ONO-5046 suppresses reactive oxidative species-associated formation of neutrophil extracellular traps

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Background: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.Methods: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).Results: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.Conclusions:The neutrophil elastase inhibitor ONO-5046 suppresses ROS-associated NETs formation, which may lead to inflammation resolution.

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