Role of Notch signaling in hypoxia-induced secretion of proinflammatory cytokine in N9 microglia / 中华神经医学杂志

ABSTRACT

Objective To investigate the role of Notch signaling in hypoxia-induced pro-inflammatory cytokine secretion in N9 microglia by inhibiting Notch signaling with a γ-secretase inhibitor,DAPT.Methods N9 cells cultured in vitro were divided into normoxia group,normoxia+10 μmol/L DAPT treatment group,hypoxia group and hypoxia+10 μmol/L DAPT treatment group;10 μmol/L DAPT was added to the media of treatment groups;normoxia group and hypoxia group were added the same amount of solvent.Then,cells in the hypoxia group and hypoxia+10 μmol/L DAPT treatment group were changed into hypoxia conditions (3％ O2) for 12 h.The mRNA and protein were extracted among the four groups;real-time PCR was used to observe the mRNA expression levels of interleukin (IL)-6,IL-1 β and tumor necrosis factor (TNF)-α,Western blotting was applied to detect the protein levels of N1ICD,Hes1 and Hey1,and ELISA was used to determine the secretion levels of IL-6,IL-1 β and TNF-α among four groups.Results DAPT had an inhibitory effecton mRNA expression and protein secretion of the inflammatory cytokines IL-6,IL-1β and TNF-α,and the inhibition effect gradually increased with the increase of DAPT doses.As compared with those in the normoxia group,the mRNA and protein secretion levels of IL-6,IL-1 β and TNF-α and Notch signaling molecules N1ICD,Hes1,Hey1 protein levels were significantly increased in the hypoxia group (P＜0.05).As compared with those in the hypoxia group,the mRNA and secretion levels of IL-6,IL-1β and TNF-α and Notch signaling molecules N1ICD,Hes1,Hey1 protein levels were significantly decreased in hypoxia+DAPT group (P＜0.05).There were no significant differences in the mRNA and protein secretion levels of IL-6,IL-1β and TNF-α and Notch signaling molecules N1ICD,Hes1,Hey1 protein levels between normoxia group and nomoxia+DAPT group (P＞0.05).Conclusion Notch signaling mediates hypoxia induced pro-inflammatory cytokine secretion in microglia.