【作者】 娄小娥；

【导师】 陈智；

【作者基本信息】 浙江大学 ， 内科学， 2009， 博士

【摘要】 病毒性肝炎（virus hepatitis）传染性强,发病率高,流行范围广,是全球最主要的传染病之一。病毒性肝炎肝损伤主要由免疫介导,在肝脏局部,肝细胞受HBV感染后激活免疫系统,产生炎症与化学趋化因子,将炎症、淋巴细胞活化并募集至肝脏,这些炎症和淋巴细胞的浸润如果导致过强的免疫反应,则最终引起肝组织的损伤。因此,调节机体免疫反应和抑制肝脏炎症是乙型肝炎治疗的重要策略之一。双环醇（6-甲氧羰基-6’-羟甲基-2,3,2’,3’-二亚甲基二氧基-4,4’-二甲氧基联苯）由中国医学科学院药物研究所刘耕陶院士和张纯贞教授合作的科研小组历经15年艰辛研制成功,该药降低转氨酶作用显著,对肝细胞具有较好的保护作用,并具有抗肝纤维化的作用,长期使用无明显不良反应。双环醇的问世为病毒性肝炎的治疗提供了一种新的安全、有效的辅助治疗药物,其药理基础和作用机制均值得继续深入研究。迄今为止,已有大量动物实验证明,双环醇对四氯化碳（CCl₄）、D-氨基半乳糖、对乙酰氨基酚、脂多糖（LPS）和刀豆蛋白A（ConA）等化学物质诱导的肝损伤均有保护作用,作用机制涉及抑制氧化和免疫损伤、细胞凋亡和细胞膜稳定等。但尚缺乏双环醇对病毒感染造成的肝脏炎症的保护作用及其机制的研究。近年来关于病原体相关分子模式（Pathogen-associated molecular patterns,PAMPs）的研究进展表明,激活高等动物天然免疫应答的PAMPs广泛存在于病原体细胞表面,识别PAMPs是以模式识别受体（Pattern recognition receptors,PPRs）,Toll样受体（Toll-like receptors,TLRs）介导的。细菌和病毒的DNA作为一种PAMPs能被机体识别并激发免疫应答,是因为其基因组中的一段以非甲基化CpG二核苷酸为核心的高保守序列,即CpG基序（CpG motif）。机体免疫系统通过TLR9（Toll-like receptor 9）特异识别细菌和病毒DNA中的CpG基序并形成受体复合物后,通过髓性分化因子88（myeloid differentiation factor 88,MyD 88）依赖途径,激活NF-κB（nuclear factor-κB）通路和应激酶通路等信号转导通路,促进机体产生Th1型细胞应答和分泌炎症因子,当炎症因子过度释放时,则造成宿主的炎症或损伤。研究证实人工合成的含一个或数个非甲基化CpG基序的寡聚脱氧核糖核酸（CpGoligodeoxynucleotides,CpG-ODNs）,也具有和细菌及病毒DNA中CpG基序一样的免疫刺激效应。因此,CpG-ODNs可用以模拟细菌和病毒等微生物病原侵入机体所引发的免疫反应状态,构建相应的疾病模型。目前已有研究小组构建了CpG-DNA/LPS以及CpG-DNA/D-GalN诱导的小鼠肝损伤模型,为我们进一步研究病毒感染后的肝脏炎症免疫病理提供了可能的模型。本研究中,我们用CpG-ODN/D-GalN造成小鼠肝损伤,模拟小鼠感染DNA病毒后所引起的肝脏炎症反应,来研究双环醇的保护作用和机制。根据CpG-ODN的免疫活性具有种属特异性的特征,第一部分研究中,我们以对小鼠具有最佳免疫效果的CpG1826构建小鼠肝损伤模型;第二部分研究中,则以对人类最具免疫效应的CpG2006模拟病毒刺激人正常细胞株L02细胞,从整体动物和离体细胞两个水平研究双环醇对CpG-ODN造成的免疫性炎症的保护作用,及其对TLR9介导的信号通路分子的活化和细胞因子分泌的作用。1.双环醇对CpG-ODN/D-GalN诱导的小鼠肝损伤的保护作用及其机制目的:用CpG-ODN诱导D-GalN致敏的小鼠肝脏炎症,研究双环醇的保护作用及可能的机制。方法:BALB/C小鼠随机分组,每组8只。第1至5天每天一次给小鼠灌胃300mg/kg和100mg/kg双环醇或溶剂;第5天给药后2小时由尾静脉注射CpG-ODN（1826）20μg/D-GalN20mg/只,模型组仅在第5天尾静脉注射CpG-ODN（1826）20μg/D-GalN20mg/只。12小时后处死小鼠并留取标本。光镜观察肝脏损伤的程度;生物化学法测定血清中ALT;ELISA法测定血清及肝脏组织匀浆中细胞因子TNF-α,IL-18和IFN-γ及趋化因子MCP-1,MIP-lα和RANTES的水平;免疫组化法检测肝脏中TLR9表达;Westernblot方法测定NF-κB及MAPKs信号途径相关分子,包括p65,p38和Erk1/2的变化。结果:模型组小鼠血清ALT为557.33±69.51（U/L）,比正常组小鼠ALT高6倍;双环醇100mg/kg和300mg/kg组的ALT分别为227.67±55.59和138.45±22.52（U/L）,与模型组相比分别降低59.1%和75.2%;肝脏组织病理切片结果显示,双环醇能减少肝脏炎症细胞浸润及肝细胞的溶解性坏死,减轻肝脏的损伤程度;双环醇100mg/kg和300mg/kg组血清中的细胞因子TNF-α、IL-18和IFN-γ的水平和趋化因子MCP-1、MIP-1α和Rantes的水平均显著低于模型组,且具有剂量相关性。其中血清中TNF-α降低35.6%和72.4%;IL-18降低21.80%和47.4%;IFN-γ降低30.90%和63.3%,MCP-1降低30.5%和72.6%,MIP-1α降低28.7%和38.2%,Rantes降低25.4%和64.5%,都具有显著差异;同时,双环醇对小鼠肝脏组织中的细胞因子和趋化因子也有类似的作用;免疫组化结果显示,双环醇处理组小鼠肝脏内TLR9表达明显少于模型组;Western blot结果则表明,双环醇组p-NF-κB-p65、p-MAPK-p38和p-MAPK-Erkl/2的表达明显低于模型组。结论:双环醇能够抑制CpG-ODN/D-GalN诱导的小鼠肝脏炎症,其主要机制可能是通过参与小鼠的免疫调节作用,抑制炎症因子TNF-α,IL-18,IFN-γ的过量表达,同时抑制TLR9介导的信号通路中的有关分子的活化,通过参与小鼠免疫调节,从多方面对CpG-ODN诱导的小鼠肝损伤起到保护作用。2.双环醇对CpG-DNA诱导的L02细胞TLR9表达及炎症、趋化因子分泌的抑制作用及其分子机制研究表明,炎症发生时,肝实质细胞也参与了机体的免疫反应,能对炎症信号作出反应,分泌一些炎症细胞因子和趋化因子,并通过作用于临近的肝细胞、枯否氏细胞、内皮细胞和浸润到肝脏的白细胞等发起、扩大和改变炎症反应。CpG-ODN可直接作用于肝实质细胞,上调肝细胞上的粘附分子和共刺激分子,从而使肝细胞具有了类似于抗原递呈细胞的功能,从而打破了机体的免疫耐受,对肝脏造成炎症性损伤。因此,本部分研究中,我们以正常人肝细胞株L02作为研究对象,研究双环醇对CpG-ODN刺激后的L02细胞分泌细胞因子、趋化因子及TLR9表达等的影响。目的:以CpG2006模拟双链DNA病毒刺激L02细胞并观察双环醇对L02细胞分泌炎症、趋化因子,以及对细胞内TLR9表达的影响,研究双环醇对与炎症作用相关的分子的调节作用和机制。方法:用不同浓度双环醇（0.01,0.1,0.5mM）与L02细胞温孵2小时后,加入浓度为10μg/ml CpG-ODN2006,继续培养18小时,模型组仅加入10μg/mlCpG-ODN2006。用ELISA法测定培养上清中TNF-α和IL-18及趋化因子MCP-1,RANTES和MIP-1α等水平;Western blot法和流式细胞术测定L02细胞内TLR9的表达;Western blot法测定细胞内p-NF-κB p65、p -p38-MAPK,pErk1/2-MAPK的变化。结果:双环醇能够剂量依赖性地抑制CpG-ODN诱导的L02细胞趋化因子的释放和TNF-α等细胞因子的分泌,其中双环醇0.5mM组和模型组相比,TNF-α、IL-18、MCP-1、MIP-1α和Rantes分别降低77.9%,88.7%,74.6%,63.3%和64.0%。Werstern blot和流式细胞术结果一致表明,双环醇0.5mM和0.1mM能够下调L02细胞内经ODN诱导表达的TLR9,同时,还能抑制相关NF-κB通路蛋白p65和MAPK通路相关蛋白p38及ERK1/2的磷酸化。结论:双环醇能明显抑制CpG-ODN诱导的人肝细胞株L02炎症因子和趋化因子的释放与分泌,并且抑制L02细胞内TLR9的表达及相关NF-κB和MAPK信号传导途径分子的磷酸化活性。综上,本课题首次研究了双环醇对CpG-ODN诱导的小鼠肝损伤的保护作用,并从整体动物水平和离体细胞水平研究了双环醇对肝细胞的保护作用机制。结果表明,双环醇能显著降低CpG-ODN诱导上升的小鼠血清转氨酶并改善肝脏病理情况。其机制可能与双环醇既参与机体的免疫调节,又对肝细胞有直接的保护作用有关。双环醇在体内能抑制小鼠血清和肝组织中炎症因子（TNF-α,IL-18,IFN-γ）和趋化因子MCP-1,MIP-1α,和RANTES的水平;同时也能直接抑制体外培养的L02细胞分泌这些因子。同时,双环醇还能下调L02细胞内TLR9的表达及其信号途径所涉及的信号转导分子如NF-κB-p65,MAPK-p38和ERK1/2的活化。因此,双环醇可以从多途径减轻CpG-ODN对肝脏造成的损伤,从而达到治疗肝炎的效果。这些研究结果也提示,双环醇抗肝细胞炎症的作用有免疫学机制的介入,为解释双环醇在临床上治疗肝炎的分子机制提供了更多的的理论依据。更多还原

【Abstract】 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.更多还原