Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades
Flagellin of Pseudomonas aeruginosa induces transforming growth factor beta 1 expression in normal bronchial epithelial cells through mitogen activated protein kinase cascades
复制标题
铜绿假单胞菌鞭毛蛋白通过丝裂原激活蛋白激酶级联诱导正常支气管上皮细胞中转化生长因子β1的表达
DOI：
10.3760/cma.j.issn.0366-6999.2011.04.021
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发表时间：
2011-02-20
影响因子：
6.1
通讯作者：
Sun Tie-ying
中科院分区：
文献类型：
--
作者：
Yang Jing-jing;Wang Dan-dan;Sun Tie-ying
铜绿假单胞菌（P aeruginosa）引起的急性肺部感染是一个严重的问题，特别是在患有结构性肺部疾病或免疫受损宿主的患者中，导致具有高发病率和死亡率的压倒性威胁。作为感染的结果，纤维化可能与慢性肺部疾病有关。但急性感染铜绿假单胞菌后可出现肺功能不可逆下降和胸部影像学纤维带等纤维化表现。由铜绿假单胞菌引起的急性肺部感染引起的纤维生成/重塑很少有报道。本研究旨在探讨铜绿假单胞菌体外急性感染与纤维化/重塑的关系。我们利用急性铜绿假单胞菌肺部感染的关键启动物——铜绿假单胞菌鞭毛蛋白来阐明急性铜绿假单胞菌肺部感染导致纤维生成/重塑的机制。方法研究铜绿假单胞菌鞭毛蛋白（以下简称鞭毛蛋白）对转化生长因子β 1 （tgf - β 1）和白介素-8 （IL-8）表达的影响，以及可能参与的信号通路肿瘤坏死因子受体相关因子6 (TRAF6)/丝裂原活化蛋白激酶（MAPK）通路。从铜绿假单胞菌标准菌株PAO1中纯化鞭毛蛋白。用不同浓度的鞭毛蛋白刺激正常支气管上皮细胞BEAS-2B，用细胞计数试剂盒-8评估细胞活力。BEAS-2B细胞与含有特异性MAPK抑制剂或TRAF6 siRNA的鞭毛蛋白孵育。收集细胞裂解液和培养上清液。采用酶联免疫吸附试验（ELISA）检测tgf - β 1和IL-8水平。Western blotting检测MAPK信号蛋白p38、c-Jun nh2末端激酶（c-Jun NH2-terminal kinase， JNK）和细胞外蛋白水平。调节激酶（ERK）结果与对照组相比，鞭毛蛋白使BEAS-2B细胞中tgf - β 1的表达升高((104.3 +/- 20.8)vs (44.6 +/- 4.4) pg/ml (P < 0.01))， p38或JNK抑制剂使其表达降低(分别为（45.1 +/- 18.8）vs (104.3 +/- 20.8) pg/ml和（48.1 +/- 20.8）vs （104.3 +/- 20.8) pg/ml (P < 0.05)）。与对照组相比，鞭毛蛋白也提高了BEAS-2B细胞中IL-8的表达((554.9 +/- 57.7)vs (51.4 +/- 22.9) pg/ml (P < 0.01))， p38 MAPK抑制剂减弱了鞭毛蛋白的表达((301.1 +/- 155.1)vs （554.9 +/- 57.7) pg/ml (P < 0.05)）。Western blotting结果显示，MAPK蛋白p38、JNK和ERK均在鞭毛蛋白攻击后的早期阶段被激活，分别在15分钟（P < 0.01）、30分钟（P < 0.01）和15分钟（P < 0.01）。经鞭毛蛋白处理后，TRAF6 siRNA降低了TRAF6的表达，改变了JNK、p38、ERK的活化，但对tgf - β 1、IL-8表达的影响无统计学意义。结论铜绿假单胞菌PAO1鞭毛蛋白在体外通过MAPK信号级联诱导正常支气管上皮细胞BEAS-2B中tgf - β 1的表达。提示铜绿假单胞菌引起的急性肺部感染可能在早期就开始了纤维化/重塑过程。中华医学杂志2011；124 (4): 599 - 605
Background Acute lung infection due to Pseudomonas aeruginosa (P aeruginosa) is a serious problem, especially in patients with structural lung conditions or immune compromised hosts, leading to an overwhelming threat with a high risk of morbidity and mortality. As an outcome of infection, fibrosis can be linked with chronic lung diseases. But some fibrotic manifestations, such as an irreversible decrease of lung function and fibrous bands seen on chest imaging, have been found after an acute infection with P aeruginosa. Fibrogenesis/remodeling resulting from acute lung infection by P aeruginosa is rarely reported. This study was designed to explore the relation between fibrogenesis/remodeling and acute infection by P aeruginosa in vitro. We used flagellin protein from P aeruginosa, a key initiator of acute P aeruginosa lung infection, to elucidate mechanisms by which acute lung infection with P aeruginosa can cause fibrogenesis/remodeling.Methods We studied the effect of flagellin from P aeruginosa (flagellin for short) on the transforming growth factor beta 1 (TGF-beta 1) and interleukin-8 (IL-8) expression, and the possible involvement of the signaling pathway, tumor necrosis factor receptor-associated factor 6 (TRAF6)/mitogen activated protein kinase (MAPK) pathway. Flagellin was purified from the P aeruginosa standard strain, PAO1. Normal bronchial epithelial cells BEAS-2B were challenged with different concentrations of flagellin, and cell viability assessment was performed by cell counting kit-8. BEAS-2B cells were incubated with flagellin with the specific MAPK inhibitors or TRAF6 siRNA. Cell lysates and the cultured supernatant were collected. The level of TGF-beta 1 and IL-8 were detected by enzyme-linked immunosorbant assay (ELISA). Western blotting was used to detect the protein levels of MAPK signal proteins p38, c-Jun NH2-terminal kinase (JNK) and extracellular. regulated kinase (ERK).Results Expression of TGF-beta 1 in BEAS-2B cells was elevated by flagellin vs. control groups ((104.3 +/- 20.8) vs. (44.6 +/- 4.4) pg/ml (P < 0.01)) and was ablated by either p38 or JNK inhibitors compared with flagellin treatment ((45.1 +/- 18.8) vs. (104.3 +/- 20.8) pg/ml and (48.1 +/- 20.8) vs. (104.3 +/- 20.8) pg/ml, respectively (P < 0.05)). Flagellin also elevated the expression of IL-8 in BEAS-2B cells vs. the control groups ((554.9 +/- 57.7) vs. (51.4 +/- 22.9) pg/ml (P < 0.01)), and p38 MAPK inhibitors weaken the expression by flagellin ((301.1 +/- 155.1) vs. (554.9 +/- 57.7) pg/ml (P < 0.05)). Western blotting revealed that all three MAPK proteins, p38, JNK and ERK were activated by flagellin challenge in an early phase, respectively in 15 minutes (P < 0.01), 30 minutes (P < 0.01) and 15 minutes (P < 0.01). TRAF6 siRNA which decreased expression of TRAF6, altered the activation of JNK, p38, and ERK following flagellin treatment, but its influence on the expression of TGF-beta 1 and IL-8 has no statistical significance.Conclusions Flagellin from P.aeruginosa PAO1 induces TGF-beta 1 expression in normal bronchial epithelial cells, BEAS-2B, through the MAPK signal cascade in vitro. It suggests that the fibrogenesis/remodeling process may be initiated from an early stage of acute lung infection due to P aeruginosa. Chin Med J 2011;124(4):599-605