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. 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.
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. sinensisEPSs, 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. 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.