Objective To investigate the role of non–receptor tyrosine kinase Tec in the production of TNF–α and IL–1β from macrophages induced by LPS and its related mechanism.
Methods RAW264.7 mononuclear–macrophages cultured in 6–well plates were divided into 4 groups according to the random number table, with 24 wells in each group. Cells in blank group were routinely cultured (cultured with DMEM medium containing 10% FBS) for 2 hours. Cells in LFM–A13 group were pretreated with 75 μmol/L Tec specific inhibitor LFM–A13 for 1 hour and then routinely cultured for 1 hour. Cells in LPS group were routinely cultured for 1 hour and then treated with 0.1 μg/mL LPS for 1 hour. Cells in LPS+ LFM–A13 group were pretreated with 75 μmol/L LFM–A13 for 1 hour and then treated with 0.1 μg/mL LPS for 1 hour. The content of TNF–α and IL–1β in culture supernatant of cells was determined with ELISA. The mRNA expressions of TNF–α and IL–1β in cells were assayed with real–time fluorescent quantitative RT–PCR. The activity of intracellular Tec, p38 MAPK, and transforming growth factor activated kinase 1 (TAK1) was determined with Western blotting. Data were processed with one–way analysis of variance and LSD test.
Results The content of TNF–α and IL–1β in culture supernatant of cells in LFM–A13 group was close to that in blank group (with
P values above 0.05). The mRNA expressions of TNF–α and IL–1β in the cells of LFM–A13 group were close to those of blank group (with
P values above 0.05). The content of TNF–α and IL–1β in culture supernatant of cells in LPS group was respectively (1 213±154) and (636±90) pg/mL, which was higher than that in blank group [(330±44) and (211±31) pg/mL, with
P values below 0.01]. The mRNA expressions of TNF–α and IL–1β in the cells of LPS group were respectively 1.57±0.22 and 1.44±0.24, which were significantly higher than those of blank group (1.00±0.18 and 1.00±0.19, with
P values below 0.01). The content of TNF–α and IL–1β in culture supernatant of cells in LPS+ LFM–A13 group was respectively (787±109) and (453±64) pg/mL, which was significantly lower than that in LPS group (with
P values below 0.05). The mRNA expressions of TNF–α and IL–1β in the cells of LPS+ LFM–A13 group were respectively 1.21±0.15 and 1.21±0.22, and they were significantly lower than those of LPS group (with
P values below 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS+ LFM–A13 group was close to that in blank group (with
P values above 0.05). The activity of intracellular Tec, TAK1, and p38 MAPK of cells in LPS group was respectively 2.69±0.41, 3.99±0.65, and 2.07±0.31, which was significantly higher than that in blank group (1.00±0.17, 1.00±0.16, and 1.00±0.18, with
P values below 0.01) and LPS+ LFM–A13 group (1.02±0.17, 1.18±0.20, and 1.58±0.28,
P<0.05 or
P<0.01).
Conclusions Tec promotes the production and release of pro–inflammatory cytokines TNF–α and IL–1β from macrophages induced by LPS via TAK1–p38 MAPK signaling pathway.
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