Kinetics Of The Activation Of NF-κ B And AP-1 And Their Regulation On TNF-α Expression Induced By Lipopolysaccharide In Rat Alveolar Macrophages

Objective This study was designed to investigate the kinetics of the activation of the NF-κB and AP-1 ,and elucidate their role in TNF-αexpression induced by lipopolysaccharide(LPS) in rat alveolar macrophages(AM).Methods 1.The dynamic changes of the activity of NF-κB and AP-1 were determined with Electrophoretic mobility shift assay(EMSA).2.The phosphorothioate ODN (S-ODN) decoy were transfected into AM prior to LPS stimulation.3.The level of TNF-α in culture supernatants were measured with ELISA kit.Results 1. The simultaneous remarkable activation of NF-κB and AP-1 could be detected after 0.5 hour LPS stimulation, but after that , the kinetics of NF-κB and AP-1 became different. The activity of NF-κB reached the highest level at 1 hour after LPS stimulation and gradually decreased at 3 hours, 5 hours,8 hours after LPS stimulation, while the activity of AP-1 peaked at 0.5 hour, rapidly decreased at 1 hour, then increased at 3 hours again and reduced at 5 hours,8 hours after LPS stimulation. Both the activation of NF-κB and AP-1 could persist at least 8 hours,but the activation of AP-1 was more fluctuant than that of NF-κB. 2.The results showed the effect of LPS on the activation of NF-κB and AP-1 was dose dependent within 1000ng/ml.3. NF-κB and AP-1 S-ODN decoy both could markedly inhibit the LPS induced TNF-α production by rat AM. 4.The effect of simultaneous inhibition of NF-κB and AP-1 on the TNF-αproduction was stronger than that of single inhibition of NF-κB and AP-1, but they both could not completely inhibit the production of TNF-α induced by LPS in rat AM. Conclusions It showed that LPS could simultaneously induce the activation of NF-κB and AP-1 in rat AM,and they both played an important role in LPS induced inflammatory response. It also suggested that NF-κB and AP-1 both involved in the regulation on LPS induced TNF-α production by rat AM. However, simultaneous inhibition of the activity of NF-κB and AP-1 could not completely prevent the TNF-αproduction by rat AM .It implied that other nuclear factors might also play an important role in the regulation on LPS induced TNF-αexpression by rat AM.