LPS Up-regulate Adipophilin And Promote Release Of Macrophage Inflammatory Cytokines Through TLR4-AP-1 Pathway

[Objective]To elucidate the utility of adipophilin in the process of LPS-induced cell secretion of inflammatory cytokines,and to investigate the relationship between adipophilin and M1-macrophages 。[Methods] First of all,observe adipophilin expression status in LPS induce RAW264.7 macrophages,detected protein level of intracellular adipophilin,Arg-1,iNOS by Western Blotting,and detected pro-inflammatory cytokines IL-6,MCP-1,TNF-α secretion in cell supernatants fluid by ELISA.Then,respectively preincubate RAW264.7 cells 6 hours byTAK242 or curcumin,specific inhibitors of TLR4 or AP-1,with 150 ng / ml LPS culture cells,Western Blotting measure the protein expression level of TLR4,AP-1,iNOS,adipophilin,and detect the concentration of TNF-α,MCP-1,IL-6 by ELISA.Finally,the use of a retroviral vector transfection technique,build a stable silence adipophilin RAW264.7 cell models,then add to LPS culture cell,detect related proteins and cytokines in the conditions of using the above methods.[Results] When the RAW264.7 macrophages are culture with different concentrations of LPS for 6 hours,the expression of adipophilin protein is increase at the concentration of 150 ng / ml;When LPS culture RAW264.7 macrophages at different times,the adipophilin protein level is firstly increase and then reduce,as the same as reach the largest level in 6 hours.Meanwhile,the inflammatory cytokines concentration of IL-6,MCP-1,TNF-α is accordant with adipophilin,reach the bigest level in 12 hours;When using TLR4 inhibitor TAK242 culture cells,compared with the control group,adipophilin,AP-1 protein expression and inflammatory cytokines IL-6,MCP-1,TNF-α concentrations are significantly decrease in inhibiting group;And when cells are treate with a specific inhibitor curcumin of AP-1,compared with the unsuppressed group,TLR4 activity does not change,but adipophilin expression and inflammatory cytokines IL-6,MCP-1,TNF-α concentrations are significantly increase;When LPS is added to silence adipophilin cells,compared to non-silent group,TLR4 activity and protein status expression of AP-1 are no changes,but the inflammatory cytokines IL-6,MCP-1,TNF-α concentration is remarkable reduce.In addition,throughout the process of the experiment,we detecte adipophilin changes is similiart with the M1 macrophages markers iNOS and TNF-α,but when add to TLR4 inhibitor,the protein level of iNOS was no difference.[Conclusions] LPS up-regulate adipophilin protein expression through TLR4-AP-1 pathway,then promote the inflammatory cytokinesrelease of IL-6,MCP-1,TNF-α.