脱氧雪腐镰刀菌烯醇与黄曲霉毒素B_1单一及联合染毒对小鼠神经递质分泌和钙稳态的影响
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摘要
本试验旨在研究脱氧雪腐镰刀菌烯醇(DON)与黄曲霉毒素B_1(AFB_1)单一及联合染毒对小鼠神经递质分泌和钙稳态的影响。选取96只18日龄昆明系雄性小鼠,随机分成4组,每组24只,每天分别进行灌胃染毒,对照组灌服生理盐水,DON组灌服500μg/kg DON,AFB_1组灌服200μg/kg AFB_1,DON+AFB_1组灌服200μg/kg AFB_1+500μg/kg DON。试验期45 d。分别于试验第0天(试验开始前)、第15天、第30天、第45天每组选取6只小鼠进行剖杀,取脑组织,采用酶联免疫吸附测定(ELISA)试剂盒检测神经递质、钙离子(Ca~(2+))和钙调蛋白(CaM)含量,实时荧光定量PCR(qRT-PCR)检测CaM mRNA的表达量,Western blotting法检测钙调蛋白依赖性蛋白激酶(CaMKⅡ)蛋白表达量。结果显示:在各时间点与对照组相比,DON+AFB_1组小鼠脑组织中5-羟色胺(5-HT)、谷氨酸(Glu)、乙酰胆碱(ACH)含量显著或极显著上升(P<0.05或P<0.01),多巴胺(DA)、Ca~(2+)、CaM含量极显著下降(P<0.01),CaM mRNA表达量(第45天除外)极显著下降(P<0.01),磷酸化钙调蛋白依赖性蛋白激酶(p-CaMKⅡ)蛋白表达量/CaMKⅡ蛋白表达量极显著升高(P<0.01);DON组和AFB_1组小鼠脑组织中5-HT、ACH含量显著或极显著上升(P<0.05或P<0.01),Ca~(2+)含量极显著下降(P<0.01),CaM mRNA表达量(第45天除外)显著或极显著下降(P<0.05或P<0.01),p-CaMKⅡ蛋白表达量/CaMKⅡ蛋白表达量极显著升高(P<0.01)。试验结果表明,DON与AFB_1单一及联合染毒均可造成小鼠脑组织中神经递质分泌紊乱和钙稳态失衡,DON和AFB_1的联合毒性大于单一毒性,两者具有协同效应。
The aim of this experiment was to study the effects of single and combined exposure of deoxynivalenol(DON) and aflatoxin B1(AFB_1) on neurotransmitter secretion and calcium homeostasis in mice.Ninetysix Kunming male mice were randomly divided into 4 groups(24 mice each group).Each group was administered by gavage.Mice in control group were given physiological saline,mice in DON group were given500 μg/kg DON,mice in AFB_1 group were given 200 μg/kg AFB_1 and mice in DON+AFB_1 group were given200 μg/kg AFB_1+500 μg/kg DON.The trial was conducted for 45 days.On days 0(before the trial),15,30 and 45,six mice were selected from each group and slaughtered,brain tissues were removed and the contents of neurotransmitters,calcium ion(Ca~(2+)) and calmodulin(CaM) were detected by enzyme-linked immunosorbent assay(ELISA) kits.The content of CaM mRNA expression level was detected by quantitative realtime PCR(qRT-PCR) and the protein expression level of calmodulin-dependent protein kinase(CaMKⅡ)was detected by Western blotting.The results showed that compared with the control group on the each time point,the contents of 5-hydroxytryptamine(5-HT),glutamic acid(Glu),acetylcholine(ACH) were significantly increased(P<0.05 or P<0.01),while the contents of dopamine(DA),Ca~(2+) and CaM in DON+AFB_1 group were significantly decreased(P<0.01),as well as the CaM mRNA expression level(expect day 45)was significantly reduced(P<0.01).Meanwhile,the phosphorylated-CaMKⅡ(p-CaMKⅡ) protein expression level/CaMKⅡ protein expression level in DON+AFB_1 group was significantly higher than that in control group(P<0.01).Compared with the control group on the each time point,the contents of 5-HT and ACH in DON group and AFB_1 group were significantly increased(P<0.05 or P<0.01),while the content of Ca~(2+) was significantly decreased(P<0.01),and the CaM mRNA expression level(expect day 45) was significantly reduced(P<0.05 or P<0.01).However,the of p-CaMKⅡ protein expression level/CaMKⅡ protein expression level in DON group and AFB_1 group was significantly higher than that in control group(P<0.01).These results indicate that single and combined exposure of DON and AFB_1 all can cause disturbance of neurotransmitter secretion and imbalance of calcium homeostasis.The combined neurotoxic effect of DON and AFB_1 is greater than any of single exposure and they have synergistic effect.
The aim of this experiment was to study the effects of single and combined exposure of deoxynivalenol(DON) and aflatoxin B1(AFB_1) on neurotransmitter secretion and calcium homeostasis in mice.Ninetysix Kunming male mice were randomly divided into 4 groups(24 mice each group).Each group was administered by gavage.Mice in control group were given physiological saline,mice in DON group were given500 μg/kg DON,mice in AFB_1 group were given 200 μg/kg AFB_1 and mice in DON+AFB_1 group were given200 μg/kg AFB_1+500 μg/kg DON.The trial was conducted for 45 days.On days 0(before the trial),15,30 and 45,six mice were selected from each group and slaughtered,brain tissues were removed and the contents of neurotransmitters,calcium ion(Ca~(2+)) and calmodulin(CaM) were detected by enzyme-linked immunosorbent assay(ELISA) kits.The content of CaM mRNA expression level was detected by quantitative realtime PCR(qRT-PCR) and the protein expression level of calmodulin-dependent protein kinase(CaMKⅡ)was detected by Western blotting.The results showed that compared with the control group on the each time point,the contents of 5-hydroxytryptamine(5-HT),glutamic acid(Glu),acetylcholine(ACH) were significantly increased(P<0.05 or P<0.01),while the contents of dopamine(DA),Ca~(2+) and CaM in DON+AFB_1 group were significantly decreased(P<0.01),as well as the CaM mRNA expression level(expect day 45)was significantly reduced(P<0.01).Meanwhile,the phosphorylated-CaMKⅡ(p-CaMKⅡ) protein expression level/CaMKⅡ protein expression level in DON+AFB_1 group was significantly higher than that in control group(P<0.01).Compared with the control group on the each time point,the contents of 5-HT and ACH in DON group and AFB_1 group were significantly increased(P<0.05 or P<0.01),while the content of Ca~(2+) was significantly decreased(P<0.01),and the CaM mRNA expression level(expect day 45) was significantly reduced(P<0.05 or P<0.01).However,the of p-CaMKⅡ protein expression level/CaMKⅡ protein expression level in DON group and AFB_1 group was significantly higher than that in control group(P<0.01).These results indicate that single and combined exposure of DON and AFB_1 all can cause disturbance of neurotransmitter secretion and imbalance of calcium homeostasis.The combined neurotoxic effect of DON and AFB_1 is greater than any of single exposure and they have synergistic effect.
引文
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[3]BROEKAERT N,DEVREESE M,DEMEYERE K,et al.Comparative in vitro cytotoxicity of modified deoxynivalenol on porcine intestinal epithelial cells[J].Food and Chemical Toxicology,2016,95:103-109.
[4]HAN J,WANG Q C,ZHU C C,et al.Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation[J].Toxicology and Applied Pharmacology,2016,300:70-76.
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[7]Centers for Disease Control and Prevention.Outbreak of aflatoxin poisoning-eastern and central provinces,Kenya,January-July 2004[J].Morbidity and Mortality Weekly Report,2004,53(34):790-793.
[8]许伟,范梦雪,孟婷婷,等.脱氧雪腐镰刀菌烯醇对PC12细胞凋亡的影响[J].南京农业大学学报,2016,39(5):838-844.
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[10]陈鑫.谷氨酸、钙离子与脑损伤后神经元缺失[J].国外医学神经病学(神经外科学分册),2002,29(2):185-187.
[11]何成华,樊彦红,王莹,等.饲料中黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇对鲤鱼的联合毒性研究[J].南京农业大学学报,2010,33(6):85-89.
[12]李斌,郭红卫.镰刀菌毒素与黄曲霉毒素的联合毒性研究---AFB1和DON的RDS实验[J].卫生研究,2000,29(6):393-395.
[13]GIRISH C K,ACDONALD E J,SCHEININ M,et al.Effects of feedborne Fusarium mycotoxins on brain regional neurochemistry of turkeys[J].Poultry Science,2008,87(7):1295-1302.
[14]ROTTER B A,PRELUSKY D B,PESTKA J J.Toxicology of deoxynivalenol(vomitoxin)[J].Journal of Toxicology and Environmental Health,1996,48(1):1-34.
[15]王德山,单德红,于化新,等.呕必宁对顺铂所致脑组织5-HT含量变化的影响[J].中华中医药学刊,2007,25(5):869-871.
[16]文袅.硒对亚急性砷中毒鸡脑损伤影响的研究[D].硕士学位论文.成都:四川农业大学,2011.
[17]顾仁骏,张秀明,尹景岗,等.5-羟色胺、乙酰胆碱和多巴胺测定在急性一氧化碳中毒后迟发性脑病中的意义[J].中华神经科杂志,1999,32(3):191.
[18]MASLIAH E,RABER J,ALFORD M,et al.Amyloid protein precursor stimulates excitatory amino acid transport.Implications for roles in neuroprotection and pathogenesis[J].Journal of Biological Chemistry,1998,273(20):12548-12554.
[19]赵吉清,吴强,董兆君,等.谷氨酸在模拟高原梭曼中毒性脑损伤的作用研究[J].第三军医大学学报,2001,23(6):630-632.
[20]耿芳芳,范梦雪,姜云晶,等.DON暴露对雏鸡神经递质及钙调蛋白含量的影响[J].西北农林科技大学学报(自然科学版),2017,45(2):56-62.
[21]李时光,王冀康,李文强,等.急性一氧化碳中毒后迟发性脑病患者血及脑脊液中乙酰胆碱水平变化及其意义[J].临床神经病学杂志,2010,23(4):248-250.
[22]CHERNORUDSKIY A L,ZITO E.Regulation of calcium homeostasis by ER redox:a close-up of the ER/mitochondria connection[J].Journal of Molecular Biology,2017,429(5):620-632.
[23]石广滨.胰岛素样生长因子-1对痴呆模型大鼠保护作用及其机制的实验研究[D].博士学位论文.长春:吉林大学,2006.
[24]冯征,张均田.中枢神经系统钙稳态失调和老龄脑功能[J].生理科学进展,2000,31(2):102-108.
[25]吴伟.钙离子相关机制在慢性颞叶癫痫发生中作用机制的实验研究[D].博士学位论文.济南:山东大学,2005.
[26]李志光,江泉观.细胞内钙稳态失调与中毒性损伤-中毒机理的钙流学说[J].国外医学(卫生学分册),1987(2):80-85.
[27]彭双清,杨进生.镰刀菌毒素脱氧雪腐镰刀菌烯醇对心肌细胞Ca2+通道的阻滞作用[J].中国预防医学杂志,2004,5(4):241-243.
[2]JUAN C,RAIOLA A,MAES J,et al.Presence of mycotoxin in commercial infant formulas and baby foods from Italian market[J].Food Control,2014,39:227-236.
[3]BROEKAERT N,DEVREESE M,DEMEYERE K,et al.Comparative in vitro cytotoxicity of modified deoxynivalenol on porcine intestinal epithelial cells[J].Food and Chemical Toxicology,2016,95:103-109.
[4]HAN J,WANG Q C,ZHU C C,et al.Deoxynivalenol exposure induces autophagy/apoptosis and epigenetic modification changes during porcine oocyte maturation[J].Toxicology and Applied Pharmacology,2016,300:70-76.
[5]SAVARD C,PINILLA V,PROVOST C,et al.In vitro effect of deoxynivalenol(DON)mycotoxin on porcine reproductive and respiratory syndrome virus replication[J].Food and Chemical Toxicology,2014,65:219-226.
[6]MITCHELL N J,BOWERS E,HURBURGH C,et al.Potential economic losses to the US corn industry from aflatoxin contamination[J].Food Additives&Contaminants:Part A,2016,33(3):540-550.
[7]Centers for Disease Control and Prevention.Outbreak of aflatoxin poisoning-eastern and central provinces,Kenya,January-July 2004[J].Morbidity and Mortality Weekly Report,2004,53(34):790-793.
[8]许伟,范梦雪,孟婷婷,等.脱氧雪腐镰刀菌烯醇对PC12细胞凋亡的影响[J].南京农业大学学报,2016,39(5):838-844.
[9]GAIGS,BONNET M S,TARDIVEL C,et al.c-Fos immunoreactivity in the pig brain following deoxynivalenol intoxication:focus on NUCB2/nesfatin-1 expressing neurons[J].NeuroToxicology,2013,34:135-149.
[10]陈鑫.谷氨酸、钙离子与脑损伤后神经元缺失[J].国外医学神经病学(神经外科学分册),2002,29(2):185-187.
[11]何成华,樊彦红,王莹,等.饲料中黄曲霉毒素B1和脱氧雪腐镰刀菌烯醇对鲤鱼的联合毒性研究[J].南京农业大学学报,2010,33(6):85-89.
[12]李斌,郭红卫.镰刀菌毒素与黄曲霉毒素的联合毒性研究---AFB1和DON的RDS实验[J].卫生研究,2000,29(6):393-395.
[13]GIRISH C K,ACDONALD E J,SCHEININ M,et al.Effects of feedborne Fusarium mycotoxins on brain regional neurochemistry of turkeys[J].Poultry Science,2008,87(7):1295-1302.
[14]ROTTER B A,PRELUSKY D B,PESTKA J J.Toxicology of deoxynivalenol(vomitoxin)[J].Journal of Toxicology and Environmental Health,1996,48(1):1-34.
[15]王德山,单德红,于化新,等.呕必宁对顺铂所致脑组织5-HT含量变化的影响[J].中华中医药学刊,2007,25(5):869-871.
[16]文袅.硒对亚急性砷中毒鸡脑损伤影响的研究[D].硕士学位论文.成都:四川农业大学,2011.
[17]顾仁骏,张秀明,尹景岗,等.5-羟色胺、乙酰胆碱和多巴胺测定在急性一氧化碳中毒后迟发性脑病中的意义[J].中华神经科杂志,1999,32(3):191.
[18]MASLIAH E,RABER J,ALFORD M,et al.Amyloid protein precursor stimulates excitatory amino acid transport.Implications for roles in neuroprotection and pathogenesis[J].Journal of Biological Chemistry,1998,273(20):12548-12554.
[19]赵吉清,吴强,董兆君,等.谷氨酸在模拟高原梭曼中毒性脑损伤的作用研究[J].第三军医大学学报,2001,23(6):630-632.
[20]耿芳芳,范梦雪,姜云晶,等.DON暴露对雏鸡神经递质及钙调蛋白含量的影响[J].西北农林科技大学学报(自然科学版),2017,45(2):56-62.
[21]李时光,王冀康,李文强,等.急性一氧化碳中毒后迟发性脑病患者血及脑脊液中乙酰胆碱水平变化及其意义[J].临床神经病学杂志,2010,23(4):248-250.
[22]CHERNORUDSKIY A L,ZITO E.Regulation of calcium homeostasis by ER redox:a close-up of the ER/mitochondria connection[J].Journal of Molecular Biology,2017,429(5):620-632.
[23]石广滨.胰岛素样生长因子-1对痴呆模型大鼠保护作用及其机制的实验研究[D].博士学位论文.长春:吉林大学,2006.
[24]冯征,张均田.中枢神经系统钙稳态失调和老龄脑功能[J].生理科学进展,2000,31(2):102-108.
[25]吴伟.钙离子相关机制在慢性颞叶癫痫发生中作用机制的实验研究[D].博士学位论文.济南:山东大学,2005.
[26]李志光,江泉观.细胞内钙稳态失调与中毒性损伤-中毒机理的钙流学说[J].国外医学(卫生学分册),1987(2):80-85.
[27]彭双清,杨进生.镰刀菌毒素脱氧雪腐镰刀菌烯醇对心肌细胞Ca2+通道的阻滞作用[J].中国预防医学杂志,2004,5(4):241-243.