Department of Respiratory Diseases, the First Affiliated Hospital of Liaoning Medical University, Jinzhou, Liaoning, 121001, China.
BMC Microbiol. 2014 Feb 6;14:26. doi: 10.1186/1471-2180-14-26.
Pyocyanin (PCN), an extracellular product of Pseudomonas aeruginosa and a blue redox active secondary metabolite, plays an important role in invasive pulmonary infection. However, the detailed inflammatory response triggered by PCN infection in inflammatory cells (particularly macrophages), if present, remains to be clarified. To investigate the effects of PCN on macrophages, the ability of PCN to induce inflammation reaction and the signaling pathway for IL-8 release in PCN-induced differentiated U937 cells were examined.
It was found that PCN increased IL-8 release and mRNA expression in Phorbol 12-myristate 13-acetate (PMA) differentiated U937 cells in both a concentration- and time-dependent manner by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). P38 and ERK MAPKs were activated after 10 min of induction with PCN and their levels returned to baselines after 30 min by Western blotting. It was also found that within 10 min of PCN incubation, the level of p-I-κBα in the cytosol was increased, which returned to baseline level after 60 min. Meanwhile, the level of p-p65 was increased in the nuclear extract and cytosol, and maintained high in total cell lysates. The results were further confirmed by the observation that p38, ERK1/2 and NF-κB inhibitors inhibited PCN-induced NF-κB activation and attenuated PCN-induced IL-8 expression in U937 cells as a function of their concentrations. Moreover, it was shown that PCN induced oxidative stress in U937 cells and N-acetyl cysteine, an antioxidant, was able to inhibit PCN-induced IL-8 protein expression.
It is concluded that PCN induces IL-8 secretion and mRNA expression in PMA-differentiated U937 cells in a concentration- and time- dependent manner. Furthermore, p38 and ERK MAPKs and NF-κΒ signaling pathways may be involved in the expression of IL-8 in PCN-incubated PMA-differentiated U937 cells.
绿脓菌素（PCN）是铜绿假单胞菌的一种细胞外产物，也是一种蓝色氧化还原活性的次级代谢产物，在侵袭性肺部感染中发挥重要作用。然而，PCN 感染炎症细胞（尤其是巨噬细胞）所引发的详细炎症反应，如果存在的话，仍有待阐明。为了研究 PCN 对巨噬细胞的影响，我们检测了 PCN 诱导 PMA 分化的 U937 细胞中炎症反应和白细胞介素 8（IL-8）释放的信号通路。
通过逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA），我们发现 PCN 呈浓度和时间依赖性地增加 PMA 分化的 U937 细胞中 IL-8 的释放和 mRNA 表达。PCN 诱导 10 分钟后，p38 和 ERK MAPKs 被激活，Western blot 分析显示，30 分钟后它们的水平恢复到基线。还发现，PCN 孵育 10 分钟内，细胞质中 p-I-κBα的水平增加，60 分钟后恢复到基线水平。同时，核提取物和细胞质中 p-p65 的水平增加，总细胞裂解物中保持高水平。通过观察 p38、ERK1/2 和 NF-κB 抑制剂抑制 PCN 诱导的 NF-κB 激活和减弱 U937 细胞中 PCN 诱导的 IL-8 表达，进一步证实了这一点，其作用呈浓度依赖性。此外，研究表明 PCN 诱导 U937 细胞产生氧化应激，抗氧化剂 N-乙酰半胱氨酸能够抑制 PCN 诱导的 IL-8 蛋白表达。
PCN 以浓度和时间依赖的方式诱导 PMA 分化的 U937 细胞中 IL-8 的分泌和 mRNA 表达。此外，p38 和 ERK MAPKs 和 NF-κB 信号通路可能参与了 PCN 孵育的 PMA 分化的 U937 细胞中 IL-8 的表达。
