利用斑马鱼模型研究琼胶寡糖抗氧化机制
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摘要
为进一步实现琼胶寡糖的高值化利用,探究琼胶寡糖(Agaro-oligosaccharides,AOS)对2,2-偶氮二(2-甲基丙基咪)二盐酸盐(AAPH)诱导损伤后斑马鱼的抗氧化作用及机制。采用高效液相色谱法对酸法制备的琼胶寡糖组分进行分析。优化建立AAPH诱导的斑马鱼模型,并以斑马鱼胚胎存活率、活性氧生成率和细胞死亡率评价不同浓度琼胶寡糖的抗氧化活性。结果表明,琼胶寡糖主要由琼二糖(51.60%)、琼四糖(28.40%)和琼六糖(14.50%)组成,对斑马鱼胚胎添加琼胶寡糖浓度低于200μg/m L时无致畸、致死毒性;琼胶寡糖(25、50、100μg/m L)对最优AAPH诱导浓度(15 mmol/L)引起的氧化应激损伤斑马鱼模型有保护作用,且呈剂量依赖性。其中最优浓度100μg/m L琼胶寡糖能显著提高斑马鱼胚胎存活率(96.88%,p <0.05),显著降低AAPH引起的斑马鱼体内活性氧生成率(100.53%,p <0.05)和细胞死亡率(101.69%,p <0.05)。本研究通过斑马鱼模型首次揭示了琼胶寡糖可通过清除体内大量活性氧生成和阻止细胞死亡损伤的抗氧化机制实现提高其体内抗氧化活性,为其作为保健功能食品等应用提供了理论基础。
In order to further realize the high value utilization of agaro-oligosaccharides( AOS),antioxidant activities and mechanisms of AOS,2,2-azobis( 2-methylpropylimidazole) dihydrochloride( AAPH)-induced zebrafish model were studied.The composition of AOS was determined by HPLC.The optimal AAPH induced zebrafish oxidative stress model was established,and the antioxidant activities of AOS with different concentrations were investigated by determination of embryo survival rate,reactive oxygen production rate,and cell death rate. The results showed that AOS was mainly composed of agarobiose( 51.60%),agarotetraose( 28.40%) and agarohexaose( 14.50%).Zebrafish embryos with AOS concentration below 200 μg/m L had no teratogenic and lethal toxicity.AOS( 25,50,100 μg/m L) had a protective effect on oxidative stress induced by AAPH( 15 mmol/L) in a dose-dependent manner. The optimal concentration of 100 μg/m L AOS could significantly increase the survival rate of zebrafish embryos( 96.88%,p < 0.05),and could significantly reduce the reactive oxygen species production rate( 100.53%,p < 0.05) and cell death rate( 101.69%,p < 0.05) of AAPH-induced zebrafish.In this study,the zebrafish model was used to reveal for the first time. AOS had antioxidant activity in vivo by eliminating a large number of reactive oxygen species( ROS) production and preventing cell death and injury,which provided a theoretical basis for the application of AOS as a health food.
In order to further realize the high value utilization of agaro-oligosaccharides( AOS),antioxidant activities and mechanisms of AOS,2,2-azobis( 2-methylpropylimidazole) dihydrochloride( AAPH)-induced zebrafish model were studied.The composition of AOS was determined by HPLC.The optimal AAPH induced zebrafish oxidative stress model was established,and the antioxidant activities of AOS with different concentrations were investigated by determination of embryo survival rate,reactive oxygen production rate,and cell death rate. The results showed that AOS was mainly composed of agarobiose( 51.60%),agarotetraose( 28.40%) and agarohexaose( 14.50%).Zebrafish embryos with AOS concentration below 200 μg/m L had no teratogenic and lethal toxicity.AOS( 25,50,100 μg/m L) had a protective effect on oxidative stress induced by AAPH( 15 mmol/L) in a dose-dependent manner. The optimal concentration of 100 μg/m L AOS could significantly increase the survival rate of zebrafish embryos( 96.88%,p < 0.05),and could significantly reduce the reactive oxygen species production rate( 100.53%,p < 0.05) and cell death rate( 101.69%,p < 0.05) of AAPH-induced zebrafish.In this study,the zebrafish model was used to reveal for the first time. AOS had antioxidant activity in vivo by eliminating a large number of reactive oxygen species( ROS) production and preventing cell death and injury,which provided a theoretical basis for the application of AOS as a health food.
引文
[1]欧阳茜茜,陈法锦,李思东,等.琼胶及琼胶寡糖的加工与应用研究进展[J].山东化工,2016,45(23):58-60.
[2]胡斌.新琼寡糖的益生元效应研究[D].青岛:中国海洋大学,2006.
[3]张伟宾,刘丹,许竞男,等.琼胶寡糖鉴定方法的研究进展[J].中国海洋药物,2017(6):96-102.
[4]郭晓凤.新琼寡糖的益生元作用研究[D].青岛:中国海洋大学,2014.
[5]宋香凝,郑毅,姜泽东,等.江蓠琼胶寡糖抗氧化性及对罗非鱼肉的保鲜作用[J].食品科技,2017(2):149-154.
[6]Wang W,Liu P,Hao C,et al.Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways[J].Scientific Reports,2017,7(44252):1-11.
[7]Higashimura Y,Naito Y,Takagi T,et al.Oligosaccharides from agar inhibit murine intestinal inflammation through the induction of heme oxygenase-1 expression[J].Journal of Gastroenterology,2013,48(8):897-909.
[8]Enoki T,Tominaga T,Takashima F,et al.Anti-tumorpromoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis[J].Biological and Pharmaceutical Bulletin,2012,35(7):1145-1149.
[9]Higashimura Y,Naito Y,Takagi T,et al.Protective effect of agaro-oligosaccharides on gut dysbiosis and colon tumorigenesis in high-fat diet-fed mice[J].American Journal of Physiology Gastrointestinal and Liver Physiology,2016,310(6)):367-375.
[10]Sun J H,Lee J H,Kim E J,et al.Anti-obesity and antidiabetic effect of neoagarooligosaccharides on high-fat dietinduced obesity in mice[J].Marine Drugs,2017,15(4):1-12.
[11]Chen H M,Zheng L,Lin W,et al.Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC[J].Talanta,2004,64(3):773-777.
[12]Chen H M,Yan X J.Antioxidant activities of agarooligosaccharides with different degrees of polymerization in cellbased system[J].Biochimica Et Biophysica Acta,2005,1722(1):103-111.
[13]徐强,薛长湖,赵雪,等.酸解法制备琼胶低聚糖及其抗氧化性评价[J].中国海洋药物,2002,21(1):19-22.
[14]薛长湖,徐强,赵雪,等.琼胶低聚糖清除自由基的活性[J].水产学报,2003,27(3):283-288.
[15]陈海敏.琼二糖寡聚体特效降解、标记分析、结构特征与生物活性的研究[D].青岛:中国科学院研究生院,2004.
[16]Chen Haimin,Yan Xiaojun,Zhu Peng,et al.Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro,and in vivo[J].Nutrition Journal,2006,5(1):31-31.
[17]Caro M,Iturria I,Martinez-Santos M,et al.Zebrafish dives into food research:Effectiveness assessment of bioactive compounds[J].Food and Function,2016,7(6):2615-2623.
[18]许冰洁,张立将,李春启,等.斑马鱼胚胎评价5种药物的发育毒性与模型验证[J].中国药理学通报,2016(1):74-79.
[19]Lee S H,Ko C I,Jee Y,et al.Anti-inflammatory effect of fucoidan extracted from Ecklonia cava in zebrafish model[J].Carbohydrate Polymers,2013,92(1):84-89.
[20]Kang M C,Kim K N,Kang S M,et al.Protective effect of dieckol isolated from Ecklonia cava against ethanol caused damage in vitro and in zebrafish model[J].Environmental Toxicology and Pharmacology,2013,36(3):1217-1226.
[21]Kang M C,Cha S H,Wijesinghe W A,et al.Protective effect of marine algae phlorotannins against AAPH-induced oxidative stress in zebrafish embryo[J].Food Chemistry,2013,138(2-3):950-955.
[22]Kim E A,Lee S H,Ko C I,et al.Protective effect of fucoidan against AAPH-induced oxidative stress in zebrafish model[J].Carbohydrate Polymers,2014,102(1):185-191.
[23]Kang M C,Kim S Y,Kim Y T,et al.In vitro and in vivo antioxidant activities of polysaccharide purified from Aloe vera(Aloe barbadensis)gel[J].Carbohydrate Polymers,2014,99(1):365-371.
[24]Rosenkranz A R,Schmaldienst S,Stuhlmeier K M,et al.Amicroplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate[J].Journal of Immunological Methods,1992,156(1):39-45.
[25]Hu Y R,Ma H,Zou Z Y,et al.Activation of Akt and JNK/Nrf2/NQO1 pathway contributes to the protective effect of coptisine against AAPH-induced oxidative stress[J].Biomedicine and Pharmacotherapy,2017,85:313-322.
[26]陈海敏,严小军,郑立,等.琼胶的降解及其产物的分析[J].河南工业大学学报:自然科学版,2003,24(3):41-44.
[27]Westerfield M.The zebrafish book:A guide for the laboratory use of zebrafish(Danio rerio)[M].Eugene:Univ of Oregon Press,2007:6-13.
[28]Lee J H,Han J W,Ko J Y,et al.Protective effect of a freshwater alga,Spirogyra sp.against lipid peroxidation in vivo zebrafish and purification of antioxidative compounds using preparative centrifugal partition chromatography[J].Journal of Applied Phycology,2015,28(1):1-9.
[29]Wijesinghe W A J P,Kim E A,Kang M C,et al.Assessment of anti-inflammatory effect of 5β-hydroxypalisadin B isolated from red seaweed Laurencia snackeyi,in zebrafish embryo in vivo model[J].Environmental Toxicology and Pharmacology,2014,37(1):110-117.
[2]胡斌.新琼寡糖的益生元效应研究[D].青岛:中国海洋大学,2006.
[3]张伟宾,刘丹,许竞男,等.琼胶寡糖鉴定方法的研究进展[J].中国海洋药物,2017(6):96-102.
[4]郭晓凤.新琼寡糖的益生元作用研究[D].青岛:中国海洋大学,2014.
[5]宋香凝,郑毅,姜泽东,等.江蓠琼胶寡糖抗氧化性及对罗非鱼肉的保鲜作用[J].食品科技,2017(2):149-154.
[6]Wang W,Liu P,Hao C,et al.Neoagaro-oligosaccharide monomers inhibit inflammation in LPS-stimulated macrophages through suppression of MAPK and NF-κB pathways[J].Scientific Reports,2017,7(44252):1-11.
[7]Higashimura Y,Naito Y,Takagi T,et al.Oligosaccharides from agar inhibit murine intestinal inflammation through the induction of heme oxygenase-1 expression[J].Journal of Gastroenterology,2013,48(8):897-909.
[8]Enoki T,Tominaga T,Takashima F,et al.Anti-tumorpromoting activities of agaro-oligosaccharides on two-stage mouse skin carcinogenesis[J].Biological and Pharmaceutical Bulletin,2012,35(7):1145-1149.
[9]Higashimura Y,Naito Y,Takagi T,et al.Protective effect of agaro-oligosaccharides on gut dysbiosis and colon tumorigenesis in high-fat diet-fed mice[J].American Journal of Physiology Gastrointestinal and Liver Physiology,2016,310(6)):367-375.
[10]Sun J H,Lee J H,Kim E J,et al.Anti-obesity and antidiabetic effect of neoagarooligosaccharides on high-fat dietinduced obesity in mice[J].Marine Drugs,2017,15(4):1-12.
[11]Chen H M,Zheng L,Lin W,et al.Product monitoring and quantitation of oligosaccharides composition in agar hydrolysates by precolumn labeling HPLC[J].Talanta,2004,64(3):773-777.
[12]Chen H M,Yan X J.Antioxidant activities of agarooligosaccharides with different degrees of polymerization in cellbased system[J].Biochimica Et Biophysica Acta,2005,1722(1):103-111.
[13]徐强,薛长湖,赵雪,等.酸解法制备琼胶低聚糖及其抗氧化性评价[J].中国海洋药物,2002,21(1):19-22.
[14]薛长湖,徐强,赵雪,等.琼胶低聚糖清除自由基的活性[J].水产学报,2003,27(3):283-288.
[15]陈海敏.琼二糖寡聚体特效降解、标记分析、结构特征与生物活性的研究[D].青岛:中国科学院研究生院,2004.
[16]Chen Haimin,Yan Xiaojun,Zhu Peng,et al.Antioxidant activity and hepatoprotective potential of agaro-oligosaccharides in vitro,and in vivo[J].Nutrition Journal,2006,5(1):31-31.
[17]Caro M,Iturria I,Martinez-Santos M,et al.Zebrafish dives into food research:Effectiveness assessment of bioactive compounds[J].Food and Function,2016,7(6):2615-2623.
[18]许冰洁,张立将,李春启,等.斑马鱼胚胎评价5种药物的发育毒性与模型验证[J].中国药理学通报,2016(1):74-79.
[19]Lee S H,Ko C I,Jee Y,et al.Anti-inflammatory effect of fucoidan extracted from Ecklonia cava in zebrafish model[J].Carbohydrate Polymers,2013,92(1):84-89.
[20]Kang M C,Kim K N,Kang S M,et al.Protective effect of dieckol isolated from Ecklonia cava against ethanol caused damage in vitro and in zebrafish model[J].Environmental Toxicology and Pharmacology,2013,36(3):1217-1226.
[21]Kang M C,Cha S H,Wijesinghe W A,et al.Protective effect of marine algae phlorotannins against AAPH-induced oxidative stress in zebrafish embryo[J].Food Chemistry,2013,138(2-3):950-955.
[22]Kim E A,Lee S H,Ko C I,et al.Protective effect of fucoidan against AAPH-induced oxidative stress in zebrafish model[J].Carbohydrate Polymers,2014,102(1):185-191.
[23]Kang M C,Kim S Y,Kim Y T,et al.In vitro and in vivo antioxidant activities of polysaccharide purified from Aloe vera(Aloe barbadensis)gel[J].Carbohydrate Polymers,2014,99(1):365-371.
[24]Rosenkranz A R,Schmaldienst S,Stuhlmeier K M,et al.Amicroplate assay for the detection of oxidative products using 2',7'-dichlorofluorescin-diacetate[J].Journal of Immunological Methods,1992,156(1):39-45.
[25]Hu Y R,Ma H,Zou Z Y,et al.Activation of Akt and JNK/Nrf2/NQO1 pathway contributes to the protective effect of coptisine against AAPH-induced oxidative stress[J].Biomedicine and Pharmacotherapy,2017,85:313-322.
[26]陈海敏,严小军,郑立,等.琼胶的降解及其产物的分析[J].河南工业大学学报:自然科学版,2003,24(3):41-44.
[27]Westerfield M.The zebrafish book:A guide for the laboratory use of zebrafish(Danio rerio)[M].Eugene:Univ of Oregon Press,2007:6-13.
[28]Lee J H,Han J W,Ko J Y,et al.Protective effect of a freshwater alga,Spirogyra sp.against lipid peroxidation in vivo zebrafish and purification of antioxidative compounds using preparative centrifugal partition chromatography[J].Journal of Applied Phycology,2015,28(1):1-9.
[29]Wijesinghe W A J P,Kim E A,Kang M C,et al.Assessment of anti-inflammatory effect of 5β-hydroxypalisadin B isolated from red seaweed Laurencia snackeyi,in zebrafish embryo in vivo model[J].Environmental Toxicology and Pharmacology,2014,37(1):110-117.