Studies On Signaling Pathways In HIF-1α Expression Induced With LPS & TNF-α In Murine Peritoneal Macrophages

Objective To investigate the signaling pathways in expression of HIF-1αin Murine peritoneal macrophages induced with lipopolysaccharide (LPS) & tumor necrosis factor-α(TNF-α).Method The peritoneal macrophages from BALB/c mice were collected and incubated in 10%FBS RPMI1640 with LPS or TNF-αcombined with or without NF-κB inhibitor SSZ or NOS inhibitor L-NMMA. The levels of HIF-1αmRNA of those cells were measured by RT-PCR. The expression of HIF-1α& VEGF protein was determined by immunocytochemical staining method, and the level of nitrite in cultural supernatant was measured by Griess reaction.Result After the peritoneal macrophages being treated with LPS or TNF-αfor 10h, the level of HIF-1αmRNA did not exhibit significant change than those of control group (P>0.05); however, the expression levels of HIF-1α& VEGF protein were significantly higher than those of control group(P<0.05). After treated with NF-κB inhibitor SSZ or NOS inhibitor L-NMMA, the expression levels of HIF-1α& VEGF protein were significantly decreased than those of LPS or TNF-α-treated groups (P<0.05). The expression level of HIF-1αprotein of SSZ+L-NMMA-treated group has no significant change than that of SSZ or L-NMMA-treated groups (P>0.05), but the expression level of VEGF protein was significantly decreased (P<0.05). There was a low level of nitrite in cultural supernatant of control group, LPS and TNF-αcould increase the production of nitrite than those of control group (P<0.05), and after the macrophages were treated with SSZ or L-NMMA, the level of nitrite in cultural supernatant was significantly lower than those of the LPS or TNF-α-treated groups (P<0.05). After the peritoneal macrophages being treated with DETA-NO for 4h, the expression levels of HIF-1α& VEGF protein were significantly increased than those of control group(P<0.05).Conclusion LPS and TNF-αcan up-regulate the expression of HIF-1αand its target gene VEGF by post-transcription mechanism via NF-κB and NOS.