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目的观察Notch1和TLR4信号交叉调控NF-κB激活并探讨其可能的作用机制。方法体外培养RAW264.7细胞,LPS刺激RAW264.7细胞后检测Notch1信号激活以及Notch1信号抑制剂DAPT对TNF-α、IL-6和IL-1β表达的影响,Western blot检测DAPT对IKKα/β磷酸化以及NF-κB p65活化的影响,并检测DAPT对TLR4信号通路IKKα/β上游信号My D88和TRAF6表达的影响。结果 LPS刺激RAW264.7细胞后能显著提高Notch1信号蛋白NICD和目的蛋白Hes1表达,DAPT能明显抑制NICD和Hes1的表达。LPS诱导TNF-α、IL-6和IL-1β表达和释放能被DAPT抑制但被Notch1信号激动剂jagged1增强。DAPT能够抑制LPS介导的TRAF6表达和IKKα/β磷酸化,促进NF-κB p65核转位,但DAPT对LPS诱导My D88的表达没有明显的影响。结论 Notch1和TLR4信号存在交叉对话,Notch1通过TRAF6调控NF-κB活化参与TLR4介导的免疫应答。
Objective To observe the effects of Notch1 and TLR4 signaling on the activation of NF-κB and its possible mechanism. Methods RAW264.7 cells were cultured in vitro. After RAW264.7 cells were stimulated by LPS, the effect of Notch1 signaling and the expression of Notch1 signal inhibitor DAPT on TNF-α, IL-6 and IL-1β were detected. Western blot was used to detect the effect of DAPT on IKKα / And the activation of NF-κB p65. The effect of DAPT on the expression of MyD88 and TRAF6, the upstream signal of IKKα / β in TLR4 signaling pathway, was also examined. Results LPS stimulation of RAW264.7 cells significantly increased Notch1 signaling protein NICD and target protein Hes1 expression, DAPT can significantly inhibit NICD and Hes1 expression. LPS induced TNF-α, IL-6 and IL-1β expression and release can be DAPT inhibition but Notch1 signal agonist jagged1 enhanced. DAPT could inhibit LPS-mediated TRAF6 and IKKα / β phosphorylation and promote nuclear translocation of NF-κB p65, but DAPT had no obvious effect on LPS-induced My D88 expression. Conclusion There is a cross-talk between Notch1 and TLR4 signaling. Notch1 is involved in the TLR4-mediated immune response through the activation of NF-κB mediated by TRAF6.