Study Of The Mechanism And Effect Of Extracellular HMGB1 On Acute Hepatic Injury And Lung Injury In Mice

Study of the Mechanism and Effect of Extracellular HMGB1on Acute Hepatic Injury and Lung Injury in MicePh.D.Candidate:Gong QuanSuperviser:Prof.Gong FeiliDepartment of Immunology,Tongji Medical College of HuazhongUniversity of Science and TechnologyLiver diseases,including viral hepatitis,autoimmune hepatitis,alcoholic liver injury,liver fibrosis and hepatic cirrhosis represent a worldwide health problem in humans.Theimmune-mediated liver injury plays an important role in various liver diseases.The ConA-induced hepatitis in mice,being characterized by an activation of CD4~+T cells and naturalkiller T(NKT) cells and markedly increased production of roinflammatory cytokine,such asTNF-a,IFN-γ,is similar to human immune-mediated hepatitis,Therefore,the Con A-inducedhepatitis in mice is one of model for studying hepatitis,especially viral hepatitis andautoimmune hepatitis.High mobility group box 1 protein(HMGB1,previously HMG-1,amphoterin) has 215residues in its primary amino acid sequence,it is a highly conserved protein with 99% aminoacid identity between rodents and that of humans.HMGB1 has 2 DNA binding motifs:A boxand B box.Structure-function analyses revealed that the active cytokine domain of HMGB1is localized to the DNA-binding B box,whereas the A box competes with HMGB1 forbinding sites on the surface of activated macrophages and attenuates the biologic function ofthe full length HMGB1,so the recombinant A box(rA-box) acts as a specific antagonist ofHMGB1.Intracellular HMGB1 has been implicated in the regulation of gene transcriptionand in stabilizing nucleosome formation.Extracellular HMGB1,released from injured or necrotic cells and monocytes/ macrophages stimulated with endotoxin or proinflammatory cytokines,has recently beenproposed as a nonclassical proinflammatory cytokine,and has been found to play a pivotalrole in the pathogenesis of proinflammatory diseases such as sepsis,acute lung injury,autoimmune diseases such as systemic lupus erythematosus(SLE),rheumatoid arthritis(RA),and acute allograft rejection.Anti-HMGB1 treatment using HMGB1 inhibitors has shownbeneficial effects in experimental model of sepsis,hepatic ischemia/reperfusion injury,rheumatoid arthritis and cardiac transplantation.In current study,we have explored the change of HMGB1 in the Con A induced liverinjury in mice,Data presented here demonstrated that mRNA level of HMGB1 is increased asearly as 2 h after Con A injection,and reaches the peak value 2 h post Con A treatment.Andthe upregulation of HMGB1 is also confirmed by immunohistochemistry analysis.Furthermore,administration of exogenous recombinant HMGB1 worsens Con-A inducedhepatitis.These results suggest that HMGB1 contributes to the pathogenesis of Con Ainduced liver injury.Based on the above observations,HMGB1 specific antagonists anti-HMGB1 polyclonalantibody and r-A box are produced,and given intraperitoneally 30 min before Con A injectionat the dose of 0.6 mg/mouse.As expected,specific antagonist of HMGB1 treatmentsignificantly reduces the increase of serum level of aminotransferase(ALT),and alsoattenuates the massive necrosis and infiltration of inflammatory cells induced by Con A.Furthermore,anti-HMGB1 antibody pretreatment protected mice from the lethality associatedwith lethal dose of Con A(25μg/g).These results suggest that HMGB1 specific antagonistsanti-HMGB1 polyclonal antibody and r-A box confer protective effect on Con A inducedhepatitis.We next explored the possible mechanisms by which anti-HMGB1 treatment reducesliver injury induced by Con A.Our results shows that anti-HMGB1 treatment markedlyinhibits the increased production of proinflammatory cytokine TNF-a and IFN-γboth in vivoand in vitro and activation of NF-κB induced by Con A,and inhibits the Con A-inducedhepatocytes apoptosis,anti-HMGB1 treatment also dramatically suppressed the activation ofT cells(CD3~+NK1.1~-),NKT cells(CD3~+NK1.1~+) in liver,but fails to decrease ConA-induced subsets of T cells and NKT cells in liver as determined by flow cytometry. Interestingly,our data shows that,compared with TLR4-intact mice TLR4-defective micewere partly protected from Con A induced liver injury,suggests that HMGBl-mediated ConA induced hepatitis involves TLR4 system.In addition,we investigate whether administration of CoPPⅨ,an HO-1 inducer,couldsignificantly inhibit TNF-αand Hmgbl expression and thus attenuate the acute lung injury(ALI) induced by lipopolysaccharide(LPS) in mice.Acute lung injury was inducedsuccessfully by intratracheal administration of LPS(0.5 mg/kg) in male BALB/c mice.CoPPⅨor ZnPPⅨ(an HO-1 inhibitor) was administered to mice 24 h prior to LPS exposure.It was found that CoPPⅨ(5,10 mg/kg,i.p.) caused a significant reduction in the total cellsand neutrophils in BALF,a significant reduction in the W/D ratio and EBA leakage at 24 hafter LPS challenge.Furthermore,the histopathologic findings indicated that alveolitis withleukocyte infiltration in the alveolar space was less severe in the CoPPⅨ-treated mice than inthe mice treated with LPS alone.In addition,CoPPⅨwas also believed to havedown-regulated the expression of LPS-induced proinflammatory cytokines,including earlyproinflammatory cytokine TNF-a,and late proinflammatory cytokine Hmgbl.In contrast,noobvious difference was observed between the ZnPPⅨgroup and the LPS group.Thesefindings demonstrate the significant protection of CoPPⅨagainst LPS-induced ALI,and theeffect mechanism of CoPPⅨwas associated with decreasing the expression of TNF-a andHmgbl.In summary,our results demonstrate that Extracellular HMGB1 plays a pivotal role inCon A-induced hepatic injury.Administration of Neutralizing antibody to HMGB1significantly attenuates Con A-induced liver injury,and this protective effect is associatedwith reduced hepatocytes apoptosis,pro-inflammatory cytokines,and impaired activation of Tand NKT cells.The detrimental effect of HMGB1 is partly dependent on the activation ofTLR4 signaling.And we also find that Heme oxygenase-1 upregulation significantly inhibitsTNF-αand Hmgb1 releasing and attenuates lipopolysaccharide-induced acute lung injury inmice.