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
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摘要
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50μg/ml LPS处理)、LPS+siNC组(转染siRNA-NC后给予50μg/ml LPS处理)和LPS+siTLR4组(分别转染设计的3条siRNA-TLR4后给予50μg/ml LPS处理),采用RT-PCR检测TLR4、IL-6和TNF-αmRNA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-αmRNA(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-6mRNA、TNF-αmRNA、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。
Objective To investigate the effect of TLR4 on LPS-induced bronchial epithelial 16 HBE cell injury and its mechanism. Methods Three siRNA-TLR4-1, siRNA-TLR4-2 and siRNATLR4-3 were transfected into 16 HBE 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 16 HBE cell injury by inhibiting activation of NF-κB pathway, inhibiting LPS-induced apoptosis and inflammation.
Objective To investigate the effect of TLR4 on LPS-induced bronchial epithelial 16 HBE cell injury and its mechanism. Methods Three siRNA-TLR4-1, siRNA-TLR4-2 and siRNATLR4-3 were transfected into 16 HBE 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 16 HBE cell injury by inhibiting activation of NF-κB pathway, inhibiting LPS-induced apoptosis and inflammation.
引文
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.
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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 CCN1production 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 Nú?ez.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 TLR4signalling 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 MAPKsignaling 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.
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 factorβ1(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 CCN1production 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 Nú?ez.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 TLR4signalling 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 MAPKsignaling 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.