Department of Anesthesiology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China.
Department of Anesthesiology, Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong 510630, China; Department of Anesthesiology, The Seventh Affiliated Hospital of Sun Yat-sen University, Shenzhen City, Guangdong Province, People's Republic of China.
Life Sci. 2018 Oct 1;210:243-250. doi: 10.1016/j.lfs.2018.09.008. Epub 2018 Sep 6.
Neutrophil extracellular traps (NETs) have been identified as a non-apoptosis cell death pattern that leads to the release of granular contents into the extracellular space and subsequent excessive inflammatory response. The present study aims to investigate whether ONO-5046, a novel neutrophil elastase inhibitor, could affect NETs formation and promote inflammation resolution.
Neutrophils were separated and identified. Cell survival rate was analyzed using the trypan blue stain-resistance method. Different concentrations of lipopolysaccharide (LPS) (0.01 μg/mL, 0.1 μg/mL, and 1.0 μg/mL) were used to stimulate NETs formation. ONO-5046 (0.1 μg/mL and 1 μg/mL) was administered after high-dose LPS stimulation and NETs formation. Moreover, tBHP was used to further investigate the relationships between the effects of ONO-5046 and reactive oxygen species (ROS) release. ROS was detected by DCFH-DA fluorescent staining and NETs formation was demonstrated via immunofluorescence staining for neutrophil elastase and citrullinated histone H3 (H3Cit).
NETs formation was stimulated by LPS in a dose-dependent manner. High doses of LPS induced ROS generation and decreased cellular survival. ONO-5046 reduced LPS-induced NETs formation in a dose-dependent manner, as evidenced by immunofluorescence staining for neutrophil elastase and H3Cit whereby the fluorescence intensity decreased and neutrophil ROS generation was attenuated. However, the effects of ONO-5046 on NETs reduction were reversed by ROS inducer tBHP.
The neutrophil elastase inhibitor ONO-5046 suppresses ROS-associated NETs formation, which may lead to inflammation resolution.
中性粒细胞胞外诱捕网（NETs）已被确定为一种非细胞凋亡的细胞死亡模式，其导致颗粒内容物释放到细胞外空间并随后引发过度的炎症反应。本研究旨在探讨新型中性粒细胞弹性蛋白酶抑制剂 ONO-5046 是否会影响 NETs 的形成并促进炎症的消退。
分离并鉴定中性粒细胞。采用台盼蓝拒染法分析细胞存活率。使用不同浓度的脂多糖（LPS）（0.01μg/mL、0.1μg/mL 和 1.0μg/mL）刺激 NETs 的形成。在高剂量 LPS 刺激和 NETs 形成后给予 ONO-5046（0.1μg/mL 和 1μg/mL）。此外，使用 tBHP 进一步研究 ONO-5046 的作用与活性氧（ROS）释放之间的关系。通过 DCFH-DA 荧光染色检测 ROS，通过免疫荧光染色检测中性粒细胞弹性蛋白酶和瓜氨酸化组蛋白 H3（H3Cit）来显示 NETs 的形成。
LPS 以剂量依赖性方式刺激 NETs 的形成。高剂量 LPS 诱导 ROS 的产生并降低细胞存活率。ONO-5046 以剂量依赖性方式减少 LPS 诱导的 NETs 的形成，这表现在中性粒细胞弹性蛋白酶和 H3Cit 的免疫荧光染色中，荧光强度降低且中性粒细胞 ROS 的产生受到抑制。然而，ROS 诱导剂 tBHP 逆转了 ONO-5046 对 NETs 减少的作用。
中性粒细胞弹性蛋白酶抑制剂 ONO-5046 抑制与 ROS 相关的 NETs 的形成，这可能导致炎症的消退。
