摘要
目的:探讨小窝蛋白1(Cav-1)对脂多糖(LPS)诱导的气道黏液高分泌的影响。方法:体外培养人气道上皮细胞(16HBE),用LPS刺激细胞构建黏液高分泌模型,以Toll样受体4(TLR4)抑制剂E5564、核转录因子-κB(NF-κB)抑制剂PDTC、转染Cav-1质粒和siRNA为干预因素,将细胞随机分为对照组、LPS刺激组、LPS+Cav-1质粒组、LPS+Cav-1 siRNA组、LPS+阴性siRNA组、LPS+空质粒组、LPS+E5564组及LPS+PDTC组。四甲基偶氮唑盐法(MTT)检测各组细胞的活力;RT-PCR检测黏蛋白(MUC)5AC的转录水平;Western blot检测Cav-1、TLR4、磷酸化IκBα(p-IκBα)蛋白的相对含量;ELISA检测MUC5AC的分泌水平;激光共聚焦技术检测细胞内MUC5AC蛋白的分布和含量。结果:LPS刺激组细胞内TLR4、p-IκBα、NF-κB、MUC5AC转录及蛋白水平显著高于对照组(P值均<0.05),过表达Cav-1可进一步增加上述指标的表达量,而下调Cav-1及给予E5564、PDTC可以抑制LPS引起的上述效应(P<0.05)。结论:Cav-1可通过上调TLR4/NF-κB信号通路而加重LPS诱导的MUC5AC的表达量。
Objective： To explore the effect of caveolin-1 （Cav-1）on lipopolysaccharide （LPS）-induced airway mucous hypersecretion. Methods： 16HBE human airway epithelial ceils with Toll-like receptor 4 （TLR4） inhibitor, nuclear factor-kappa B （NF- KB） inhibitor, Car-1 siRNA or plasmid pr-treated, further stimulated with LPS. The cells were divided into 8 groups：the control group, the LPS group,the LPS＋ Cav-I expression group,the LPS＋ Cav-1 siRNA group,the LPS ＋ negative siRNA group,the LPS ＋ empty vector group,the LPS ＋ E5564 group, the LPS ＋ PDTC group. Cell survival rate was detected by MTT assay. Transcription level of mucin（ MUC）5AC was evaluated with RT-PCR. The level of MUC5AC protein was measured by ELISA. The expression of TLR4, Cav-1 and phosphorylated IKBot （p-IKBct）were measured by Western blot. MUC5AC protein changes were observed by immunofluorescence and confocal laser technology. Results： LPS remarkably increased MUC5AC, as well as TLR4, p-IKBα（ P〈0.05 ）. These effects were prevented by E5564 and PDTC. We found that the overexpression of Cav-1 further enhanced the expression of TLR4, p-IKBot and MUC5AC. However,downregulation of Cav-1 inhibited the expression of TLR4, p-IKBα, MUC5AC. Conclusion ： Cav-1 enhances LPS- induced MUC5AC hyperseeretion through TLR4/NF-KB signaling pathway.
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2016年第1期19-22,共4页
Chinese Journal of Immunology
基金
国家自然科学基金(81370111
31171346)资助项目
