Objective: To investigate the role and mechanism of nonreceptor tyrosine kinase Tec in the production of pro-inflammatory cytokine interleukin-8 (IL-8) induced by endotoxin/lipopolysaccharide (LPS) in human alveolar epithelial cells A549. Methods: Human alveolar epithelial cells A549 were routinely cultured and passaged in Roswell Park Memorial Institute-1640 medium containing 10% fetal bovine serum. The second or third passage of cells were collected for subsequent experiments. (1) Cells were collected and divided into 6 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in simple LPS group were routinely cultured for 1 h and then stimulated by 1 μg/mL LPS for 1 h. Cells in simple LFM-A13 group were cultured with conventional culture medium adding 75 μmol/L LFM-A13 for 1 h and then cultured with replaced conventional culture medium for 1 h. Cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, and 100 μmol/L LFM-A13+ LPS group were cultured with conventional culture medium adding 25, 75, and 100 μmol/L LFM-A13 respectively for 1 h and then all stimulated by 1 μg/mL LPS added into the replaced conventional culture medium for 1 h. The protein expression of Tec in cells of each group was detected by Western blotting, and the content of IL-8 in cell culture supernatant of each group was determined by enzyme-linked immunosorbent assay. (2) Cells were collected and divided into 5 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in small interfering RNA (siRNA) control+ LPS group were transfected with empty lentivirus for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec mus-298 RNA interference (RNAi)+ LPS group, Tec mus-299 RNAi+ LPS group, and Tec mus-300 RNAi+ LPS group were transfected with lentivirus loaded with Tec mus-298 RNAi, Tec mus-299 RNAi, and Tec mus-300 RNAi respectively for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expression of Tec in cells of each group was detected by Western blotting to screen Tec-siRNA with the best silencing effect on Tec gene. (3) Cells were collected and divided into 4 groups with 4 wells in each group according to the random number table. Cells in blank control group were routinely cultured for 2 h. Cells in virus control group were transfected with empty lentivirus for 10 h and then routinely cultured for 2 h. Cells in simple LPS group were stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. Cells in Tec-siRNA+ LPS group were transfected with lentivirus loaded with Tec-siRNA with the best silencing effect on Tec gene for 10 h and then stimulated by 1 μg/mL LPS added into the conventional culture medium for 2 h. The protein expressions of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) MAPK of cells in each group were detected by Western blotting. Data were processed with one-way analysis of variance and the least significant difference-t test. Results: (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (t=9.72, P<0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (t=4.31, P=0.05). Compared with that of simple LPS group, the protein expression of Tec of cells in 25 μmol/L LFM-A13+ LPS group, 75 μmol/L LFM-A13+ LPS group, or 100 μmol/L LFM-A13+ LPS group was obviously decreased (t=9.72, 9.07, 16.33, P<0.05 or P<0.01). Compared with (189±22) pg/mL of blank control group, the content of IL-8 in culture supernatant of cells in simple LPS group was obviously increased [(214±10) pg/mL, t=2.18, P<0.05], but the content of IL-8 in culture supernatant of cells in simple LFM-A13 group was not obviously changed [(173±43) pg/mL, t=0.64, P>0.05]. Compared with that of simple LPS group, the content of IL-8 in culture supernatant of cells in 25 μmol/L LFM-A13+ LPS group was not obviously changed [(204±38) pg/mL, t=0.54, P>0.05], but the content of IL-8 in culture supernatant of cells in 75 μmol/L LFM-A13+ LPS group and 100 μmol/L LFM-A13+ LPS group was obviously decreased [(144±44), (137±51) pg/mL, t=3.63, 2.55, P<0.05 or P<0.01]. (2) Compared with that of blank control group, the protein expression of Tec of cells in siRNA control+ LPS group was obviously increased (t=14.24, P<0.01). Compared with that of siRNA control+ LPS group, the protein expression of Tec of cells in Tec mus-298 RNAi+ LPS group or Tec mus-299 RNAi+ LPS group was obviously decreased (t=36.03, 18.23, P<0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (t=4.08, P>0.05). The protein expression of Tec was the lowest in cells of Tec mus-298 RNAi+ LPS group, so Tec mus-298 RNAi was used in subsequent experiment. (3) Compared with 1.16±0.16 and 0.78±0.11 of blank control group, the protein expressions of p38 MAPK and ERK MAPK of cells in virus control group were not obviously changed (1.66±0.13, 0.89±0.11, t=11.09, 3.60, P>0.05), but the protein expressions of p38 MAPK and ERK MAPK of cells in simple LPS group were obviously increased (2.83±0.29, 1.86±0.37, t=9.70, 7.23, P<0.05). Compared with those of simple LPS group, the protein expression of p38 MAPK and protein expression of ERK MAPK of cells in Tec-siRNA+ LPS group were obviously decreased (0.69±0.16, 1.03±0.24, t=13.78, 4.12, P<0.05 or P<0.01). Conclusions: Tec may mediate the production and release of pro-inflammatory cytokine IL-8 from human alveolar epithelial cells A549 induced by LPS via the p38 MAPK and ERK MAPK signal pathways.
目的： 探讨非受体酪氨酸激酶Tec在内毒素/脂多糖(LPS)诱导人肺泡上皮细胞A549促炎性细胞因子白细胞介素8(IL-8)产生中的作用及机制。 方法： 将人肺泡上皮细胞A549用含体积分数10%胎牛血清的RPMI-1640培养液常规培养传代，取第2或第3代细胞进行后续实验。(1)取细胞，按随机数字表法分为6组，每组4孔。空白对照组细胞常规培养2 h；单纯LPS组细胞常规培养1 h后加入1 μg/mL的LPS刺激1 h；单纯LFM-A13组细胞于常规培养液中加入75 μmol/L的LFM-A13处理1 h后更换培养液常规培养1 h；25 μmol/L LFM-A13＋LPS组、75 μmol/L LFM-A13＋LPS组、100 μmol/L LFM-A13＋LPS组细胞分别于常规培养液中加入25、75、100 μmol/L的LFM-A13处理1 h后，均更换培养液并加入1 μg/mL的LPS刺激1 h。采用蛋白质印迹法检测各组细胞内Tec的蛋白表达情况，采用酶联免疫吸附测定法检测各组细胞培养上清液中IL-8的含量。(2)取细胞，按随机数字表法分为5组，每组4孔。空白对照组细胞常规培养2 h；小干扰RNA(siRNA)对照＋LPS组细胞用空的慢病毒转染10 h后，于常规培养液中加入1 μg/mL的LPS刺激2 h；Tec mus-298 RNAi＋LPS组、Tec mus-299 RNAi＋LPS组、Tec mus-300 RNAi＋LPS组细胞分别用搭载Tec mus-298 RNAi、Tec mus-299 RNAi、Tec mus-300 RNAi的慢病毒转染10 h后，均于常规培养液中加入1 μg/mL的LPS刺激2 h。采用蛋白质印迹法检测各组细胞内Tec的蛋白表达情况，筛选沉默Tec基因效果最佳的Tec-siRNA。(3)取细胞，按随机数字表法分为4组，每组4孔。空白对照组细胞常规培养2 h；病毒对照组细胞转染空的慢病毒10 h后，常规培养2 h；单纯LPS组细胞于常规培养液中加入1 μg/mL的LPS刺激2 h；Tec-siRNA＋LPS组细胞转染搭载沉默Tec基因效果最佳的Tec-siRNA的慢病毒10 h后，于常规培养液中加入1 μg/mL的LPS刺激2 h。采用蛋白质印迹法检测各组细胞内p38丝裂原活化蛋白激酶(MAPK)和细胞外信号调节激酶(ERK)MAPK的蛋白表达。对数据行单因素方差分析和LSD-t检验。 结果： (1)与空白对照组比较，单纯LPS组细胞中Tec蛋白表达明显升高(t＝9.72，P<0.05)，单纯LFM-A13组细胞中Tec蛋白表达无明显变化(t＝4.31，P＝0.05)。与单纯LPS组比较，25 μmol/L LFM-A13＋LPS组、75 μmol/L LFM-A13＋LPS组、100 μmol/L LFM-A13＋LPS组细胞中Tec蛋白表达明显降低(t＝9.72、9.07、16.33，P<0.05或P<0.01)。与空白对照组的(189±22)pg/mL比较，单纯LPS组细胞培养上清液中IL-8含量明显升高[(214±10)pg/mL，t＝2.18，P<0.05]，单纯LFM-A13组细胞培养上清液中IL-8含量无明显变化[(173±43)pg/mL，t＝0.64，P>0.05]。与单纯LPS组比较，25 μmol/L LFM-A13＋LPS组细胞培养上清液中IL-8含量无明显变化[(204±38)pg/mL，t＝0.54，P>0.05]，75 μmol/L LFM-A13＋LPS组、100 μmol/L LFM-A13＋LPS组细胞培养上清液中IL-8含量明显降低[(144±44)、(137±51)pg/mL, t＝3.63、2.55，P<0.05或P<0.01]。(2)与空白对照组比较，siRNA对照＋LPS组细胞中Tec蛋白表达明显升高(t＝14.24，P<0.01)。与siRNA对照＋LPS组比较，Tec mus-298 RNAi＋LPS组、Tec mus-299 RNAi＋LPS组细胞中Tec蛋白表达明显下降(t＝36.03、18.23，P<0.01)，Tec mus-300 RNAi＋LPS组细胞中Tec蛋白表达无明显变化(t＝4.08，P>0.05)。Tec mus-298 RNAi＋LPS组细胞中Tec蛋白表达最低，遂将Tec mus-298 RNAi用于后续实验。(3)与空白对照组的1.16±0.16、0.78±0.11比较，病毒对照组细胞内p38 MAPK和ERK MAPK蛋白表达无明显变化(1.66±0.13、0.89±0.11，t＝11.09、3.60，P>0.05)，单纯LPS组细胞内p38 MAPK和ERK MAPK蛋白表达明显升高(2.83±0.29、1.86±0.37，t＝9.70、7.23，P<0.05)。与单纯LPS组比较，Tec-siRNA＋LPS组细胞内p38 MAPK和ERK MAPK蛋白表达明显下降(0.69±0.16、1.03±0.24，t＝13.78、4.12，P<0.05或P<0.01)。 结论： Tec可能通过p38 MAPK、ERK MAPK信号通路介导LPS诱导人肺泡上皮细胞A549促炎性细胞因子IL-8的产生和释放。.
Keywords: Extracellular signal-regulated MAP kinases; Human alveolar epithelial cells; Interleukin-8; Lipopolysaccharides; Nonreceptor tyrosine kinase Tec; p38 mitogen-activated protein kinases.