关键词: 哮喘, 中性粒细胞胞外诱捕网, 一氧化氮供体, 一氧化氮合酶

Abstract: Objective To investigate the inductive effect of nitric oxide donor on asthmatic children′s neutrophil extracellular traps (NETs),in order to find new monitoring indicators and therapeutic targets that may be used in childhood asthma. Methods Totally 38 children with bronchial asthma in the Pediatric Department of the Fifth People′s Hospital of Shanghai were enrolled in this study from September to November in 2017. Percoll density gradient centrifugation was used to isolate neutrophils from the peripheral blood of asthmatic children, and nitric oxide donor (SNP) was added for in vitro incubation.Fluorescent staining was used to detect the double-stranded DNA (the main component of NETs) in the cell supernatant for quantitative analysis.Meantime, the cell slides were prepared to observe the main structures of NETs by fluorescence microscopy.In addition, inhibitors of nitric oxide synthase (NOS) were used to interfere the induction of SNP. Results The neutrophils were successfully isolated from the peripheral blood of asthmatic children, and the recovery rate was over 95%, the cell viability was over 80%.The extracellular mesh-like NETs structures were obviously observed in 3 hours after induction in PMA and SNP group, but NETs structure was not observed in PBS, SNP + L-NAME and SNP + SMT groups.The relative amounts of NETs in the intervention group (PMA 160 nmol/L, LPS 100 ng/ml and SNP 400 μmol/L) were significantly higher than those in the negative control PBS group (P＜0.05).The relative amounts of NETs in SNP + NOS blockers group (SMT 10 mmol/L, L-NAME 500 mol/L and L-NMMA 1 mmol/L) were less than simple SNP group (P＜0.05).NOS inhibitor could reduce but could not completely block the generation of NETs (P＜0.05).The relative generation of NETs at 1 h, 2 h, 3 h induced by SNP kept increasing while the formation decreased at 3 h with PBS(P＜0.05).With the prolongation of L-NAME, the relative amount of NETs induced by SNP decreased continuously(P＜0.05). Conclusion SNP can induce the formation of NETs in asthmatic children, and inhibitors of NOS can reduce the generation of NETs, which provides a new idea for the monitoring and treatment of childhood asthma.

Key words: asthma, neutrophil extracellular traps, nitric oxide donor, nitric oxide synthase