Objective: To explore the role of heat shock protein 90α (HSP90α) and endoplasmic reticulum (ER) stress pathway in allergic airway inflammation induced by house dust mite (HDM) in bronchial epithelial cells.
Methods: A HDM- induced asthmatic cell model was established in human bronchial epithelial (HBE) cells by exposure to a concentration gradient (200, 400 and 800 U/mL) of HDM for 24 h. To test the effect of siHSP90α and HSP90 inhibitor 17-AAG on HDM-induced asthmatic inflammation, HBE cells were transfected with siHSP90α (50 nmol, 12 h) or pretreated with 17-AAG (900 nmol, 6 h) prior to HDM exposure (800 U/mL) for 24 h, and the changes in the expression of HSP90α and ER stress markers were assessed. We also tested the effect of nasal drip of 17-AAG, HDM, or their combination on airway inflammation and ER stress in C57BL/6 mice.
Results: In HBE cells, HDM exposure significantly up-regulated the expression of HSP90α protein (P=0.011) and ER stress markers XBP-1 (P=0.044), ATF-6α (P=0.030) and GRP-78 (P=0.027). Knocking down HSP90α and treatment with 17-AAG both significantly inhibited HDM-induced upregulation of XBP-1 (P=0.008). In C57BL/6 mice, treatment with 17-AAG obviously improved HDM-induced airway inflammation and significantly reduced the number of inflammatory cells in the airway (P=0.014) and lowered the levels of IL-4 (P=0.030) and IL-5 (P=0.035) in alveolar lavage fluid. Immunohistochemical staining showed that the expressions of XBP-1 and GRP-78 in airway epithelial cells decreased significantly after the treatment of 17-AAG.
Conclusions: HSP90α promotes HDM-induced airway allergic inflammation possibly by upregulating ER stress pathway in bronchial epithelial cells.
目的: 探讨热休克蛋白90（HSP90α）参与屋尘螨（HDM）诱导的哮喘气道炎症的作用及调控气道上皮细胞内质网应激的机制。
方法: 人气道上皮细胞HBE体外培养，由HDM诱导哮喘体外模型，浓度梯度刺激24 h，分为正常对照组；200 U/mL组；400 U/mL组；800 U/mL组。随后分别使用siHSP90α干扰序列转染及HSP90抑制剂17-AAG干预，分为HBE正常对照组、HBE+HDM组（800 U/mL，24 h）、HBE+HDM（800 U/mL，24 h）+siHSP90α（50 nmol，12 h）组、HBE+HDM（800 U/mL，24 h）+17- AAG（900 nmol，6 h）组，测定HSP90α以及内质网应激标志物的表达水平。C57BL/6小鼠由HDM诱导哮喘体内模型，分为正常对照组、17-AAG滴鼻组、HDM滴鼻组、HDM+17-AAG滴鼻组，测定气道炎症水平与内质网应激标志物的表达水平。
结果: 在HDM诱导的HBE细胞哮喘体外模型中，蛋白电泳结果显示HSP90α（P=0.011）表达上调，琼脂糖凝胶电泳结果显示内质网应激标志物XBP-1（P=0.044）、ATF-6α（P=0.030）和GRP-78（P=0.027）表达水平显著上调；而敲低HSP90α和使用17-AAG会显著抑制HDM诱导的XBP-1（P=0.008）上调；在HDM诱导的哮喘小鼠模型中，相较于HDM组，H&E染色和瑞氏-姬萨姆染色显示HDM+17-AAG组的气道炎症改善，炎症细胞聚集明显减少。肺泡灌洗液计数显示，炎症细胞数量显著减少（P=0.014）。ELISA法显示肺泡灌洗液中炎症因子IL-4（P=0.030）、IL-5（P=0.035）显著降低。免疫组织化学染色显示气道上皮细胞XBP-1和GRP- 78表达明显减少。
结论: HSP90α加重HDM诱导的哮喘气道炎症，可能通过上调内质网应激实现的。
Keywords: asthma; endoplasmic reticulum stress; heat shock protein 90α; house dust mite.