Wnt信号通路对铅暴露引起的斑马鱼胚胎发育毒性的影响
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摘要
铅是环境中常见的污染物,为探讨Wnt信号通路在铅暴露引起的胚胎发育毒性中的作用,将斑马鱼胚胎随机分为10组,每组100个,以铅(0、6、12、24、48μmol/L)和Wnt信号通路的激活剂HLY78(5μmol/L)单独及联合处理斑马鱼胚胎4-144 h,利用实时定量PCR技术进行分析.结果显示,铅单独暴露能够引起wnt3a表达量降低,而HLY78处理能够抑制wnt3a表达量的进行改变,修复Wnt信号通路.利用EthoVision XT软件获取并分析斑马鱼的自主运动、心率和运动行为等毒性试验的有效参数.与对照组相比,单独铅暴露组可致斑马鱼胚胎死亡率和畸形率增高,以及孵化率、心率和仔鱼平均活力降低(P<0.05);加入Wnt信号通路的激活剂HLY78,与单独暴露组相比,死亡率和畸形率显著降低,孵化率、心率和仔鱼平均活力显著升高(P <0.05).各组间胚胎的自主运动没有显著差异.本研究结果表明铅暴露可影响斑马鱼胚胎的发育,使用HLY78激活Wnt信号通路能够显著地缓解铅暴露诱导的这些影响,预示破坏Wnt信号通路可能与铅诱导的发育毒性相关.(图5参28)
The goal of this study was to investigate the role of Wnt signaling in mediating the developmental toxicity induced by lead(Pb) exposure using zebrafish(Danio rerio) embryos. Zebrafish embryos were randomly divided into 10 groups. Pb(0,6, 12, 24, 48 μmol/L) were respectively exposure to zebrafish embryos for 4–144 h post fertilization, with or without HLY78,an activator of the Wnt signaling pathway(5 μmol/L). The expression of wnt3 a was detected using quantitative real-time PCR(qRT-PCR). Pb exposure significantly decreased the expression of wnt3 a, and supplementation with HLY78 was able to reduce this effect. Mortality, hatchability, and malformation were observed and recorded. Spontaneous movement, heart rate, and locomotive behavior in larval zebrafish were recorded and analyzed using the Vision XT software. Pb exposure significantly decreased hatchability, heart rate, and swimming activities(P < 0.05). Furthermore, mortality and malformation were significantly increased(P < 0.05). Compared to individual Pb exposure, supplementation with HLY78 significantly increased hatchability, heart rate, and swimming activities(P < 0.05), and the mortality and malformation of larvae were significantly decreased(P < 0.05). However, there was no significant difference in the spontaneous movement of embryos between groups.Overall, Pb exposure induced a significant increase in developmental toxicity and decreased the expression of wnt3 a. Activation of Wnt signaling alleviated Pb-exposure induced developmental toxicity. Results in this study allow us to further understand the molecular mechanisms of Pb-induced developmental toxicity, which potentially involves the inhibition of Wnt signaling.
The goal of this study was to investigate the role of Wnt signaling in mediating the developmental toxicity induced by lead(Pb) exposure using zebrafish(Danio rerio) embryos. Zebrafish embryos were randomly divided into 10 groups. Pb(0,6, 12, 24, 48 μmol/L) were respectively exposure to zebrafish embryos for 4–144 h post fertilization, with or without HLY78,an activator of the Wnt signaling pathway(5 μmol/L). The expression of wnt3 a was detected using quantitative real-time PCR(qRT-PCR). Pb exposure significantly decreased the expression of wnt3 a, and supplementation with HLY78 was able to reduce this effect. Mortality, hatchability, and malformation were observed and recorded. Spontaneous movement, heart rate, and locomotive behavior in larval zebrafish were recorded and analyzed using the Vision XT software. Pb exposure significantly decreased hatchability, heart rate, and swimming activities(P < 0.05). Furthermore, mortality and malformation were significantly increased(P < 0.05). Compared to individual Pb exposure, supplementation with HLY78 significantly increased hatchability, heart rate, and swimming activities(P < 0.05), and the mortality and malformation of larvae were significantly decreased(P < 0.05). However, there was no significant difference in the spontaneous movement of embryos between groups.Overall, Pb exposure induced a significant increase in developmental toxicity and decreased the expression of wnt3 a. Activation of Wnt signaling alleviated Pb-exposure induced developmental toxicity. Results in this study allow us to further understand the molecular mechanisms of Pb-induced developmental toxicity, which potentially involves the inhibition of Wnt signaling.
引文
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2张浩,刘汝涛,魏云波.重金属铅诱发机体氧化应激效应的研究进展[J].科学与管理,2016,36(5):49-57[Zhang H,Liu RT,Wei YB.Current perspectives on lead-induced oxidative stress[J].Sci Manage,2016,36(5):49-57]
3葛玲玲,宋佳坤,范纯新.斑马鱼中Wnt信号通过调控侧线基板形成影响侧线发育机制[J].上海海洋大学学报,2018,27(1):16-23[Ge LL,Song JK,Fan CX.The Wnt signaling affects development of the lateral line by regulating the specification of lateral line placode in the zebrafish[J].J Shanghai Ocean Univ,2018,27(1):16-23]
4裴哲,曾中秋,陈雨文,李文华,Tang YJ,唐亚雄.肝癌Wnt通路可溶性跨膜受体Frizzled-7稳定表达细胞株的构建与鉴定[J].应用与环境生物学报,2015,21(6):1076-1079[Pei Z,Zeng ZQ,Chen YW,Li WH,Tang YJ,Tang YX.Establishment and identification of a stable hepatocellular carcinoma cell line of soluble Frizzled-7,a Frizzled receptor of Wnt signaling[J].Chin J Appl Environ Biol,2015,21(6):1076-1079]
5 Rong XZ,Chen C,Zhou P,Zhou YM,Li M,Lu L,Liu YZ,Zhou JF,Duan CM.R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos[J].PLoS ONE,2014,9(6):e99514
6 Wang S,Yin J,Chen D,Nie F,Song X,Fei C,Miao H,Jing C,Ma W,Wang L,Xie S,Li C,Zeng R,Pan W,Hao X,Li L.Small-molecule modulation of Wnt signaling via modulating the Axin-LRP5/6 interaction[J].Nat Chem Biol,2013,9(9):579-585
7 Guo X,Zhang L,Fan Y,Zhang D,Qin L,Dong S,Li G.Oxysterolbinding protein-related protein 8 inhibits gastric cancer growth through induction of er stress,inhibition of Wnt signaling,and activation of apoptosis[J].Oncol Res,2017,25(5):799-808
8 Venkatadri R,Iyer AKV,Kaushik V,Azad N.A novel resveratrolsalinomycin combination sensitizes ER-positive breast cancer cells to apoptosis[J].Pharmacol Rep,2017,69(4):788-797
9 Xia Q,Luo J,Mei X,Wang Y,Huang W,Wang J,Yang R,Ma Z,Lin R.Adevelopmental toxicity assay of Carpesii Fructus on zebrafish embryos/larvae[J].Toxicol Res(Camb),2017,6(4):460-467
10 Padilla S,Hunter DL,Padnos B,Frady S,MacPhail RC.Assessing locomotor activity in larval zebrafish:influence of extrinsic and intrinsic variables[J].Neurotoxicol Teratol,2011,33(6):624-630
11 Riehl R,Kyzar E,Allain A,Green J,Hook M,Monnig L,Rhymes K,Roth A,Pham M,Razavi R,Dileo J,Gaikwad S,Hart P,Kalueff AV.Behavioral and physiological effects of acute ketamine exposure in adult zebrafish[J].Neurotoxicol Teratol,2011,33(6):658-667
12张红,徐凤兰,周蓉,胡晓娅,罗冰芝,张哲,刘小琴,桂芳,司婧.铅和萘联合暴露对斑马鱼胚胎发育的影响[J].环境与职业医学,2018,35(2):95-101[Zhang H,Xu FL,Zhou R,Hu XY,Luo BZ,Zhang Z,Liu XQ,Gui F,Si J.Effects of combined exposure to lead and naphthalene on development of zebrafish embryos[J].J Environ Occupat Med,2018,35(2):95-101]
13张小晶,贾聪聪,林林,郭小娟,方周溪,白承连,黄陈平.铅对斑马鱼胚胎发育的毒性作用[J].解剖学报,2012,43(5):662-666[Zhang XJ,Jia CC,Lin L,Guo XJ,Fang ZX,Bai CL,Huang CP.Toxic effects of lead on embryonic development of zebrafish[J].Acta Anatom Sin,2012,43(5):662-666]
14陈香平,黄长江,陈元红,刘滨,刘翠平,朱亚先,张勇.铅和得克隆联合暴露对斑马鱼胚胎的神经毒性作用[J].生态毒理学报,2017,12(3):309-316[Chen XP,Huang CJ,Chen YH,Liu B,Liu CP,Zhu YX,Zhang Y.Neurotoxic effects of co-exposure to lead and dechlorane plus on zebrafish(Danio rerio)embryos[J].Asian J Ecotoxicol,2017,12(3):309-316]
15汪慧琼,黄永平,刘建文,颜崇淮.铅神经毒性机制及神经保护药物的研究进展[J].毒理学杂志,2015,29(3):222-226[Wang HQ,Huang YP,Liu JW,Yan CH.Research progress on the mechanism of lead neurotoxicity and neuroprotective drugs[J].J Toxicol,2015,29(3):222-226]
16张遥,任秀智,韩金祥,鲁艳芹.Wnt信号通路与人类疾病相关性的研究进展[J].中国生物制品学杂志,2018,31(1):81-86[Zhang Y,Ren XZ,Han JX,Lu YQ.Progress in study on relationship between Wnt signaling pathway and human diseases[J].Chin J Biol,2018,31(1):81-86]
17葛玲玲.斑马鱼中Wnt信号对神经丘特异表达基因Six2b的调控[D].上海:上海海洋大学,2017[Ge LL.The Regulation of Wnt signaling on expression of neuromast specific gene Six2b in the zebrafish[D].Shanghai:Shanghai Ocean University,2017]
18 Hikasa H,Sokol SY.Wnt signaling in vertebrate axis specification[J].Cold Spring Harb Perspect Biol,2013,5(1):a007955
19徐扬.R-spondin及其受体在Wnt信号通路中相互作用的研究[D].吉林:吉林大学,2013[Xu Y.The research on the interaction between R-spondin and its receptors in Wnt signaling[D].Jilin:Jilin University,2013]
20 Yao H,Xu X,Zhou Y,Xu C.Impacts of isopyrazam exposure on the development of early-life zebrafish(Danio rerio)[J].Environ Sci Pollut Res,2018,25(24):23799-23808
21 Li H,Yu S,Cao F,Wang C,Zheng M,Li X,Qiu L.Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish(Danio rerio)[J].Ecotoxicol Environ Saf,2018,151:1-9
22 Suvarchala G,Philip GH.Toxicity of 3,5,6-trichloro-2-pyridinol tested at multiple stages of zebrafish(Danio rerio)development[J].Environ Sci Pollut Res Int,2016,23(15):15515-15523
23 Sasakura Y,Makabe KW.Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells[J].De Growth Differ,2001,43(5):573-582
24 Schneider J,Skelton RL,Von Stetina SE,Middelkoop TC,van Oudenaarden A,Korswagen HC,Miller DM.UNC-4 antagonizes Wnt signaling to regulate synaptic choice in the C.elegans motor circuit[J].Development,2012,139(12):2234-2245
25杨阳.噻呋酰胺对斑马鱼的毒性及作用机制研究[D].北京:中国农业大学,2017[Yang Y.The toxic effects and mechanisms of thifluzamide on zebrafish(Danio rerio)[D].Beijing:China Agricultural University,2017]
26 Ueno S,Weidinger G,Osugi T,Kohn AD,Golob JL,Pabon L,Reinecke H,Moon RT,Murry CE.Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells[J].PNAS,2007,104(23):9685-9690
27 Barik A,Zhang B,Sohal GS,Xiong WC,Mei L.Crosstalk between Agrin and Wnt signaling pathways in development of vertebrate neuromuscular junction[J].Dev Neurobiol,2014,74(8):828-838
28 Huraskin D,Eiber N,Reichel M,Zidek LM,Kravic B,Bernkopf D,von Maltzahn J,Behrens J,Hashemolhosseini S.Wnt/beta-catenin signaling via Axin2 is required for myogenesis and,together with YAP/Taz and Tead1,active in IIa/IIx muscle fibers[J].Development,2016,143(17):3128-3142
2张浩,刘汝涛,魏云波.重金属铅诱发机体氧化应激效应的研究进展[J].科学与管理,2016,36(5):49-57[Zhang H,Liu RT,Wei YB.Current perspectives on lead-induced oxidative stress[J].Sci Manage,2016,36(5):49-57]
3葛玲玲,宋佳坤,范纯新.斑马鱼中Wnt信号通过调控侧线基板形成影响侧线发育机制[J].上海海洋大学学报,2018,27(1):16-23[Ge LL,Song JK,Fan CX.The Wnt signaling affects development of the lateral line by regulating the specification of lateral line placode in the zebrafish[J].J Shanghai Ocean Univ,2018,27(1):16-23]
4裴哲,曾中秋,陈雨文,李文华,Tang YJ,唐亚雄.肝癌Wnt通路可溶性跨膜受体Frizzled-7稳定表达细胞株的构建与鉴定[J].应用与环境生物学报,2015,21(6):1076-1079[Pei Z,Zeng ZQ,Chen YW,Li WH,Tang YJ,Tang YX.Establishment and identification of a stable hepatocellular carcinoma cell line of soluble Frizzled-7,a Frizzled receptor of Wnt signaling[J].Chin J Appl Environ Biol,2015,21(6):1076-1079]
5 Rong XZ,Chen C,Zhou P,Zhou YM,Li M,Lu L,Liu YZ,Zhou JF,Duan CM.R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos[J].PLoS ONE,2014,9(6):e99514
6 Wang S,Yin J,Chen D,Nie F,Song X,Fei C,Miao H,Jing C,Ma W,Wang L,Xie S,Li C,Zeng R,Pan W,Hao X,Li L.Small-molecule modulation of Wnt signaling via modulating the Axin-LRP5/6 interaction[J].Nat Chem Biol,2013,9(9):579-585
7 Guo X,Zhang L,Fan Y,Zhang D,Qin L,Dong S,Li G.Oxysterolbinding protein-related protein 8 inhibits gastric cancer growth through induction of er stress,inhibition of Wnt signaling,and activation of apoptosis[J].Oncol Res,2017,25(5):799-808
8 Venkatadri R,Iyer AKV,Kaushik V,Azad N.A novel resveratrolsalinomycin combination sensitizes ER-positive breast cancer cells to apoptosis[J].Pharmacol Rep,2017,69(4):788-797
9 Xia Q,Luo J,Mei X,Wang Y,Huang W,Wang J,Yang R,Ma Z,Lin R.Adevelopmental toxicity assay of Carpesii Fructus on zebrafish embryos/larvae[J].Toxicol Res(Camb),2017,6(4):460-467
10 Padilla S,Hunter DL,Padnos B,Frady S,MacPhail RC.Assessing locomotor activity in larval zebrafish:influence of extrinsic and intrinsic variables[J].Neurotoxicol Teratol,2011,33(6):624-630
11 Riehl R,Kyzar E,Allain A,Green J,Hook M,Monnig L,Rhymes K,Roth A,Pham M,Razavi R,Dileo J,Gaikwad S,Hart P,Kalueff AV.Behavioral and physiological effects of acute ketamine exposure in adult zebrafish[J].Neurotoxicol Teratol,2011,33(6):658-667
12张红,徐凤兰,周蓉,胡晓娅,罗冰芝,张哲,刘小琴,桂芳,司婧.铅和萘联合暴露对斑马鱼胚胎发育的影响[J].环境与职业医学,2018,35(2):95-101[Zhang H,Xu FL,Zhou R,Hu XY,Luo BZ,Zhang Z,Liu XQ,Gui F,Si J.Effects of combined exposure to lead and naphthalene on development of zebrafish embryos[J].J Environ Occupat Med,2018,35(2):95-101]
13张小晶,贾聪聪,林林,郭小娟,方周溪,白承连,黄陈平.铅对斑马鱼胚胎发育的毒性作用[J].解剖学报,2012,43(5):662-666[Zhang XJ,Jia CC,Lin L,Guo XJ,Fang ZX,Bai CL,Huang CP.Toxic effects of lead on embryonic development of zebrafish[J].Acta Anatom Sin,2012,43(5):662-666]
14陈香平,黄长江,陈元红,刘滨,刘翠平,朱亚先,张勇.铅和得克隆联合暴露对斑马鱼胚胎的神经毒性作用[J].生态毒理学报,2017,12(3):309-316[Chen XP,Huang CJ,Chen YH,Liu B,Liu CP,Zhu YX,Zhang Y.Neurotoxic effects of co-exposure to lead and dechlorane plus on zebrafish(Danio rerio)embryos[J].Asian J Ecotoxicol,2017,12(3):309-316]
15汪慧琼,黄永平,刘建文,颜崇淮.铅神经毒性机制及神经保护药物的研究进展[J].毒理学杂志,2015,29(3):222-226[Wang HQ,Huang YP,Liu JW,Yan CH.Research progress on the mechanism of lead neurotoxicity and neuroprotective drugs[J].J Toxicol,2015,29(3):222-226]
16张遥,任秀智,韩金祥,鲁艳芹.Wnt信号通路与人类疾病相关性的研究进展[J].中国生物制品学杂志,2018,31(1):81-86[Zhang Y,Ren XZ,Han JX,Lu YQ.Progress in study on relationship between Wnt signaling pathway and human diseases[J].Chin J Biol,2018,31(1):81-86]
17葛玲玲.斑马鱼中Wnt信号对神经丘特异表达基因Six2b的调控[D].上海:上海海洋大学,2017[Ge LL.The Regulation of Wnt signaling on expression of neuromast specific gene Six2b in the zebrafish[D].Shanghai:Shanghai Ocean University,2017]
18 Hikasa H,Sokol SY.Wnt signaling in vertebrate axis specification[J].Cold Spring Harb Perspect Biol,2013,5(1):a007955
19徐扬.R-spondin及其受体在Wnt信号通路中相互作用的研究[D].吉林:吉林大学,2013[Xu Y.The research on the interaction between R-spondin and its receptors in Wnt signaling[D].Jilin:Jilin University,2013]
20 Yao H,Xu X,Zhou Y,Xu C.Impacts of isopyrazam exposure on the development of early-life zebrafish(Danio rerio)[J].Environ Sci Pollut Res,2018,25(24):23799-23808
21 Li H,Yu S,Cao F,Wang C,Zheng M,Li X,Qiu L.Developmental toxicity and potential mechanisms of pyraoxystrobin to zebrafish(Danio rerio)[J].Ecotoxicol Environ Saf,2018,151:1-9
22 Suvarchala G,Philip GH.Toxicity of 3,5,6-trichloro-2-pyridinol tested at multiple stages of zebrafish(Danio rerio)development[J].Environ Sci Pollut Res Int,2016,23(15):15515-15523
23 Sasakura Y,Makabe KW.Ascidian Wnt-5 gene is involved in the morphogenetic movement of notochord cells[J].De Growth Differ,2001,43(5):573-582
24 Schneider J,Skelton RL,Von Stetina SE,Middelkoop TC,van Oudenaarden A,Korswagen HC,Miller DM.UNC-4 antagonizes Wnt signaling to regulate synaptic choice in the C.elegans motor circuit[J].Development,2012,139(12):2234-2245
25杨阳.噻呋酰胺对斑马鱼的毒性及作用机制研究[D].北京:中国农业大学,2017[Yang Y.The toxic effects and mechanisms of thifluzamide on zebrafish(Danio rerio)[D].Beijing:China Agricultural University,2017]
26 Ueno S,Weidinger G,Osugi T,Kohn AD,Golob JL,Pabon L,Reinecke H,Moon RT,Murry CE.Biphasic role for Wnt/beta-catenin signaling in cardiac specification in zebrafish and embryonic stem cells[J].PNAS,2007,104(23):9685-9690
27 Barik A,Zhang B,Sohal GS,Xiong WC,Mei L.Crosstalk between Agrin and Wnt signaling pathways in development of vertebrate neuromuscular junction[J].Dev Neurobiol,2014,74(8):828-838
28 Huraskin D,Eiber N,Reichel M,Zidek LM,Kravic B,Bernkopf D,von Maltzahn J,Behrens J,Hashemolhosseini S.Wnt/beta-catenin signaling via Axin2 is required for myogenesis and,together with YAP/Taz and Tead1,active in IIa/IIx muscle fibers[J].Development,2016,143(17):3128-3142
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