摘要/Abstract

摘要： 目的 ·探讨内质网应激（ endoplasmic reticulum stress，ERS）反应在中性粒细胞弹力蛋白酶（ neutrophil elastase，NE）诱导人气道上皮细胞黏蛋白 5AC（mucin 5AC，MUC5AC）分泌中的作用及机制。方法 ·培养人气道上皮细胞 HBE16，分别给予活性氧（reactive oxygen species，ROS）抑制剂 N-乙酰半胱氨酸（ N-acetylcysteine，NAC）、ERS抑制剂 4-苯基丁酸（ 4-phenylbutyrate， 4-PBA）预处理，或分别转染需肌醇酶 1α（inositol-requiring kinase 1α，IRE-1α）小干扰 RNA（siRNA）、X盒结合蛋白 1（X-box binding protein 1，XBP-1）siRNA，再予以 NE刺激；同时设单纯 NE组和空白对照组。试剂盒检测 ROS的生成水平； Western blotting检测干预后 ERS相关信号分子葡萄糖调节蛋白 78（glucose-regulated protein 78，GRP78）、磷酸化蛋白激酶 R样内质网激酶（phosphorylated protein kinase R-like endoplasmic reticulum kinase，pPERK）、活化转录因子 6（activating transcription factor 6，ATF6）、磷酸化 IRE-1α（pIRE-1α）及 XBP-1蛋白表达量的变化；实时荧光定量 PCR检测剪切的 XBP-1（XBP-1s）mRNA水平； ELISA和免疫荧光检测 MUC5AC在各组细胞中的蛋白表达情况。结果 ·与空白对照组相比， NE刺激 24 h后细胞 ROS含量增加， GRP78、 ATF6、pPERK及 pIRE-1α蛋白水平均显著升高（均 P<0.05），XBP-1s mRNA及蛋白表达也明显增加（均 P<0.05），同时 MUC5AC蛋白表达增加（均 P<0.05）；NAC及 4-PBA预处理后均使上述 ERS相关蛋白表达较单纯 NE组降低（均 P<0.05），MUC5AC蛋白水平降低（均 P<0.05）；转染 IRE-1α siRNA或 XBP-1 siRNA，细胞中 MUC5AC蛋白表达也较单纯 NE组明显降低，同时 XBP-1s mRNA及蛋白表达也有明显下降（均 P<0.05）。结论 · NE通过产生 ROS引起 ERS反应，诱导 MUC5AC蛋白表达及分泌增加， ERS通路 IRE-1α/XBP-1在其中发挥着一定的作用。
关键词: 内质网应激, 需肌醇酶1&, alpha, X盒结合蛋白1, 中性粒细胞弹力蛋白酶, 活性氧, 黏蛋白5AC
Abstract:
Objective · To explore the effect of endoplasmic reticulum stress (ERS) on neutrophil elastase (NE) induced mucin 5AC (MUC5AC) production in human airway epithelial cells. Methods · HBE16 airway epithelial cells were cultured and pretreated with reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) or ERS inhibitor 4-phenylbutyrate (4-PBA), or transfected with small interfering RNA (siRNA) against inositolrequiring kinase 1α (IRE-1α) or X-box binding protein 1 (XBP-1), respectively before incubation with NE. NE group and blank control group were also set up. ROS production was assayeddetection kit; of glucose-regulated protein 78 (GRP78), phosphorylated protein kinase R-like endoplasmic reticulum kinase (pPERK), activating transcription factor 6 (ATF6), phosphorylated IRE-1α (pIRE-1α), and XBP-1 protein was detectedWestern blotting; spliced XBP-1 (XBP-1s) mRNA was measuredreal-time PCR; levels of MUC5AC protein in culture supernatant and cytoplasm were assayedELISA and immunofluorescence. Results · There was an obvious increase of ROS production with strong elevation of GRP78, ATF6, pPERK, and pIRE-1α protein in NE group cells after 24 h, compared with blank control group (P<0.05). The protein and mRNA of XBP-1s, and MUC5AC production also increased obviously (P<0.05). NAC and 4-PBA reduced ERS-related protein and MUC5AC production and secretion (P<0.05). Further studies showed that MUC5AC secretion was also bluntedIRE-1α siRNA or XBP-1 siRNA, accompanied with decreased of XBP-1s mRNA and protein (P<0.05). Conclusion · NE induces ERSproducing ROS, and increases MUC5AC protein production and secretion; IRE-1α/XBP-1 play a certain role in this process.
Key words: endoplasmic reticulum stress (ERS), inositol-requiring kinase 1&, alpha, (IRE-1&, alpha, ), X-box binding protein 1 (XBP-1), neutrophil elastase (NE), reactive oxygen species (ROS), mucin 5AC (MUC5AC)


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