下调TLR4对LPS诱导的支气管上皮16HBE细胞损伤的影响及其机制

doi:10.3877/cma.j.issn.2095-1221.2019.03.007

目的

探讨TLR4对脂多糖（LPS）诱导的支气管上皮16HBE细胞损伤的影响及其机制。

方法

将3条siRNA-TLR4-1、siRNA-TLR4-2和siRNA-TLR4-3转染至16HBE细胞中，筛选出最佳的siRNA-TLR4干扰序列进行实验。实验分为对照组（未处理）、LPS组（给予50 μg/ml LPS处理）、LPS+siNC组（转染siRNA-NC后给予50 μg/ml LPS处理）和LPS+siTLR4组（分别转染设计的3条siRNA-TLR4后给予50 μg/ml LPS处理），采用RT-PCR检测TLR4、IL-6和TNF-α mRNA的表达，MTT法检测细胞活力，流式细胞仪检测细胞凋亡率，Western Blot检测Bcl-2、Bax、Cleaved Caspase-3、NF-κB p65和IκBα蛋白的表达。多组间比较使用单因素方差分析，组间多重比较采用SNK-q，两组间比较采用独立样本t检验。

结果

LPS组与LPS+siTLR4-2组TLR4 mRNA（2.05±0.12，3.28±0.15）和蛋白表达（0.38±0.03，0.77±0.05）比较，差异具有统计学意义（t?= 11.091，11.585，P均< 0.001）；LPS组与LPS+siTLR4-3组TLR4 mRNA（1.39±0.09，3.28±0.15）和蛋白表达（0.31±0.02，0.77±0.05）比较，差异具有统计学意义（t?= 20.857，12.650，P均?< 0.001）且siRNA-TLR4-3的效果最为显著。与对照组相比，LPS组、LPS+siNC组和LPS+siTLR4组细胞中IL-6 mRNA（11.42±1.05，11.38±1.34，6.22±0.35，0.97±0.06，F?= 98.803，P均< 0.01）、TNF-α mRNA（15.76±1.12，15.69±0.87，7.54±0.41，1.03±0.09，F?= 278.064，P均< 0.01）、Cleaved Caspase-3蛋白（0.75±0.06，0.77±0.05，0.38±0.03，0.13±0.02，F?= 154.851，P均< 0.01）、Bax蛋白（0.48±0.05，0.52±0.05，0.33±0.02，0.11±0.02，F?= 71.310，P均< 0.01）、NF-κBp65蛋白（0.64±0.05，0.67±0.05，0.45±0.04，0.28±0.02，F?= 56.571，P?均?< 0.01）的表达水平和细胞凋亡率[（21.36±2.85）﹪，（20.94±3.22）﹪，（13.08±1.16）?﹪，（7.25±1.28）﹪，F?= 25.685，P均< 0.01]均明显升高，而细胞活力（0.53±0.04，0.51±0.04，0.78±0.06，1.15±0.08，F?= 80.811，P均< 0.01）和Bcl-2蛋白（0.28±0.03，0.25±0.03，0.40±0.03，0.69±0.06，F?= 76.762，P均< 0.01）、IκBα蛋白（0.38±0.03，0.35±0.03，0.44±0.03，0.72±0.06，F?= 53.635，P均< 0.01）的表达水平均明显降低；与LPS组相比，LPS+siTLR4组中IL-6 mRNA、TNF-α mRNA、Cleaved Caspase-3蛋白、Bax蛋白、NF-κBp65蛋白的表达水平和细胞凋亡率均明显降低，差异有统计学意义（t = 8.138，11.937，9.553，4.825，5.140，4.661，P均< 0.01），而细胞活力和Bcl-2蛋白、IκBα蛋白的表达水平均明显升高，差异有统计学意义（t = 6.005，4.899，3.674，P < 0.05），而LPS组和LPS+siNC组间差异无统计学意义（P > 0.05）。

结论

下调TLR4可通过抑制NF-κB通路激活抑制LPS诱导的细胞凋亡和炎症反应减轻16HBE细胞损伤。

关键词: Toll样受体4, 脂多糖, 支气管上皮细胞, 炎症, 凋亡

Objective

To investigate the effect of TLR4 on LPS-induced bronchial epithelial 16HBE cell injury and its mechanism.

Methods

Three siRNA-TLR4-1, siRNA-TLR4-2 and siRNA-TLR4-3 were transfected into 16HBE cells, and the best interference sequence was screened for experiment. The experiment was divided into a control group (untreated) , LPS group (treated with 50?μg/ml LPS) , LPS + siNC group (treated with 50?μg/mL LPS after transfection of siRNA-?NC) and LPS + siTLR4 group (treated with 50?μg/ml LPS after transfection of siRNA-TLR4) . The expression of TLR4, IL-6 and TNF-α was detected by RT-PCR, cell viability was detected by MTT, apoptotic rate was detected by flow cytometry, and the expression of Bcl-2, Bax, Cleaved Caspase-?3, NF-κBp65 and IκBα proteins were tested by Western Blot. One-way analysis of variance was used for comparison between groups. SNK-q was used for multiple comparisons between groups, and independent sample t test was used for comparison between the two groups.

Results

The TLR4 mRNA (2.05±0.12 vs 3.28±0.15) and protein expression (0.38±0.03 vs 0.77±0.05) in the LPS group and the LPS+siTLR4-2 group were statistically significant (t?= 11.091, 11.585, P?< 0.001) ; the expressions of TLR4 mRNA (1.39±0.09 vs 3.28±0.15) and protein (0.31±0.02 vs 0.77±0.05) in LPS group and LPS+siTLR4-3 group were statistically significant (t?= 20.857, 12.650, P?< 0.001) . The effect of siRNA-TLR4-3 was more significant. Compared with the control group, the expression levels of IL-6 mRNA (11.42±1.05, 11.38±1.34, 6.22±0.35 vs 0.97±0.06, F?= 98.803, P?< 0.01) , TNF-α mRNA (15.76±1.12, 15.69±0.87, 7.54±0.41 vs 1.03±0.09, F?=278.064, P?< 0.01) , Cleaved Caspase-3 protein (0.75±0.06, 0.77±0.05, 0.38±0.03 vs 0.13±0.02; F?= 154.851, P?< 0.01) , Bax protein (0.48±0.05, 0.52±0.05, 0.33±0.02 vs 0.11±0.02; F?= 71.310, P?< 0.01) , NF-?κBp65 protein (0.64±0.05, 0.67±0.05, 0.45±0.04 vs 0.28±0.02; F?= 56.571, P?< 0.01) and apoptotic rate[ (21.36±2.85) ﹪, (20.94±3.22) ﹪, (13.08±1.16) ﹪ vs (7.25±1.28) ﹪; F?= 25.685, P?< 0.01] in LPS group, LPS+siNC group and LPS+siTLR4 group were significantly higher, while the cell viability (0.53±0.04, 0.51±0.04, 0.78±0.06 vs 1.15±0.08; F?= 80.811, P?< 0.01) and expression levels of Bcl-2 (0.28±0.03, 0.25±0.03, 0.40±0.03 vs 0.69±0.06; F?= 76.762, P?< 0.01) and IκBα (0.38±0.03, 0.35±0.03, 0.44±0.03 vs 0.72±0.06; F?= 53.635, P?< 0.01) proteins were significantly lower (P?< 0.05) ; compared with LPS+siTLR4 group, the expression levels of IL-6 mRNA, TNF-α mRNA, Cleaved Caspase-3 protein, Bax protein, NF-κBp65 protein and apoptotic rate in LPS+ siTLR4 group were significantly lower, with a significant difference (t?= 8.138, 11.937, 9.553, 4.825, 5.140, 4.661, P?< 0.01) , while the expression levels of cell viability and Bcl-2 protein and IκBα protein were significantly increased , with a significant difference (t?= 6.005, 4.899, 3.674, P?< 0.05) , while there was no significant difference between LPS group and LPS+siNC group (P?> 0.05) .

Conclusion

Downregulation of TLR4 can alleviate 16HBE cell injury by inhibiting activation of NF-κB pathway, inhibiting LPS-induced apoptosis and inflammation.

Key words: Toll-like Receptor 4, Lipopolysaccharide, Bronchial epithelial cells, Inflammation, Apoptosis

表1 各组16HBE细胞中TLR4 mRNA和蛋白的表达（n = 3，±s）

分组	TLR4 mRNA	TLR4蛋白
对照组	1.02±0.08	0.25±0.03
LPS组	3.28±0.15^a	0.77±0.05^a
LPS+siNC组	3.22±0.12^a	0.75±0.06^a
LPS+siTLR4-1组	3.18±0.09^a	0.79±0.06^a
LPS+siTLR4-2组	2.05±0.12^ab	0.38±0.03^ab
LPS+siTLR4-3组	1.39±0.09^ab	0.31±0.02^ab
F值	248.002	97.437
P值	< 0.01	< 0.01

表1 各组16HBE细胞中TLR4 mRNA和蛋白的表达（n = 3，±s）

分组	TLR4 mRNA	TLR4蛋白
对照组	1.02±0.08	0.25±0.03
LPS组	3.28±0.15^a	0.77±0.05^a
LPS+siNC组	3.22±0.12^a	0.75±0.06^a
LPS+siTLR4-1组	3.18±0.09^a	0.79±0.06^a
LPS+siTLR4-2组	2.05±0.12^ab	0.38±0.03^ab
LPS+siTLR4-3组	1.39±0.09^ab	0.31±0.02^ab
F值	248.002	97.437
P值	< 0.01	< 0.01

图1 Western Blot检测TLR4蛋白的表达结果

表2 各组16HBE细胞中IL-6和TNF-α mRNA的表达（n = 3，±s）

分组	IL-6	TNF-α
对照组	0.97±0.06	1.03±0.09
LPS组	11.42±1.05^a	15.76±1.12^a
LPS+siNC组	11.38±1.34^a	15.69±0.87^a
LPS+siTLR4组	6.22±0.35^ab	7.54±0.41^ab
F值	98.803	278.064
P值	< 0.01	< 0.01

表2 各组16HBE细胞中IL-6和TNF-α mRNA的表达（n = 3，±s）

分组	IL-6	TNF-α
对照组	0.97±0.06	1.03±0.09
LPS组	11.42±1.05^a	15.76±1.12^a
LPS+siNC组	11.38±1.34^a	15.69±0.87^a
LPS+siTLR4组	6.22±0.35^ab	7.54±0.41^ab
F值	98.803	278.064
P值	< 0.01	< 0.01

表3 各组16HBE细胞的OD值和凋亡率（n = 3，±s）

分组	OD值	凋亡率（﹪）
对照组	1.15±0.08	7.25±1.28
LPS组	0.53±0.04^a	21.36±2.85^a
LPS+siNC组	0.51±0.04^a	20.94±3.22^a
LPS+siTLR4组	0.78±0.06^ab	13.08±1.16^ab
F值	80.811	25.658
P值	< 0.01	< 0.01

表3 各组16HBE细胞的OD值和凋亡率（n = 3，±s）

分组	OD值	凋亡率（﹪）
对照组	1.15±0.08	7.25±1.28
LPS组	0.53±0.04^a	21.36±2.85^a
LPS+siNC组	0.51±0.04^a	20.94±3.22^a
LPS+siTLR4组	0.78±0.06^ab	13.08±1.16^ab
F值	80.811	25.658
P值	< 0.01	< 0.01

图2 流式细胞仪检测细胞凋亡

表4 各组16HBE细胞中凋亡相关蛋白的表达水平（n = 3，±s）

分组	Bcl-2	Bax	Cleaved Caspase-3
对照组	0.69±0.06	0.11±0.02	0.13±0.02
LPS组	0.28±0.03^a	0.48±0.05^a	0.75±0.06^a
LPS+siNC组	0.25±0.03^a	0.52±0.05^a	0.77±0.05^a
LPS+siTLR4组	0.40±0.03^ab	0.33±0.02^ab	0.38±0.03^ab
F值	76.762	71.310	154.851
P值	< 0.01	< 0.01	< 0.01

表4 各组16HBE细胞中凋亡相关蛋白的表达水平（n = 3，±s）

分组	Bcl-2	Bax	Cleaved Caspase-3
对照组	0.69±0.06	0.11±0.02	0.13±0.02
LPS组	0.28±0.03^a	0.48±0.05^a	0.75±0.06^a
LPS+siNC组	0.25±0.03^a	0.52±0.05^a	0.77±0.05^a
LPS+siTLR4组	0.40±0.03^ab	0.33±0.02^ab	0.38±0.03^ab
F值	76.762	71.310	154.851
P值	< 0.01	< 0.01	< 0.01

图3 Western Blot检测Bcl-2、Bax和Cleaved Caspase-3蛋白的表达

表5 各组16HBE细胞中NF-κB信号通路相关蛋白表达（n = 3，±s）

分组	IκBα	NF-κBp65
对照组	0.72±0.06	0.28±0.02
LPS组	0.38±0.03^a	0.64±0.05^a
LPS+siNC组	0.35±0.03^a	0.67±0.05^a
LPS+siTLR4组	0.48±0.03^ab	0.45±0.04^ab
F值	53.635	56.571
P值	< 0.01	< 0.01

表5 各组16HBE细胞中NF-κB信号通路相关蛋白表达（n = 3，±s）

分组	IκBα	NF-κBp65
对照组	0.72±0.06	0.28±0.02
LPS组	0.38±0.03^a	0.64±0.05^a
LPS+siNC组	0.35±0.03^a	0.67±0.05^a
LPS+siTLR4组	0.48±0.03^ab	0.45±0.04^ab
F值	53.635	56.571
P值	< 0.01	< 0.01

图4 Western Blot检测IκBα和NF-κBp65蛋白的表达

[1]	Cornelia B,Jonathan D,Rachel B, et al. Modulation of human airway barrier functions during burkholderia thailandensis and francisella tularensis infection running title: airway barrier functions during bacterial infections[J]. Pathogens, 2016, 5(3):53.
[2]	Jeong JH,Kim J,Kim J, et al. ACN9 regulates the inflammatory responses in human bronchial epithelial cells[J]. Tuberc Respir Dis (Seoul), 2017, 80(3):247-254.
[3]	Ling KM,Sutanto EN,Iosifidis TA, et al. Reduced transforming growth factor β1（TGF-β1）in the repair of airway epithelial cells of children with asthma[J]. Respirology, 2016, 21(7):1219-1226.
[4]	Sugita K,Steer CA,Martinezgonzalez I, et al. Type 2 innate lymphoid cells disrupt bronchial epithelial barrier integrity by targeting tight junctions via IL-13 in asthma[J]. J Allergy Clin Immunol, 2017, 141(1): 300-310.
[5]	Li CL,Xu ZB,Fan XL, et al. MicroRNA-21 mediates the protective effects of mesenchymal stem cells derived from iPSCs to human bronchial epithelial cell injury under hypoxia[J]. Cell Transplant, 2018, 27(3):571-583.
[6]	Shi L,Dong N,Ji DX, et al. Lipopolysaccharide-induced CCN1 production enhances interleukin-6 secretion in bronchial epithelial cells[J]. Cell Biol Toxicol, 2018, 34(1):39-49.
[7]	Ru Q,Xiong Q,Chen L, et al. Lipopolysaccharide accelerates fine particulate matter-induced cell apoptosis in human lung bronchial epithelial cells[J]. Int J Occup Med Environ Health, 2018, 31(2):173-183.
[8]	Murdock JL, Gabriel Núñez. TLR4: The winding road to the discovery of the LPS receptor[J]. J Immunol, 2016, 197(7):2561-2562.
[9]	Ding SM,Hou XE,Yuan JR, et al. Wedelolactone protects human bronchial epithelial cell injury against cigarette smoke extract-induced oxidant stress and inflammation responses through Nrf2 pathway[J]. Int Immunopharmacol, 2015, 29(2):648-655.
[10]	Li C,Zhihong H,Wenlong L, et al. The NLRP3 inflammasome regulates bronchial epithelial cell injury and pro-apoptosis after exposure to biomass fuel smoke[J]. Am J Resp Cell Mol, 2016, 55(6): 815.
[11]	Kawamoto Y,Morinaga Y,Kimura Y, et al. TNF-alpha inhibits the growth of Legionella pneumophila in airway epithelial cells by inducing apoptosis[J]. J Infect Chemother, 2017, 23(1):51-55.
[12]	Perrin-Cocon L,Aublin-Gex A,Sestito SE, et al. TLR4 antagonist FP7 inhibits LPS-induced cytokine production and glycolytic reprogramming in dendritic cells, and protects mice from lethal influenza infection[J]. Sci Rep, 2017, 7:40791.
[13]	闫鹏展,赵礼金,涂奎, 等. TLR4在LPS诱导的大鼠肝内胆管组织损伤中的作用及机制[J]. 山东医药, 2017 (24):15-18.
[14]	吴乙偲,郑世翔,陈星华, 等. TLR4在脂多糖诱导肾小管上皮细胞MAPK信号通路中的作用及其机制[J]. 武汉大学学报（医学版）, 2016, 37(5):708-713.
[15]	Choy KW,Lau YS,Murugan D, et al. Paeonol attenuates LPS-induced endothelial dysfunction and apoptosis by inhibiting BMP4 and TLR4 signalling simultaneously but independently[J]. J Pharmacol Exp Ther, 2018, 364(3):420-432.
[16]	刘翠翠,赵龙,石晓岚. 等.茶多酚对脂多糖诱导的支气管上皮细胞损伤的保护作用及其机制[J]. 中国儿童保健杂志, 2017, 25(10): 1015-1018.
[17]	Lai JL,Liu YH,Liu C, et al. Indirubin inhibits LPS-Induced inflammation via TLR4 abrogation mediated by the NF-kB and MAPK signaling pathways[J]. Inflammation, 2017, 40(1):1-12.
[18]	Zhang Z,Liang Z,Li H, et al. Perfluorocarbon reduces cell damage from blast injury by inhibiting signal paths of NF-κB, MAPK and Bcl-2/Bax signaling pathway in A549 cells[J]. PLoS One, 2017, 12(3): e0173884.
[19]	Wei HY,Ma X. Tamoxifen reduces infiltration of inflammatory cells, apoptosis and inhibits IKK/NF-kB pathway after spinal cord injury in rats[J]. Neurol Sci, 2014, 35(11):1763-1768.

[1]	钟雅雯, 王煜, 王海臻, 黄莉萍. 肌苷通过抑制线粒体通透性转换孔开放缓解缺氧/复氧诱导的人绒毛膜滋养层细胞凋亡[J/OL]. 中华妇幼临床医学杂志(电子版), 2024, 20(05): 525-533.
[2]	王振宇, 张洪美, 荆琳, 何名江, 闫奇. 膝骨关节炎相关炎症因子与血浆代谢物间的因果关系及中介效应[J/OL]. 中华损伤与修复杂志(电子版), 2024, 19(06): 467-473.
[3]	张洁, 罗小霞, 余鸿. 系统性免疫炎症指数对急性胰腺炎患者并发器官功能损伤的预测价值[J/OL]. 中华普外科手术学杂志(电子版), 2025, 19(01): 68-71.
[4]	唐梅, 周丽, 牛岑月, 周小童, 王倩. ICG荧光导航的腹腔镜肝切除术临床意义[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 655-658.
[5]	付成旺, 杨大刚, 王榕, 李福堂. 营养与炎症指标在可切除胰腺癌中的研究进展[J/OL]. 中华普外科手术学杂志(电子版), 2024, 18(06): 704-708.
[6]	杜贵伟, 陆勇, 成博, 贺薏, 梁爽. 钬激光碎石术术后联合坦索罗辛治疗对输尿管结石患者的影响分析[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 491-496.
[7]	郑俊, 吴杰英, 谭海波, 郑安全, 李腾成. EGFR-MEK-TZ三联合分子的构建及其对去势抵抗性前列腺癌细胞增殖与凋亡的影响[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(05): 503-508.
[8]	高娟, 徐建庆, 闫芳, 丁盛华, 刘霞. Rutkow、TAPP、TEP 手术治疗单侧腹股沟疝患者的临床疗效及对血清炎症因子水平的影响[J/OL]. 中华疝和腹壁外科杂志(电子版), 2024, 18(06): 675-680.
[9]	邢嘉翌, 龚佳晟, 祝佳佳, 陆群. 肺癌化疗患者继发肺部感染的病原菌耐药性及炎症因子变化分析[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 714-718.
[10]	李智, 冯芸. NF-κB 与MAPK 信号通路及其潜在治疗靶点在急性呼吸窘迫综合征中的研究进展[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(05): 840-843.
[11]	孙璐, 蒋亚玲, 陈凌君. 布托啡诺对脑缺血再灌注损伤大鼠神经炎症和JAK2/STAT3信号通路的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(06): 344-350.
[12]	黄程鑫, 陈莉, 刘伊楚, 王水良, 赖晓凤. OPA1 在乳腺癌组织的表达特征及在ER阳性乳腺癌细胞中的生物学功能研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 275-284.
[13]	王国强, 张纲, 唐建坡, 张玉国, 杨永江. LINC00839 调节miR-17-5p/WEE1 轴对结直肠癌细胞增殖、凋亡和迁移的影响[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 491-499.
[14]	陈利, 杨长青, 朱风尚. 重视炎症性肠病和代谢相关脂肪性肝病间的串话机制研究[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(05): 385-389.
[15]	王湛, 李文坤, 杨奕, 徐芳, 周敏思, 苏珈仪, 王亚丹, 吴静. 炎症指标在早发性结直肠肿瘤中的应用[J/OL]. 中华临床医师杂志(电子版), 2024, 18(09): 802-810.

阅读次数

全文

摘要

选择文件类型/文献管理软件名称

选择包含的内容

Effect of TLR4 on LPS-induced bronchial epithelial 16HBE cell injury

模态框（Modal）标题

选择文件类型/文献管理软件名称

选择包含的内容

Effect of TLR4 on LPS-induced bronchial epithelial 16HBE cell injury