NF-κB信号通路的激活在胃食管反流破坏食管黏膜上皮屏障功能机制中的作用

Role of NF-κB pathway activation in attenuation of esophageal barrier function via gastroesophageal reflux in mice

目的:本研究旨在探讨肿瘤坏死因子κB(nuclear factorκB,NF-κB)在反流破坏食管黏膜屏障发病机制中的作用,为寻求有效的干预措施提供理论依据。方法:建立单纯十二指肠和混合反流小鼠模型,检测食管黏膜跨上皮电阻抗以及细胞间间隙,使用基因芯片和生物信息学的方法筛选出反流食管黏膜差异表达的基因。运用免疫组化检测食管黏膜NF-κB亚基p50、p65和紧密连接蛋白Cldn1、Cldn4的表达,运用qRT-PCR和Western blot检测食管黏膜上皮Cldn1、Cldn4的表达,运用ELISA检测食管黏膜NF-κB靶基因白细胞介素(interleukin,IL)-1β、IL-6和IL-8的表达,通过特殊细胞染色检测并计数食管黏膜中性粒细胞、肥大细胞以及嗜酸性粒细胞。反流小鼠模型予以腹腔注射NFκB抑制剂Bay11-7085[20 mg/(kg·d)10 d,观察用药组以及未用药组小鼠食管黏膜跨上皮电阻抗以及IL-1β、IL-6和IL-8的表达差异。结果:反流食管黏膜跨上皮电阻抗下降,细胞间间隙增宽;基因富集分析和显著性分析指出反流食管黏膜NF-κB及其下游靶基因表达被激活;基因表达实验指出反流食管黏膜内NF-κB亚基p50和p65以及NF-κB下游靶基因IL-1β、IL-6和IL-8表达上调,紧密连接Cldn1和Cldn4的表达和分布发生失调;反流食管黏膜中性粒细胞、肥大细胞以及嗜酸性粒细胞计数均明显增加;NF-κB抑制实验指出给药组反流黏膜的跨上皮电阻抗值较未给药组显著上调,而炎症因子的表达水平则显著下调。结论:胃食管反流激活小鼠食管黏膜NF-κB信号通路,导致上皮屏障功能的损伤。抑制NF-κB通路具有潜在增强食管黏膜抗反流的能力。

Objective：To investigate the role of nuclear factor κB（NF-κB）in impairment of esophageal barrier function viagastroesophageal reflux（GER） and to provide theoretical guidance for treatment. Methods：Surgical models of duodenal and mixed re flux were developed in mice. Trans-epithelial electrical resistance（TEER）and intercellular space of esophageal mucosa were detected by mini-Ussing chamber and Toluidine Blue stain.Mouse esophageal epithelium was analyzed by gene micro-array. The expressions of NF-κB subunits（p50 and p65） and tight junction proteins（Cldn1 and Cldn4） were detected by immunohistochemistry. Cldn1 and Cldn4 were also quantified by qRT-PCR and Western blot. NF-κB target genes（IL-1β,IL-6 and IL-8）were detected by ELISA.Neutrophils,mast cells and eosinophils within mucosa were quantified. The duodenal and mixed reflux models were treated with an NF-κB inhibitor,Bay11-7085 [20 mg/（kg·d） i.p.] for 10 days. The TEER and cytokines（IL-1β,IL-6,IL-8） were measured.Results：Decrease of TEER and dilated intercellular space were observed in mucosa via reflux.Activation of NF-κB pathway was confirmed by gene set enrichment analysis and significance analysis of micro-arrays. Cldn1 and Cldn4 were down-regulated and mislocalized by qRT-PCR,Western blot and immunohistochemistry.Increase of neutrophils,mast cells and eosinophils within refluxed mucosa were observed. Treatment with Bay11-7085 counteracted the effect of reflux on TEER and cytokines. Conclusion：GER activates NF-κB pathway and attenuates esophageal barrier function in mice. Targeting NF-κB pathway is a potential way to strengthen esophageal barrier function.