呋虫胺暴露对斑马鱼幼鱼的免疫毒性研究

doi:10.19586/j.2095-2341.2025.0015

生物技术进展 ›› 2025, Vol. 15 ›› Issue (3): 446-455.DOI: 10.19586/j.2095-2341.2025.0015

呋虫胺暴露对斑马鱼幼鱼的免疫毒性研究

程云硕(), 李子旭, 茆广华(), 吴向阳()

江苏大学环境与安全工程学院，江苏镇江 212013

收稿日期:2025-02-13 接受日期:2025-03-10 出版日期:2025-05-25 发布日期:2025-07-01
通讯作者: 茆广华,吴向阳
作者简介:程云硕 E-mail: 2916579457@qq.com
基金资助:
国家自然科学基金项目(22276079)

Studies on the Immunological Effects of Dinotefuran Exposure on Zebrafish Juveniles

Yunshuo CHENG(), Zixu LI, Guanghua MAO(), Xiangyang WU()

School of Environment and Safety Engineering，Jiangsu University，Jiangsu Zhenjiang 212013，China

Received:2025-02-13 Accepted:2025-03-10 Online:2025-05-25 Published:2025-07-01
Contact: Guanghua MAO,Xiangyang WU

摘要/Abstract

摘要：

为探讨呋虫胺（dinotefuran，DIN）对斑马鱼早期发育阶段的免疫毒性及作用机制，以斑马鱼幼鱼为研究对象，在不同浓度的DIN（2、200和2 000 μg?L^-1）中暴露5 d，检测氧化应激、免疫细胞、免疫相关参数及免疫相关通路中基因转录水平4个方面的变化。结果表明，DIN可显著减少中性粒细胞、巨噬细胞、胸腺T细胞的数量（P<0.01），降低溶菌酶（lysozyme，LYS）、IgM和补体C3等免疫因子的含量（P<0.01），提高白细胞介素1β（interleukin 1β，IL-1β）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）等炎症因子的含量（P<0.01）；可剂量依赖性的增加幼鱼体内的活性氧水平（P<0.01），抑制过氧化氢酶（catalase，CAT）、超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathion peroxidase，GSH-Px）等抗氧化酶活性（P<0.01）。同时，DIN暴露改变了TLR4/NF-κB、JAK-STAT和Nrf2-Keap1信号通路中关键基因的转录水平。研究表明，DIN暴露可致斑马鱼幼鱼免疫毒性，且TLR4/NF-κB、JAK-STAT和Nrf2-Keap1通路在其中发挥重要作用。

关键词: 呋虫胺, 斑马鱼, 免疫毒性, 分子机制

Abstract:

In order to investigate the immunotoxicity and mechanism of action of dinotefuran （DIN） on early developmental stages of zebrafish， zebrafish larvae were exposed to different concentrations of DIN （2， 200 and 2 000 μg?L^-1） for 5 days to investigate changes in oxidative stress， immune cells， immune-related parameters and immune-related pathways. The results demonstrated that DIN significantly reduced the number of neutrophil， macrophage and thymic T cells （P<0.01）， decreased the levels of immune factors （LYS， IgM and C3）（P<0.01）， and increased the levels of inflammatory factors （IL-1β， IL-6， and TNF-α）（P<0.01）. DIN dose-dependently increased the levels of reactive oxygen species （ROS） in juvenile fish （P<0.01） and inhibited antioxidant enzyme activities （CAT， SOD and GSH-Px）（P<0.01）. Concurrently， DIN exposure modulated the transcript levels of pivotal genes within the TLR4/NF-κB， JAK-STAT and Nrf2-Keap1 signalling pathways. The study demonstrated that DIN exposure could induce immunotoxicity in zebrafish larvae and that the TLR4/NF-κB， JAK-STAT and Nrf2-Keap1 pathways play a significant role in this process.

Key words: dinotefuran, zebrafish, immunotoxicity, molecular mechanism

中图分类号:

Q965.9

程云硕, 李子旭, 茆广华, 吴向阳. 呋虫胺暴露对斑马鱼幼鱼的免疫毒性研究[J]. 生物技术进展, 2025, 15(3): 446-455.

Yunshuo CHENG, Zixu LI, Guanghua MAO, Xiangyang WU. Studies on the Immunological Effects of Dinotefuran Exposure on Zebrafish Juveniles[J]. Current Biotechnology, 2025, 15(3): 446-455.

图/表 9

表1 实时荧光定量 PCR 引物序列

Table 1 The sequences of primer list used in this study

基因	引物序列(5'→3')
tlr4a	F: 5'-CAATGGCTTGGGTACTTTGC-3'
tlr4a	R: 5'-GATTTGAGGAGTGCCGGATA-3'
myd88	F: 5'-TCCGAAAGAAACTGGGTCTG-3'
myd88	R: 5'-CGGAATAACGGAGTTCAGCTTGTG-3'
irak4	F: 5'-CAGAGAGGATTGTGGGAACGA-3'
irak4	R: 5'-GAGGAAGCCCAGACAAAACCT-3'
traf6	F: 5'-AAGCCGGTCAGCCTATTGTC-3'
traf6	R: 5'-CTGGCTGTCAAACTCACCCT-3'
ikkβ	F: 5'-GTGGCGGTGGATTATTGG-3'
ikkβ	R: 5'-GCACGGGTTGCCAGTTTG-3'
il-6	F: 5'-TGGACGTAAAGAGTCTCCTTGG-3'
il-6	R: 5'-TCATGTTCACCATCTCTCTGAAA-3'
il-1β	F: 5'-CATTTGCAGGCCGTCACA-3'
il-1β	R: 5'-GGACATGCTGAAGCGCACTT-3'
tnf-α	F: 5'-GCTGGATCTTCAAAGTCGGGTGTA-3'
tnf-α	R: 5'-TGTGAGTCTCAGCACACTTCCATC-3'
nfκb p65	F: 5'-TATGAAGCAGACCTACAG-3'
nfκb p65	R: 5'-TCTTGGCATCAGGAATA-3'
keap1	F: 5'-TGTGATCTGGTTCTGCATGTC-3'
keap1	R: 5'-ACTCCTTGAAGTTGCTGGTG-3'
ho-1	F: 5'-ATGCCCTTGTTTCCAGTCAGC-3'
ho-1	R: 5'-CTCGGAGGAGATGGAAGGAAG-3'
cat	F:5'-AGGGCAACTGGGATCTTACA-3'
cat	R: 5'-TTTATGGGACCAGACCTTGG-3'
sod	F: 5'-GTCGTCTGGCTTGTGGAGTG-3’
sod	R: 5'-TGTCAGCGGGCTAGTGCTT-3'
jak1	F: 5'-GCAGGCAACTGTGTGTGAAG-3'
jak1	R: 5'-AGAACTCGAGCTGGTGTGTG-3'
jak2	F: 5'-TCGCTGCTTCTTCTGTCAGG-3'
jak2	R: 5'-ACAGCCGTCCATTTTGGCTT-3'
socs3b	F: 5'-CCTTCCATACCCACCGAGAC-3'
socs3b	R: 5'-GCGCTGTCAAGCCTACTATG-3'
stat3	F: 5'-TGTGACACCAACGACCTGC-3'
stat3	R: 5'-CCAAACTGCATCAATGAATCTA-3'
gapdh	F: 5'-GTGGAGTCTACTGGTGTCTTC-3'
gapdh	R: 5'-GTGCAGGAGGCATTGCTTACA-3'

图1 DIN对斑马鱼幼鱼存活率的影响注：ns表示处理组与对照组相比无显著差异。

Fig. 1 Effect of DIN on the survival of zebrafish larvae

图2 DIN对斑马鱼幼鱼免疫系统的影响A：嗜中性粒细胞图（100×）；B：巨噬细胞图（11.5×）；C：斑马鱼尾鳍去除后中性粒细胞采集图；D：胸腺T细胞荧光图（100×）；E：嗜中性粒细胞组；F：巨噬细胞数；G：剪尾后嗜中粒性细胞数；H：胸腺T细胞数；**表示与对照组相比，处理组在P<0.01水平上具有统计学意义。

Fig. 2 Toxicity of DIN to the zebrafish larvae on innate and adaptive immune systems

图3 DIN对斑马鱼幼鱼免疫功能的影响注：*、**表示与对照组相比，处理组在P<0.05和P<0.01水平上具有统计学意义。

Fig. 3 Effects of DIN exposure to zebrafish larvae on the immune function

图4 DIN暴露对斑马鱼幼鱼氧化应激水平的影响A:ROS荧光图像(11.2×)； B:DIN暴露对斑马鱼幼鱼氧化应激水平的影响。*、**分别表示在P<0.05和P<0.01水平上具有统计学意义。

Fig. 4 The effect of DIN exposure on levels of oxidative stress in zebrafish larvae

图5 DIN暴露激活NF-κB信号通路A:NF-κB信号通路转录水平示意图； B:NF-κB信号通路中相关基因的表达量。*、**表示与对照组相比，处理组分别在P<0.05和P<0.01水平上具有统计学意义。

Fig. 5 DIN exposure activates the NF-κB signalling pathway

图6 DIN暴露对JAK-STAT信号通路相关基因表达的影响注：*、**表示与对照组相比，处理组在P<0.05和P<0.01水平上具有统计学意义。

Fig. 6 Expression of JAK-STAT signalling pathway related genes

图7 DIN暴露对Nrf2-Keap1信号通路相关基因表达的影响注：*、**表示与对照组相比，处理组在P<0.05和P<0.01水平上具有统计学意义。

Fig. 7 Expression of Nrf2-Keap1 signalling pathway related genes

图8 DIN暴露致斑马鱼幼鱼免疫毒性机制图

Fig. 8 Mechanism of zebrafish larval immunotoxicity from DIN exposure

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呋虫胺暴露对斑马鱼幼鱼的免疫毒性研究

Studies on the Immunological Effects of Dinotefuran Exposure on Zebrafish Juveniles

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