铜绿假单胞菌PA3611蛋白诱导支气管上皮细胞-间充质细胞转化的机制研究

Pseudomonas aeruginosa (PA) is one of the important pathogens, which has been proven to colonize and cause infection in the respiratory tract of patients with structural lung diseases, and further lead to bronchial fibrosis. Epithelial-Mesenchymal Transition (EMT) of bronchial epithelial cells plays a vital role in the process of bronchial fibrosis. Up to the present, the research on bronchial epithelial EMT caused by secreted virulence factors of PA has not been reported. In our previous studies, we found that PA3611 protein stimulation induced the morphologic change of cultured HBECs from polygon to fusiform or spindle, as well as the up-regulation of mesenchymal cell markers and down-regulation of epithelial cell markers. Meantime, TGF-β1 secretion was markedly increased, IκBα expression was significantly decreased, and NF-κB p65 subunit phosphorylation was markedly enhanced. In addition, the levels of miR-144 and miR-642 expression and p38 MAPK phosphorylation were obviously increased in HBECs after PA3611 stimulation. Therefore, we attempt to confirm the following mechanism via experiments in vivo and in vitro: after PA infection in the respiratory tracts, PA3611 protein could up-regulate the expression of miR-144 and miR-642 through p38 MAPK activation in HBECs. These two microRNAs target and inhibit the IκBα gene and then activate NF-κB p65 subunit, which enhances TGF-β1 synthesis and consequently induces EMT of bronchial epithelia.

铜绿假单胞菌(PA)极易在结构性肺病患者气道中定植与感染，导致支气管纤维化形成。气道上皮细胞-间充质细胞转化(EMT)在支气管纤维化过程中起关键作用。有关PA分泌毒力因子致支气管上皮细胞EMT的相关研究未见文献报道。我们前期研究发现：PA分泌的PA3611蛋白刺激支气管上皮细胞后，细胞形态及标记物水平的显著变化均提示EMT发生；且刺激组TGF-ß1显著提高、IκBα显著降低、NF-κB p65亚基磷酸化显著增强；此外，刺激组细胞 miRNA-144 及 miRNA-642 表达显著上升，p38 MAPK 磷酸化显著增高。据此，我们拟通过体内外研究证实如下机制：PA感染支气管上皮细胞后，PA3611 蛋白通过激活p38 MAPK信号分子，刺激miRNA-144及miRNA-642表达上调，后两者靶向抑制 IκBα，促进NF-κB p65活化与TGF-ß1合成，最终诱导支气管上皮细胞EMT。

结构性肺病可增加慢性感染的机会，包括铜绿假单胞菌(PA)感染，而铜绿假单胞菌的感染则可进一步促进肺结构损伤及气道重塑，从而进一步影响肺功能，形成恶性循环，最终导致肺纤维化。支气管上皮细胞-间充质细胞转化（EMT）在支气管纤维化过程中起重要作用。然而，PA感染导致肺结构破坏与支气管上皮细胞EMT的关系及其机制尚不清楚。PA3611是PA感染状态下分泌的一种毒性蛋白，蛋白质组学分析显示，PA3611可能是一种受群体感应系统调节的毒力因子。在本研究中，我们发现：PA分泌蛋白PA3611能通过上调EMT间充质细胞标志物ɑ-SMA和vimentin，下调上皮细胞标志物E-cadherin 和zonula occludens-1表达，进而促进EMT的发生；同时，我们研究还发现：PA3611刺激上皮细胞可导致TGF-β1分泌明显增加、p38MAPK磷酸化显著增强，支气管上皮细胞中miR-3065-5p、miR-6802-3p表达水平显著升高，而IκBα磷酸化表达明显降低、NF-κB p65磷酸化则明显增强。而在加入TGF-β1单克隆抗体后，则能观察到PA3611对EMT诱导效应受到明显抑制，且p38MAPK磷酸化受到明显抑制、两种miR表达升高得以逆转，NF-κB p65磷酸化增强的现象也得到了显著抑制；分别过表达或沉默p38则能观察到TGF-β1分泌水平不受影响，但两种miR表达及NF-κB p65磷酸化则分别出现上调或下调，EMT也随之增强或削弱；过表达或沉默两种miR后，TGF-β1分泌及p38MAPK磷酸化不受影响，但NF-κB p65及EMT则受到相应调控；过表达或沉默p65则可观察到TGF-β1分泌，而p38磷酸化及两种miR表达皆不受影响，但PA3611诱导的EMT得到增强或削弱；此外，体内实验证实，在PA野生株PAO1及PA3611敲除株PA△3611感染大鼠模型中，在4-6周观察到其病理变化、菌落计数及肺间质纤维化的相关标志物水平PAO1组显著强于PA△3611组。本研究结果表明：PA分泌的毒力因子PA3611通过TGF-β1诱导的p38/miRNANF-κB通路促进了EMT的发生。该研究结果探明了PA 慢性感染导致气道重塑的主要毒力蛋白PA3611，揭示了PA感染诱导支气管上皮细胞EMT发生的基本机制，为临床干预PA慢性感染致支气管EMT提供潜在靶标。