As an important immune mechanism of neutrophils, the release of Web‐like chromatin structures known as neutrophil extracellular traps (NETs) can rapidly locate and capture invading pathogens, which has received sustained attention. There are still some fundamental questions surrounding established studies on the mechanism of balance between reactive oxygen species (ROS) dependent release and neutrophil antioxidant response. Glutathione peroxidase 3 (GPx3) is an important antioxidant protein and has been identified can regulate the immune response. However, the effect of GPx3 on the NETs formation and microRNA in this process remain poorly understood. In the present study, we used chicken peripheral blood neutrophils treated with Phorbol‐12‐myristate‐13‐acetate (PMA) for 3 h as NETs formation model. The result of morphological observation showed that GPx3 inactivation compromised the release of NETs. Further analysis revealed that knockdown of GPx3 significantly disturbed oxidative balance by inhibiting antioxidant enzymes activity and increasing H₂O₂ content. Quantitative analysis of NETs‐related genes found that the phosphorylation level of mitogen‐activated protein kinase (MAPK) pathway genes (ERK, JNK, and p38) and expression of phosphoinositide‐3‐kinase (PI3K)/AKT pathway genes (PI3K and AKT) were suppressed with the downregulation of GPx3. Meanwhile, we identified that miR‐1696 can target GPx3 expression by using dual luciferase reporter system. Additionally, overexpression of miR‐1696 can not only inhibit the formation of NETs by restraining the expression of GPx3, interfering with the generation of ROS and activation of the MAPK and PI3K/AKT pathways, but also reducing the release of PMA‐induced NETs promoted by overexpression of GPx3. These results provide evidence that miR‐1696 targeted GPx3 activities in neutrophils could be used to regulate the NETs formation stimulated by PMA.

中文翻译：

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MiR-1696/GPx3轴参与氧化应激介导的鸡中性粒细胞胞外陷阱抑制

作为中性粒细胞的重要免疫机制，被称为中性粒细胞胞外陷阱（NETs）的网状染色质结构的释放可以快速定位和捕获入侵的病原体，这一直受到关注。关于活性氧 (ROS) 依赖性释放和中性粒细胞抗氧化反应之间的平衡机制的既定研究仍然存在一些基本问题。谷胱甘肽过氧化物酶 3 (GPx3) 是一种重要的抗氧化蛋白，已被确定可以调节免疫反应。然而，GPx3 在这个过程中对 NETs 形成和 microRNA 的影响仍然知之甚少。在本研究中，我们使用 Phorbol-12-myristate-13-acetate (PMA) 处理 3 小时的鸡外周血中性粒细胞作为 NETs 形成模型。形态学观察结果表明 GPx3 失活影响了 NETs 的释放。进一步的分析表明，GPx3 的敲低通过抑制抗氧化酶活性和增加 H₂ O ₂内容。NETs相关基因的定量分析发现，丝裂原活化蛋白激酶（MAPK）通路基因（ERK、JNK和p38）的磷酸化水平和磷酸肌醇3-激酶（PI3K）/AKT通路基因（PI3K和AKT）的表达) 被 GPx3 的下调所抑制。同时，我们通过使用双荧光素酶报告系统确定了 miR-1696 可以靶向 GPx3 表达。此外，miR-1696的过表达不仅可以通过抑制GPx3的表达来抑制NETs的形成，干扰ROS的产生以及MAPK和PI3K/AKT通路的激活，而且还可以减少PMA诱导的NETs的释放。通过 GPx3 的过度表达。这些结果提供了证据，证明 miR-1696 靶向中性粒细胞中的 GPx3 活性可用于调节 PMA 刺激的 NETs 形成。