基础医学与临床 ›› 2017, Vol. 37 ›› Issue (10): 1363-1367.

• 研究论文 • 上一篇    下一篇

LPS触发的TLR4对NF-κB和IRF3信号通路的调控差异

宋晓琦,刘玮,刘硕,姜明红   

  1. 中国医学科学院基础医学研究所
  • 收稿日期:2016-08-04 修回日期:2016-11-28 出版日期:2017-10-05 发布日期:2017-09-25
  • 通讯作者: 姜明红 E-mail:junyi2@21cn.com
  • 基金资助:
    国家自然基金

Different regulation profiles of LPS-activated TLR4 on the NF-κB and IRF3 signaling pathway

  • Received:2016-08-04 Revised:2016-11-28 Online:2017-10-05 Published:2017-09-25

摘要: 目的 探讨脂多糖(LPS)触发的Toll样受体4(TLR4)对NF-κB和干扰素调节因子3(IRF3)信号通路的调控差异。方法 LPS刺激小鼠原代腹腔巨噬细胞,用免疫荧光法检测TLR4及两种转录因子p65及IRF3的定位,用Western Blot方法检测转录因子p65和IFR3的磷酸化水平。结果 巨噬细胞经LPS刺激30 min内,细胞膜上的TLR4荧光强度增加(P < 0.01),细胞浆中TLR4荧光强度显著增加(P < 0.01),且与早期内体抗原1(Early endosome antigen 1, EEA1)共定位;当LPS刺激90和180 min时,胞膜和胞浆内的TLR4信号均显著下降(P < 0.01),与EEA1共定位的信号也明显减少(P < 0.01)。静息状态下,TLR4的下游信号通路分子p65和IRF3的荧光信号均出现在细胞浆中;LPS刺激后,两者的荧光信号在细胞核中逐渐增加(P < 0.05),但p65荧光信号比IRF3增强得更早,且更持久。p65磷酸化的修饰也明显早于IRF3,且持续时间更长。结论 TLR4活化后的定位变化导致其下游IRF3信号通路的传递时间明显延后,且比NF-κB信号通路短暂。

关键词: LPS, TLR4, p65, IRF3

Abstract: Objective To investigate the different regulation of TLR4 between NF-κB and IRF3 signaling pathways which are triggered by LPS. Methods LPS was used to stimulate mouse peritoneal macrophages. Immunofluorescence was performed to observe the translocation of TLR4 and the transcription factors p65 and IRF3. The phosphorylation level of transcription factors was examined by Western Blot. Results With stimulation of LPS in 30 minutes, the fluorescence intensity of TLR4 on the cytomembrane was increased (P < 0.01), while its fluorescence intensity on the endosome in cytoplasm was increased more robustly (P < 0.01), and TLR4 in cytoplasm was found to co-locate with EEA1. When the stimulation reached 90 minutes and 180 minutes, the fluorescence intensity of TLR4 in both cytomembrane and cytoplasm was decreased obviously (P < 0.01), and the co-location with EEA1 was attenuated (P < 0.01). In rest state, the fluorescence intensity of the downstream molecule p65 and IRF3 was almost totally in the cytoplasm. After the stimulation of LPS, fluorescence intensity both in nucleus was increased gradually (P < 0.05), but the fluorescence intensity of p65 in nucleus was enhanced earlier and lasted longer than that of IRF3. The phosphorylation of p65 was also earlier and lasted longer than that of IRF3. Conclusions The changed location of TLR4 after being activated can make the transfer time of IRF3 pathway longer and the lasting time shorter than that of NF-κB robustly.

Key words: LPS, TLR4, p65, IRF3