The total terpenoids of Celastrus orbiculatus (TTC) inhibit NOX-dependent formation of PMA-induced neutrophil extracellular traps (NETs)

Abstract

Previously, we identified that Celastrus orbiculatus, a traditional Chinese herbal medicine, exhibited prominent anti-inflammatory and anti-tumor activities. More recently, the formation of neutrophil extracellular traps (NETs) or NETosis has been recognized as a critical pathological event in the development of inflammatory and autoimmune diseases. The present study is aimed to explore the pharmacological effect of the total terpenoids from the stems of C. orbiculatus (TTC) on NETosis and underlying mechanisms, which may provide fundamental knowledge for future utilization of the Chinese medicine. Human neutrophils were isolated by density gradient centrifugation; lactase dehydrogenase (LDH) assay was used to detect cytotoxic effect of TTC on neutrophils. Moreover, we established phorbol-12-myristate-13-acetate (PMA)-induced NETosis. Quantitative and qualitative study of PMA-induced NET release was labeled by SYTOX™ Green. ROS production was determined by flow cytometry. The neutrophil NADPH oxidase (NOX) activity was assessed by lucigenin chemiluminescence assay, and the phosphorylation of NOX subunit was analyzed by immunoblot assay. TTC (5–80 μg.mL−1) had no predominant neutrophil cytotoxicity after 4 h exposure. PMA (200 ng.mL−1) significantly induced the formation of NETs after 4 h stimulus, whereas TTC dose-dependently (5–80 μg.mL−1) inhibited the process. TTC (40 μg.mL−1) blocked neutrophil elastase (NE) and myeloperoxidase (MPO) translocation from cytoplasm to nucleus and disrupted the formation of NET-associated deoxyribonucleic acid (DNA)–MPO and DNA–NE complexes. Moreover, TTC dose-dependently blocked PMA-mediated ROS production, and inhibited the NOX enzymatic activity of neutrophils upon PMA stimulus for 1 h. Finally, TTC suppressed PMA-induced phosphorylation of NOX subunit p40phox on Thr154 residue. TTC inhibited PMA-induced NOX phosphorylation, thereby suppressing NOX enzymatic activity and ROS generation in neutrophils undergoing NETosis. Consequently, TTC disrupted NETosis in the early stage of NOX-dependent NETs formation, which might serve as a promising anti-inflammatory agent by targeting suicidal NETosis.