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-BlueTM 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.  Conclusion  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.

Key words: Clonorchis sinensis；Excretory-secretory product；Pathogen-associated molecular pattern；NO；NF-&kappa, B