OBJECTIVETo 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.

METHODSRAW264.7 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.

RESULTSThe 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).

CONCLUSIONSTec 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.