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

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

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/mlLPS处理)、LPS+siNC组(转染siRNA-NC后给予50μg/mlLPS处理)和LPS+siTLR4组(分别转染设计的3条siRNA-TLR4后给予50μg/mlLPS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,WesternBlot检测Bcl-2、Bax、CleavedCaspase-3、NF-κBp65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TLR4mRNA(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TLR4mRNA(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-6mRNA(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)、CleavedCaspase-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、CleavedCaspase-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 16HBE cell injury and its mechanism. Methods Three siRNA-TLR4-1, siRNA-TLR4-2 and siRNATLR4- 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.