Role of non–receptor tyrosine kinase Tec in the production of pro–inflammatory cytokines from macrophages induced by endotoxin/lipopolysaccharide
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摘要: 目的 探讨非受体型酪氨酸激酶Tec在LPS诱导巨噬细胞产生TNF–α和IL–1β中的作用和相关机制。 方法 按随机数字表法将培养于6孔板的RAW264.7小鼠单核巨噬细胞分为4组,每组24孔。空白对照组细胞用含体积分数10%FBS的DMEM培养液常规培养2 h;LFM–A13组细胞先用75 μmol/L的Tec特异性抑制剂LFM–A13处理1 h,再同前常规培养1 h;LPS组细胞先同前常规培养1 h,再用0.1 μg/mL LPS刺激1 h;LPS+LFM–A13组细胞先用75 μmol/L的LFM–A13处理1 h,再用0.1 μg/mL LPS刺激1 h。培养结束后,采用ELISA法测定细胞培养上清液中TNF–α和IL–1β的含量,实时荧光定量RT–PCR法检测细胞内TNF–α和IL–1β mRNA的表达,蛋白质印迹法检测细胞内Tec、转化生长因子激活激酶1(TAK1)和p38 MAPK的活性。对数据行单因素方差分析、LSD检验。 结果 LFM–A13组与空白对照组细胞培养上清液中的TNF–α、IL–1β含量相近(
P 值均大于0.05),2组细胞内TNF–α、IL–1β mRNA表达量亦相近(P 值均大于0.05)。LPS组细胞培养上清液中的TNF–α、IL–1β含量分别为(1 213±154)、(636±90)pg/mL,显著高于空白对照组的(330±44)、(211±31)pg/mL(P 值均小于0.01);其细胞内TNF–α和IL–1β mRNA表达量分别为1.57±0.22、1.44±0.24,显著高于空白对照组的1.00±0.18、1.00±0.19(P 值均小于0.01)。LPS+LFM–A13组细胞培养上清液中的TNF–α、IL–1β含量分别为(787±109)、(453±64)pg/mL,显著低于LPS组(P 值均小于0.05);其细胞内TNF–α和IL–1β mRNA表达量分别为1.21±0.15、1.21±0.22,也显著低于LPS组(P 值均小于0.05)。LFM–A13组与空白对照组细胞内Tec、TAK1以及p38 MAPK活性相近(P 值均大于0.05)。LPS组细胞内Tec、TAK1、p38 MAPK活性分别为2.69±0.41、3.99±0.65、2.07±0.31,明显高于空白对照组的1.00±0.17、1.00±0.16、1.00±0.18(P 值均小于0.01);LPS+LFM–A13组细胞内Tec、TAK1和p38 MAPK活性分别为1.02±0.17、1.18±0.20、1.58±0.28,较LPS组显著下降(P <0.05或P <0.01)。 结论 Tec通过TAK1—p38 MAPK途径,促进了LPS诱导巨噬细胞促炎性细胞因子TNF–α和IL–1β的产生和释放。-
关键词:
- 脂多糖类 /
- 巨噬细胞 /
- 细胞因子类 /
- p38丝裂原活化蛋白激酶类 /
- Tec /
- 转化生长因子激活激酶
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 (withP 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 (withP 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, withP 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, withP 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 (withP 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 (withP 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 (withP 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, withP 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 orP <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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