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目的研究白细胞介素-1β(IL-1β)对肺II型上皮A549细胞分泌白细胞介素-8(IL-8)的诱导作用,探讨相关的细胞内信号通道的激活和传导的机理。方法以1ng/ml终浓度的IL-1β干预经核转录因子-κB(NF-κB)的IKK2复合物抑制物(AS602868)预处理的A549细胞,Western blot检测IL-1β干预后细胞内磷酸化IκBα(p-IκBα)和IκBα蛋白的表达水平;激光扫描共焦显微镜(LSCM)检测NF-κB的p65和p50蛋白的核转移过程;ELISA检测NF-κB的p65和p50蛋白与DNA结合活性及IL-8蛋白的释放。结果IL-1β干预后细胞内p-IκBα蛋白明显升高,IκBα蛋白明显下降;LSCM扫描显示p65和p50蛋白从胞浆向胞核转移,同时p65和p50与DNA结合活性明显升高(P<0.01);IL-8的蛋白表达水平明显升高(P<0.01)。细胞经AS602868的预处理,阻断了细胞内p-IκBα蛋白升高和IκBα蛋白降解;减少了p65和p50蛋白的核转移和与DNA结合活性(P<0.01);降低了IL-8蛋白的表达水平(P<0.01)。结论IL-1β通过激活NF-κB介导了A549细胞分泌IL-8,结果提示可能通过抑制NF-κB信号通道的方法,减轻呼吸机相关肺损伤(VALI)的生物性损伤。
Objective To investigate the induction of interleukin-1β (IL-1β) on the secretion of interleukin-8 (IL-8) by human lung epithelial-type A549 cells and to explore the mechanisms involved in the activation and transduction of relevant intracellular signaling pathways. Methods IL-1β at a final concentration of 1ng / ml was used to pre-treat A549 cells pretreated with IKK2 inhibitor of nuclear factor-κB (AS602868), and Western blot was used to detect the phosphorylation of IL-1β The nuclear translocation of p65 and p50 in NF-κB was detected by laser scanning confocal microscopy (LSCM). The DNA binding activities of p65 and p50 proteins in NF-κB IL-8 protein release. Results The expression of p-IκBα protein was significantly increased and IκBα protein was significantly decreased after IL-1β treatment. LSCM showed that p65 and p50 protein migrated from cytoplasm to nucleus, and the binding activities of p65 and p50 to DNA were significantly increased (P < 0.01). The protein expression of IL-8 was significantly increased (P <0.01). Cells pretreated with AS602868 blocked the increase of intracellular p-IκBα protein and IκBα protein degradation; decreased the nuclear translocation and DNA binding activity of p65 and p50 proteins (P <0.01); decreased the expression of IL-8 protein Expression level (P <0.01). Conclusion IL-1β can mediate the secretion of IL-8 by A549 cells through activation of NF-κB. The results suggest that it may reduce the biological damage of ventilator-associated lung injury (VALI) by inhibiting NF-κB signaling pathway.