Liver X Receptor Induces P65 NF-?B Ubiquitin-degradation,which Negatively Regulates Inflammatory Responses

The liver X receptors(LXRs)are members of the nuclear receptor superfamily of ligand-activated transcription factors.Massive studies have shown that LXRs have anti-inflammatory properties after activation.Our previous study has shown that pretreatment with LXRs agonist T0901317 can inhibit the expression of p65 NF-?B in rats with acute lung injury(ALI)induced by lipopolysaccharide(LPS),thereby decreasing the expression of inflammatory cytokines.However,it is still uncertain how activated LXRs regulate p65 NF-?B protein expression.Ubiquitination modification is one of the protein posttranslational regulation mechanisms,and the subunit p65 of NF-?B can be degraded by the ubiquitination pathway.Based on the above background,we established the inflammatory model by stimulating RAW264.7 cells with LPS,and investigated whether LXRs play a role in inhibiting the macrophage inflammatory response by promoting the p65 NF-?B ubiquitination.Methods: Firstly,RAW264.7 cells were pretreated with LXRs agonist T0901317(10ummol / L)for 12 h and then stimulated with LPS(10ng / ml)for 6h.The expression of TNF-?,IL-1?,IL-6 and p65 NF-?B gene and protein were analysed by ELISA,PCR and Western blot respectively.Secondly,cells were treated with Cycloheximide(CHX)to stop translation,p65 NF-?B protein content were evaluated by western blot at 0h,2h and 4h after treatment of CHX.Then,equal amounts of p65 NF-?B protein were quantified by semi-quantitative analyse.Thirdly,T0901317 and LPS pretreated RAW264.7 cells were treated with MG-132,the expression of p65 NF-?B protein and polyubiquitination were quantified by Western blot,and the expression of TNF-?,IL-1? and IL-6 were observed by ELISA.Result:T0901317 pretreatment efficiently reduced the production of TNF-?,IL-1?,IL-6 and p65 NF-?B protein in LPS-induced RAW264.7 cells,but had no effect on the expression of p65 NF-?B m RNA.T0901317 reduced p65 NF-?B protein at 0h,2h and 4h after treatment of CHX.The results indicate that LXRs activation promotes p65 NF-?B protein degradation at post-translational level.MG-132 significantly down-regulated p65 NF-?B protein degradation in RAW264.7 cells pretreated with T0901317,and increases p65 NF-?B poly-ubiquitination,thereby reversing the effect of T0901317 on the expression of TNF-?,IL-1? and IL-6.Conclusion: T0901317 negatively regulates the expression of IL-1?,IL-6 and TNF-? via NF-?B signaling pathway,the possible mechanism is that LXRs activation promotes p65 NF-?B ubiquitin-degradation.