Abstract：
Raf kinase inhibitor protein (RKIP) is an inflammation‑inhibiting mediator that is involved in several diseases; however, the potential mechanism of action of RKIP on the inflammatory response induced by influenza A virus (IAV) remains unclear. The present study aimed to investigate whether RKIP regulated the inflammatory response via the ERK/MAPK pathway. The present study detected the expression levels of RKIP and alterations in the inflammatory response in human normal bronchial epithelial BEAS‑2B cells, diseased human bronchial epithelial cells and primary human bronchial epithelial cells infected with IAV. Cells were treated with locostatin to inhibit the expression of RKIP. RKIP was overexpressed by lentivirus transduction and the small molecule inhibitor SCH772984 was applied to specifically inhibit activation of the ERK/MAPK pathway. In addition, C57BL/6 mice were infected with IAV to further confirm the role of RKIP in regulation of the inflammatory response via ERK/MAPK in vivo. Western blotting, reverse transcription‑quantitative PCR, ELISA, 5‑ethynyl‑-2\'‑deoxyuridine assay, immunofluorescence staining, Cell Counting Kit‑8, cell cycle assay, hematoxylin and eosin staining, and immunohistochemistry were used to detect all of the changes. Notably, RKIP attenuated the inflammatory response that was triggered by IAV infection in airway epithelial cells, which was characterized by augmented inflammatory cytokines and cell cycle arrest. Furthermore, the ERK/MAPK pathway was revealed to be activated by IAV infection and downregulation of RKIP aggravated the airway inflammatory response. By contrast, overexpression of RKIP effectively ameliorated the airway inflammatory response induced by IAV. These findings demonstrated that RKIP may serve a protective role in airway epithelial cells by combating inflammation via the ERK/MAPK pathway. Collectively, the present findings suggested that RKIP may negatively regulate airway inflammation and thus may constitute a promising therapeutic strategy for airway inflammatory‑related diseases that are induced by IAV.
摘要：
Raf激酶抑制剂蛋白（RKIP）是一种炎症抑制介质，与多种疾病有关；然而，RKIP对甲型流感病毒(IAV)诱导的炎症反应的潜在作用机制尚不清楚.本研究旨在探讨RKIP是否通过ERK/MAPK通路调节炎症反应。本研究检测了人正常支气管上皮BEAS-2B细胞中RKIP的表达水平和炎症反应的变化，感染IAV的患病人支气管上皮细胞和原代人支气管上皮细胞。将细胞用洛司他汀处理以抑制RKIP的表达。RKIP通过慢病毒转导过表达,并且应用小分子抑制剂SCH772984来特异性抑制ERK/MAPK途径的激活。此外,用IAV感染C57BL/6小鼠以进一步证实RKIP在体内通过ERK/MAPK调节炎症反应中的作用。西方印迹,逆转录-定量PCR，ELISA,5-乙炔基-2-脱氧尿苷测定，免疫荧光染色，细胞计数试剂盒-8，细胞周期测定，苏木精和伊红染色，和免疫组织化学用于检测所有的变化。值得注意的是,RKIP减弱了气道上皮细胞中由IAV感染引发的炎症反应，其特征是炎症细胞因子增强和细胞周期停滞。此外，ERK/MAPK通路被IAV感染激活,RKIP下调加重了气道炎症反应.相比之下,RKIP的过表达可有效改善IAV诱导的气道炎症反应。这些发现表明，RKIP可能通过ERK/MAPK途径抵抗炎症而在气道上皮细胞中起保护作用。总的来说，本研究结果表明,RKIP可能负向调节气道炎症,因此可能构成IAV诱导的气道炎症相关疾病的有前景的治疗策略.
