Liver Injury Mediated By The UII/Ut System Is Possibly Associated With Autophagy Increase And Apoptosis Decrease Of Kupffer Cells In Acute Liver Failure

Objective Using urotensin II receptor(UT)specific antagonist,urantide,to determine the effects of urotensin II(UII)/ UT system on autophagy in liver tissue and Kupffer cell(KC)with acute liver injury,and to investigate the underling mechanisms of UII/UT system mediating acute liver injuries.Methods In vivo experiment,acute liver injury mice model were induced by Lipopolysaccharide and D-galactosamine(LPS/D-GalN)via intraperitoneal injection.Male BALB/c mice were randomly divided into 4 groups.Group A: LPS/DGalN(-)urantide(-);group B: LPS/D-GalN(-)urantide(+);group C: LPS/DGalN(+)urantide(-)and group D: LPS/D-GalN(+)urantide(+).The groups B and D received urantide by a caudal vein injection.Pretreatment 30 min later,the groups C and D were treated with LPS/D-GalN by intraperitoneal injection.Serum and liver tissue samples were collected at 6 h.Serum alanine transaminase(ALT)and aspartate aminotransferase(AST)levels were detected.The Beclin-1,autophagy related gene 5(Atg5),microtubule-associated protein 1 light chain 3(LC3)and sequestosome 1(Sqstm1/p62)mRNA level was detected by Quantitative Real-time PCR(qRT-PCR).The autophagic protein LC3 and p62 was tested by Western blot.In vitro experiment,primary rat KCs were isolated and cultured by collagenase digestion,discontinuous density gradient centrifugation and selective wall-pasting.The KCs were randomly divided into 4 groups,including group A: LPS(-)urantide(-),group B: LPS(-)urantide(+),group C: LPS(+)urantide(-)and group D: LPS(+)urantide(+).Similarly,KCs were pretreated with 20 nm urantide solution before LPS stimulation.the mRNA levels of TNF Receptor-Associated Factor 6(TRAF6),LC3 and p62 were detected by qRT-PCR,the protein levels of TRAF6,LC3,p62,B-cell lymphoma-2(Bcl-2)and cleaved caspase-3 were determined by Western blot,and the reactive oxygen species(ROS)levels were detected by 2',7'-Dichlorodihydrofluorescein diacetate(DCFH-DA)ROS Assay Kit.Results In vivo experiments,the serum AST and AST levels in group C were higher than in group A and B,whereas those in group D were lower than in group C(all P<0.01).The relative expression levels of Beclin-1,Atg5,LC3 and p62 mRNA as well as LC3-II and p62 protein in liver tissue in group C were lower than in group A and B(all P<0.01),while those in group D,urantide pretreatment,had no significant difference compared with those in group C(all P>0.05).In vitro experiments,the relative expression levels of cellular TRAF6 m RNA and protein as well as ROS production in group C were higher than in group A and B,whereas those in group D were lower than in group C(all P<0.01).Furthermore,the same trend were showed in LC3 and p62 m RNA as well as LC3-II protein(all P<0.01).However,the relative expression levels of p62 protein in group C were higher than in group A and B(all P<0.01),and those in group D were higher than in group C(P<0.01).Additionally,the relative expression levels of Bcl-2 protein in group C were lower than in group A and B(all P<0.01),and those in group D were down regulative than in group C(P<0.01);The relative expression levels of cleaved caspase-3 protein in group C were higher in group A and B(all P<0.01),and those in group D were up regulative than in group C(P<0.01).Conclusion In vivo,the liver autophagy with ALF induced by LPS/D-Gal N was inhibited,and urantide pretreatment didn't affect it.In vitro,LPS stimulation induced TRAF6 activation,reactive oxygen species(ROS)production,autophagy upregulation and apoptosis induction,whereas urantide pretreatment significantly inhibited TRAF6 activation,ROS production and autophagy upregulation,but promoted apoptosis.Therefore,this study revealed that liver injury mediated by the UII/UT system has no relation to the inhibited autophagy of whole-liver tissue,but possibly associated with autophagy increase and apoptosis decrease of KCs in acute liver failure.