内质网应激通路需肌醇酶1α/X盒结合蛋白1在中性粒细胞弹力蛋白酶诱导的气道黏液分泌中的作用

Role of inositol-requiring kinase 1α/X-box binding protein 1 in airway mucus secretion induced by neutrophil elastase

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-boxbinding 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;Westernblotting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-1αsiRNA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。

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 assayed by detection kit;expression 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 detected by Western blotting;spliced XBP-1(XBP-1s)mRNA was measured by real-time PCR;levels of MUC5AC protein in culture supernatant and cytoplasm were assayed by ELISA and immunofluorescence.Results·There was an obvious increase of ROS production with strong elevation of GRP78,ATF6,pPERK,and pIRE-1αprotein 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 expression and MUC5AC production and secretion(P<0.05).Further studies showed that MUC5AC secretion was also blunted by IRE-1αsiRNA or XBP-1 siRNA,accompanied with decreased expression of XBP-1s mRNA and protein(P<0.05).Conclusion·NE induces ERS by producing ROS,and increases MUC5AC protein production and secretion;IRE-1α/XBP-1 play a certain role in this process.