【作者】 沈海容；

【导师】 吕志平；

【作者基本信息】 南方医科大学 ， 中西医结合临床， 2014， 博士

【摘要】 研究背景：乙肝是由乙型肝炎病毒(HBV)引起的,以肝脏损害为主的一种严重危害人类健康的传染性疾病,具有传染性强,流行面广和发病率高等特点。HBV感染呈世界性分布,流行区域包括亚洲、南太平洋、澳大利亚、新西兰、南美、中东、亚撒哈拉非洲和北极圈的土著。全世界大约有20亿乙型肝炎病毒感染者,每年由于急慢性HBV感染而死亡的人数高达100万,至2004年全球HBV慢性感染者已达4亿,我国有1.3亿HBV携带者,占世界乙肝感染者的30%。研究发现,“无症状乙肝病毒携带者”中60%-70%肝活检病理报告为慢性迁延性肝炎或慢性活动性肝炎,长期的乙肝病毒感染可发展为肝硬化,甚至肝癌。因此抗乙肝病毒治疗,阻止疾病的进一步发展是关键,而研究开发抗病毒效果好、副作用低的新药是势在必行。目前抗乙肝病毒药物的筛选主要通过体内动物和体外细胞模型实验进行。体内动物模型用得最多的是鸭乙型肝炎病毒(DHBV)模型。因家鸭易于喂养,因此该动物模型已广泛应用。该动物模型分为先天感染模型和后天感染模型两种,国外多采用前者,而国内则多采用后者。DHBV模型主要用于病毒复制试验、免疫学试验以及抗病毒药物的试验等三方面。在抗乙肝病毒药效学的研究中,DHBV模型一直是较为公认的动物模型。体外实验模型主要是HepG2.2.15细胞。HepG2.2.15细胞株系由美国The Mount Sinai Medical Center于1986年建立,并且已成功地应用于抗HBV药物的筛选,作为抗HBV作用的指标,被中国卫生部收载于治疗肝炎的中药新药药效学研究指南。此模型对中药成分抗病毒研究比较合适,已成功的对叶下株、水芹、大黄等数十种成分进行了实验研究。肝损伤是肝病的病理基础。肝损伤的发生机制颇为复杂,可分为化学性和免疫性。化学机制主要通过细胞色素P450及结合反应产生的中间代谢产物产生损伤,如改变质膜的完整性、线粒体功能失调细胞内离子浓度变化、降解酶的活性和自由基的作用等；免疫机制则通过细胞因子、一氧化氮、补体及免疫变态反应等产生损伤。肝炎病毒是最为常见的致肝损伤原因。目前,多数学者认为乙型肝炎病毒感染引起的肝损伤主要与机体免疫应答有关。同时自身免疫性刺激、病毒或寄生虫感染也是肝损伤特别是肝炎的主要的原因。四氯化碳、D-氨基半乳糖或硫代乙酰胺诱导的急性化学肝损伤,以及慢性模型所造成肝损伤已广泛应用于筛选保肝药。然而,这些损伤显然是外部化学因素而不是由人类的宿主防御而诱导的肝炎,因此不适用于评价肝免疫调节剂。研究肝损伤的发病机制和保肝药物的作用原理需要建立合适的实验性肝损伤动物模型。因此,在抗肝炎药物研究中可采取损伤机制不同的多个模型上观察药物的保护作用,以综合判断药物疗效和作用机制。Con-A诱导肝损伤模型是近年来发展起来的由T淋巴细胞介导肝损伤模型。刀豆蛋白a(Concanavalin-A, Con-A)是一种在体内对肝细胞具有特异性毒性作用的植物凝集素,它进入循环后,引起CD4+T淋巴细胞为主的炎症细胞浸润肝细胞实质,继而激活TNF-a和白介素等细胞因子,引发炎症反应,通过肝细胞凋亡等多种途径损害肝细胞,造成免疫性肝损伤。该模型只需一次性尾静脉注射Con-A,最主要特点是发病迅速,肝脏损害明显。其病理生理过程,与人类慢性乙型肝炎中T淋巴细胞介导的肝细胞损伤极为相似,具有肝脏依赖性、剂量依赖性等特点。这一实验模型很好地模拟了人类病毒性肝炎、自身免疫性肝病等疾病,是筛选急性肝损伤和暴发性肝衰竭治疗药物比较理想的动物模型。我国是乙肝发生率较高的国家之一,寻找和研究既具有抗病毒又具有保肝作用的有效天然药物,一直是中医药科研工作的重要方向。西医用于肝炎的治疗多见于抗病毒药、免疫调节药及护肝降酶类等药物。临床治疗结果表明,这些药物多具有良好的抑制病毒复制,恢复正常肝功能,保护肝细胞等作用,并且疗效迅速,不足之处为远期疗效不稳定,易反复,HBsAg转阴率低,有些药品价格昂贵和表现一定的副作用。另一方面,现代医学在干预肝损伤的治疗方面并无特异性药物,多采用休息加强营养补充维生素和对症治疗等,严重者甚至被迫终止用药。中医中药运用整体观念,辨证论治的特色,发挥中药的多途径、多层次、多靶点,价格低廉、副作用少的优势,在抗乙肝病毒和抗肝损伤治疗中取得了不错的疗效,越来越为广大医务工作者所重视。红背叶根是岭南地区常用的中草药之一,为双子叶植物药大戟科植物红背山麻杆Alchornea trewioides(Benth.)Muell.-Arg.的根。功效清热解毒,祛风除湿,散瘀止血,平喘,杀虫止痒。在民间保肝应用历史悠久,疗效确切。本课题组的前期研究结果首次表明红背叶根具有保肝、降酶、抗肝纤维化的作用,并申请了专利保护。为进一步探讨筛选红背叶根抗病毒及保肝作用的有效部位,我们提取了4种红背叶根提取物,然后对它们进行实验研究,为其抗病毒、保肝作用提供确切的依据。研究目的：通过使用HepG2.2.15细胞模型,先天感染DHBV的鸭乙肝模型和Con-A致小鼠肝损伤模型,对红背叶根水提物(WE),石油醚提取物(PE),乙酸乙酯提取物(EE),正丁醇提取物(BE)等4种提取物进行抗乙肝病毒(HBV)和抗急性免疫性肝损伤及其免疫调节机制的实验研究,探讨其抗病毒和抗免疫性肝损伤及免疫调节的作用机制,为红背叶根的进一步开发利用奠定实验基础。研究方法：第一部分红背叶根4种提取物抗乙肝病毒的实验研究实验一：红背叶根不同提取物体外抑制HBsAg和IBeAg的实验研究方法：通过四甲基偶氮唑盐(MTT)法观察红背叶根不同提取物对HepG2.2.15细胞的影响,采用ELISA法检测分析红背叶根不同提取物对HepG2.2.15细胞分泌HBsAg和HBeAg的抑制作用。实验二：红背叶根提取物体外对乙肝病毒复制的影响方法：采用荧光定量PCR-探针法检测分析红背叶根4种提取物对HepG2.2.15细胞分泌HBV-DNA的抑制作用。实验三：红背叶根提取物体内抗鸭乙肝病毒的实验研究方法：常规PCR法筛选先天性感染的DHBV雏鸭。将筛选出的先天感染鸭随机分组。给药组每天一次性灌胃,共灌胃14d。对照组未做任何处理。每组动物分别于给药前1d、给药后7d、给药后14d、停药后5d自胫静脉采血,离心后收集血清。运用斑点杂交法对鸭血清进行DHBV-DNA检测。第二部分红背叶根4种提取物对Con-A致小鼠急性肝损伤的保护作用研究实验四：红背叶根提取物对Con-A致小鼠急性肝损伤的转氨酶和肝组织形态学的影响方法：随机分组后,模型组和正常对照组每天灌胃同体积生理盐水；各给药组连续给药3d,末次给药1h后,模型组及各给药组尾静脉注射Con-A溶液,诱导小鼠急性免疫性肝损伤,正常对照组注射等量的无菌生理盐水,禁食,不禁水,8h后摘眼球采血,处死小鼠后取肝脏同一部位,10%福尔马林固定。所采集的血液经离心后收集血清,待测血清谷丙转氨酶、谷草转氨酶；固定好的肝组织进行病理学检查,经HE染色后,观察小鼠肝脏组织结构变化情况。实验五：红背叶根提取物对Con-A致小鼠急性肝损伤的免疫调节作用研究方法：采用WST-1法检测肝组织SOD活力；TBA法检测肝组织MDA含量,比色法测定肝组织GSH-PX活力；ELISA法检测血清炎性细胞因子IL-6、TNF-α、IFN-γ;流式细胞术检测脾巨噬细胞上TLR4、Tim-4表达百分比。统计处理以上各实验结果用均数±标准差(mean±SD)表示,多个样本比较用one-way ANOVA进行分析处理；重复测量数据采用重复测量方差分析,多组等级／频数表资料用非参数检验中的K Independent Samples Tests进行数据处理。两个变量之间的相关关系采用双变量相关分析(Bivariate)。采用统计软件SPSS13.0进行分析,以P<0.05定为差异有统计学意义。结果1、BE在无毒浓度下对HepG2.2.15分泌的HBsAg、HBeAg的抑制率分别达65.2%、94.4%,治疗指数分别为>9.8和>67.1；EE对HBeAg抑制率达53.4%,治疗指数>2,而对HBsAg的无抑制作用；其他两种提取物种对HBsAg、HBeAg则无抑制效果。2、结果显示,F=9.479,P=-0.000。与细胞对照组相比,WE、PE、EE以及BE对HepG2.2.15分泌的HBV-DNA的病毒载量均有明显的抑制作用,差异有统计学意义(P<0.05)。3、结果显示,总体上,不同时间点DHBV-DNA变化无显著性差异(F=2.296,P=0.110)；各组间DHBV-DNA却存在显著差异(F=12.416,P=0.000)；时间与分组间存在交互效应(F=6.393,P=0.000)。红背叶根4种提取物只有PE高剂量组在治疗14d后血清DHBV-DNA有下降趋势,差异有统计学意义(P<0.01),停药5天后仍保持下降趋势,差异有统计学意义(P<0.05),其他组无抑制血清DHBV-DNA的作用。4、结果显示,ALT方面,F=34.616,P=0.000；AST方面,F=37.658,P=0.000。肝细胞变性方面,X2=57.935,P=0.000；肝细胞坏死方面,X2=41.751,P=0.000；表明各组疗效存在显著性差异。红背叶根4种提取物都可显著降低血清中谷丙转氨酯、谷草转氨酶,差异有统计学意义(P<0.01)。在肝脏组织形态学方面,与正常组相比较,模型组肝脏变性及坏死等病理变化特别显著(P<0.05/14),动物模型建立成功。在肝细胞变性方面,各给药组改善肝脏变性的效果无显著差异。在肝细胞坏死方面,PE的中剂量组,EE的高剂量组、BE的低、中剂量组以及甘利欣组减轻肝细胞坏死的效果差异显著(P<0.05/14)；镜下观察结果见表4-2和图4-(1-15)(HE染色×200)。5、结果显示,SOD的F=34.961,P＝0.000:MDA的F=23.613,P=0.000；GSH-PX的F=37.089,P=0.000。IL-6的F=74.279,P,=0.000:TNF-α的F=28.023,P=0.000；IFN-γ的F＝374.800,P＝0.000.TLR4的F=4.552,P=0.015:Tim-4的F=6.763,P=0.003。红背叶根4种提取物不仅都可以显著降低肝脏MDA含量(P<0.01),而且还可以显著提高肝脏SOD活力,差异均有统计学意义(P<0.01)；至于GSH-PX,除了EE之外,其他三种提取物都可以显著提高肝脏GSH-PX活力,差异均有统计学意义(P<0.05)。同时,红背叶根4种提取物既可显著抑制炎性细胞因子IL-6、TNF-α、IFN-γ,差异有统计学意义(P<0.05)；又可提高脾巨噬细胞上的TLR4和Tim-4的表达百分率,差异有统计学意义(P<0.05),提示4种提取物可上调脾巨噬细胞上的TLR4和Tim-4的水平。TLR4和Tim-4呈正相关关系(r=0.538,P=0.021)(P<0.05)。结论在乙肝病毒方面,体外研究结果显示只有EE和BE可以抑制HBV抗原,而BE的效果较EE好；4种红背叶根提取物都可以抑制HBV-DNA；体内研究结果显示只有PE高剂量组在治疗14d后血清DHBV-DNA有下降趋势,停药5天后仍保持下降趋势,其他组无抑制血清DHBV-DNA的作用。由此可见HBV抗原和HBV-DNA是红背叶根抗HBV的两个作用靶点。研究结果表明,红背叶根4种提取物中正丁醇提取物既可以抑制HBV两个抗原,又可抑带HBVDNA,因此红背叶根正丁醇提取物抗乙肝病毒效果最好的。在急性肝损伤方面,红背叶根提取物可以降低转氨酶,改善肝脏组织变性坏死形态,还可以降低肝脏MDA含量,提高肝脏SOD和GSH-PX活力。同时,红背叶根4种提取物不仅都可显著抑制炎性细胞因子IL-6、TNF-α、IFN-γ,而且还上调了脾巨噬细胞上TLR4和Tim-4的表达水平,TLR4和Tim-4呈显著正相关关系,由此可见降低转氨酶,改善肝脏组织形态,抑制脂质过氧化反应,抑制炎性细胞因子,上调TLR4和Tim-4的表达是红背叶根4种提取物保肝的几种可能机制；另外,TLR4和Tim-4在免疫调节方面发挥着重要的作用,我们发现两者存在正相关关系,可推断脾巨噬细胞上的TLR4和Tim-4表达的上调可能与抗乙肝病毒和抗肝损伤有关。可见,红背叶根对急性肝损伤具有较好的免疫调节作用。4种红背叶根提取物中,以BE抗乙肝病毒和抗肝损伤的作用最好,我们推测BE通过免疫调控作用,上调了TLR4和Tim-4水平,一方面,BE可能做为TLR4的激动剂,进而抑制HBsAg、HBeAg和HBVDNA,从而起到抗乙肝病毒的作用,另一方面,Tim-4水平的上调,抑制了脂质过氧化反应和炎性细胞因子,进而降低转氨酶,改善肝脏组织变性坏死形态,从而起到抗肝损伤的作用。更多还原

【Abstract】 BackgroundHepatitis B is caused by the hepatitis B virus (HBV) and features in liver damage.It is such a kind of infectious diseases that mainly a serious hazard to human health,with characteristics of strong infection, a wide range of popularity and high incidence. HBV infection distributes worldwide, popular region including indigenous Asia, South Pacific, Australia, New Zealand, South America, the Middle East, sub Saharan Africa and Aboriginal of Arctic Circle.There are about2billion people suffer from hepatitis B virus infection in the world, the number of annual death due to acute and chronic HBV infection was as high as1million, to2004Global HBV chronic infection has reached400,000,000. China has120,000,000HBV carriers of hepatitis B infection, accounting for30%of the world. Liver injury is the pathological basis of all the liver diseases. The study found,60%-70%liver biopsy pathology reports of "asymptomatic hepatitis B virus carriers" present chronic persistent hepatitis or chronic active hepatitis;and long-term chronic hepatitis B virus infection may develop into liver cirrhosis, and even hepatocellular carcinoma. Therefore, anti-HBV treatment is the key to prevent the further development of the disease;and the research and development of new antiviral drugs of good effect and low side effect is to be imperative.The screening of anti HBV drugs needs to perform first in animal and cell model experiment. The most used animal models in vivo is duck hepatitis B virus (DHBV) model. For the reason of easy feeding, the DHBV model has been widely used. These models were divided into congenital infection model and acquired infection model, foreign countries use the former, while the latter is used in our country. The DHBV model is mainly used for three aspects:viral replication test, immunological tests and antiviral drug test. As for the research on anti-HBV pharmacodynamics, DHBV model has been a widely accepted animal model.The cell models are mainly HepG2.2.15cells. HepG2.2.15cell line, which was established by the The Mount Sinai Medical Center of America in1986, has been successfully applied to the screening of anti-HBV drugs. China Health Department has contained it as the anti-HBV effect index in the treatment of hepatitis in the pharmacodynamics guide of new traditional Chinese medicine. This model is suitable for studies of antiviral medicines.And it has been successful used in the experimental studies on dozens of ingredients of common leafflower herb, cress, rhubarb.Liver injury is the pathological basis of liver disease. The mechanism of liver injury is rather complicated, which can be divided into chemical and immunity.The chemical mechanism of injury is mainly through intermediate metabolites produced by cytochrome P450and conjugation reaction,such as the change of plasma membrane integrity, mitochondrial dysfunction in the intracellular concentration changes, the activity of enzymes and free radicals; while the immune mechanism by cytokines, nitric oxide, complement and immune allergic reaction and damage. Hepatitis B virus is the most common cause of liver injury.At present, most scholars think that immune response is mainly related with the liver injury induced the hepatitis B virus infection. At the same time, autoimmune stimulation, viral or parasitic infection are the main reasons for liver injury, especially hepatitis. Acute chemical liver injury induced by carbon tetrachloride, D-galactosamine or thioacetamide, and liver injury caused by chronic model have been widely used in screening medicine for liver protection. However, the damage is obviously external chemical factors rather than by the human host defense induced hepatitis, therefore they are not suitable for evaluation of liver immune modulators. Studies on the mechanism of liver injury pathogenesis and liver-protective drugs needs to establish the suitable animal models. Therefore, in the study of anti-hepatitis drug,multiple models of different damage mechanisms can be taken to observe the protective effect of drug, with the purpose of comprehensive judgment of therapeutic effect and mechanism of action.Con-A induced liver injury model, whose liver injury is mediated by T lymphocyte,has been developed in recent years. Concanavalin A (Con-A) is a plant lectin that has the effect of specific toxicity to liver cells in vivo.It induces the infiltration of inflammatory cell,predominantly CD4+T lymphocyte in liver cells after moving into the circulation, and than activates cytokines such as TNF-a and interleukin, and causes inflammation reaction,and damages live cell through a variety of ways such as liver cell apoptosis, resulting in immunological liver injury. This model requires only a single intravenous injection of Con-A, its main character is a rapid onset, obvious liver damage. The pathophysiology of liver cell damage is very similar with that mediated by T lymphocyte in human chronic hepatitis B, with the characteristics of liver-dependent and dose-dependent. The model well simulates the human viral hepatitis, autoimmune liver disease, and is an ideal animal model for the screening of drugs for the treatment of acute liver injury and fulminant hepatic failure. China is one of the countries with high incidence of liver disease. It has always been an important direction of research of traditional Chinese medicine to search for and study effective natural medicines which has both antiviral and liver-protective effect. Anti-viral, immunomodulatory drugs and protecting liver and lowering protein drugs are several drugs that usually used in the treatment of liver diseases by western medicine. Clinical results show that, these drugs are good at the control of virus replication, the restoration of normal liver function, and the protection of liver cells, and so on. In the mean time, their curative effects are rapid. However, the disadvantages of antiviral drugs are unstable long-term efficacy, easy relapse, and low HBsAg negative rate. Besides, some drugs are expensive and have certain side effects. On the other hand, modern medicine in the treatment of liver injury has no specific drug. It usually suggests to take a rest, strengthen nutrition, and supplement and symptomatic treatment. Severe cases were even forced to discontinue their medication. Whereas Traditional Chinese Medicine, with its unique characteristics of the overall concept, the harmony between man and nature,and treatment based on syndrome differentiation, gives full play to the advantages of Chinese traditional medicine which include multi-targets, multi-ways, multi-levels, low price, less side effect, good curative effect in the treatment of hepatitis and liver injury,thus more and more medical researchers pay great attention to the development of it.The root of Alchornea trewioides(Benth.)Muell.-Arg (ATR),which belongs to dicotyledonous plants botanicals Euphorbiaceae, is one of the commonly used Chinese herb in south of the Five Ridges area. In the civil application It has a long history and curative effect in the protection of liver. The previous results of our research group suggested for the first time that ATR has the functions of protecting liver, reducing enzyme, anti-liver fibrosis. And It has been applied for patent protection. In order to further investigate and screen the effective parts of ATR, and provide exact mechanism for its liver-protective, antiviral effect,we extracted and isolated4kinds of ATR extracts, do experimental research on them.ObjectiveTo study the mechanism of ATR against hepatitis B virus (HBV) and acute immunological liver injury and its mechanism of immune regulation, we used four extracts water extract (WE), petroleum ether extract (PE), ethyl acetate extract (EE), n-butanol extract (BE) of ATR and do experiments on anti-hepatitis B virus and anti-immune liver injury and immune regulation action by HepG2.2.15cell model,duckling models congenitally infected with DHBV and Con-A induced acute liver injury mice model, with the purpose of laying a foundation for the further development and utilization of ATR.Methods:Part one Anti-hepatitis B virus experimentsExperiment1:the experimental study of ATR extracts on the inhibition of HBsAg and HBeAg in vitro.Methods:the four methyl thiazolyl tetrazolium (MTT) method was used to observe the toxic effects of ATR extracts on HepG2.2.15cells; and the inhibitory effects of ATR extracts on the HBsAg and HBeAg secreted by HepG2.2.15cells was detected by ELISA analysis.Experiment2:The inhibitory effect of ATR extracts on the replication of the hepatitis B virusMethods:detecting the inhibitory effect of ATR extracts on HBV-DNA secretion of HepG2.2.15cells by fluorescence quantitative PCR-probe analysis. Experiment3:Experimental study of ATR extracts against duck hepatitis B virus in vivoMethods:Screening the duckling models congenitally infected with DHBV by the conventional PCR method.And randomly grouping the screened ducks. Drug groups were administered drugs once a day by intragastric gavage, totally14d. The control group did not do any processing. Ducklings in each group were drawn blood from tibial vein1d before administration,7d and14d after administration,5d after drug cessation, collecting the serum after centrifugation.Using dot blot hybridization to detect DHBV-DNA in duck serum.Part two Studies of the protective effects of4ATR extracts on Con-A induced acute liver injury in miceExperiment4:the influence of ATR extracts on the transaminases and hepatic histopathology of Con-A induced acute liver injury in miceMethods:after grouping randomly, the model group and normal control group were gavaged daily with the same volume of normal saline; each drug group was continually administrated for3d.lh after the last administration, the model group and the drug groups were injected with Con-A solution, which induces acute immune hepatic injury in mice,while the normal control group was injected equal saline, fasting, but not water deprivation for8h.Then drew blood from eyeballs of mice,and took the same part in the livers of mice after the mice were killed,10%formalin fixed. The collected blood is centrifuged to collect serum preparing for the measure of alanine aminotransferase, aspartate aminotransferase; the formalin-fixed liver tissue were used to do further pathological examination and observing the change of structure of liver tissue after HE staining.Experiment5:immunological regulation of ATR extracts on Con-A induced acute liver injury in mice Methods:the WST-1method was used to detect the activity of SOD in liver tissue; the TBA method was to measure the content of MDA in liver, and the activity of GSH-PX in liver tissue was detected by colorimetric assay; the ELISA method was used to analyze the contents of serum inflammatory cytokines IL-6, TNF-α, IFN-γ; the flow cytometry was used to detect TLR4, Tim-4expression on splenic macrophages.Statistical processingThe experimental results were presented by mean±standard deviation (mean±SD), multiple samples were analyzed by one-way ANOVA; repeated measurement data using repeated measures analysis of variance, multiple level/frequency table data with a non parametric test of K Independent Samples. Correlation between the two variables by using bivariate correlation analysis (Bivariate).Using the statistical software SPSS13.0to carry on the analysis, and P<0.05is confirmed as the difference has statistical significance.Results1. The inhibition rates of BE on the secretion of HBsAg, HBeAg on HepG2.2.15reached65.2%,94.4%respectively under non-toxic concentrations,and the therapeutic index were>9.8and>67.1respectively;the inhibition rate of EE on HBeAg was53.4%, the therapeutic index>2, while no inhibitory effect on HBsAg; whereas WE and PE had no effect on the inhibition of HBsAg, HBeAg.2. Results demonstrated that F=9.479, P=0.000. Compared with the control cells, WE, PE, EE and BE all could significantly inhibit the viral load of HBV-DNA secreted by HepG2.2.15, the difference was statistically significant (P<0.05).3. The results showed that, overall, no significant difference of DHBVDNA changes at different time points (F=2.296, P=0.110); there are significant differences among the groups DHBVDNA (F=12.416, P=0.000); a significant interaction existed between time and group (P=6.393, P=0.000).In the duckling models congenitally infected with DHBV, among the ATR extracts groups, only the high-dose of PE could inhibit DHBVDNA after two weeks’ treatment and5ds of drug cessation,the difference was statistically significant(T14and P5, P<0.05).4. The results showed that, ALT, F=34.616, P=0.000; AST, F=37.658, P=0.000. Degeneration of liver cells, X2=57.935, P=0.000; necrosis of liver cells, X2=41.751, P=0.000;the results above indicated that there was a significant difference in the curative effect of each group.4ATR extracts could significantly reduced the serum alanine transaminase, aspartate transaminase,the difference was statistically significant (P<0.01).In the liver tissue morphology, Compared with the normal group, model group liver degeneration and necrosis pathological changes were especially significant (P<0.05/14), which indicated that our animal model was established successfully. As for the degeneration of liver cells, there was no significant difference among drug groups in improving the liver degeneration. In the necrosis of liver cells, medium dose of PE group, EE high dose group, BE in low, middle dose group and the GLX group reduced necrosis of liver cells significantly (P<0.05/14); microscopy results are shown in Table4-2and figure4-(1-15)(HE staining,*200).5. The results showed that, SOD, F=34.961, P=0.000; MDA F=23.613P=0.000; GSH-PX, F=37.089, P=0.000. IL-6F=74.279, P=0.000; TNF-α F=28.023, P=0.000; IFN-γ F=374.800, P=0.000. TLR4F=4.552, P=0.015; Tim-4F=6.763, P=0.003.The4ATR extracts not only could significantly decreased the liver MDA content (P<0.01), but also could significantly improve the SOD activity of liver (P<0.05); as for the GSH-PX, except for EE, other three extracts could significantly improve the GSH-PX activity of liver (P<0.01). At the same time,4ATR extracts species not only could significantly inhibit the inflammatory cytokines IL-6, TNF-α, IFN-γ, the difference was statistically significant (P<0.05); but also could improve the percentage of expression of TLR4and Tim-4on spleen macrophages, the difference was statistically significant (P<0.05), suggesting that4ATR extracts could raised TLR4and Tim-4levels on splenic macrophages. TLR4and Tim-4were positively correlated (r=0.538, P=0.021)(P<0.05).ConclusionsAs for hepatitis B virus, study results in vitro showed that only EE and BE could inhibit the HBV antigen, and BE is better than EE;4ATR extracts could inhibit HBV-DNA;study results in vivo showed that only PE of high dose group in the treatment of7d,14d could reduce the level of serum DHBV-DNA, and the DHBV-DNA level remained decreased and showed no rebound after drug cessation for5days(P<0.05), other groups had no inhibitory effect of serum DHBV-DNA. Thus we can conclude that the HBV antigen and HBV-DNA are two targets of ATR against HBV.The results showed that, among the4ATR extracts, n-butanol extract not only could inhibit two HBV antigens, but also could inhibit HBVDNA level, so the n-butanol extract of ATR is the best in the effect of anti-HBV. As for acute liver injury, ATR extracts not only could reduce transaminase, improving liver tissue morphology, but also could reduce the content of liver MDA, liver SOD and GSH-PX activity increased. At the same time,4ATR extracts not only could significantly inhibit the inflammatory cytokines IL-6, TNF-α, IFN-gamma, but also increased the expression level of TLR4and Tim-4on splenic macrophages, there was a significant positive correlation between TLR4and Tim-4, thus we can conclude that reducing transaminase, improving liver histology, inhibiting lipid peroxidation, and inflammation cytokines, up-regulating the expression of TLR4and Tim-4are several liver-protective mechanisms for4ATR extracts.In addition, as it is known that TLR4 and Tim-4plays an important role in immune regulation, we found that there was a positive correlation between the two, so it could be inferred that the up-regulation of TLR4and Tim-4expression on splenic macrophages may be associated with the activity of anti-hepatitis B virus and anti-hepatic injury. And ATR has a rather good immune-regulating effect on acute liver injury. Among the4kinds of ATR extracts,BE was the best at the effect of anti-hepatitis B virus and anti-hepatic injury.We hypothesized that BE up-regulate the levels of TLR4and Tim-4through its immune regulation function. On the one hand, BE may act as a TLR4agonist, and inhibit the secretion of HBsAg, HBeAg and HBVDNA, thus playing the role of anti hepatitis B virus,;on the other hand, its up-regulation of Tim4lead to the inhibition of lipid peroxidation and inflammatory cytokines, thereby reducing transaminase, improving liver tissue degeneration and necrosis, thus playing the role of anti-hepatic injury.更多还原