AIM:To investigate the effects of hypoxia on the secretions of proinflammatory cytokines TNF-alpha and IL-6 and to inquire into the mechanism.METHODS:Separated mice abdominal macrophages which were identified with non-specific esterase dye method, and created the hypoxic cultured model. The levels of TNF-alpha and IL-6 in the medium were determined by ELISA method. The mRNA expressions of TNF-alpha and IL-6 were measured by RT-PCR method. NF-kappaB activation was assayed by Western blot method. Finaly, we added cortone (5 microg/ml) to the medium, then observed the secretion levels of TNF-alpha and IL-6 during hypoxia.RESULTS:The secretions of TNF-alpha and IL-6 from Mphi exposed to hypoxia for 12 h were increased significantly compared with control (P < 0.01). The expressions of TNF-alpha mRNA and IL-6 mRNA were enhanced obviously contrasted with control (P < 0.01). NF-kappaB activation in Mphi nuclei was raised at 2 h during hypoxia and persisted to 5 h. We added cortone to the medium and found no significant change in secretion of TNF-alpha and IL-6 during hypoxia.CONCLUSION:Hypoxia could activate NF-kappaB and make it shift to nucleus which promoted the transcriptions and expressions of TNF-alpha and IL-6.

The effects of hypoxia on the level of reactive oxygen species (ROS), IkappaBalpha tyrosine phosphorylation, transcription of P65 mRNA and NF-kappaB 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. IkappaBalpha 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-kappaB activation began to increase after 3 h hypoxia, and reached a peak after 4 h hypoxia. (2) When antioxidant NAC (500 mmol/L) was added into the medium, the level of IkappaBalpha phosphorylation showed no significant changes during hypoxia. After adding protein tyrosine kinase inhibitor genistein (200 micromol/L), NF-kappaB 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 IkappaBalpha phosphorylation and NF-kappaB activation through intracellular ROS, and that the regulation of NF-kappaB activity may involve IkappaBalpha phosphorylation and the expressions of each subunit gene of NF-kappaB.