HNE诱导下PKCδ/θ-Duox1-ROS对气道黏液高分泌影响的体外实验研究

Effect of HNE-induced PKCδ/θ-Duox 1-ROS on airway mucus hypersecretion:A vitro experimental study

目的:探讨蛋白激酶C(PKC)δ/θ-双重功能氧化酶1(Duox1)-活性氧(ROS)信号通路对人气道黏蛋白(MUC)5AC的调控作用及机制,为气道黏液高分泌治疗提供新靶点。方法:PKC及其亚基PKCδ/θ抑制剂、Duox1抑制剂或自由基清除剂DMTU预处理人气道上皮细胞16HBE,给予人中性粒细胞弹性蛋白酶(HNE)刺激建立体外气道炎症细胞模型。试剂盒测定各组细胞ROS生成水平,实时荧光定量PCR检测Duox1和MUC5AC mRNA水平,Western blot测定各组细胞干扰因素对Duox1蛋白水平的影响,ELISA和免疫荧光检测各组细胞MUC5AC蛋白表达。结果:与对照组相比,HNE组ROS生成明显增多,Duox1、MUC5AC mRNA和蛋白表达增加(P<0.05);给予Duox1抑制剂、自由基清除剂或PKC抑制剂及PKCδ/θ抑制剂后,ROS生成受到明显抑制,Duox1和MUC5AC mRNA及蛋白表达减少(P<0.05);给予PKCα/β后,ROS生成、Duox1和MUC5AC mRNA及蛋白表达与HNE组比较无明显变化(P>0.05)。结论:体外细胞模型实验中,HNE可通过PKCδ/θ-Duox1-ROS介导MUC5AC高表达,在气道黏液高分泌发展过程中起重要作用。

Objective:To investigate regulatory effect and mechanism of protein kinase C(PKC)δ/θ-dual functional oxidase 1(Duox1)-reactive oxygen species(ROS)signaling pathway on human airway mucin(MUC)5AC,to provide a new target for treatment of high secretion of airway mucus.Methods:Human airway epithelial cells 16HBE were pretreated with PKC and its subunit PKCδ/θinhibitor,Duox1 inhibitor or free radical scavenger DMTU,respectively,and then human neutrophil elastase(HNE)stimulation to establish an in vitro airway inflammatory cell model.Generation level of ROS in each group of cells was determined by kit,mRNA levels of Duox1 and MUC5AC were detected by real-time fluorescent quantitative PCR,influence of interfering factors of each group of cells on Duox1 protein level was determined by Western blot,and protein expression of MUC5AC in each group of cells was detected by ELISA and immunofluorescence.Results:Compared with control group,ROS production in HNE group was increased significantly,expressions of Duox1 and MUC5AC mRNA and protein were also increased(P<0.05).After administration of Duox1 inhibitors,free radical scavengers or PKC inhibitors and PKCδ/θinhibitors,ROS production was significantly inhibited,Duox1 and MUC5AC mRNA and protein expressions were decreased(P<0.05),while after giving PKCα/β,ROS generation,Duox1 and MUC5AC mRNA and pro⁃tein expressions were not significantly changed compared with HNE group(P>0.05).Conclusion:HNE can mediate high expression of MUC5AC through PKCδ/θ-Duox1-ROS,which plays an important role in development of high secretion of airway mucus in vitro cell model experiment.