Protective Effect And Its Mechanism Of Bicyclol Against Liver Inflammation Induced By CpG-ODN

Chronic viral hepatitis is a worldwide disease,in particular,the incidence of chronic viral hepatitis B(CHB) in China is the highest in the world.Around 25%to 40% of the CHB population die from hepatocirrhosis or liver cancer leaded by hepatitis. Although many drugs have been used in the treatment of CHB and chronic viral hepatitis C patients,among them,interferon -2αand Lamivudine are widely used. However,there is a high rebound rate of HBV-DNA after withdrawal of lamivudine, and the side effects of interferon-2αand Lamivudine are unacceptable.Therefore,it is an urgent need to develop effective and safe new drugs to treat chronic viral hepatitis in China.Virus of hepatitis B does not damage hepatocyte directly.However,T cell-mediated immune responses play an essential role in hepatocellular injury induced by autoimmune hepatitis,viral infection,and hepatotoxines.When effection happened, chemokines and cytokines were produced at the sites of inflammation and play an essential role in the recruitment of particular cell types that infiltrate and participate in the pathologic lesions,then hepatitis injury develops with infiltratin and/or proliferation of T cells and macrophages.Therefore,to modulate and inhibit inflammation in liver is one of the most important therapy strategies of treatment of CHB.Bicyclol,4,4'-dimethoxy-5,6,5',6'-bis(dimethylene-dioxy)-2-hydroxymethyl-2'-m ethoxy carbonyl biphenyl) is a novel synthetic anti-hepatitis drug for the treatment of chronic hepatitis B patients in China.Numerous reports have shown that Bicyclol has protective effect against various forms of acute and chronic experimental liver injury, such as liver injury induced by carbon tetrachloride,acetaminophen,D-galactosamine, concanavalin and alcohol.Bicyclol excerts its anti-inflammatory effect thgough reflecting the function of neutrophils,monocytes or macrophages,It has been reported that the hepatoprotective mechanisms of Bicyclol involve the clearance of reactive oxygen species,regulation of cytokine secretion,and inhitition of apotosis induced by immunological injury,etc.The purpose of the present study was to determine whether bicyclol had protective effect on liver injury induced by CpG-ODN,a rodent model represents early pathological changes in viral hepatitis,and investigate related mechanisms on cytokine expression,TLR9 expression and NF-κB& MAPK signal pathway.The results in vivo and in vitro demonstrated that oral administration of bicyclol significantly ameliorated liver injury by reducing the production of inflammatory cytokines and chemokines,and inhititing TLR9 and NF-κB& MAPK signal transduction caused by CpG-ODN.1.Bicyelol protects mice from CpG-ODN -induced hepatitis and its mechanismThe innate immune cells recognize conserved molecular patterns present in microbes(pathogen-associated molecular patterns through a family of proteins known as toll-like receptors(TLRs) that function as pattern recognition receptors and are therby activated to exert various effector functions,including secretion of pro-inflammatory cytokines and mediators such as TNF-α,IL-6,IL-12,and nitric oxide.Production of inflammatory mediators and pro-inflammatory cytokines by innate immune cells is indispensable for the efficient control of growth and dissemination of invading pathogens.However,excessive and uncontrolled production of inflammatory medicators and pro-inflammatory cytokines caused by bacterial infection is potentially harmful to the host and may lead to sever systematic inflammatory complications.Unmethylated CpG dinucleotides in particular sequence contexts(CpG motifs, GACGTT for murine and GTCGTT for human) in bacterial DNA are capable of activationg innate immune cells and are one of the well studied pathogen-associated molecular patterns.The ability of bacterial DNA to activate innate immunity can be mimicked by synthetic oligodexynucleotides containing the unmethylated CpG motif. This unmethylated CpG motif containing bacterial DNA and synthetic oligodeoxynucleotides(CpG DNA) are endocytosed and then recognized as a conserved molecular patern by a pattern recognition receptor TLR9 in an endosomal compartment of antigen-presenting cells in the vertebrate innate immune system.Upon recognition of CpG DNA,TLR recruits the adaptor molecule,myeloid differentiation factor 88(MyD88),through interation between their C-tterminal toll0IL-1 receptor(TIR) domains.This recruitment of MyD88 to toll/IL-1 receptor domains of TLR9 initiates a signaling pathway that sequentially involves IL-1R-associated kinase(IRAK) family proteins and tumor necrosis factor-a receptor-associated factor(TRAF) 6 and TRAF3.CpG-DNA can be recognized by TLR9 as its ligand,and subsequent produce proinflammatory cytokines,chemokines,and modulators in innate immune cells.Its beneficial effects on our immune system,as well as its therapeutic applications have been extensively studied.However,excenssive CpG DNA will induce a severe acute liver injury in a sequence-specific manner and subsequent shock-mediated death in mice treated with the hepatocyte-specifice transcription inhibitor D-GalN.The present study attempts to address questions of whether bicyclol protect mice from CpG DNA-induced hepatitis and its possible mechanism.We pretreat mice with bicyclol before CpG-ODN,and then measured alanine aminotransferase(ALT) levels in plasma,inflammatory infiltration and hepatocyte necrosis in liver.Potential therapeutic mechanisms were elucidated further by measuring several inflammatory mediators,chemotokines,TLR9 and signal transduction.Mice pretreated with bicyclol exhibited much less incresase ALT levels in plasma,reduced inflammatory infiltration and hepatocyte necrosis in liver compared with control mice pretreated with vehicle solutions.We further investigated the mechanisms of the protective effects of bicyclol.In bicyclol-pretreated mice,we found abrogated tumor necrosisi factor(TNF)-α,interferon(IFN)-γ,and interferon 18 at both serum and liver tissue.At the same time,the concentratin of MCP-1,MIP-1αand Rantes in plasma as well as in liver tissue were down-regulated in these mice.Moreover, Toll-like receptor 9 expression,p-NF-κB-p65 and p-p38 MAPK,pErk1/2 expressions in liver were decrease significantly.Therefore,bicyclol is capable of regulating immune-mediated liver injury in vivo.The protective effect depended on its suppressive effect on the production of important inflammatory medicators and TLR9 and its associated signal transductor.Conclusion:Our study shows that bicyclol can alleviate liver injury induced by CpG-ODN.The suppressant effect was associated with inhibition of several inflammatory medicators,including TNF-α,IFN-γ,IL-18 and chemokines. 2.Inhibit effect and its mechanism of bicyclol on the production of chemokines and cytokines induced by CpG-OND in L02 cell lineBacterial DNA and synthetic oligodeoxynucleotides(ODN) containing unmethylated CpG motifs stimulate the cells of the innate immune system trough a specifice receptor called Toll-like receptor-9(TLR9).Here we report on the potential molecular mechanism of anti-inflammation induced by Bicyclol in L02 cells.The treatment of Bicyclol reduced the TLR9 ligand CpG-ODN induced the protein levels of chemokine(MCP-1,MIP-1α,Rantes ) and1 inflammation cytokines(TNF-α,IL-18),and weaken the release of CKs in a dose-dependent manner,suggesting Bicyclol impaired the onset of TLR9-mediated inflammation.Through ELISA,the activity of p65-NF-κB and p38-MAPK was found to be attenuated after Bicyclol treatment.Furthermore,the expression of phosphated NF-κB-p65 and the p38-MAPK,Erk1/2 were significantly altered by Bicyclol treatment,as demonstrated in western analysis.All the above observations suggest that Bicyclol reduce TLR9 ligand-induced inflammation in L02 cells probably through suppressing the NF-κB and MAPK pathway.Our results show for the first time that bicyclol exerts a protective effect on CpG-ODN/D-GalN induced hepatitis,with the evidence of a remarkable decrease of plasma level of alanine transaminase(ALT) 8-24 hours after injection and simulataneous amend of infiltration of the liver with neutrophils,macrophages and T tells,necrosis of the hepatocytes.The mechanism of its protective effect is possibly through suppression of inflammatory medicators and inhititing hepatocyte inflammation.Thus,bicyclol might be used to treat T-cell-mediated liver injury,such as autoimmune hepatitis, alcoholic hepatitis and chronic viral hepatitis.