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

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

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