连翘花黄色素对秀丽隐杆线虫在氧化应激下的保护作用
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摘要
自由基过度引起的氧化应激是多种疾病发生的因素。连翘花黄色素(forsythia flower yellow pigment, FFYP)中含有大量的抗氧化活性物质,但其对氧化应激的抵抗性仍不清楚。本文首先通过化学方法检测FFYP的体外抗氧化活性;用细胞内抗氧化活性(cellular antioxidant activity,CAA)方法检测FFYP细胞内抗氧化活性;然后以秀丽隐杆线虫(Caenorhabditis elegans,C.elegans)为模型,检测FFYP对线虫氧化应激抵抗力及体内抗氧化指标的影响;用Daf-16和Skn-1突变体线虫和qRT-PCR实验探究其作用机制。研究结果表明,FFYP具有1,1-二苯基-2-三硝基苯肼(1,1-diphenyl-2-picrylhydrazyl, DPPH)自由基清除能力,铁离子还原能力和活性氧自由基(reactive oxygen species, ROS)清除能力,并且具有浓度依赖性。用500μmol/L的胡桃醌提供氧化应激压力时,FFYP能显著提高线虫在氧化应激下的寿命,表明FFYP可以提高线虫对氧化应激的抵抗力。进一步研究发现,FFYP可显著降低线虫体内ROS自由基含量,提高超氧化物歧化酶(superoxide dismutase, SOD)和过氧化氢酶(catalase, CAT)活性,增加还原型谷胱甘肽(glutathione, GSH)含量,表明FFYP通过提高线虫体内抗氧化防御系统活性清除自由基来提高线虫对氧化应激的抵抗力。突变体线虫实验显示,FFYP对线虫延长氧化应激下寿命的效应在Skn-1突变体线虫中完全消失,在Daf-16突变体中效应被减弱。qRT-PCR实验也显示,Daf-16和Skn-1靶基因的表达量均被提高。表明FFYP对线虫氧化应激抵抗力提高的作用是通过Daf-16和Skn-1共同作用。这预示着FFYP具有很好的抗氧化及抗应激药用价值,有潜力成为一种新的有生物活性的天然色素。
Oxidative stress caused by excessive free radicals is a factor in the occurrence of various diseases. A large number of components in forsythia flower yellow pigment(FFYP) have antioxidant activity, but its resistance mechanism to oxidative stress is still unclear. In this paper, the antioxidant activity of FFYP in vitro was detected by chemical method. The anti-oxidation activity of FFYP in cells was detected by Cellular Antioxidant Activity(CAA) assay. The Caenorhabditis elegans(C. elegans) model was used to detect the effects of FFYP on oxidative stress resistance and antioxidant index in vivo; the mechanism of action was analyzed by Daf-16 and Skn-1 mutant nematodes and qRT-PCR. The experimental results showed that FFYP could remove 1, 1-diphenyl-2-picrylhydrazyl(DPPH) radicals, reduce iron ions and scavenge reactive oxygen species(ROS). All the above effects were concentration dependent. FFYP significantly increased the lifespan of nematodes under oxidative stress when 500 μmol/L of juglone was added. This indicated that FFYP increased the resistance of nematodes to oxidative stress. Further studies have found that FFYP could significantly reduce the content of ROS in nematodes, increase the activity of superoxide dismutase(SOD) and catalase(CAT), and increase the levels of glutathione(GSH). This study demonstrated that FFYP enhanced the resistance of nematodes to oxidative stress by increasing the activity of the antioxidant defense system to scavenge free radicals. Mutant nematode experiments showed that the promotion effect of FFYP in the lifespan of nematodes under oxidative stress completely disappeared in the Skn-1 mutant nematode, and was attenuated in the Daf-16 mutant. qRT-PCR experiments also showed that the expression levels of Daf-16 and Skn-1 target genes were both increased. These results indicated that FFYP enhanced the resistance of nematodes to oxidative stress through the interaction of Daf-16 and Skn-1. This indicated that FFYP, as an antioxidant that could prevent oxidative damage, has the potential to become a new bioactive natural pigment.
Oxidative stress caused by excessive free radicals is a factor in the occurrence of various diseases. A large number of components in forsythia flower yellow pigment(FFYP) have antioxidant activity, but its resistance mechanism to oxidative stress is still unclear. In this paper, the antioxidant activity of FFYP in vitro was detected by chemical method. The anti-oxidation activity of FFYP in cells was detected by Cellular Antioxidant Activity(CAA) assay. The Caenorhabditis elegans(C. elegans) model was used to detect the effects of FFYP on oxidative stress resistance and antioxidant index in vivo; the mechanism of action was analyzed by Daf-16 and Skn-1 mutant nematodes and qRT-PCR. The experimental results showed that FFYP could remove 1, 1-diphenyl-2-picrylhydrazyl(DPPH) radicals, reduce iron ions and scavenge reactive oxygen species(ROS). All the above effects were concentration dependent. FFYP significantly increased the lifespan of nematodes under oxidative stress when 500 μmol/L of juglone was added. This indicated that FFYP increased the resistance of nematodes to oxidative stress. Further studies have found that FFYP could significantly reduce the content of ROS in nematodes, increase the activity of superoxide dismutase(SOD) and catalase(CAT), and increase the levels of glutathione(GSH). This study demonstrated that FFYP enhanced the resistance of nematodes to oxidative stress by increasing the activity of the antioxidant defense system to scavenge free radicals. Mutant nematode experiments showed that the promotion effect of FFYP in the lifespan of nematodes under oxidative stress completely disappeared in the Skn-1 mutant nematode, and was attenuated in the Daf-16 mutant. qRT-PCR experiments also showed that the expression levels of Daf-16 and Skn-1 target genes were both increased. These results indicated that FFYP enhanced the resistance of nematodes to oxidative stress through the interaction of Daf-16 and Skn-1. This indicated that FFYP, as an antioxidant that could prevent oxidative damage, has the potential to become a new bioactive natural pigment.
引文
[1] 毛文星,李冰,王志梅,等.氧化应激、血管内皮功能障碍与高血压[J].现代生物医学进展(Mao WX,Li B,Wang ZM,et al.Oxidative stress and endothelial dysfunction in hypertension[J].Progr Mod Biomed),2014,14(19):3770-3774
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[5] Zhu X,Yue H,Guo X,et al.The preconditioning of berberine suppresses hydrogen peroxide-induced premature senescence via regulation of sirtuin 1[J].Oxid Med Cell Longev,2017,2017:2391820
[6] Ishikado A,Sono Y,Matsumoto M,et al.Willow bark extract increases antioxidant enzymesand reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans[J].Free Radic Biol Med,2013,65:1506-1515
[7] 阮佼.芦丁对脂多糖诱导急性肺损伤小鼠的抗氧化应激作用[J].中国现代应用药学(Ruan J.Effect of rutin on oxidative stress of acute lung injury induced by lipopolysaccharide[J].Chin J Mod Appl Pharm),2015,32(9):1059-1062
[8] Huang C,Lin Y,Su H,et al.Forsythiaside protects against hydrogen peroxide-induced oxidative stress and apoptosis in PC12 cell[J].Neurochem Res,2015,40(1):27-35
[9] Wei T,Tian W,Yan H,et al.Protective effects of phillyrin on H2O2-induced oxidative stress and apoptosis in PC12 cells[J].Cell Mol Neurobiol,2014,34(8):1165-1173
[10] Kampk?tter A,Timpel C,Zurawski RF,et al.Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin[J].Comp Biochem Physiol B Biochem Mol Biol,2008,149(2):314-323
[11] 李兴泰,李洪成,刘泽.连翘花醇提物保护线粒体及抗氧化研究[J].中成药(Li XT,Li HC,Liu Z.Protective effects of ethanol extract of Forsythia suspense flower on mitochondria and antioxidant activity[J].Chin Trad Patent Med),2009,31(6):839-843
[12] 盛锋,李长城,付蕾,等.乙醇提取连翘花黄色素溶液稳定性的研究[J].食品与发酵工业(Sheng F,Li CC,Fu L,et al.Study on the stability of yellow pigment ethanol solution extracted from Forsythia Flower[J].Food Ferment Ind),2002,28(1):43-47
[13] 原江锋,邱智军,呼丽君,等.连翘花提取物活性成分及其稳定性[J].食品科学(Yuan JF,Qiu ZJ,Hu LJ,et al.Active compounds and stability of Forsythia suspensa flower extract[J].Food Sci),2015,36(14):130-134
[14] 王金亭,李秋凤.微波辅助提取连翘花黄色素及其抗氧化活性的研究[J].中国酿造(Wang JT,Li QF.Extraction of the yellow pigment from Forsythia with microwave assisted method and study on its antioxidant activity[J].China Brew),2010,(11):100-103
[15] 张飞.连翘花和叶中清除自由基成分分析及其含量测定[D].河北农业大学(Zhang F.Qualitative and quantitative analyses of the major bioactive compounds on scavenging free radical in flower and leaves of Forsythia suspense[D].Agric Univ Hebei Prov),2011
[16] 李丹凤,李石飞,张立伟.连翘不同部位活性成分含量分析[J].山西医科大学学报(Li DF,Li SF,Zhang LW.Analysis of the contents of active constituents in different parts of Forsythia suspense[J].J Shanxi Med Univ),2015,46(11):1097-1100
[17] Sugawara T,Sakamoto K.Killed Bifidobacterium longum enhanced stress tolerance and prolonged lifespan of Caenorhabditis elegans via DAF-16 [J].Br J Nutr,2018,120(8):872-880
[18] Schulenburg H,Felix MA.The natural biotic environment of Caenorhabditis elegans[J].Genetics,2017,206(1):55-86
[19] Wolfe KL,Liu RH.Cellular antioxidant activity (CAA) assay for assessing antioxidants,foods,and dietary supplements[J].J Agric Food Chem,2007,55(22):8896-8907
[20] Beckman KB,Ames BN.The free radical theory of aging matures[J].Physiol Rev,1998,78(2):547-581
[21] Bokov A,Chaudhuri A,Richardson A.The role of oxidative damage and stress in aging[J].Mech Ageing Dev,2004,125(10-11):811-826
[22] Hanschmann EM,Godoy JR,Berndt C,et al.Thioredoxins,glutaredoxins,and peroxiredoxins molecular mechanisms and health significance:from cofactors to antioxidants to redox signaling[J].Antioxid Redox Signal,2013,19(13):1539-1605
[23] Marengo B,Nitti M,Furfaro AL,et al.Redox homeostasis and cellular antioxidant systems:crucial players in cancer growth and therapy[J].Oxid Med Cell Longev,2016,2016:6235641.The oxidative stress in allelopathy:participation of prenyllipid antioxidants in the response to juglone in chlamydomonas reinhardtii[J].Phytochemistry,2017,144:171-179
[25] 张超,陈圣慧,何承伟,等.毒物兴奋效应与神经保护[J].科技导报(Zhang C,Chen SH,He CW,et al.Hormesis and neuroprotection[J].Sci Tech Rev),2015,33(3):110-113
[26] 刘天天,靳洪涛,王爱平.毒物兴奋效应及其对毒理学和医药研究的影响[J].毒理学杂志(Liu TT,Jin HT,Wang AP.Hormesis and its impact on toxicology and medical research[J].J Toxicol Feb),2016,30(1):78-83
[27] Friedman DB,Johnson TE.A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility[J].Genetics,1988,118(1):75-86
[28] Wu JZ,Huang JH,Khanabdali R,et al.Pyrroloquinoline quinone enhances the resistance to oxidative stress and extends lifespan upon DAF-16 and SKN-1 activities in C.elegans[J].Exp Gerontol,2016,80:43-50
[29] 李娇,崔晓东,马晓丽,等.重组荞麦胰蛋白酶抑制剂延长C.elegans 寿命的作用机制[J].中国生物化学与分子生物学报(Li J,Cui XD,Ma XL,et al.Mechanism underlying prolongevity induced by rBTI in Caenorhabditis elegans[J].Chin J Biochem Mol Biol),2016,32(10):1112-1120
[30] Ghazi A,Henis-Korenblit S,Kenyon C.Regulation of Caenorhabditis elegans lifespan by a proteasomal E3 ligase complex[J].Proc Natl Acad Sci U S A,2007,104(14):5947-5952
[31] 詹成修,熊阳杰,张涛,等.崇明拟异小杆线虫daf-16基因的功能鉴定[J].昆虫学报(Zhan CX,Xiong YJ,Zhang T,et al.Functional identification of daf-16 in Heterorhabditidoides chongmingensis[J].Acta Entomol Sin),2018,61(8):932-940
[2] 任春久,张瑶,崔为正,等.氧化应激在2型糖尿病发病机制中的作用研究进展[J].生理学报(Ren CJ,Zhang Y,Cui WZ,et al.Progress in the role of oxidative stress in the pathogenesis of type 2 diabetes[J].Acta Physiol Sin),2013,65(6):664-673
[3] Brabdes RP,Kreuzer J.Vascular NADPH oxidases:molecular mechanisms of activation [J].Cardiovasc Res,2005,65(1):16-27
[4] 张忠佩,秦元华,任一鑫,等.氧化应激反应对肝纤维化形成研究的进展[J].中国病原生物学杂志(Zhang ZP,Qin YH,Ren YX,et al.Advances in research on the oxidative stress response to hepatic fibrosis[J].J Pathogen Biol),2017,12(11):1130-1133
[5] Zhu X,Yue H,Guo X,et al.The preconditioning of berberine suppresses hydrogen peroxide-induced premature senescence via regulation of sirtuin 1[J].Oxid Med Cell Longev,2017,2017:2391820
[6] Ishikado A,Sono Y,Matsumoto M,et al.Willow bark extract increases antioxidant enzymesand reduces oxidative stress through activation of Nrf2 in vascular endothelial cells and Caenorhabditis elegans[J].Free Radic Biol Med,2013,65:1506-1515
[7] 阮佼.芦丁对脂多糖诱导急性肺损伤小鼠的抗氧化应激作用[J].中国现代应用药学(Ruan J.Effect of rutin on oxidative stress of acute lung injury induced by lipopolysaccharide[J].Chin J Mod Appl Pharm),2015,32(9):1059-1062
[8] Huang C,Lin Y,Su H,et al.Forsythiaside protects against hydrogen peroxide-induced oxidative stress and apoptosis in PC12 cell[J].Neurochem Res,2015,40(1):27-35
[9] Wei T,Tian W,Yan H,et al.Protective effects of phillyrin on H2O2-induced oxidative stress and apoptosis in PC12 cells[J].Cell Mol Neurobiol,2014,34(8):1165-1173
[10] Kampk?tter A,Timpel C,Zurawski RF,et al.Increase of stress resistance and lifespan of Caenorhabditis elegans by quercetin[J].Comp Biochem Physiol B Biochem Mol Biol,2008,149(2):314-323
[11] 李兴泰,李洪成,刘泽.连翘花醇提物保护线粒体及抗氧化研究[J].中成药(Li XT,Li HC,Liu Z.Protective effects of ethanol extract of Forsythia suspense flower on mitochondria and antioxidant activity[J].Chin Trad Patent Med),2009,31(6):839-843
[12] 盛锋,李长城,付蕾,等.乙醇提取连翘花黄色素溶液稳定性的研究[J].食品与发酵工业(Sheng F,Li CC,Fu L,et al.Study on the stability of yellow pigment ethanol solution extracted from Forsythia Flower[J].Food Ferment Ind),2002,28(1):43-47
[13] 原江锋,邱智军,呼丽君,等.连翘花提取物活性成分及其稳定性[J].食品科学(Yuan JF,Qiu ZJ,Hu LJ,et al.Active compounds and stability of Forsythia suspensa flower extract[J].Food Sci),2015,36(14):130-134
[14] 王金亭,李秋凤.微波辅助提取连翘花黄色素及其抗氧化活性的研究[J].中国酿造(Wang JT,Li QF.Extraction of the yellow pigment from Forsythia with microwave assisted method and study on its antioxidant activity[J].China Brew),2010,(11):100-103
[15] 张飞.连翘花和叶中清除自由基成分分析及其含量测定[D].河北农业大学(Zhang F.Qualitative and quantitative analyses of the major bioactive compounds on scavenging free radical in flower and leaves of Forsythia suspense[D].Agric Univ Hebei Prov),2011
[16] 李丹凤,李石飞,张立伟.连翘不同部位活性成分含量分析[J].山西医科大学学报(Li DF,Li SF,Zhang LW.Analysis of the contents of active constituents in different parts of Forsythia suspense[J].J Shanxi Med Univ),2015,46(11):1097-1100
[17] Sugawara T,Sakamoto K.Killed Bifidobacterium longum enhanced stress tolerance and prolonged lifespan of Caenorhabditis elegans via DAF-16 [J].Br J Nutr,2018,120(8):872-880
[18] Schulenburg H,Felix MA.The natural biotic environment of Caenorhabditis elegans[J].Genetics,2017,206(1):55-86
[19] Wolfe KL,Liu RH.Cellular antioxidant activity (CAA) assay for assessing antioxidants,foods,and dietary supplements[J].J Agric Food Chem,2007,55(22):8896-8907
[20] Beckman KB,Ames BN.The free radical theory of aging matures[J].Physiol Rev,1998,78(2):547-581
[21] Bokov A,Chaudhuri A,Richardson A.The role of oxidative damage and stress in aging[J].Mech Ageing Dev,2004,125(10-11):811-826
[22] Hanschmann EM,Godoy JR,Berndt C,et al.Thioredoxins,glutaredoxins,and peroxiredoxins molecular mechanisms and health significance:from cofactors to antioxidants to redox signaling[J].Antioxid Redox Signal,2013,19(13):1539-1605
[23] Marengo B,Nitti M,Furfaro AL,et al.Redox homeostasis and cellular antioxidant systems:crucial players in cancer growth and therapy[J].Oxid Med Cell Longev,2016,2016:6235641.The oxidative stress in allelopathy:participation of prenyllipid antioxidants in the response to juglone in chlamydomonas reinhardtii[J].Phytochemistry,2017,144:171-179
[25] 张超,陈圣慧,何承伟,等.毒物兴奋效应与神经保护[J].科技导报(Zhang C,Chen SH,He CW,et al.Hormesis and neuroprotection[J].Sci Tech Rev),2015,33(3):110-113
[26] 刘天天,靳洪涛,王爱平.毒物兴奋效应及其对毒理学和医药研究的影响[J].毒理学杂志(Liu TT,Jin HT,Wang AP.Hormesis and its impact on toxicology and medical research[J].J Toxicol Feb),2016,30(1):78-83
[27] Friedman DB,Johnson TE.A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility[J].Genetics,1988,118(1):75-86
[28] Wu JZ,Huang JH,Khanabdali R,et al.Pyrroloquinoline quinone enhances the resistance to oxidative stress and extends lifespan upon DAF-16 and SKN-1 activities in C.elegans[J].Exp Gerontol,2016,80:43-50
[29] 李娇,崔晓东,马晓丽,等.重组荞麦胰蛋白酶抑制剂延长C.elegans 寿命的作用机制[J].中国生物化学与分子生物学报(Li J,Cui XD,Ma XL,et al.Mechanism underlying prolongevity induced by rBTI in Caenorhabditis elegans[J].Chin J Biochem Mol Biol),2016,32(10):1112-1120
[30] Ghazi A,Henis-Korenblit S,Kenyon C.Regulation of Caenorhabditis elegans lifespan by a proteasomal E3 ligase complex[J].Proc Natl Acad Sci U S A,2007,104(14):5947-5952
[31] 詹成修,熊阳杰,张涛,等.崇明拟异小杆线虫daf-16基因的功能鉴定[J].昆虫学报(Zhan CX,Xiong YJ,Zhang T,et al.Functional identification of daf-16 in Heterorhabditidoides chongmingensis[J].Acta Entomol Sin),2018,61(8):932-940
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