脱氧雪腐镰刀菌烯醇与黄曲霉毒素B1单一及联合染毒对小鼠神经递质分泌和钙稳态的影响

刘秦, 储小燕, 刘锦芳, 张娅菲, 朱雷, 张咪, 冯士彬, 李玉, 吴金节, 王希春

doi:10.3969/j.issn.1006-267x.2019.02.048

动物营养学报 >

2019 , Vol. 31 >Issue 2: 912 - 921

DOI: https://doi.org/10.3969/j.issn.1006-267x.2019.02.048

饲料科学与技术 Feed science and technology

脱氧雪腐镰刀菌烯醇与黄曲霉毒素B₁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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安徽农业大学动物科技学院, 合肥 230036

刘秦(1993-),男,江苏如皋人,硕士研究生,研究方向为畜禽营养代谢病与中毒病。E-mail:liuqinjit@163.com

收稿日期: 2018-07-02

网络出版日期: 2019-02-18

基金资助

安徽省生猪产业技术体系资助项目（AHCYJSTX-05）

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Effects of Single and Combined Exposure of Deoxynivalenol and Aflatoxin B₁ on Neurotransmitter Secretion and Calcium Homeostasis in Mice

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College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

Received date: 2018-07-02

Online published: 2019-02-18

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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B₁（AFB₁）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500 μg/kg DON，AFB₁组灌服200 μg/kg AFB₁，DON+AFB₁组灌服200 μg/kg AFB₁+500 μg/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（Ca²⁺）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+AFB₁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）、Ca²⁺、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AFB₁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），Ca²⁺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AFB₁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AFB₁的联合毒性大于单一毒性，两者具有协同效应。

关键词： 脱氧雪腐镰刀菌烯醇; 黄曲霉毒素B₁; 神经递质; 钙调蛋白; 小鼠

本文引用格式

刘秦, 储小燕, 刘锦芳, 张娅菲, 朱雷, 张咪, 冯士彬, 李玉, 吴金节, 王希春 . 脱氧雪腐镰刀菌烯醇与黄曲霉毒素B₁单一及联合染毒对小鼠神经递质分泌和钙稳态的影响[J]. 动物营养学报, 2019 , 31(2) : 912 -921 . DOI: 10.3969/j.issn.1006-267x.2019.02.048

Abstract

The aim of this experiment was to study the effects of single and combined exposure of deoxynivalenol (DON) and aflatoxin B₁ (AFB₁) on neurotransmitter secretion and calcium homeostasis in mice. Ninety-six 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 given 500 μg/kg DON, mice in AFB₁ group were given 200 μg/kg AFB₁ and mice in DON+AFB₁ group were given 200 μg/kg AFB₁+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 (Ca²⁺) and calmodulin (CaM) were detected by enzyme-linked immunosorbent assay (ELISA) kits. The content of CaM mRNA expression level was detected by quantitative real-time 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), Ca²⁺ and CaM in DON+AFB₁ 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+AFB₁ 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 AFB₁ group were significantly increased (P<0.05 or P<0.01), while the content of Ca²⁺ 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 AFB₁ group was significantly higher than that in control group (P<0.01). These results indicate that single and combined exposure of DON and AFB₁ all can cause disturbance of neurotransmitter secretion and imbalance of calcium homeostasis. The combined neurotoxic effect of DON and AFB₁ is greater than any of single exposure and they have synergistic effect.

Key words： deoxynivalenol; aflatoxin B₁; neurotransmitter; calmodulin; mice

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