联苯菊酯对皱纹盘鲍血细胞胞外陷阱形成的影响
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Q 786;S 944.4+5

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山东省农业良种工程(2017LZGC009);山东省自然科学基金(ZR2020QD097);国家自然科学基金(42176141,41806196)


Effects of bifenthrin on the formation of extracellular traps in hemocytes of Haliotis discus hannai
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    摘要:

    为探究贝类细胞胞外陷阱对联苯菊酯(BF)胁迫的响应,实验以皱纹盘鲍血细胞为研究对象,探讨不同浓度BF (0、0.01、0.10、1.00 mg/L)对血细胞细胞活力、胞外陷阱(ETs)、活性氧(ROS)产量以及ROS、糖酵解相关基因表达量的影响。结果显示,皱纹盘鲍血细胞的细胞活力随着BF浓度升高分别降低至90.40%、80.22%和72.28%,具有一定的剂量依赖性特征。BF降低了皱纹盘鲍血细胞活力,且具有一定的剂量依赖性特征。不同浓度BF刺激后,pi3k 表达量极显著上调,分别为对照的2.16 、3.32 和3.32 倍。在0.01 mg/L BF 刺激下,akt表达量极显著上调,在1.00 mg/L BF刺激下达到最高,为对照的5.34 倍。在0.01 mg/L BF刺激下,hif-1α的表达量与对照组无显著差异,在1.00 mg/L BF刺激时升高最为显著,为对照的11.63 倍。在BF诱导形成ETs过程中,血细胞ROS产量增加,且ROS相关基因表达量显著上调。通过添加烟酰胺腺嘌呤二核苷酸磷酸 (NADPH)氧化酶抑制剂(DPI),发现ETs的形成受到抑制,表明ROS参与了ETs形成过程。同时,在ETs形成的过程中,糖酵解相关基因表达量显著提升,初步表明糖酵解反应参与到ETs的形成过程。与对照组相比,pk的表达量显著升高,在BF浓度为1.00 mg/L时表达量最高;hk的表达量在所有刺激浓度下均极显著升高,且在BF浓度为1.00 mg/L时表达量最高。研究表明,ROS和糖酵解反应参与了BF诱导的皱纹盘鲍细胞胞外陷阱发生过程,BF可能会通过干扰血细胞发挥正常的细胞免疫反应,继而对细胞造成一定的免疫毒害作用。

    Abstract:

    Bifenthrin (BF) is widely used in agricultural production due to its excellent environmental stability and insecticidal activity, resulting in its widespread residue in the environment. In recent years, BF has been frequently detected in coastal areas of China, which would harm aquatic organisms. Extracellular traps play an important role in innate immunity in molluscs. However, knowledge of the formation of ETs is still limited in the marine mollusks induced by environmental contaminants so far. This study aimed to investigate the effects of bifenthrin on the formation of extracellular traps in hemocytes of Haliotis discus hannai. Here, H. discus hannai, with an average shell length of approximately 60 mm, was collected from a local farm and maintained in aerated seawater (temperature 20-22 °C; salinity 28-30) for a week before processing. Then hemocytes were obtained and stimulated by BF (0, 0.01, 0.10 and 1.00 mg/L) for an hour. The results showed that the cell viability of hemocytes decreased to 90.40%, 80.22% and 72.28%, respectively, in a dose-dependent manner. Meanwhile, observed formation of ETs outside hemocytes induced by BF, quantitative analysis revealed the amount of ETs formation significantly increases in a dose-dependent manne, compared to the control group. A more abundant structure of ETs could be induced by 1 mg/L BF. Furthermore, the mRNA expressions of phosphatidylinositol 3 kinase (PI3K) and protein kinase B (Akt) were significantly upregulated in a similar trend in response to the BF-induced reactive oxygen species (ROS) burst and hypoxia inducible factor 1α (HIF-1α) increased significantly (except 0.10 mg/L). Notably, we detected an increase of ROS production during ETs. Quantitative analysis revealed that ROS production increased significantly with the increased BF concentration, as well as in a dose-dependent manner. Furthermore, the formation of ETs was blocked with the inhibition of ROS production by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor (DPI). Meanwhile, the mRNA expression of pyruvate kinase (PK) and hexokinase (HK) was significantly elevated, indicating that during the formation of extracellular traps, part of the energy was supplied by the glycolytic pathway. In summary, stimulation by bifenthrin causes toxic effects on H. discus hannai hemocytes, which in turn exert immune functions through the formation of ETs. The ROS-mediated mediates the formation of ETs, and the PI3K/AKT signaling pathway. HIF-1α, is involved in the formation of ETs, meanwhile energetically supplied by glycolysis. This study preliminarily explored the response of hemocytes of H. discus hannai under BF stress to ETs, to provied a reference for further research on the immune response of hemocytes in shellfish and expand the understanding of environmental pesticide residues on cellular immune toxicity.

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化少蒙,陈丽竹,杨顶珑,吕晓静,刘相全,何金霞,李旋.联苯菊酯对皱纹盘鲍血细胞胞外陷阱形成的影响[J].水产学报,2024,48(8):089416

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  • 收稿日期:2022-12-06
  • 最后修改日期:2023-03-22
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  • 在线发布日期: 2024-08-14
  • 出版日期: 2024-08-01