Mu Xinwei, Pan Chen, Zheng Shuyun, Alhamdi Yasir, Sun Bingwei, Shi Qiankun, Wang Xiang, Sun Zhiwei, Toh Chenghock, Wang Guozheng
Department of Critical Care Medicine, Nanjing First Hospital, Nanjing Medical University, Nanjiang, Jiangsu Province, PR China.
Department of Critical Care Medicine, Nanjing Children,s Hospital, Nanjing Medical University, Nanjiang, Jiangsu Province, PR China.
PLoS One. 2014 Aug 7;9(8):e104032. doi: 10.1371/journal.pone.0104032. eCollection 2014.
To investigate the protective effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on barrier function of intestinal epithelial cells.
After pre-incubation with CORM-2 for 1 hour, cultured intestinal epithelial IEC-6 cells were stimulated with 50 µg/ml lipopolysaccharides (LPS). Cytokines levels in culture medium were detected using ELISA kits. Trans-epithelial electrical resistance (TER) of IEC-6 cell monolayers in Transwells were measured with a Millipore electric resistance system (ERS-2; Millipore) and calculated as Ω/cm2 at different time points after LPS treatment. The permeability changes were also measured using FITC-dextran. The levels of tight junction (TJ) proteins (occludin and ZO-1) and myosin light chain (MLC) phosphorylation were detected using Western blotting with specific antibodies. The subsequent structural changes of TJ were visualized using transmission electron microscopy (TEM).
CORM-2 significantly reduced LPS-induced secretion of TNF-α and IL-1β. The LPS-induced decrease of TER and increase of permeability to FITC-dextran were inhibited by CORM-2 in a concentration dependent manner (P<0.05). LPS-induced reduction of tight junction proteins and increase of MLC phosphorylation were also attenuated. In LPS-treated cells, TEM showed diminished electron-dense material and interruption of TJ and desmosomes between the apical lateral margins of adjoining cells, which were prevented by CORM-2 treatment.
The present study demonstrates that CORM-2, as a novel CO-releasing molecule, has ability to protect the barrier function of LPS-stimulated intestinal epithelial cells. Inhibition of inflammatory cytokines release, restoration of TJ proteins and suppression of MLC phosphorylation are among the protective effects of CORM-2.
探讨一氧化碳释放分子-2（CORM-2）对肠上皮细胞屏障功能的保护作用及其机制。
用CORM-2预孵育1小时后，用50μg/ml脂多糖（LPS）刺激培养的肠上皮IEC-6细胞。使用ELISA试剂盒检测培养基中的细胞因子水平。用密理博电阻系统（ERS-2；密理博）测量Transwells中IEC-6细胞单层的跨上皮电阻（TER），并在LPS处理后的不同时间点计算为Ω/cm²。还使用异硫氰酸荧光素标记的葡聚糖（FITC-葡聚糖）测量通透性变化。使用特异性抗体通过蛋白质免疫印迹法检测紧密连接（TJ）蛋白（闭合蛋白和ZO-1）水平以及肌球蛋白轻链（MLC）磷酸化水平。使用透射电子显微镜（TEM）观察TJ的后续结构变化。
CORM-2显著降低LPS诱导的肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）分泌。CORM-2以浓度依赖性方式抑制LPS诱导的TER降低和FITC-葡聚糖通透性增加（P<0.05）。LPS诱导的紧密连接蛋白减少和MLC磷酸化增加也得到减轻。在LPS处理的细胞中，TEM显示相邻细胞顶端侧缘之间的电子致密物质减少以及TJ和桥粒中断，而CORM-2处理可防止这种情况发生。
本研究表明，作为一种新型的CO释放分子，CORM-2具有保护LPS刺激的肠上皮细胞屏障功能的能力。抑制炎性细胞因子释放、恢复TJ蛋白以及抑制MLC磷酸化是CORM-2的保护作用之一。
