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

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

Abstract

Abstract:

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

Guangfu Shen, Changqin Luo, Bo Huang, Jing Bi, Xuan Zhang, Rui Yu, Jun Yang, Jun Xie, Pei Du. Effect of TLR4 on LPS-induced bronchial epithelial 16HBE cell injury[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2019, 09(03): 166-172.

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Figures/Tables 9

References 19

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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

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

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

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

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

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