Roles Of U?/UT System Played In Innate Immune Inflammatory Signal Pathway TLR4-IRF3 In Lipopolysaccharide(LPS)-Stimulated Primary Kupffer Cells

Objective: To investigate effects of urotensin ?(U?)/UT system on innate immune inflammatory signal pathway TLR4-IRF3 in the lipopolysaccharide(LPS)-stimulated Kupffer cells(KCs).Methods: Rat KCs are isolated and purified by means of in situ perfusion,density gradient centrifugation and early changing medium.Isolated cells are identified by ink phagocytosis and ED2 staining test.The cells were randomly divided into six groups according to random number table,and treated as follows: group A: U?(-)urantide(-)LPS(-);group B: U?(+)urantide(-)LPS(-);group C: U?(-)urantide(+)LPS(-);group D: U?(-)urantide(-)LPS(+);group E: U?(+)urantide(-)LPS(+);group F: U?(-)urantide(+)LPS(+).Pro-inflammatory cytokines including IL-6,IFN-? and IFN-? were assayed by ELISA in the culture supernatant of KCs.Cell surface TLR4 were tested by flow cytometry technique.Expression of IRF3 were tested through real-time PCR and Western blot.Results: Rat KCs were successfully isolated and purified,and confirmed by ink phagocytosis and ED2 staining test.Levels of IL-6,IFN-? and IFN-? were higher in group D and E than in group A,B and C(all P?0.001),and lower in group F than in group D and E(all P?0.001).No difference was statistically found between group D and group E or among group A,B and C in IL-6,IFN-? and IFN-? secretion levels(all P>0.05).More TLR4 positive KCs were shown in group D and E than in group A,B and C(all P<0.001),but group F had less TLR4(+)cells than group D and E(all P<0.001).No difference was statistically found between group D and group E or among group A,B and C in TLR4(+)cells(all P>0.05);Levels of IRF3 mRNA were significantly higher in group D and E than in group A,B and C(all P?0.001),and lower in group F than in group D and E(all P?0.001).No difference was statistically found between group D and group E or among group A,B and C in IRF3 mRNA levels(all P>0.05);Levels of cytoplasm IRF3 protein were significantly lower in group D and E than in group A,B and C(all P?0.001),and higher in group F than in group D and E(all P?0.001).No difference was statistically found between group D and group E or among group A,B and C in cytoplasm IRF3 protein levels(all P>0.05);Levels of nuclear IRF3 protein were higher in group D and E than in group A,B and C(all P?0.001),and lower in group F than in group D and E(all P?0.001).No difference was statistically found between group D and group E or among group A,B and C in nuclear IRF3 protein levels(all P>0.05).Conclusions: Significant increases were showed in the secretion levels of cytokines including IL-6?IFN-? and IFN-?,the expression of TLR4 protein and IRF3 mRNA and the nuclear translocation of IRF3 protein in KCs after stimulated by LPS,which were inhibited via urantide pretreatment.In conclusions,U?/UT system mediates immune inflammatory response through(or at least in part through)activating TLR4-IRF3 pathway in LPS-stimulated KCs.