双环醇对小鼠急性酒精性肝损伤和肝细胞凋亡的保护作用及机制研究

【作者】 赵菁；

【导师】 李燕； 程桂芳； 陈晓光； 申竹芳；

【作者基本信息】 中国协和医科大学 ， 药理学， 2009， 博士

【摘要】 酒精性肝病（alcoholic liver disease,ALD）为长期过量饮酒所致肝脏损伤,是西方国家肝脏疾病的主要诱因,在我国发病率也有逐年上升的趋势。ALD的临床表现包括以肝脏脂肪变为主要特征的轻度损伤和炎症、纤维化/肝硬化等中/重度损伤。此外,约15%酒精性肝硬化患者可发展为肝细胞癌。ALD的发生、发展是涉及遗传、营养和环境因素的多因素过程,其早期发病机制与氧化应激、内毒素介导的细胞因子释放、线粒体损伤、肝细胞凋亡等密切相关。已知ALD的早期损伤如经及时治疗多可逆转,因此寻找治疗ALD的药物具有重要的临床意义。双环醇为我国自行研制的具有自主知识产权的抗肝炎新药,已用于治疗慢性病毒性肝炎。临床资料显示,双环醇可明显改善慢性乙型、丙型病毒性肝炎的症状和肝功能损伤,兼有一定抑制乙型肝炎病毒复制的作用,具有反跳率低、不良反应小、服用方便的特点。以往药理研究表明,双环醇对四氯化碳、D-半乳糖胺、刀豆蛋白A和扑热息痛等多种化学毒物诱发的实验性肝损伤均有保护作用,其保护机制与清除活性氧自由基、调控细胞因子分泌、抑制免疫损伤诱发的凋亡等相关。一项开放、随机、对照临床研究表明,双环醇对ALD也有较好的治疗效果,提示双环醇可能在治疗ALD方面有一定的应用前景。本实验室既往研究也显示,双环醇对酒精性脂肪肝动物有显著保护作用,但相关机制未作深入探讨。因此本研究选用两种简单、易行的急性酒精性肝损伤模型,模拟ALD早期损伤,观察双环醇的保护作用,并针对ALD早期致病因素探讨双环醇的相关作用机制,为其临床应用提供科学可靠的实验依据。1.双环醇对急性酒精中毒小鼠肝损伤的保护作用及机制研究小鼠单次酒精（6g/kg）灌胃可导致急性肝损伤,表现为血清丙氨酸氨基转移酶（alanine transaminase,ALT）水平升高至对照组的2.3倍、肝脏甘油三酯蓄积达3.6倍以及中央静脉和小叶间静脉周围肝细胞肿胀、水样变性等病理变化。双环醇预处理（200,300mg/kg）剂量依赖性显著抑制血清ALT升高（分别降低60%和71%）和肝脏甘油三酯蓄积（分别降低18%和35%）,并明显改善上述肝组织病理变化。氧化应激是急性酒精性肝损伤的主要诱因之一。酒精灌胃后6h时脂质过氧化产物硫代巴比妥酸反应性底物（thiobarbituric acid-reactive substance,TBARS）水平开始升高,12h时为对照组的2.7倍;而非酶抗氧化物谷胱甘肽（glutathion,GSH）含量从1.5h即开始下降,6h时降至对照组的40%。双环醇（300mg/kg）可显著抑制TBARS水平的升高（32%）,并防止GSH耗竭,使其维持在正常水平。此外,酒精灌胃后1.5h时肝脏抗氧化物酶超氧化物歧化酶（superoxide dismutase,SOD）、过氧化氢酶（catalase,CAT）、谷胱甘肽还原酶（glutathion reductase,GR）和谷胱甘肽过氧化物酶（glutathion peroxidase,GSH-px）活性发生显著变化,与对照组相比分别降低35%、18%、49%、45%。双环醇预处理（200,300mg/kg）可显著抑制SOD和GSH-px活性的降低,使SOD活性分别恢复至对照组的94%和99%,GSH-px活性分别恢复至对照组1.3倍和.5倍。双环醇（300mg/kg）对CAT和GR活性的降低也有一定改善作用,但无统计学意义。肿瘤坏死因子-α（tumor necorsis factor-α,TNF-α）和白细胞介素-1β（interleukin-1β,IL-1β）是介入ALD损伤的重要炎症因子。酒精灌胃后1.5h肝脏TNF-α和IL-1β蛋白水平开始升高,12h达峰,分别为正常对照组的2.4倍和1.7倍。双环醇（200,300mg/kg）可明显抑制肝脏TNF-α和IL-1β水平升高,使TNF-α蛋白水平分别降低33%和47%,IL-1β蛋白水平分别降低26%和46%。酒精灌胃后12h,TNF-α和IL-1βmRNA表达分别增至对照组的2倍、2.4倍,而双环醇可明显抑制这一变化。此外,双环醇（300mg/kg）还可减少Kupffer细胞TNF-α的表达。内毒素是激活Kupffer细胞的主要因素。酒精灌胃后1.5h血浆内毒素含量显著升高（达到对照组的9.6倍）,6h后可恢复至正常水平。双环醇（200,300mg/kg）可显著抑制血浆内毒素含量升高,抑制率分别为79%、60%。CD14是内毒素活化Kupffer细胞的关键受体。酒精灌胃后12h可诱导Kupffer细胞CD14的高表达。双环醇（300mg/kg）可显著抑制CD14的过表达。由此可见,双环醇对急性酒精性肝损伤有显著的保护作用,其肝保护机制可能与减轻氧化应激、抑制细胞因子表达、降低血浆内毒素水平和CD14表达以抑制Kupffer细胞活化相关。2.双环醇对酒精中毒小鼠肝脏细胞凋亡的保护作用及机制研究小鼠连续三次酒精（6g/kg）灌胃后可导致明显肝损伤,变现为血清ALT水平升高（为对照组的2.2倍）,肝脏炎症相关蛋白环氧合酶-2（cyclooxygenase-2,COX-2）表达增加（为对照组的2.2倍）,肝组织出现髓样变性、小空泡变性和炎性细胞浸润。酒精中毒小鼠还出现明显的肝细胞凋亡,表现为脱氧核苷酸末端转移酶介导的缺口末端标记（terminal deoxynucleotidyl transferase（TdT）nick-rnd labeling,TUNEL）染色阳性细胞数显著增加。双环醇给药可明显降低酒精诱导的血清酶学改变,还可抑制COX-2蛋白的过表达,减轻肝脏病理改变,同时具有抑制肝细胞凋亡的保护作用。酒精在体内的代谢过程中可生成毒性代谢产物-乙醛,同时伴随产生大量活性氧自由基（reactive oxygen species,ROS）,是后续氧化/硝化应激的始发因素。模型组参与乙醇代谢的胞浆乙醇脱氢酶（alcohol dehydrogenase,ADH）活性升高至对照组的1.5倍,微粒体细胞色素P450 2E1（cytochrome P450 2E1,CYP2E1）活性和蛋白表达也分别升高至对照组的2.2倍和2.6倍。双环醇（300mg/kg）可使ADH和CYP2E1活性分别降低20%和25%,CYP2E1蛋白的过表达可基本恢复至正常水平。此外,模型组线粒体和胞浆乙醛脱氢酶（acetaldehyde dehydrogenase,ALDH）活性分别升高27%和66%,双环醇（300mg/kg）可显著抑制胞浆ALDH活性的增加。酒精引起的氧化应激可导致模型组动物肝脏氧化蛋白含量升高至对照组的1.7倍,胞浆NAD⁺/NADH则降低30%,而线粒体GSH含量降低10%。双环醇给药（200,300mg/kg）可显著抑制酒精诱导的氧化蛋白含量升高,改善NAD⁺/NADH下降,并使GSH维持在正常水平。除氧化应激外,酒精还可诱发硝化应激。酒精连续三次灌胃后,小鼠肝脏一氧化氮（nitric oxide,NO）含量升高至对照组的1.5倍,诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）活性和蛋白表达分别升高至对照组的2.6和3.2倍,硝基酪氨酸（nitrotyrosine,NT）蛋白表达升高至对照组的2.5倍。双环醇200,300mg/kg给药可显著降低酒精引发的NO含量升高,使之接近正常水平,300mg/kg明显抑制iNOS和NT蛋白的过表达（40%和53%）。ALD中氧化/硝化应激可引发线粒体损伤,启动内源性凋亡信号通路。模型组小鼠肝脏线粒体对罗丹明123（rhodamine 123,R123）的摄取量明显少于对照组,对高钙浓度诱发肿胀的敏感性降低（50%）,提示线粒体发生了膜渗透性转换。双环醇（200,300mg/kg）可显著改善受损的线粒体功能,表现为线粒体对R123的摄取量以及高钙浓度引发的吸光度下降幅度均明显增加。此外,模型组线粒体呼吸链功能受到明显抑制,线粒体呼吸链复合物（mitochondrial respitatory complex,MRC）Ⅰ和MRCⅣ活性分别下降为正常对照组的42%和70%。双环醇300mg/kg给药后MRCⅠ和MRCⅣ活性较模型组均有明显升高,分别恢复至对照组的65%和85%。线粒体损伤最终导致细胞色素C释放入胞浆,表现为模型组肝脏胞浆细胞色素C蛋白含量增加至正常对照组的2倍。双环醇给药组胞浆细胞色素C蛋白含量几乎接近正常水平。Bax和Bcl-X_S/L分别为重要的促凋亡蛋白和抗凋亡蛋白,模型组两种蛋白表达均上调,分别达到对照组的1.6倍和3.1倍。双环醇300mg/kg对Bax和Bcl-X_S/L的过表达均有一定抑制作用。Fas/Fas配体（Fas ligangd,FasL）参与死亡受体介导的外源性凋亡通路。模型组肝脏Fas、膜结合型FasL（membrane-bond FasL,mFasL）、可溶性FasL（solubleFasL,sFasL）蛋白表达分为上调为对照组的1.5倍、4和2倍。双环醇300mg/kg可使Fas表达降低为模型组的45%,mFasL和sFasL的表达恢复正常水平。模型组肝脏细胞核中核因子-κB（nuclear factor-κB,NF-κB）的蛋白表达也有明显变化,即降至对照组的16%,此外胞浆抑制蛋白-κB（inhibitor of NF-kB,ⅠκB）的蛋白表达较对照组增加15%。双环醇300mg/kg可使NF-κB蛋白表达维持在正常水平,同时抑制ⅠκB的过表达（50%）。由此可见,双环醇对酒精所致肝细胞凋亡有明显的保护作用,其机制与影响酒精代谢酶活性和/或表达、抑制氧化/硝化应激,改善线粒体损伤,减轻Fas/FasL过表达,从而同时抑制内源性、外源性凋亡信号通路有关。综上所述,双环醇对酒精引起的小鼠肝脏损伤具有明显的保护作用。其不仅可抑制血清转氨酶升高、肝脏甘油三酯蓄积,还可改善肝细胞肿胀、水样和髓样变性、炎性细胞浸润等病理学损伤。此外,双环醇对肝细胞凋亡也有显著的抑制作用,其保护作用的机制可归纳为:1)酒精代谢酶调控:抑制ADH和ALDH活性,抑制CYP2E1活性并下调蛋白表达。2)抑制氧化应激:减轻脂质过氧化,恢复GSH含量,改善抗氧化物酶SOD、GSH-px、GR、CAT的活性,减少氧化蛋白含量,恢复NAD⁺/NADH3)抑制硝化应激:降低NO水平,抑制iNOS和NT蛋白过表达4)调节炎症细胞因子表达:下调TNF-α、IL-1β蛋白和mRNA表达5)抑制Kupffer细胞活化:降低内毒素水平,抑制CD-14过表达6)减轻线粒体损伤:维持线粒体膜完整性和正常膜电位,恢复线粒体GSH含量,增加MRCⅠ和Ⅳ活性,减少细胞色素C释放7)调节凋亡相关蛋白:下调Fas、mFasL、sFasL、Bax、Bcl-X_S/L、I-κB蛋白表达,上调NF-κB蛋白表达上述研究为进一步了解双环醇的肝保护作用特点以及临床治疗ALD提供了有参考价值的实验依据。更多还原

【Abstract】 ALD is induced by chronic alcohol abuse and represents a major cause of morbidity and mortality in western counties.The incidence of ALD is increasing with the improvement of living conditions in China recently.The clinical manifestation of ALD ranges from minor injury including steatosis to moderate and severe injury characterized by hepatitis and fibrosis/cirrhosis.Moreover,approximately 15%of the patients with established alcoholic cirrhosis develop to hepatocellular carcinoma.The development of ALD is a multifactoral process involving various genetic,nutritional and environmental factors,and its early pathogenesis is closely related with oxidative stress,endotoxin-mediated cytokine release,and apoptosis.Most injuries in early stage of ALD are reversible,so that further aggravation can be prevented by abstinence and symptomatic treatment.Therefore,development of drugs targeting at early injuries of ALD will have great clinical significance.Bicyclol is a novel anti-hepatitis drug for the treatment of chronic viral hepatitis. Clinical data showed that bicyclol can significantly improve the symptoms and damaged liver function in chronic type B and type C viral hepatitis in addition to inhibit the replication of type B hepatitis virus,featured by low rebounding rate,few adverse effects and convenient usage.Previous pharmacological studies have demonstrated that bicyclol offer protective effect on experimental liver injury induced by various chemical toxicants,such as carbon tetrachloride,D-galactosamine, concanavalin A and acetaminophen.The hepatoprotective mechanisms of bicyclol include the clearance of reactive oxygen species,regulation of cytokine secretion,and inhibition of apoptosis induced by immunological injury,etc.An open-labeled,randomized and controlled clinical study has reported that bicyclol had therapeutic effect on ALD,indicating its application perspective in the treatment of ALD.Previous study in our laboratory has also demostrated its role in protecting experimental animals from alcohol-induced steatosis,however,in-depth investigation on related mechanisms are still needed.Therefore,two simple and convenient models of acute alcohol-induced liver injury were established in the present study to observe the protective effect of bicyclol and related mechanisms,so as to provide experimental evidences for its clinical application. PartⅠ:Protective effect of bicyclol on acute alcohol-induced liver injury in mice and related mechanismsSingle intragastic administration of alcohol（6g/kg）induced acute liver injury,as evidenced by elevation of serum ALT（2.3 times of control at 6h）and hepatic triglyceride levels（3.6 times of control at 6h）,swelling and hydropic degeneration of hepatocytes,which reflected early biochemical and pathological changes in ALD. Bicyclol pretreatment（200,300mg/kg）significantly alleviated ALT elevation（60% and 71%）and hepatic triglyceride accumulation（18%and 35%）in a dose dependent manner.Moreover,bicyclol markedly improve the above liver pathological changes.Oxidative stress is the major pathogenetic factor in acute alcohol-induced liver injury.Hepatic TBARS content was elevated from 6h after alcohol administration and reached 2.7 fold of the control at 12 h.On the contrary,hepatic GSH content was decreased as early as 1.5 h after alcohol treatment and only 40%of control was found at 6 h.Pretreatment with bicyclol（300mg/kg）significantly inhibited the increase of TBARS content（32%）and prevented GSH depletion.In addition,a notable change of liver SOD,CAT,GR and GSH-px was observed as indicated by the decreasing of enzyme activity（35%,18%,49%and 45%）at 1.5 h after alcohol administration. Pretreatment with bicyclol（200,300mg/kg）significantly inhibited the decrease of SOD（returned to 94%and 99%of control,）and GSH-px（returned to 1.3 and 1.5 fold of control）dose-dependently.In addition,bicyclol（300mg/kg）also showed slight protective effect against alcohol-induced decrease of CAT and GR activity,although there was no statistical significance.TNF-αand IL-1βare two key inflammatory cytokines in ALD.Hepatic TNF-αand IL-1βlevels were elevated at 1.5 h and reached peak level at 12 h after alcohol administration.Bicyclol（200,300mg/kg）significantly alleviated hepatic TNF-αproduction by 33%and 47%,and IL-1βby 26%and 46%,respectively.The mRNA expression of TNF-αand IL-1βwas increased to 2 and 2.4 times of control by alcohol administration and bicyclol significantly inhibited such changes of two cytokines. Furthermore,bicyclol（300mg/kg）pretreatment can decrease the positive TNF-αstaining on Kupffer cells. Endotoxin is the main factor responsible for the activation of Kupffer cell.It was found that plasma endotoxin level was markedly increased at 1.5 h（9.6 fold of control） and declined to the normal level at 6 h after alcohol treatment in the present study. Bicyclol（200,300 mg/kg）significantly inhibited the elevation of plasma endotoxin level by 79%and 60%,respectively.CD-14 is the critical receptor in activation of Kupffer cell by endotoxin.Expression of CD-14 can be greatly induced at12h after alcohol intoxication and bicyclol（300mg/kg）reduced the over-expression of CD-14 significantly.In conclusion,bicyclol showed significant protective effect on acute alcohol-induced liver injury.The hepatoprotective action of bicyclol is mostly mediated by its ability to attenuate oxidative stress,suppress cytokine expression at both protein and gene level,and inhibit the activation of Kupffer cell by decreasing plasma endotoxin level and CD 14 expression.PartⅡ:Effect of bicyclol on hepatocyte apoptosis in alcohol-intoxicated mice and related mechanismsThree consecutive intragastric administration of alcohol（6g/kg）in 12h interval resulted in an obvious liver injury and inflammation in mice,as indicated by the elevation of serum alanine transaminase（2.2 fold of control）,increased hepatic COX-2 protein expression（2.2 fold of control）,as well as liver pathological changes characterized by myelonic degeneration,vacuolar degeneration and inflammatory infiltration.Alcohol-intoxicated mice also showed obvious hepatocyte apoptosis,as positive cells in TUNEL staining were significantly increased.Bicyclol pretreatment can markedly decrease elevated serum transaminase,inhibit over-expression of COX-2 protein,and alleviate pathological changes.Furthermore,bicyclol diminished the number of TUNEL positive cells and staining intensity,suggesting its role in protection against alcohol-induced hepatocyte apoptosis.The metabolism of alcohol produces toxic metabolite-acetaldehyde and generates large amount of ROS at the same time,which is the priming factor for successive oxidative and nitrosative stress.Compared with control group,liver cytosolic ADH activity was increased by 46%after alcohol administration,while liver microsomal CYP2E1 activity and protein expression was increased to 2.2 fold and 2.6 fold of control,respectively.Pretreatment with bicyclol（300mg/kg）can decrease ADH and CYP2E1 activity by 20%and 25%respectively,and return CYP2E1 protein expression to approximate normal level.Moreover,liver mitochondrial and cytosolic ALDH activity in model group was elevated by 27%and 66%,respectively.Bicyclol （300mg/kg）can significantly regulate the increased cytosolic ALDH activity.Oxidative stress is an important pathogenetic factor in early stage of ALD.Liver oxidized protein content was increased to 1.7 fold,while cytosolic NAD⁺/NADH and mitochondrial GSH content was decreased by 30%and 10%by alcohol,when comparing with control group.Bicyclol pretreatment（200,300mg/kg）notably inhibited the elevation of protein carbonyl content,antagonized the change in NAD⁺/NADH,and maintained GSH at normal level.In addition to oxidative stress,alcohol can also induce nitrosative stress.After three consecutive alcohol administration,hepatic NO content,iNOS activity and protein expression,and NT protein expression was increased to 1.5,2.6,3.2 and 2.5 fold of control,respectively.Bicyclol pretreatment（200,300mg/kg）can effectively reversed the elevated NO content to normal level,and inhibited the over-expression of iNOS and NT by 40%and 53%respectively.Both oxidative and nitrosative stress in ALD can induce liver mitochondrial injury,leading to the activation of endogenous apoptosis signaling pathway.The uptake of R123 by liver mitochondria in model group was less than that in control group,and the sensitivity to high level of calcium-induced swelling was also decreased（about 50%of control group）,suggesting the occurrence of membrane permeability transition.Bicyclol（300mg/kg）markedly alleviated injured mitochondrial function,as manifested by recovery of R123 uptake and increased changing amplitude in response to high calcium concentration.In addition,the function of mitochondrial respiratory chain was affected.MRCⅠandⅣactivity was decreased to 58%and 30%of control by alcohol administration,which was recovered to 65%and 85%of control by bicyclol（300mg/kg）.Mitochondrial injury will ultimately lead to the release of cytochrome C into cytoplasm,as reflected by the two-fold increase of cytochrome C protein content in alcohol group.Bicyclol showed inhibitive effect on the release of cytochrome C.The expression of Bax and Bcl-X_S/L, as the pro-apoptotic and anti-apoptotic protein,were both up-regulated in alcohol-intoxicated mice（1.6 and 3.1 fold of control）.Bicyclol（300mg/kg） pretreatment suppressed the over-expression of both Bax and Bcl-X_s/L.Fas and FasL are believed to be involved in death-receptor mediated exogenous apoptosis pathway.Protein expression of Fas,mFasL,and sFasL were all elevated in alcohol-intoxicated mice（1.5,4 and 2 fold of control）.Bicyclol（300mg/kg）decreased the expression of Fas by 55%and recovered the expression of mFasL and sFasL to normal level.Furthermore,protein level of NF-κB in nucleus was decreased to 16% of control,while cytosolic ptotein level of I-κB was increased by 15%compared with control.Bicyclol（300mg/kg）can maintain the protein expression of NF-κB at normal level,and inhibit the over-expression of I-κB simultaneously（50%）.In summary,bicyclol had significant protective effect on alcohol-induced hepatocyte apoptosis.Its mechanisms were related to its influence on alcohol metabolizing enzymes,inhibition on oxidative/nitrosative stress,improvement of mitochondrial injury,and alleviation of Fas/FasL over-expression,thereby suppressing both endogenous and exogenous apoptosis signaling pathways.In conclusion,bicyclol had a notable protective effect on alcohol-induced liver injury and hepatocyte apoptosis in mice.It can not only significantly inhibit elevation of serum transaminase and accumulation of hepatic triglyceride,but also improve histopathological changes,including hepatocyte swelling,hydropic degeneration, myelonic degeneration,vacuolar degeneration and inflammatory infiltration.In addition,bicyclol markedly decreased the number of TUNEL positive cells and attenuated staining intensity.The possible mechanisms included:1)Regulation on alcohol metabolizing enzyme:inhibit ADH and ALDH activity, inhibit CYP 2E1 activity as well as its protein expression2)Inhibition of oxidative stress:attenuate lipid peroxidation,restore GSH content,improve the activity of antioxidant enzymes,including SOD,GSH-px,GR, and CAT,decrease the content of oxidized protein,and restore NAD⁺/NADH3)Inhibition of nitrosative stress:reduce NO content,and inhibit over-expression of iNOS and nitrotyrosine4)Regulation on inflammatory cytokines:down-regulate both protein and mRNA expression of TNF-αand IL-1β5)Suppression of Kupffer cell activation:decrease endotoxin level,and inhibit CD-14 over-expression6)Attenuation of mitochondrial injury:maintain mitochondrial membrane integrity and normal membrane potential,restore mitochondrial GSH content, increase MRCⅠand MRCⅣactivity,and reduce cytochrome C release7)Regulation on expression of apoptosis-related proteins:down-regulate Fas, mFasL,sFasL,Bax,Bcl-X_S/L,IκB,while up-regulate NF-κBWith the results mentioned above,the present study will provide valuable experimental evidences for further investigation on the hepatoprotective effect of bicyclol and the possibility of clinical application in the treatment of ALD.更多还原