Chronic toxic effects of BDE-209 on the intestinal tract of zebrafish (Danio Rerio)
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摘要:
以模式生物斑马鱼为研究对象，探究十溴联苯醚（BDE-209）慢性暴露对肠道组织的毒性效应及分子机制。将斑马鱼暴露于不同浓度的BDE-209〔6、60和600 μg/L，二甲基亚砜溶剂对照〕溶液中28 d，利用苏木精-伊红染色对斑马鱼肠道组织进行病理学检查，并通过生物化学指标和ELISA试验对肠道内氧化应激和炎症反应相关生物标志物含量进行分析，通过实时荧光定量 PCR 分析肠道屏障、炎症反应和细胞凋亡相关基因的相对表达量。结果表明：BDE-209暴露造成了斑马鱼肠壁变薄、肠绒毛和肠壁外纵肌内空泡化增多以及肠壁和肠绒毛纤毛破损，同时通过下调肠道内ZO-1、Claudin-2和Tjp2a的mRNA相对表达量影响了肠道的物理屏障功能。BDE-209暴露导致肠道内活性氧、丙二醛、过氧化氢酶和超氧化物歧化酶的含量增加，表明BDE-209暴露造成了肠道内氧化应激。此外，BDE-209暴露导致促炎细胞因子肿瘤坏死因子α和白细胞介素1β的含量以及肠道内脂多糖含量升高，加重了肠道的炎症反应，并导致p53、Bax、Caspase3基因表达上调和Bcl2基因表达下调，促进了斑马鱼肠道的细胞凋亡。
Abstract:Taking the model organism zebrafish (Danio Rerio) as the research object, the toxic effects and molecular mechanism of chronic exposure of decabromodiphenyl ether (BDE-209) on intestinal tissue were explored. Zebrafish were exposed to different concentrations of BDE-209 (6, 60, and 600 μg/L, dimethyl sulfoxide solvent control) for 28 d. The intestinal tissue of zebrafish was pathologically examined by hematoxylin-eosin (H & E) staining. The contents of biomarkers related to oxidative stress and inflammatory response in the intestine were analyzed by biochemical indicators and ELISA experiments. The relative expression of genes related to the intestinal barrier, inflammatory response, and apoptosis was analyzed by real-time qPCR. The results showed that BDE-209 exposure resulted in thinning of the intestinal wall, increase of vacuolation in intestinal villi and external longitudinal muscle, damage of intestinal wall and cilia, and down-regulation of intestinal ZO-1, Claudin-2, and Tjp2a mRNA relative expression to affect intestinal physical barrier function. BDE-209 exposure increased the contents of reactive oxygen species (ROS), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) in the intestine, indicating that BDE-209 exposure caused intestinal oxidative stress. In addition, BDE-209 exposure up-regulated the contents of pro-inflammatory cytokines tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) and the content of lipopolysaccharide (LPS) in the intestine, leading to increased intestinal inflammatory response, and increased the expression of p53, Bax, Caspase3 gene and down-regulated Bcl2 gene expression, promoting the apoptosis of zebrafish intestine.
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图 1 斑马鱼肠道切片苏木精-伊红 (H & E) 染色后的组织形态
注：红色箭头标注的为肠壁；蓝色箭头标注的为肠道出现空泡化现象；绿色箭头标注的为肠绒毛出现破损现象。
Figure 1. Tissue morphology after haematoxylin-eosin (H & E) staining in intestinal sections from zebrafish
图 2 BDE-209暴露后斑马鱼肠道内ROS、MDA、SOD和CAT含量变化
注：*表示P< 0.05；**表示P< 0.01；***表示P< 0.001。
Figure 2. Changes of ROS, MDA, SOD and CAT contents in the intestine of zebrafish after exposure to BDE-209
图 3 BDE-209暴露后斑马鱼肠道内TNF-α、IL-1β和LPS含量变化
注：*表示P < 0.05；**表示P< 0.01；***表示P< 0.001。
Figure 3. Changes of TNF-α, IL-1β and LPS contents in the intestine of zebrafish after exposure to BDE-209
图 4 BDE-209暴露后斑马鱼肠道内屏障功能、炎症通路和细胞凋亡相关基因表达量的变化
注：*表示P< 0.05；**表示P< 0.01。
Figure 4. Expression changes of genes related to barrier function, inflammatory pathway and apoptosis in the intestine of zebrafish after exposure to BDE-209
表 1 实时荧光定量PCR分析中使用的目标基因引物的序列
Table 1. The sequences of target genes primer pairs used in the RT-qPCR analysis
基因名称 引物序列(5’-3’) 序列注册号 Rpl8 前置引物：
AACAGAGCCGTTGTTGGTGTT
后置引物：
GAAGGGATGCTCAACAGGGTTNM_200713.1 ZO-1 前置引物：
GAAAGCTCCCGCTCCATAGAA
后置引物：
ATCTACATCGGGTTGCCCAGXM_009303250.3 Claudin-2 前置引物：
TATCGTTGATTCCCGTCGCC
后置引物：
TCATCGCAACAGGATGCACTNM_001004559.2 Tjp2 前置引物：
AACGGCAGTTTCTGTGTAGA
后置引物：
ATCTTGGAGTCCCGCTGTANM_001201570.1 Muc2.1 前置引物：
CCCGTCCATGTGTCAGGAAT
后置引物：
TGAGTCCAGTTCCACCATGACXM_021470771.1 Akt2 前置引物：
CACAAAGTCCCGCACCAAAG
后置引物：
GTGCAACTTCGTCCTTAGCGNM_198146.2 Tlr-4 前置引物：
CACATGCGGGACTTTCAAGC
后置引物：
TGTTGGCATTGCGTTCCATCNM_001131051.1 Nfkb2 前置引物：
GCTGGAGCACTAAGGATGGA
后置引物：
GCACAAAGGGCTCATGCTTCNM_001001840.3 IL-10 前置引物：
GACCATTCTGCCAACAGCTC
后置引物：
GACCCCCTTTTCCTTCATCTTTNM_001020785.2 p53 前置引物：
TACTTGCCGGGATCGTTTGA
后置引物：
CAGGTCCGGTGAATAAGTGCNM_001271820.1 Bcl2 前置引物：
AAATGGAGGTTGGGATGCCT
后置引物：
AAAAGGCTCCGATGGTCACTNM_001030253.2 Bax 前置引物：
GCCCGTGAGATCTTCTCTGA
后置引物：
CCCTGGTTGAAATAGCCTTGANM_131562.2 Caspase3 前置引物：
CGTGTGGATACAACAGATGCTA
后置引物：
CCTGTCCTGCGATCAAAGTTNM_131877.3 
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