Department of Otorhinolaryngologic Head and Neck Surgery, First Hospital of Shanxi Medical University, Taiyuan, 030001 Shanxi, China.
Biomed Res Int. 2021 Jan 6;2021:8844119. doi: 10.1155/2021/8844119. eCollection 2021.
Otitis media (OM) is a common inflammatory disease of the middle ear cavity and mainly occurs in children. As a critical regulator of inflammation response, the nuclear factor kappa B (NF-B) pathway has been found to play an essential role in the pathogenesis of various human diseases. The aim of this study was to explore the potential mechanism under the inflammatory response of human middle ear epithelial cells (HMEECs). We established models of OM by treating HMEECs with lipopolysaccharide (LPS) or interleukin 17A (IL-17A). Enzyme-linked immunosorbent assay and western blot analysis were used to measure the inflammatory response of HMEECs under LPS or IL-17A stimulation. The results revealed that the concentrations of proinflammatory cytokines ( < 0.001) and protein levels of mucin (MUC) (for MUC5AC, = 0.002, = 0.004; for MUC8, = 0.004, < 0.001) were significantly elevated by LPS or IL-17A stimulation in HMEECs. Moreover, we found that LPS or IL-17A treatment promoted the phosphorylation of IB (for p-IB, = 0.018, = 0.002; for IB, = 0.238, = 0.057) and the translocation of p65 from cytoplasm to nucleus in HMEECs (for nucleus p65, = 0.01; for cytoplasm p65, < 0.001). In addition, RT-qPCR analysis revealed that long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was verified to be upregulated in LPS- or IL-17A-stimulated HMEECs ( < 0.001). Western blot analysis and immunofluorescence staining assay revealed that that MALAT1 knockdown significantly suppressed the activation of the NF-B pathway by reducing phosphorylated IB levels and inhibiting the nuclear translocation of p65 ( < 0.001) in LPS- or IL-17A-stimulated HMEECs (for p-IB, < 0.001; for IB, = 0.242, = 0.647). Silence of MALAT1 decreased the proinflammatory cytokine production and MUC protein levels ( < 0.001). Furthermore, rescue assays revealed that the increase of proinflammatory cytokine production (for TNF-, = 0.002, = 0.015; for IL-1, < 0.001, = 0.006; for IL-6, = 0.002, < 0.001) and MUC protein levels (for MUC5AC, = 0.001, < 0.001; for MUC8, < 0.001, = 0.001) induced by MALAT1 overexpression was neutralized by 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) treatment in LPS- or IL-17A-stimulated HMEECs. In conclusion, MALAT1 promotes inflammatory response in LPS- or IL-17A- stimulated HMEECs via the NF-B signaling pathway, which may provide a potential novel insight for the treatment of OM.
中耳炎（OM）是中耳腔的一种常见炎症性疾病，主要发生在儿童中。核因子 kappa B（NF-B）途径作为炎症反应的关键调节剂，已被发现在各种人类疾病的发病机制中发挥重要作用。本研究旨在探讨人中耳上皮细胞（HMEEC）炎症反应的潜在机制。我们通过用脂多糖（LPS）或白细胞介素 17A（IL-17A）处理 HMEEC 来建立 OM 模型。酶联免疫吸附试验和 Western blot 分析用于测量 LPS 或 IL-17A 刺激下 HMEEC 的炎症反应。结果显示，促炎细胞因子的浓度（ < 0.001）和粘蛋白（MUC）的蛋白水平（对于 MUC5AC， = 0.002， = 0.004；对于 MUC8， = 0.004， < 0.001）在 LPS 或 IL-17A 刺激的 HMEEC 中显著升高。此外，我们发现 LPS 或 IL-17A 处理促进了 IB 的磷酸化（对于 p-IB， = 0.018， = 0.002；对于 IB， = 0.238， = 0.057）和 p65 从细胞质向细胞核的易位（对于细胞核 p65， = 0.01；对于细胞质 p65， < 0.001）。此外，RT-qPCR 分析显示，长链非编码 RNA（lncRNA）转移相关肺腺癌转录本 1（MALAT1）在 LPS 或 IL-17A 刺激的 HMEEC 中被证实上调（ < 0.001）。Western blot 分析和免疫荧光染色试验显示，沉默 MALAT1 通过降低磷酸化 IB 水平和抑制 p65 的核易位（ < 0.001），显著抑制 LPS 或 IL-17A 刺激的 HMEEC 中 NF-B 途径的激活（对于 p-IB， < 0.001；对于 IB， = 0.242， = 0.647）。沉默 MALAT1 降低了促炎细胞因子的产生和 MUC 蛋白水平（ < 0.001）。此外，挽救实验显示，促炎细胞因子产生的增加（对于 TNF-， = 0.002， = 0.015；对于 IL-1， < 0.001， = 0.006；对于 IL-6， = 0.002， < 0.001）和 MUC 蛋白水平（对于 MUC5AC， = 0.001， < 0.001；对于 MUC8， < 0.001， = 0.001）由 MALAT1 过表达引起的在 LPS 或 IL-17A 刺激的 HMEEC 中被 4-N-[2-（4-苯氧基苯基）乙基]喹唑啉-4,6-二胺（QNZ）处理所中和。总之，MALAT1 通过 NF-B 信号通路促进 LPS 或 IL-17A 刺激的 HMEEC 中的炎症反应，这可能为 OM 的治疗提供新的见解。
