基于Toll样受体4/核因子κB通路观察香烟烟雾溶液刺激对人支气管上皮样细胞相关基因表达的影响

Effects of cigarette smoke solution stimulation on gene expression in human bronchial epithelial-like cells based on the Toll-like receptor 4/nuclear factor κB pathway

目的 观察Toll样受体4(TLR4)基因沉默或过表达后香烟烟雾溶液(CSE)对人支气管上皮样细胞(16HBE)相关基因表达的影响。方法 2020年8—12月,用常规培养的人支气管上皮样细胞16HBE作为空白对照组,实验设干扰小RNA(siRNA)TLR4-1组(hs-TLR4-si-1+16HBE)、siRNA TLR4-2组(hs-TLR4-si-2+16HBE)、siRNA TLR4-3组(hs-TLR4-si-3+16HBE),另设阴性对照组(NC+16HBE),实时荧光定量逆转录聚合酶链反应(qRT-PCR)筛选TLR4基因最佳沉默效果;自制CSE作用于16HBE,实验设常规培养的16HBE作为空白对照组、CSE组(CSE+16HBE)、siRNA TLR4组(CSE+hs-TLR4-si-2+16HBE)、siRNA TLR4-NC组(CSE+NC+16HBE)、Lv-TLR4组(CSE+Lv-TLR4+16HBE)、Lv-TLR4-NC组(CSE+Lv-TLR4-NC+16HBE),qRT-PCR检测TLR4、核因子κB(NF-κB)以及黏蛋白MUC5AC、MUC7、MUC8基因mRNA的表达。结果 与空白对照组相比,siRNA TLR4-2组TLR4 mRNA表达显著降低(1.00±0.22比0.47±0.04,P<0.05),因此选择hs-TLR4-si-2作为TLR4基因沉默的最佳siRNA。与空白对照组相比,CSE组TLR4(1.00±0.09比1.81±0.18)、NF-κB(1.00±0.04比1.65±0.11)、MUC5AC(1.00±0.12比2.09±0.24)、MUC7(1.00±0.20比2.21±0.26)、MUC8(1.00±0.11比1.75±0.06)mRNA表达均升高(均P<0.05);与CSE组相比,CSE+siRNA TLR4组TLR4(1.81±0.18比1.43±0.17)、NF-κB(1.65±0.11比1.12±0.05)、MUC5AC(2.09±0.24比1.38±0.13)、MUC7(2.21±0.26比1.46±0.30)、MUC8(1.75±0.06比1.23±0.03)mRNA表达均降低(均P<0.05),CSE+Lv-TLR4组TLR4(1.81±0.18比2.42±0.06)、NF-κB(1.65±0.11比1.94±0.07)、MUC5AC(2.09±0.24比2.56±0.19)、MUC7(2.21±0.26比2.72±0.33)、MUC8(1.75±0.06比2.10±0.10)mRNA表达均升高(均P<0.05)。结论 TLR4/NF-κB信号通路与CSE所产生的气道炎症有关,且其下游相关基因及黏蛋白表达受TLR4基因调控,TLR4的过度表达会加重CSE导致的气道炎症及慢性阻塞性肺疾病(COPD),抑制TLR4/NF-κB信号通路可在一定程度上减轻CSE所导致的气道炎症及COPD。

Objective To observe the effect of cigarette smoke solution(CSE) on the expression of related genes in human bronchial epithelial-like cells after Toll-like receptor 4(TLR4) gene silencing or overexpression.Methods From August to December 2020,conventionally cultured human bronchial epithelial-like cells with 16 HBE were used as a blank control group,and experiments were set up for the small interfering RNA(siRNA) TLR4-1 group(hs-TLR4-si-1+16 HBE),siRNA TLR4-2 group(hs-TLR4-si-2+16 HBE),siRNA TLR4-3 group(hs TLR4-si-3+16 HBE),and another negative control group(NC+16 HBE).Real-time fluorescence quantitative reverse transcription polymerase chain reaction(qRT-PCR) was used to determine the best silencing effect of the TLR4 gene.Homemade CSE was applied to 16 HBE cells,and experiments were performed with conventional cultured 16 HBE cells as a blank control group,CSE group(CSE+16 HBE),siRNA TLR4 group(CSE+hs-TLR4-si-2+16 HBE),siRNA TLR4-NC group(CSE+NC+16 HBE),Lv-TLR4 group(CSE+Lv-TLR4-NC+16 HBE),Lv-TLR4 group(CSE+Lv-TLR4-NC+16 HBE),and Lv-TLR4-NC group(CSE+Lv-TLR4-NC+16 HBE).qRT-PCR was performed to detect the expression of TLR4,nuclear factor-κB(NF-κB),and mucin MUC5 AC,MUC7,and MUC8 mRNAs.Results Compared with the blank control group,TLR4 mRNA expression was significantly lower in the siRNA TLR4-2 group(1.00±0.22 vs.0.47±0.04)(P<0.05),so hs-TLR4-si-2 was selected as the best siRNA for TLR4 gene silencing.Compared with the blank control group,TLR4(1.00±0.09 vs.1.81±0.18),NF-κB(1.00±0.04 vs.1.65±0.11),MUC5 AC(1.00±0.12 vs.2.09±0.24),MUC7(1.00±0.20 vs.2.21±0.26),and MUC8(1.00±0.11 vs.1.75±0.06) mRNA expression were elevated(P<0.05).Compared with the CSE group,TLR4(1.81±0.18 vs.1.43±0.17),NF-κB(1.65±0.11 vs.1.12±0.05),MUC5 AC(2.09±0.24 vs.1.38±0.13),MUC7(2.21±0.26 vs.1.46±0.30),and MUC8(1.75±0.06 vs.1.23±0.03) mRNA expression was reduced in the CSE+siRNA TLR4 group(P<0.05),and TLR4(1.81±0.18 vs.2.42±0.06),NF-κB(1.65±0.11 vs.1.94±0.07),MUC5 AC(2.09±0.24 vs.2.56±0.19),MUC7(2.21±0.26 vs.2.72±0.33),and MUC8(1.75±0.06 vs.2.10±0.10) mRNA expression was elevated in the CSE+Lv-TLR4 group(P<0.05).Conclusions TLR4/NF-κB signaling pathway is related to airway inflammation produced by CSE,and its downstream related genes and mucin expression are regulated by the TLR4 gene.TLR4 overexpression can aggravate airway inflammation and chronic obstructive pulmonary disease(COPD)caused by CSE,and inhibition of the TLR4/NF-κB signaling pathway can reduce airway inflammation and COPD caused by CSE to some extent.