Effects And Mechanism Of Lipoxin On LPS-Induced Inflammatory Mediators In RAW264.7 Macrophages

Objective:To investigate the effects of lipoxin A₄ on LPS-induced inflammatory responses in RAW264.7 macrophages and its underlying molecular mechanism.Methods:Well-cultured RAW 264.7 cells were used and randomly divided into 4 groups:(1) Control group:cells were cultured with PBS(2) LPS group: cells were treated with 1μg/ml LPS(3) LPS+Lipoxin A₄ group:cells were treated with 1μg/ml LPS and 100ng/ml Lipoxin A₄(4) LPS+Lipoxin A₄ and ZnPPIX group:cells were treated with 1μg/ml LPS and 100ng/ml Lipoxin A₄ and 100μM ZnPPIX.24 hours after RAW 264.7 cells were collected,the levels of TNF-α,IL-10,and PGE2 from the cells supernatant were assayed by ELISA,COX-2 and HO-1 proteins from RAW 264.7 cells were determined by western blotting,HO-1 activity in the RAW264.7 cells were measured by spectrophotometer,and TLR4 on the membrane of RAW264.7 macrophages were detected Flow cytometry.Results:1.Administration of LPS obviously increased the level of TNF-αin RAW264.7 cells;Lipoxin A₄ inhibited LPS-induced level of TNF-αin RAW264.7 cells;ZnPPIX partially abolished the suppressive effect of lipoxin A₄ on level of TNF-αinduced by LPS in RAW264.7 cells.2.Administration of LPS increased the level of IL-10 in RAW264.7 cells; Lipoxin A₄ markedly increased LPS-induced level of IL-10 in RAW264.7 cells;ZnPPIX inhibited the increasing level of IL-10 in RAW264.7 cells induced by Lipoxin A₄ and LPS.3.Administration of LPS obviously increased the production of COX-2 and the level of PGE2;Lipoxin A₄ inhibited LPS-induced COX-2 protein expression and PGE2 production;ZnPPIX partially abolished the suppressive effects of lipoxin A₄ on the COX-2 expression and PGE2 elevation induced by LPS in RAW264.7 cells.4.LPS increased the protein expression and activation of HO-1 in RAW264.7 cells;Lipoxin A₄ enhanced LPS-induced the protein expression and activation of HO-1 in RAW264.7 cells;ZnPPIX obviously weaked LPS-induced activation of HO-1 without influence on the expression of HO-1.5.LPS decreased the content of TLR4 on the membrane of RAW264.7 macrophages;Lipoxin A₄ also further reduced the content of TLR4 on the membrane of RAW264.7 macrophages;ZnPPIX reversed the suppressive effect of lipoxin A₄ on the content of TLR4 on the membrane of RAW264.7 macrophages.Conclusion:Lipoxin A₄ can inhibit LPS-induced pro-inflammatory mediators including COX-2,PGE2 and TNF-α,reduce the content of TLR4 on RAW264.7 macrophages simultaneously,increase the production of IL-10, and these effects seems to be mediated through HO-1 pathway.