title: [Effect of Excretory-secretory Products of Clonorchis sinensis on Nitric Oxide Production and NF-κB Activation in RAW264.7 Mouse Macrophages].
abstract: OBJECTIVE
  To study the role of excretory-secretory products (ESPs) from Clonorchis sinensis in the production of nitric oxide (NO) and the activation of nuclear transcription factor kappa B (NF-κB) in the macrophages of RAW264.7 mouse.
  
  
  METHODS
  20 µg/ml of C. sinensis EPSs, the organic solvent extracts of EPSs (ESP-ex), and 0.1 µg/ml of lipopolysaccharide from Salmonella minnesota (LPS-SM) were used as stimulators in co-culture with RAW264.7 mouse macrophages as experimental groups. The Hank's balanced salt solution (HBSS) served as control. At the same time the RAW264.7 macrophages were stimulated with EPSs, ESP-ex, and LPS-SM, and then added 0.3 mmol/L of SMT, a specific inhibitor of iNOS as the interference groups. After co-culture for 18 days, the concentrations of NO2- in the culture supernatants were detected with Griess regents, and the activation of NF-κB was determined by transfection with a NF-κB-inducible reporter plasmid, pNiFty2-SEAP. The activities of secreted embryonic alkaline phosphatase (SEAP) in culture supernatants were quantified by using HEK-Blue™ detection medium and expressed as the value of optical density at 620 nm (A620 value). The intercellular activities of SEAP were determined by microscopic observation.
  
  
  RESULTS
  After stimulation with both ESPs-ex and LPS-SM, the concentrations of NO2- in culture supernatants were (14.30 ± 1.62) and (14.10 ± 2.17) µmol/L, respectively, which were significantly higher than that of the control [(7.70 ± 0.95) µmol/L] (P < 0.05), and significantly decreased to (8.97 ± 0.81) and (4.96 ± 1.36) µmol/L after adding SMT, respectively (P < 0.05). However, the concentration of NO2- in ESPs stimulation group [(4.06 ± 0.62) µmol/L] was lower than that of the control (P < 0.05), and almost unchanged [(3.99 ± 0.87) µmol/L] after adding SMT (P > 0.05). SEAP activity in ESP group (0.836 ± 0.005) was significantly higher than that of the control [(0.097 ± 0.009) µmol/L] (P < 0.05). A strong blue color reaction was observed in cells of ESP group. SEAP activity of ESPs-ex and LPS groups [(0.112 ± 0.004), (0.116 ± 0.009) µmol/L] was slightly higher than that of the control (P > 0.05), and blue color reaction was observed in some cells.
  
  
  CONCLUSIONS
  ESPs from C. sinensis can stimulate NF-κB activation in RAW264.7 cells. The water-soluble components of ESPs can inhibit the NO production, while ESPs-ex and LPS-SM can promote the NO production.
authors.forename: Qing-li
authors.initials: QL
authors.lastname: Yang
authors.forename: Zhi-hua
authors.initials: ZH
authors.lastname: Jiang
authors.forename: Ji-qing
authors.initials: JQ
authors.lastname: Shen
authors.forename: Ying-dan
authors.initials: YD
authors.lastname: Chen
authors.lastname: Zhou Xiao-nong
mesh_terms: D000818:Animals; D002460:Cell Line; D003004:Clonorchis sinensis; D008070:Lipopolysaccharides; D008264:Macrophages; D051379:Mice; D016328:NF-kappa B; D009569:Nitric Oxide
chemical_list: D008070:Lipopolysaccharides; D016328:NF-kappa B; D009569:Nitric Oxide