHIKAWA M.Potentiation of the antihypertensive activity of orally administered ovokinin,a vasorelaxing peptide derived from ovalbumin,by emulsification in egg phosphatidylcholine[J].Bioscience,Biotechnology and Biochemistry,1995,59(12):2344-2345.DOI:10.1271/bbb.59.2344.
[23]RAJAPAKSE N,MENDIS E,JUNG W K,et al.Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties[J].Food Research International,2005,38(2):175-182.DOI:10.1016/j.foodres.2004.10.002.
[24]YOUNG D,FAN M Z,MINE Y.Egg yolk peptides up-regulate glutathione synthesis and antioxidant enzyme activities in a porcine model of intestinal oxidative stress[J].Journal of Agricultural and Food Chemistry,2010,58(13):7624-7633.DOI:10.1021/jf1011598.
[25]YU Z P,YIN Y G,ZHAO W Z,et al.Application and bioactive properties of proteins and peptides derived from hen eggs:opportunities and challenges[J].Journal of the Science of Food and Agriculture,2014,94(14):2839-2845.DOI:10.1002/jsfa.6670.
[26]SUN S G,NIU H H,YANG T,et al.Antioxidant and anti-fatigue activities of egg white peptides prepared by pepsin digestion[J].Journal of the Science of Food and Agriculture,2014,94(15):3195-3200.DOI:10.1002/jsfa.6671.
[27]LIU Jingbo,CHEN Zhifei,HE Jing,et al.Anti-oxidative and antiapoptosis effects of egg white peptide,Trp-Asn-Trp-Ala-Asp,against H2O2-induced oxidative stress in human embryonic kidney 293cells[J].Food and Function,2014,5(12):3179-3188.DOI:10.1039/c4fo00665h.
[28]WANG L Y,DING L,WANG Y,et al.Isolation and characterisation of in vitro and cellular free radical scavenging peptides from corn peptide fractions[J].Molecules,2015,20(2):3221-3237.DOI:10.3390/molecules20023221.
[29]AFRI M,FRIMER A A,COHEN Y.Active oxygen chemistry within the liposomal bilayer part IV:locating 2’,7’-dichlorofluorescein(DCF),2’,7’-dichlorodihydrofluorescein(DCFH)and 2’,7’-dichlorodihydro fluorescein diacetate(DCFH-DA)in the lipid bilayer[J].Chemistry and Physics of Lipids,2004,131(1):123-133.DOI:10.1016/j.chemphyslip.2004.04.006.
[30]LEBEL C P,ISCHIROPOULOS H,BONDY S C.Evaluation of the probe 2’,7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress[J].Chemical Research in Toxicology,1992,5(2):227-231.DOI:10.1021/tx00026a012.
[31]HWANG S L,YEN G C.Modulation of Akt,JNK,and p38 activation is involved in citrus flavonoid-mediated cytoprotection of PC12 cells challenged by hydrogen peroxide[J].Journal of Agricultural and Food Chemistry,2009,57(6):2576-2582.DOI:10.1021/jf8033607.
[32]YOSHIMURA T,MATSUSHIMA K,OPENHEIM J J,et al.Neutrophil chemotactic factor produced by lipopolysaccharide(LPS)-stimulated human blood mononuclear leukocytes:partial characterization and separation from interleukin 1(IL 1)[J].Journal of Immunology,1987,139(3):788-793.
[33]YOON J,LIM J W,KIM H.Alpha-lipoic acid inhibits IL-8 expression by reducing oxidative stress in ataxia telangiectasia fibroblasts[J].FASEB Journal,2017,31:lb426.
[34]KASHIWAGI Y,TAKEDACHI M,MORI K,et al.High glucoseinduced oxidative stress increases IL-8 production in human gingival epithelial cells[J].Oral Diseases,2016,22(6):578-584.DOI:10.1111/odi.12502.
[35]ASHOUR A E,AHMED A F,KUMAR A,et al.Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-kappa Bsignaling and IL-8 expression[J].Molecular and Cellular Biochemistry,2016,416(1/2):141-155.DOI:10.1007/s11010-016-2703-4.
[36]KIM N,LIM J W,KIM H.Protective role of HSP70 against oxidative stress-mediated IL-8 expression in ataxia telangiectasia fibroblasts received glutamine deficiency[J].Free Radical Biology and Medicine,2015,86:S22.DOI:10.1016/j.freeradbiomed.2015.07.085.
[2]YANG B,XU Q Y,GUO C Y,et al.MHY1485 ameliorates UV-induced skin cell damages via activating mTOR-Nrf2signaling[J].Oncotarget,2017,8(8):12775-12783.DOI:10.18632/oncotarget.14299.
[3]YOUN Y K,LALONDE C,DEMLING R.Oxidants and the pathophysiology of burn and smoke inhalation injury[J].Free Radical Biology and Medicine,1992,12(5):409-415.
[4]RICE-EVANS C A,DIPLOCK A T.Current status of antioxidant therapy[J].Free Radical Biology and Medicine,1993,15(1):77-96.DOI:10.1016/0891-5849(93)90127-g.
[5]WEI Y H,LEE H C.Oxidative stress,mitochondrial DNA mutation,and impairment of antioxidant enzymes in aging[J].Experimental Biology and Medicine,2002,227(9):671-682.
[6]MINE Y,KOVACS-NOLAN J.New insights in biologically active proteins and peptides derived from hen egg[J].Worlds Poultry Science Journal,2006,62(1):87-95.DOI:10.1079/wps200586.
[7]MEISEL H.Biochemical properties of regulatory peptides derived from milk proteins[J].Biopolymers,1997,43(2):119-128.DOI:10.1002/(sici)1097-0282(1997)43:2<119::aid-bip4>3.0.co;2-y.
[8]NAGAI T,NAGASHIMA T,ABE A.Antioxidative activities and angiotensin I-converting enzyme inhibition of extracts prepared from chum salmon(Oncorhynchus keta)cartilage and skin[J].International Journal of Food Properties,2006,9(4):813-822.DOI:10.1080/10942910600620645.
[9]LEE H S,RYU H J,SONG H J,et al.Enzymatic preparation and antioxidant activities of protein hydrolysates from Protaetia brevitarsis Larvae[J].Journal of the Korean Society of Food Science and Nutrition,2017,46(10):1164-1170.
[10]RIPOLLéS D,PARRóN J A,CALVO M,et al.Antioxidant activity of co-products from milk fat processing and their enzymatic hydrolysates obtained with different proteolytic preparations[J].International Dairy Journal,2016,60:70-77.DOI:10.1016/j.idairyj.2016.02.031.
[11]QIN L,ZHU B W,ZHOU D Y,et al.Preparation and antioxidant activity of enzymatic hydrolysates from purple sea urchin(Strongylocentrotus nudus)gonad[J].LWT-Food Science and Technology,2011,44(4):1113-1118.DOI:10.1016/j.lwt.2010.10.013.
[12]MARINOVA M D,TCHORBANOV B P.Preparation of antioxidant enzymatic hydrolysates from honeybee-collected pollen using plant enzymes[J].Enzyme Research,2010,2010:1-5.DOI:10.4061/2010/415949.
[13]DONG X P,ZHU B W,ZHAO H X,et al.Preparation and in vitro antioxidant activity of enzymatic hydrolysates from oyster(Crassostrea talienwhannensis)meat[J].International Journal of Food Science and Technology,2010,45(5):978-984.DOI:10.1111/j.1365-2621.2010.02223.x.
[14]HEMANDEZ-LEDESMA B,DAVALOS A,BARTOLOME B,et al.Preparation of antioxidant enzymatic hydrolysates from(alphalactalbumin and beta-lactoglobulin.identification of active peptides by HPLC-MS/MS[J].Journal of Agricultural and Food Chemistry,2005,53(3):588-593.DOI:10.1021/jf048626m.
[15]MINE Y.Egg proteins and peptides in human health-chemistry,bioactivity and production[J].Current Pharmaceutical Design,2007,13(9):875-884.DOI:10.2174/138161207780414278.
[16]YOU S J,UDENIGWE C C,ALUKO R E,WU J.Multifunctional peptides from egg white lysozyme[J].Food Research International,2010,43(3):848-855.DOI:10.1016/j.foodres.2009.12.004.
[17]JI N,SUN C X,ZHAO Y X,et al.Purification and identification of antioxidant peptides from peanut protein isolate hydrolysates using UHR-Q-TOF mass spectrometer[J].Food Chemistry,2014,161:148-154.DOI:10.1016/j.foodchem.2014.04.010.
[18]SAKANAKA S,TACHIBANA Y,ISHIHARA N,et al.Antioxidant activity of egg-yolk protein hydrolysates in a linoleic acid oxidation system[J].Food Chemistry,2004,86(1):99-103.DOI:10.1016/j.foodchem.2003.08.014.
[19]TANZADEHPANAH H,ASOODEH A,CHAMANI J.An antioxidant peptide derived from ostrich(Struthio camelus)egg white protein hydrolysates[J].Food Research International,2012,49(1):105-111.DOI:10.1016/j.foodres.2012.08.022.
[20]REN Y,WU H,LI X F,et al.A two-step,one-pot enzymatic method for preparation of duck egg white protein hydrolysates with high antioxidant activity[J].Applied Biochemistry and Biotechnology,2014,172(3):1227-1240.DOI:10.1007/s12010-013-0578-3.
[21]DAVALOS A,MIGUEL M,BARTOLOME B,et al.Antioxidant activity of peptides derived from egg white proteins by enzymatic hydrolysis[J].Journal of Food Protection,2004,67(9):1939-1944.DOI:10.4315/0362-028x-67.9.1939.
[22]FUJITA H,SASAKI R,YOSHIKAWA M.Potentiation of the antihypertensive activity of orally administered ovokinin,a vasorelaxing peptide derived from ovalbumin,by emulsification in egg phosphatidylcholine[J].Bioscience,Biotechnology and Biochemistry,1995,59(12):2344-2345.DOI:10.1271/bbb.59.2344.
[23]RAJAPAKSE N,MENDIS E,JUNG W K,et al.Purification of a radical scavenging peptide from fermented mussel sauce and its antioxidant properties[J].Food Research International,2005,38(2):175-182.DOI:10.1016/j.foodres.2004.10.002.
[24]YOUNG D,FAN M Z,MINE Y.Egg yolk peptides up-regulate glutathione synthesis and antioxidant enzyme activities in a porcine model of intestinal oxidative stress[J].Journal of Agricultural and Food Chemistry,2010,58(13):7624-7633.DOI:10.1021/jf1011598.
[25]YU Z P,YIN Y G,ZHAO W Z,et al.Application and bioactive properties of proteins and peptides derived from hen eggs:opportunities and challenges[J].Journal of the Science of Food and Agriculture,2014,94(14):2839-2845.DOI:10.1002/jsfa.6670.
[26]SUN S G,NIU H H,YANG T,et al.Antioxidant and anti-fatigue activities of egg white peptides prepared by pepsin digestion[J].Journal of the Science of Food and Agriculture,2014,94(15):3195-3200.DOI:10.1002/jsfa.6671.
[27]LIU Jingbo,CHEN Zhifei,HE Jing,et al.Anti-oxidative and antiapoptosis effects of egg white peptide,Trp-Asn-Trp-Ala-Asp,against H2O2-induced oxidative stress in human embryonic kidney 293cells[J].Food and Function,2014,5(12):3179-3188.DOI:10.1039/c4fo00665h.
[28]WANG L Y,DING L,WANG Y,et al.Isolation and characterisation of in vitro and cellular free radical scavenging peptides from corn peptide fractions[J].Molecules,2015,20(2):3221-3237.DOI:10.3390/molecules20023221.
[29]AFRI M,FRIMER A A,COHEN Y.Active oxygen chemistry within the liposomal bilayer part IV:locating 2’,7’-dichlorofluorescein(DCF),2’,7’-dichlorodihydrofluorescein(DCFH)and 2’,7’-dichlorodihydro fluorescein diacetate(DCFH-DA)in the lipid bilayer[J].Chemistry and Physics of Lipids,2004,131(1):123-133.DOI:10.1016/j.chemphyslip.2004.04.006.
[30]LEBEL C P,ISCHIROPOULOS H,BONDY S C.Evaluation of the probe 2’,7’-dichlorofluorescin as an indicator of reactive oxygen species formation and oxidative stress[J].Chemical Research in Toxicology,1992,5(2):227-231.DOI:10.1021/tx00026a012.
[31]HWANG S L,YEN G C.Modulation of Akt,JNK,and p38 activation is involved in citrus flavonoid-mediated cytoprotection of PC12 cells challenged by hydrogen peroxide[J].Journal of Agricultural and Food Chemistry,2009,57(6):2576-2582.DOI:10.1021/jf8033607.
[32]YOSHIMURA T,MATSUSHIMA K,OPENHEIM J J,et al.Neutrophil chemotactic factor produced by lipopolysaccharide(LPS)-stimulated human blood mononuclear leukocytes:partial characterization and separation from interleukin 1(IL 1)[J].Journal of Immunology,1987,139(3):788-793.
[33]YOON J,LIM J W,KIM H.Alpha-lipoic acid inhibits IL-8 expression by reducing oxidative stress in ataxia telangiectasia fibroblasts[J].FASEB Journal,2017,31:lb426.
[34]KASHIWAGI Y,TAKEDACHI M,MORI K,et al.High glucoseinduced oxidative stress increases IL-8 production in human gingival epithelial cells[J].Oral Diseases,2016,22(6):578-584.DOI:10.1111/odi.12502.
[35]ASHOUR A E,AHMED A F,KUMAR A,et al.Thymoquinone inhibits growth of human medulloblastoma cells by inducing oxidative stress and caspase-dependent apoptosis while suppressing NF-kappa Bsignaling and IL-8 expression[J].Molecular and Cellular Biochemistry,2016,416(1/2):141-155.DOI:10.1007/s11010-016-2703-4.
[36]KIM N,LIM J W,KIM H.Protective role of HSP70 against oxidative stress-mediated IL-8 expression in ataxia telangiectasia fibroblasts received glutamine deficiency[J].Free Radical Biology and Medicine,2015,86:S22.DOI:10.1016/j.freeradbiomed.2015.07.085.