Wang Dandan, Wang Jie
Department of Infectious Disease, Xuzhou Children's Hospital Affiliated to Xuzhou Medical University, Xuzhou, Jiangsu 221006, P.R. China.
Exp Ther Med. 2020 Sep;20(3):2561-2568. doi: 10.3892/etm.2020.8963. Epub 2020 Jun 30.
Expression of Toll-like receptor (TLR)4 and its downstream substances, myeloid differentiation factor 88 (MyD88), NF-κB p65, tumor necrosis factor-α (TNF-α) and GR in human alveolar epithelial cells type Ⅱ (AEC Ⅱ) infected with respiratory syncytial virus (RSV) were investigated, and the antiviral immune mechanism mediated by TLR4 was explored. Human AEC Ⅱ were divided into TLR4 group, normal group and TLR4 group, and also into control group, RSV group and RSV+MP (methylprednisolone) group. MTT assay was used to measure the survival of cells after TLR4 knockout and overexpression, and the survival of normal cells after treatment with MP. The concentration of TLR4, MyD88, NF-κB p65, TNF-α, and GR was measured by ELISA after TLR4 knockout and overexpression. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure the mRNA expression of the gene knockout and overexpression groups. RT-qPCR and western blot analysis were used to determine the expression of TLR4, MyD88, NF-κB p65 and GR in RSV and RSV+MP groups. The concentration of the detected substances in the TLR4 group was significantly lower than that in the normal group (P<0.01 and <0.001), and in the TLR4 group was significantly higher than that in the normal group (P<0.05, <0.01 and <0.001); the expression of RSV in the TLR4 group was significantly higher than that in the normal group (P<0.001), and in the TLR4 group was significantly lower than that in the normal group (P<0.05). The expression levels of TLR4, MyD88 and NF-κB p65 in the RSV and RSV+MP groups were significantly higher than those in the control group (P<0.05, <0.01 and <0.001), and the increase presented in the RSV+MP group was significantly lower than that in the RSV group (P<0.05 and <0.01). TLR4-mediated antiviral immunity of human AEC Ⅱ can reduce the levels of TLR4, MyD88, NF-κB p65 and TNF-α and increase the level of GR, participating in the immune defense and reducing the damage of the viral epithelial cells of human type Ⅱ alveoli, thus improving human immunity.
研究呼吸道合胞病毒（RSV）感染的人Ⅱ型肺泡上皮细胞（AECⅡ）中Toll样受体（TLR）4及其下游物质髓样分化因子88（MyD88）、核因子κB p65（NF-κB p65）、肿瘤坏死因子-α（TNF-α）和糖皮质激素受体（GR）的表达，并探讨TLR4介导的抗病毒免疫机制。将人AECⅡ分为TLR4敲除组、正常组和TLR4过表达组，又分为对照组、RSV组和RSV+甲泼尼龙（MP）组。采用MTT法检测TLR4敲除和过表达后细胞的存活率，以及MP处理后正常细胞的存活率。采用酶联免疫吸附测定（ELISA）法检测TLR4敲除和过表达后TLR4、MyD88、NF-κB p65、TNF-α和GR的浓度。采用逆转录定量聚合酶链反应（RT-qPCR）检测基因敲除和过表达组的mRNA表达。采用RT-qPCR和蛋白质免疫印迹分析检测RSV组和RSV+MP组中TLR4、MyD88、NF-κB p65和GR的表达。TLR4敲除组中检测物质的浓度显著低于正常组（P<0.01和<0.001），TLR4过表达组中检测物质的浓度显著高于正常组（P<0.05、<0.01和<0.001）；TLR4敲除组中RSV的表达显著高于正常组（P<0.001），TLR4过表达组中RSV的表达显著低于正常组（P<0.05）。RSV组和RSV+MP组中TLR4、MyD88和NF-κB p65的表达水平显著高于对照组（P<0.05、<0.01和<0.001），且RSV+MP组的升高幅度显著低于RSV组（P<0.05和<0.01）。TLR4介导的人AECⅡ抗病毒免疫可降低TLR4、MyD88、NF-κB p65和TNF-α水平，升高GR水平，参与免疫防御，减轻人Ⅱ型肺泡病毒上皮细胞损伤，从而提高人体免疫力。
