动物营养学报 >

2019 , Vol. 31 >Issue 10: 4675 - 4683

DOI: https://doi.org/10.3969/j.issn.1006-267x.2019.10.032

分子营养 Molecular Nutrition

脱氧雪腐镰刀菌烯醇诱导仔猪海马神经细胞凋亡的线粒体通路研究

  • 朱雷 ,
  • 曹利 ,
  • 申茂玉 ,
  • 陈晓芳 ,
  • 李玉 ,
  • 冯士彬 ,
  • 吴金节 ,
  • 王希春
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  • 安徽农业大学动物科技学院, 合肥 230036
朱雷(1993-),男,山东临沂人,硕士研究生,从事畜禽中毒病研究。E-mail:811415214@qq.com

收稿日期: 2019-04-02

  网络出版日期: 2019-10-17

基金资助

国家自然科学基金项目（31472250）；安徽省生猪产业技术体系（AHCYTX-05-07）

收起

Deoxynivalenol Induces Apoptosis in Piglet Hippocampal Nerve Cells via Mitochondrial Pathway

  • ZHU Lei ,
  • CAO Li ,
  • SHEN Maoyu ,
  • CHEN Xiaofang ,
  • LI Yu ,
  • FENG Shibin ,
  • WU Jinjie ,
  • WANG Xichun
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  • College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China

Received date: 2019-04-02

  Online published: 2019-10-17

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摘要

为探讨脱氧雪腐镰刀菌烯醇（DON）对神经细胞毒性的作用机理，本试验选用仔猪海马神经细胞为研究对象，体外培养至对数生长期后，分别用含0（空白）、250、500、1 000 ng/mL DON和1 000 ng/mL DON+20 μmol/L半胱天冬酶（Caspase）-8抑制剂（FMK）的培养液进行染毒培养。培养24 h后，采用流式细胞术检测细胞凋亡率、活性氧（ROS）水平以及线粒体膜电位变化；采用实时荧光定量PCR（qRT-PCR）检测细胞凋亡线粒体途径中主要凋亡基因B淋巴细胞瘤-2（Bcl-2）、Bcl-2相关X蛋白（Bax）、BH3相互作用域死亡激动剂（Bid）、Caspase-2、Caspase-3和Caspase-9 mRNA的表达量。结果显示：1）与空白组相比，250、500、1 000 ng/mL DON组的细胞凋亡率显著或极显著升高（P<0.05或P<0.01）；与1 000 ng/mL DON组相比，1 000 ng/mL DON+FMK组的细胞凋亡率极显著降低（P<0.01）。2）与空白组相比，250、500、1 000 ng/mL DON组的ROS水平极显著升高（P<0.01）；与1 000 ng/mL DON组相比，1 000 ng/mL DON+FMK组的ROS水平极显著降低（P<0.01）。3）与空白组相比，250、500、1 000 ng/mL DON组的线粒体膜电位极显著降低（P<0.01）；与1 000 ng/mL DON组相比，1 000 ng/mL DON+FMK组的线粒体膜电位极显著升高（P<0.01）。4）与空白组相比，500、1 000 ng/mL DON组的Bax、Bid、Caspase-2、Caspase-9和Caspase-3 mRNA表达量显著或极显著升高（P<0.05或P<0.01），而Bcl-2 mRNA表达量极显著降低（P<0.01）；与1 000 ng/mL DON组相比，1 000 ng/mL DON+FMK组的Bax、Bid、Caspase-2、Caspase-9和Caspase-3 mRNA表达量极显著降低（P<0.01），而Bcl-2 mRNA表达量极显著升高（P<0.01）。结果表明，DON可通过线粒体通路诱导仔猪海马神经细胞凋亡。

关键词： 脱氧雪腐镰刀菌烯醇; 仔猪海马神经细胞; 细胞凋亡; 线粒体通路

本文引用格式

朱雷 , 曹利 , 申茂玉 , 陈晓芳 , 李玉 , 冯士彬 , 吴金节 , 王希春 . 脱氧雪腐镰刀菌烯醇诱导仔猪海马神经细胞凋亡的线粒体通路研究[J]. 动物营养学报, 2019 , 31(10) : 4675 -4683 . DOI: 10.3969/j.issn.1006-267x.2019.10.032

Abstract

The aim of this experiment was to investigate the mechanism of neurotoxicity of deoxynivalenol (DON) on piglet hippocampal nerve cells. Piglet hippocampal nerve cells were selected and cultured to logarithmic phase, and exposed to 0, 250, 500, 1 000 ng/mL DON and 1 000 ng/mL DON+20 μmol/L cysteinyl aspartate-specific protease (Caspase)-8 inhibitor (FMK) for 24 h, respectively. After 24 h culture, cell apoptosis ratio, reactive oxygen (ROS) level and mitochondrial membrane potential were determined by flow cytometry, and the main apoptosis genes in mitochondrial pathway including B-cell lymphoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), BH3 interacting domain death agonist (Bid), Caspase-2, Caspase-3 and Caspase-9 mRNA expression levels were determined by fluorescence quantitative PCR (qRT-PCR). The results showed as follows:1) compared with the blank group, the cell apoptosis ratio in 250, 500 and 1 000 ng/mL DON groups was significantly increased (P<0.05 or P<0.01), however, compared with the 1 000 ng/mL DON group, the apoptosis ratio in 1 000 ng/mL DON+FMK group was significantly decreased (P<0.01). 2) Compared with the blank group, the ROS level in 250, 500 and 1 000 ng/mL DON groups was significantly increased (P<0.01), while when compared with 1 000 ng/mL DON group, the ROS level in 1 000 ng/mL DON+FMK group was significantly decreased (P<0.01). 3) Compared with the blank group, the mitochondrial membrane potential in 250, 500 and 1 000 ng/mL DON groups was significantly decreased (P<0.01), but when compared with 1 000 ng/mL DON group, the mitochondrial membrane potential in 1 000 ng/mL DON+FMK group was significantly increased (P<0.01). 4) The mRNA expression levels of Bax, Bid, Caspase-2, Caspase-9 and Caspase-3 in 500 and 1 000 ng/mL DON groups were significantly increased (P<0.05 or P<0.01), while the expression level of Bcl-2 mRNA in 500 and 1 000 ng/mL DON groups was significantly decreased when compared with the blank group (P<0.01); however, compared with 1 000 ng/mL DON group, the expression levels of Bax, Bid, Caspase-2, Caspase-9 and Caspase-3 mRNA in 1 000 ng/mL DON+FMK group were significantly decreased (P<0.01), and the expression level of Bcl-2 mRNA in 1 000 ng/mL DON+FMK group was significantly increased (P<0.01). These results suggest that DON induces apoptosis in piglet hippocampal nerve cells through the mitochondrial pathway.

Key words： deoxynivalenol; piglet hippocampal nerve cells; cell apoptosis; mitochondrial pathway

参考文献

[1] LARSEN J C,HUNT J,PERRIN I,et al.Workshop on trichothecenes with a focus on DON:summary report[J].Toxicology Letters,2004,153(1):1-22.  
[2] STREIT E,NAEHRER K,RODRIGUES I,et al.Mycotoxin occurrence in feed and feed raw materials worldwide:long-term analysis with special focus on Europe and Asia[J].Journal of the Science of Food and Agriculture,2013,93(12):2892-2899.  
[3] PESTKA J J.Deoxynivalenol:mechanisms of action,human exposure,and toxicological relevance[J].Archives of Toxicology,2010,84(9):663-679.  
[4] FLANNERY B M,WU W,PESTKA J J.Characterization of deoxynivalenol-induced anorexia using mouse bioassay[J].Food and Chemical Toxicology,2011,49(8):1863-1869.  
[5] 王希春,张娅菲,储小燕,等.DON暴露对断奶仔猪抗氧化水平、血清生化指标及小肠黏膜形态的影响[J].浙江农业学报,2018,30(10):1655-1661.
[6] WANG X C,TANG J S,GENG F F,et al.Effects of deoxynivalenol exposure on cerebral lipid peroxidation,neurotransmitter and calcium homeostasis of chicks in vivo[J].Toxicon,2018,150:60-65.
[7] TSUJIMOTO Y.Cell death regulation by the Bcl-2 protein family in the mitochondria[J].Journal of Cellular Physiology,2010,195(2):158-167.
[8] LI D T,MA H R,YE Y Q,et al.Deoxynivalenol induces apoptosis in mouse thymic epithelial cells through mitochondria-mediated pathway[J].Environmental Toxicology and Pharmacology,2014,38(1):163-171.  
[9] GAIGÉ S,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] FLANNERY B M,CLARK E S,PESTKA J J.Anorexia induction by the trichothecene deoxynivalenol (vomitoxin) is mediated by the release of the gut satiety hormone peptide YY[J].Toxicological Sciences,2012,130(2):289-297.  
[11] WANG X C,XU W,FAN M X,et al.Deoxynivalenol induces apoptosis in PC12 cells via the mitochondrial pathway[J].Environmental Toxicology and Pharmacology,2016,43:193-202.
[12] SOBROVA P,ADAM V,VASATKOVA A,et al.Deoxynivalenol and its toxicity[J].Interdisciplinary Toxicology,2010,3(3):94-99.
[13] AWAD W A,BÖHM J,RAZZAZI-FAZELI E,et al.Effects of deoxynivalenol on general performance and electrophysiological properties of intestinal mucosa of broiler chickens[J].Poultry Science,2004,83(12):1964-1972.  
[14] 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.
[15] BONDY G S,COADY L,CURRAN I,et al.Effects of chronic deoxynivalenol exposure on p53 heterozygous and p53 homozygous mice[J].Food and Chemical Toxicology,2016,96:24-34.
[16] 陈晓芳,张娅菲,储小燕,等.脱氧雪腐镰刀菌烯醇对仔猪海马神经细胞凋亡及线粒体膜电位的影响[J].南京农业大学学报,2018,41(3):526-533.
[17] LI D T,YE Y Q,LIN S Q,et al.Evaluation of deoxynivalenol-induced toxic effects on DF-1 cells in vitro:cell-cycle arrest,oxidative stress,and apoptosis[J].Environmental Toxicology and Pharmacology,2014,37(1):141-149.  
[18] PINTON P,OSWALD I P.Effect of deoxynivalenol and other type B trichothecenes on the Intestine:a review[J].Toxins,2014,6(5):1615-1643.  
[19] NAGASE M,ALAM M M,TSUSHIMA A,et al.Apoptosis induction by T-2 toxin:activation of caspase-9,caspase-3,and DFF-40/CAD through cytosolic release of cytochrome c in HL-60 cells[J].Bioscience,Biotechnology,and Biochemistry,2001,65(8):1741-1747.  
[20] SPRINGER J E,NOTTINGHAM S A,MCEWEN M L,et al.Caspase-3 apoptotic signaling following injury to the central nervous system[J].Clinical Chemistry and Laboratory Medicine,2001,39(4):299-307.
[21] OUYANG Y B,GIFFARD R G.Cellular neuroprotective mechanisms in cerebral ischemia:Bcl-2 family proteins and protection of mitochondrial function[J].Cell Calcium,2004,36(3/4):303-311.
[22] YOULE R J,STRASSER A.The BCL-2 protein family:opposing activities that mediate cell death[J].Nature Reviews Molecular Cell Biology,2008,9(1):47-59.  
[23] BEMARDI P,KRAUSKOPF A,BASSO E,et al.The mitochondrial permeability transition from in vitro artifact to disease target[J].The FEBS Journal,2010,273(10):2077-2099.
[24] CORY S,HUANG D C S,ADAMS J M.The Bcl-2 family:roles in cell survival and oncogenesis[J].Oncogene,2003,22(53):8590-8607.  
[25] MCLAUGHLIN B.The kinder side of killer proteases:caspase activation contributes to neuroprotection and CNS remodeling[J].Apoptosis,2004,9(2):111-121.  
[26] THOMBERRY N A,LAZEBNIK Y.Caspases:enemies within[J].Science,1998,281(5381):1312-1316.  
[27] VOLBRACHT C,LEIST M,KOLB S A,et al.Apoptosis in caspase-inhibited neurons[J].Molecular Medicine,2001,7:BF03401837.
[28] BUDIHARDJO I,OLIVER H,LUTTER M,et al.Biochemical pathways of caspase activation during apoptosis[J].Annual Review of Cell and Developmental Biology,15:269-290.
[29] BOUAZIZ C,MARTEL C,EL DEIN O S,et al.Fusarial toxin-induced toxicity in cultured cells and in isolated mitochondria involves PTPC-dependent activation of the mitochondrial pathway of apoptosis[J].Toxicological Sciences,2009,110(2):363-375.  
[30] VAN GURP M,FESTJENS N,VAN LOO G,et al.Mitochondrial intermembrane proteins in cell death[J].Biochemical and Biophysical Research Communications,2003,304(3):487-497.  
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