摘要目的 探讨细菌脂多糖(LPS)诱导的肺泡上皮细胞(AEC)炎症反应及可能的反应机制.方法 将人肺腺癌细胞系A549细胞株分为2组:对照组和LPS干预组,分别培养并于0.5 h、2 h、6 h和12 h时留取标本进行相关细胞因子检测.酶联免疫吸附法(ELISA)检测细胞间黏附分子-1(ICAM-1),放射免疫法检测肿瘤坏死因子-α(TNF-α)和白细胞介素-8(IL-8)的水平.实时荧光定量PCR检测TLR-4 mRNA的表达;蛋白质免疫印迹(Western blot)法检测核转录因子-κB(NF-κB)抑制蛋白IκBα和NF-κB p65蛋白水平,观察NF-κB的活性变化.结果 与对照组比较,LPS使AECs分泌的ICAM-1,TNF-α和IL-8于2 h、6 h和12 h均升高,ICAM-1,TNF-α于2 h、IL-8于12 h达分泌高峰;作用2 h后TLR-4 mRNA表达明显升高并达到峰值(27.88±13.31),6 h后(19.82±15.58)仍高于对照组(1.00±0.00),组间比较差异有统计学意义(P＜0.05),12 h时(12.86±11.45)组间比较差异无统计学意义(P＞0.05);NF-κB活性于刺激后0.5 h、2 h、6 h和12 h均明显增加,表现为抑制蛋白IκBα迅速降解,NF-κB p65蛋白同步释出并转入细胞核内.结论 LPS能够激活肺泡上皮细胞使其释放大量炎性因子,这一过程可能是通过激活TLR-4并进而激活NF-κB而诱导了AECs的炎性损伤.

abstractsObjective Lipopolysaccharide(LPS)can activate alveolar epithelial cells(AECs)and induce inflammatory injury.Toll-like receptor-4(TLR-4)is integrally involved in LPS signaling and plays a requisite role in the activation of NF-κB.NF-κB is a key intercellular signaling event that mediates cell inflammatory responses.The aim of the study wss to investigate in an in vitro model the inflammatory responses of AECs induced by LPS and the underlying mechanisms.Methods The study was performed on A549 cells(Human lung adenocarcinoma cell line).A549 cells were divided into 2 groups:a control group and a LPS stimulation group.Pminflammatory cytokines ICAM-1,TNF-α and IL-8 were detected by ELISA or radioimmunological methods.The expression of TLR-4 mRNA was detected by real time PCR.The activation of NF-κB was detected by Western blot(proteins of I-κBα and NF-κB p65).Results Compared with the control group.the ICAM-1 and TNF-α levels of the LPS-stimulated group were significantly higher,peaked after 2 h,and then gradually decreased at 6 and 12 h.IL-8 was also significantly increased after 2 h, which continued up to 12 h.The expression of TLR-4 mRNA in the LPS group was significantly higher,peaked after 2 h and gradually decreased at 6 and 12 h.NF-κB was activated after 0.5,2,6 and 12 h,indicated by the significant degradation of IκB-α and the significant release of NF-κB P65 and its subsequent translocation into the nucleus approximately synchronized.Conclusion The results demonstrate that LPS induced inflammatory injury in AECs via activating TLR-4 and subsequently NF-κB.