低氧激活巨噬细胞内NF-κB信号转导通路的机制

Nuclear factor kappa B signal transduction in macrophages during hypoxia: reactive oxygen species generation

利用离体培养的巨噬细胞(macrophage)低氧培养模型(1％O2,5％CO2),采用2’,7’-二氯荧光乙酰乙酸盐(2’,7’-dichlorofluorescein diacetate,DCFH-DA)荧光分光光度法、Western blotting法和逆转录酶链反应(reverse transcription polymerasechain reaction,RT-PCR)法,观察低氧后细胞内活性氧(reactive oxygen species,ROS)水平、IκBα的酪氨酸(Tyr)磷酸化水平、P65mRNA转录水平以及细胞核内NF-κB(nuclear factor kappa B)激活量的变化,探讨低氧激活NF-κB信号转导通路的机制。结果表明,低氧后细胞内ROS水平、IαBα的Tyr磷酸化水平和细胞核内NF-κB的激活量均高于对照组(P<0.05),并且有时间变化趋势上的先后关系,如先用抗氧化剂N-乙酰半胱氨酸(N-acetylcysteine,NAC,500 μmol/L)和Tyr蛋白激酶抑制剂genistein(200μmol/L)预处理,低氧后细胞内IκBα的Tyr磷酸化和NF-κB;活化分别比单纯低氧组下降(P<0.01);另外,低氧后P65 mRNA转录水平也明显增加(P<0.01)。以上结果提示,低氧可能通过细胞内产生ROS,使IκBα的Tyr位点磷酸化,进而使NF-κB活化;另外,NF-κB活性调节除受抑制性亚基IκBα的磷酸化调控外,还可能表现在NF-κB分子各亚基基因自身的转录调控上。

The effects of hypoxia on the level of reactive oxygen species (ROS), IκBα tyrosine phosphorylation, transcription of P65 mRNA and NF-κB activation in isolated rat peritoneal macrophages were investigated by DCFH-DA fluorescence spectrophotometry, Western blotting and RT-PCR. The results obtained are as follows. (1) During hypoxia, the levels of intracellular ROS began to increase at 1 h, then reached a peak at 2 h, and began to decrease after 3 h. IκBα tyrosine phosphorylation began to rise after 2 h hypoxia and was the highest after 3 h hypoxia. After 4 h hypoxia it decreased gradually. NF-κB activation began to increase after 3 h hypoxia, and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 μmol/L) was added into the medium, the level of IκBα phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 μmol/L), NF-κB activation induced by hypoxia was blocked significantly. (3) The expression of p65 mRNA was also elevated markedly during hypoxia. These results suggest that hypoxia may lead to IκBα phosphorylation and NF-κB activation through intracellular ROS, and that the regulation of NF-κB activity may involve IκBα phosphorylation and the expressions of each subunit gene of NF-κB.