[非受体酪氨酸激酶Tec在内毒素/脂多糖诱导巨噬细胞产生促炎细胞因子中的作用]

[Role of non-receptor tyrosine kinase Tec in the production of pro-inflammatory cytokines from macrophages induced by endotoxin/lipopolysaccharide].

作者信息

Wang Chao, Wang Fei, Zhou Bo, Qiu Le, Wang Jian, Liu Sheng, Chen Xulin

机构信息

Department of Burns, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China.

Department of Burns, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China. Email:

出版信息

Zhonghua Shao Shang Za Zhi. 2015 Feb;31(1):11-5.

DOI:

PMID:25876633

Abstract

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.

摘要

目的

探讨非受体酪氨酸激酶Tec在脂多糖（LPS）诱导的巨噬细胞产生肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）中的作用及其相关机制。

方法

将培养于6孔板中的RAW264.7单核巨噬细胞按随机数字表法分为4组，每组24孔。空白组细胞常规培养（用含10%胎牛血清的DMEM培养基培养）2小时。LFM-A13组细胞先用75 μmol/L Tec特异性抑制剂LFM-A13预处理1小时，然后常规培养1小时。LPS组细胞常规培养1小时，然后用0.1 μg/mL LPS处理1小时。LPS+LFM-A13组细胞先用75 μmol/L LFM-A13预处理1小时，然后用0.1 μg/mL LPS处理1小时。采用酶联免疫吸附测定（ELISA）法检测细胞培养上清液中TNF-α和IL-1β的含量。采用实时荧光定量逆转录聚合酶链反应（RT-PCR）法检测细胞中TNF-α和IL-1β的mRNA表达。采用蛋白质免疫印迹法检测细胞内Tec、p38丝裂原活化蛋白激酶（MAPK）和转化生长因子激活激酶1（TAK1）的活性。数据采用单因素方差分析和LSD检验进行处理。

结果

LFM-A13组细胞培养上清液中TNF-α和IL-1β的含量与空白组接近（P值均大于0.05）。LFM-A13组细胞中TNF-α和IL-1β的mRNA表达与空白组接近（P值均大于0.05）。LPS组细胞培养上清液中TNF-α和IL-1β的含量分别为（1 213±154）和（636±90）pg/mL，高于空白组[（330±44）和（211±31）pg/mL，P值均小于0.01]。LPS组细胞中TNF-α和IL-1β的mRNA表达分别为1.57±0.22和1.44±0.24，显著高于空白组（1.00±0.18和1.00±0.19，P值均小于0.01）。LPS+LFM-A13组细胞培养上清液中TNF-α和IL-1β的含量分别为（787±109）和（453±64）pg/mL，显著低于LPS组（P值均小于0.05）。LPS+LFM-A13组细胞中TNF-α和IL-1β的mRNA表达分别为1.21±0.15和1.21±0.22，显著低于LPS组（P值均小于0.05）。LPS+LFM-A13组细胞内Tec、TAK1和p38 MAPK的活性与空白组接近（P值均大于0.05）。LPS组细胞内Tec、TAK1和p38 MAPK的活性分别为2.69±0.41、3.99±0.65和2.07±0.31，显著高于空白组（1.00±0.17、1.00±0.16和1.00±0.