屋尘螨提取物激活气道上皮细胞EphA2-STAT3/p38 MAPK信号通路介导气道炎症

House dust mite extract activates EphA2-STAT3/p38 MAPK signaling pathway in airway epithelial cells to mediate airway inflammation

目的探讨受体酪氨酸激酶肝配蛋白A型受体2(EphA2)对屋尘螨提取物(HDM)诱导气道上皮细胞表达炎症细胞因子的作用及其机制。方法用EphA2小干扰RNA(siRNA)转染气道上皮细胞株16HBE细胞建立EphA2敲减的细胞模型,HDM刺激16HBE细胞后,采用实时定量PCR检测EphA2、白细胞介素6(IL-6)和IL-8 mRNA水平,细胞因子微球检测技术(CBA)检测IL-6、 IL-8、 IL-17A、 IL-17F和肿瘤坏死因子α(TNF-α)蛋白水平;Western blot法检测EphA2、磷酸化的EphA2(p-EphA2)、信号转导子与转录激活子3(STAT3)、磷酸化的STAT3(p-STAT3)、 p38丝裂原激活蛋白激酶(p38 MAPK)、磷酸化的p38 MAPK(p-p38 MAPK)、核因子κB p65(NF-κB p65)及磷酸化的NF-κB p65(p-NF-κB p65)蛋白水平。STAT3抑制剂Stattic联合HDM或p38 MAPK抑制剂SB203580联合HDM刺激16HBE细胞,实时定量PCR检测IL-6和IL-8 mRNA水平、 CBA检测IL-6和IL-8蛋白水平。结果敲低EphA2显著抑制HDM诱导16HBE细胞表达IL-6和IL-8,降低STAT3及p38 MAPK的总蛋白及磷酸化水平,但对NF-κB p65总蛋白及磷酸化水平均无明显影响。Stattic抑制STAT3表达及活化后,显著抑制HDM诱导16HBE细胞中IL-6和IL-8 mRNA和蛋白表达;而SB203580抑制p38 MAPK信号通路后,抑制HDM诱导16HBE细胞中IL-6和IL-8 mRNA表达水平,而对其蛋白水平无影响。结论 HDM通过激活EphA2-STAT3/p38 MAPK通路诱导16HBE细胞表达IL-6和IL-8参与气道炎症。

Objective To investigate the effect of ephrin type-A receptor 2(EphA2) on the expression of inflammatory cytokines in airway epithelial cells induced by house dust mite extract(HDM) and the underlying mechanism. Methods The cell model of EphA2 knockdown was established by transfection of EphA2 siRNA into airway cell line 16HBE cells. After the 16HBE cells were stimulated with HDM, the mRNA levels of EphA2, interleukin 6(IL-6) and IL-8 were determined by real-time quantitative PCR(qPCR), and the protein levels of IL-6, IL-8, IL-17A, IL-17F and tumor necrosis factor-α(TNF-α) were measured by cytometric bead array(CBA). Western blotting was used to analyze the protein expression of EphA2, phosphorylated EphA2(p-EphA2), signal transducer and activator of transcription(STAT3), phosphorylated STAT3(p-STAT3), p38 mitogen-activated protein kinases(p38 MAPK), phosphorylated p38 MAPK(p-p38 MAPK), nuclear factor κB p65(NF-κB p65) and phosphorylated NF-κB p65(p-NF-κB p65). Then, in the 16HEB cells stimulated by STAT3 inhibitor Stattic or p38 MAPK inhibitor SB203580 in combination with HDM, the mRNA and protein expression levels of IL-6 and IL-8 were detected by qPCR and CBA. Results Knockdown of EphA2 significantly inhibited the expression of IL-6 and IL-8 in HDM-induced 16HBE, and reduced the total protein and phosphorylated levels of STAT3 and p38 MAPK, but had no significant effect on the total protein and phosphorylated levels of NF-κB p65. After stattic inhibited the expression and activation of STAT3, the mRNA and protein levels of IL-6 and IL-8 significantly decreased in HDM-induced 16HBE cells. Interestingly, while SB203580 inhibited the activation of p38 MAPK signaling pathway, it only inhibited the mRNA levels of IL-6 and IL-8 inHDM-induced 16HBE cells, but had no effect on their protein levels. Conclusion HDM can induce the expression of IL-6 and IL-8 in 16HBE cells to participate in airway inflammation by activating the EphA2-STAT3/p38 MAPK pathway.