Department of Animal Anatomy, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland.
Department of Pathophysiology, Forensic Veterinary and Administration, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland.
PLoS One. 2018 Jun 1;13(6):e0198180. doi: 10.1371/journal.pone.0198180. eCollection 2018.
Pituitary adenylate cyclase-activating polypeptide (PACAP-38) is a common neuropeptide exerting a wide spectrum of functions in many fields, including immunology. In the present study, 5-day post-fertilization (dpf) zebrafish larvae of three diverse genetic lines [transgenic lines Tg(MPX:GFP) with GFP-labelled neutrophils and Tg(pou4f3:GAP-GFP) with GFP-labelled hair cells and the wild-type Tuebingen] were used to investigate an inhibitory role of PACAP-38 in inflammation associated with damaged hair cells of the lateral line. Individuals of each genetic line were assigned to four groups: (1) control, and those consisting of larvae exposed to (2) 10 µM CuSO4, (3) 10 µM CuSO4+100 nM PACAP-38 and (4) 100 nM PACAP-38, respectively. Forty-minute exposure to CuSO4 solution was applied to evoke necrosis of hair cells and consequent inflammation. The inhibitory role of PACAP-38 was investigated in vivo under a confocal microscope by counting neutrophils migrating towards damaged hair cells in Tg(MPX:GFP) larvae. In CuSO4-treated individuals, the number of neutrophils associated with hair cells was dramatically increased, while PACAP-38 co-treatment resulted in its over 2-fold decrease. However, co-treatment with PACAP-38 did not prevent hair cells from extensive necrosis, which was found in Tg(pou4f3:GAP-GFP) individuals. Real-Time PCR analysis performed in wild-type larvae demonstrated differential expression pattern of stress and inflammation inducible markers. The most significant findings showed that CuSO4 exposure up-regulated the expression of IL-8, IL-1β, IL-6 and ATF3, while after PACAP-38 co-treatment expression levels of these genes were significantly decreased. The presence of transcripts for all PACAP receptors in neutrophils was also revealed. Adcyap1r1a and vipr1b appeared to be predominant forms. The present results suggest that PACAP-38 should be considered as a factor playing an important regulatory role in inflammatory response associated with pathological processes affecting zebrafish hair cells and it cannot be excluded that this interesting property has more universal significance.
垂体腺苷酸环化酶激活肽（PACAP-38）是一种常见的神经肽，在许多领域发挥着广泛的功能，包括免疫学。在本研究中，使用了三种不同遗传系的 5 日龄受精后（dpf）斑马鱼幼虫[GFP 标记嗜中性粒细胞的转基因系 Tg(MPX:GFP)和 GFP 标记毛细胞的 Tg(pou4f3:GAP-GFP)以及野生型 Tuebingen]来研究 PACAP-38 在与侧线毛细胞损伤相关的炎症中的抑制作用。每个遗传系的个体被分配到四个组：（1）对照组，以及暴露于（2）10 µM CuSO4、（3）10 µM CuSO4+100 nM PACAP-38 和（4）100 nM PACAP-38 的组。应用 40 分钟暴露于 CuSO4 溶液来引发毛细胞坏死和随后的炎症。通过在 Tg(MPX:GFP)幼虫中计算向受损毛细胞迁移的嗜中性粒细胞的数量，在体外用共聚焦显微镜研究 PACAP-38 的抑制作用。在 CuSO4 处理的个体中，与毛细胞相关的嗜中性粒细胞数量显著增加，而 PACAP-38 共同处理导致其减少超过 2 倍。然而，PACAP-38 的共同处理并没有阻止毛细胞的广泛坏死，这在 Tg(pou4f3:GAP-GFP)个体中发现。在野生型幼虫中进行的实时 PCR 分析显示应激和炎症诱导标志物的差异表达模式。最显著的发现表明，CuSO4 暴露上调了 IL-8、IL-1β、IL-6 和 ATF3 的表达，而在 PACAP-38 共同处理后，这些基因的表达水平显著降低。还揭示了嗜中性粒细胞中所有 PACAP 受体的转录本的存在。Adcyap1r1a 和 vipr1b 似乎是主要形式。这些结果表明，PACAP-38 应被视为在与影响斑马鱼毛细胞的病理过程相关的炎症反应中发挥重要调节作用的因素，不能排除这种有趣的特性具有更普遍的意义。
