Wang Y, Hu Y, Wang F, Liu S, Wang Y J, Chen X L
Department of Burns, the First Affiliated Hospital of Anhui Medical University, Hefei 230022, China, is working at the First Affiliated Hospital of Tsinghua University, Beijing 100020, China.
Zhonghua Shao Shang Za Zhi. 2019 Aug 20;35(8):580-586. doi: 10.3760/cma.j.issn.1009-2587.2019.08.005.
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. 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- test. (1) Compared with that of blank control group, the protein expression of Tec of cells in simple LPS group was obviously increased (=9.72, <0.05), but the protein expression of Tec of cells in simple LFM-A13 group was not obviously changed (=4.31, =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 (=9.72, 9.07, 16.33, <0.05 or <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, =2.18, <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, =0.64, >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, =0.54, >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, =3.63, 2.55, <0.05 or <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 (=14.24, <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 (=36.03, 18.23, <0.01), but the protein expression of Tec of cells in Tec mus-300 RNAi+ LPS group was not obviously changed (=4.08, >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, =11.09, 3.60, >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, =9.70, 7.23, <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, =13.78, 4.12, <0.05 or <0.01). 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.
探讨非受体酪氨酸激酶Tec在人肺泡上皮细胞A549中内毒素/脂多糖（LPS）诱导的促炎细胞因子白细胞介素-8（IL-8）产生中的作用及机制。人肺泡上皮细胞A549在含10%胎牛血清的罗斯韦尔帕克纪念研究所-1640培养基中常规培养和传代。收集第二代或第三代细胞用于后续实验。（1）收集细胞，根据随机数字表将其分为6组，每组4孔。空白对照组细胞常规培养2小时。单纯LPS组细胞常规培养1小时，然后用1μg/mL LPS刺激1小时。单纯LFM-A13组细胞用添加75μmol/L LFM-A13的常规培养基培养1小时，然后用更换后的常规培养基培养1小时。25μmol/L LFM-A13 + LPS组、75μmol/L LFM-A13 + LPS组和100μmol/L LFM-A13 + LPS组细胞分别用添加25、75和100μmol/L LFM-A13的常规培养基培养1小时，然后全部用添加到更换后的常规培养基中的1μg/mL LPS刺激1小时。采用蛋白质免疫印迹法检测各组细胞中Tec的蛋白表达，采用酶联免疫吸附测定法测定各组细胞培养上清液中IL-8的含量。（2）收集细胞，根据随机数字表将其分为5组，每组4孔。空白对照组细胞常规培养2小时。小干扰RNA（siRNA）对照+ LPS组细胞用空慢病毒转染10小时，然后用添加到常规培养基中的1μg/mL LPS刺激2小时。Tec mus-298 RNA干扰（RNAi）+ LPS组、Tec mus-299 RNAi + LPS组和Tec mus-300 RNAi + LPS组细胞分别用装载Tec mus-298 RNAi、Tec mus-299 RNAi和Tec mus-300 RNAi的慢病毒转染10小时，然后用添加到常规培养基中的1μg/mL LPS刺激2小时。采用蛋白质免疫印迹法检测各组细胞中Tec的蛋白表达，以筛选对Tec基因沉默效果最佳的Tec-siRNA。（3）收集细胞，根据随机数字表将其分为4组，每组4孔。空白对照组细胞常规培养2小时。病毒对照组细胞用空慢病毒转染10小时，然后常规培养2小时。单纯LPS组细胞用添加到常规培养基中的1μg/mL LPS刺激2小时。Tec-siRNA + LPS组细胞用对Tec基因沉默效果最佳的装载Tec-siRNA的慢病毒转染10小时，然后用添加到常规培养基中的1μg/mL LPS刺激2小时。采用蛋白质免疫印迹法检测各组细胞中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。数据采用单因素方差分析和最小显著差检验进行处理。（1）与空白对照组相比，单纯LPS组细胞中Tec的蛋白表达明显升高（F = 9.72，P < 0.05），但单纯LFM-A13组细胞中Tec的蛋白表达无明显变化（F = 4.31，P = 0.05）。与单纯LPS组相比，25μmol/L LFM-A13 + LPS组、75μmol/L LFM-A13 + LPS组或100μmol/L LFM-A13 + LPS组细胞中Tec的蛋白表达明显降低（F = 9.72、9.07、16.33，P < 0.05或P < 0.01）。与空白对照组的（189±22）pg/mL相比，单纯LPS组细胞培养上清液中IL-8的含量明显升高[（214±10）pg/mL，t = 2.18，P < 0.05]，但单纯LFM-A13组细胞培养上清液中IL-8的含量无明显变化[（173±43）pg/mL，t = 0.64，P > 0.05]。与单纯LPS组相比，25μmol/L LFM-A13 + LPS组细胞培养上清液中IL-8的含量无明显变化[（204±38）pg/mL，t = 0.54，P > 0.05]，但75μmol/L LFM-A13 + LPS组和100μmol/L LFM-A13 + LPS组细胞培养上清液中IL-8的含量明显降低[（144±44）、（137±51）pg/mL，t = 3.63、2.55，P < 0.05或P < 0.01]。（2）与空白对照组相比，siRNA对照+ LPS组细胞中Tec的蛋白表达明显升高（F = 14.24，P < 0.01）。与siRNA对照+ LPS组相比，Tec mus-298 RNAi + LPS组或Tec mus-299 RNAi + LPS组细胞中Tec的蛋白表达明显降低（F = 36.03、18.23，P < 0.01），但Tec mus-300 RNAi + LPS组细胞中Tec的蛋白表达无明显变化（F = 4.08，P > 0.05）。Tec mus-298 RNAi + LPS组细胞中Tec的蛋白表达最低，因此在后续实验中使用Tec mus-298 RNAi。（3）与空白对照组的1.16±0.16和0.78±0.11相比，病毒对照组细胞中p38 MAPK和ERK MAPK的蛋白表达无明显变化（1.66±0.13、0.89±0.11，F = 11.09、3.60，P > 0.05），但单纯LPS组细胞中p38 MAPK和ERK MAPK的蛋白表达明显升高（2.83±0.29、1.86±0.37，F = 9.70、7.23，P < 0.05）。与单纯LPS组相比，Tec-siRNA + LPS组细胞中p38 MAPK的蛋白表达和ERK MAPK的蛋白表达明显降低（0.69±0.16、1.03±0.24，F = 13.78、4.12，P < 0.05或P < 0.01）。Tec可能通过p38 MAPK和ERK MAPK信号通路介导LPS诱导的人肺泡上皮细胞A549中促炎细胞因子IL-8的产生和释放。
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