红背叶根提取物抗乙肝病毒和抗肝损伤的药效学研究
|
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。
Background
Hepatitis 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.
Objective
To 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 experiments
Experiment1: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 virus
Methods: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 vivo
Methods: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 mice
Experiment4:the influence of ATR extracts on the transaminases and hepatic histopathology of Con-A induced acute liver injury in mice
Methods: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 processing
The 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.
Results
1. 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).
Conclusions
As 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.
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。
Background
Hepatitis 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.
Objective
To 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 experiments
Experiment1: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 virus
Methods: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 vivo
Methods: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 mice
Experiment4:the influence of ATR extracts on the transaminases and hepatic histopathology of Con-A induced acute liver injury in mice
Methods: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 processing
The 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.
Results
1. 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).
Conclusions
As 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.
引文
[1]MEDZHITOV R, PRESTON-HURLBURT P, JANEWAY C A Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity[J]. Nature,1997,388(6640):394-397.
[2]陈圻,邓存良.TLR4在肝脏损伤中的作用研究进展[J].医学综述,2008,14(19)：2897-2899.
[3]Beutler B. The toll-like receptors:analysis by forward genetic methods[J]. Immunogenetics,2005,57(6):385-392.
[4]Katsargyris A, Klonaris C, Alexandrou A, et al. Toll-like receptors in liver ischemia reperfusion injury:a novel target for therapeutic modulation?[J]. Expert Opin Ther Targets,2009,13(4):427-442.
[5]Akira S, Uematsu S, Takeuchi O. Pathogen lecognition and innate immunity[J]. Cell,2006,124(4):783-801.
[6]Liew FY, Xu D, Brint EK, et al. Negative regulation of toll-like preceptor-mediated immune responses[J]. Nat Rev Immunol,2005,5(6):446-458.
[7]Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol,2002,20:197-216.
[8]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell,2006,124:783-801.
[9]KURT-JONES E A, POPOVA L,KWINN L, et al. Pattem recognition receptors TLR4 and CD14 mediate response to respiratory syncytial vinIs[J]. Nat Immunol,2000,1(5):398-401.
[10]Yin Mo Bradford BU, Wheeler MD, et al. Reduced early alcohol-induced liver injury in CD14-deficient mice. J Immunol,2001,166:473-4742.
[11]Uesugi T, Froh M, Arteel GE, et al. Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice. Hepatology,2001, 34:101-108.
[12]Masanori Isogawa, Michael D. Robek, Yoshihiro Furuichi,et al. Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo. J Virol.2005 Jun;79(11):7269-7272.
[13]Evans J T, Cluff C W, Johnson D A,et al. Enhancement of antigen-specific immunity via the TLR4 ligans MPL adjuvant and Ribi.529[J]. Expert Rev.Vaccines,2003,2(2):219-229.
[14]Dupont J, Altclas J, Lepetic A,et al. A controlled clinical trial comparing the safety and immunogenicity of a new adjuvanted hepatitis B vaccine with a standard hepatitis B vaccine[J]. Vaccine,2006,24(49-50):7167-7174.
[15]O'Neill L A,Bryant C E, Doyle S L. Therapeautic targeting Toll-like receptors for infection and inflammatory diseases and cancer[J]. Pharm.Rev.,2009,61(2): 177-197.
[16]Magness ST, Bataller R, Yang L, et al. A dual reporter gene trans-genic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations[J]. Hepatology,2004,40(5):1151-1159.
[17]Seki E, De Minicis S, Osterricher CH, et al. TLR4 enhances TGF-β signaling and hepatic fibrosis[J]. Nat Med,2007,13(11):1324-1332.
[18]McIntire JJ, Umetsu SE, Akbari O, et al. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol.2001,2(12):1109-1116.
[19]Monney L, Sabatos C A, Gaglia J L, et al. Thl-specific cell surface protein TIM-3 regulates macrophage activation and severity of an autoimmune disease[J]. Nature,2002,415:536-541.
[20]Chan R C, Chan A, Jeon M, Wu T F, Pasqualone D, Rougvie A E, et al. Chromosome cohesion is regulated by a clock gene paralogue TIM-1[J]. Nature,2003,423:1002-1009.
[21]Meyer P, Saez L, Young M W. PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock[J]. Science,2006,311:226-229.
[22]Kuchroo V K, Meyers J H, Umetsu D T, DeKruyff R H. TIM family of genes in immunity and tolerance[J]. Adv Immunol,2006,91:227-249.
[23]Kuchroo V K, Umetsu D T, DeKruyff R H, Freeman G J. The TIM gene family: emerging roles in immunity and disease[J]. Nat Rev Immunol,2003,3:454-462.
[24]MEYERS J H, CHAKAVARTI S, SCHLESINGER D, et al. TIM-4 is the ligand For TIM-1,and the TIM-1-TIM-4 interaction regulates T cell proliferation[J]. Nat Immunol,2005,6:455-464.
[25]Umetsu S E, Lee W L, McIntire J J, et al. TIM-1 induces T cell activation and inhibits the development of peripheral tolerance[J]. Nat Immunol,2005,6: 447-454.
[26]Nakae S, Iwakura Y, Suto H, Galli S J. Phenotypic differences between Thl and Th17 cells and negative regulation of Thl cell differentiation by IL-17[J]. J Leukoc Biol,2007,81:1258-1268.
[27]Binn L L, Scott M L, Rennert P D. Human TIM-1 associates with the TCR complex and up-regulates T cell activation signals[J]. J Immunol,2007, 178:4342-4350.
[28]Chakravarti S, Sabatos C A, Xiao S, et al. Tim-2 regulates T helper type 2 responses and autoimmunity[J]. J Exp Med,2005,202:437-444.
[29]Koguchi K, Anderson D E, Yang L, et al. Dysregulated T cell expression of TIM3 in multiple sclerosis[J]. J Exp Med,2006,203:1413-1418.
[30]Zhao J,Lei Z,Liu Y,Li B,Zhang L,Fang H, et al. Human pregnancy up-regulates Tim 3 in innate immune cells for systemic immunity [J]. J Immunol,2009,182: 6618-6624.
[31]Mizui M, Shikina T, Arase H, Suzuki K, Yasui T, Rennert P D, et al. Bimodal regulation of T cell-mediated immune responses by TIM-4[J]. Int Immunol, 2008,20:695-708.
[32]Khademi M, Illes Z Alexander W, et al. T cell Ig-and mucin-domain-containing molecule-3(TIM-3)and TIM-1 molecules are differentially expressed on human Thl and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis[J]. J Immunol,2004,172(11):7169-7176.
[33]Cai PC, Wu Qw, Wang L, et al. Distribution characteristics and linkage disequilibrium of TIM4 promoter polymorphisms in asthma patients of Chinese Han population[J]. J Huazhong Univ Sci Teehnolog Med Sci,2008,28(4): 447-450.
[34]夏义平,崔天盆,吴健民.TIM4基因多态性与湖北汉族变应性哮喘的相关性研究[J].中华医学遗传学杂志,2007,24(2)：213-216.
[35]Monney L, et al. Thl-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002,415:536-541.
[36]Sanchez-Fueyo A, et al. Tim-3 inhibits T helper type 1-mediated auto-and alloimmune responses and promotes immunological tolerance. Nat Immunol 2003,4:1093-1101.
[37]Jones RB, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J Exp Med 2008;205:2763-2779.
[38]Golden-Mason L,et al. Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+T cells. J Virol 2009;83:9122-9130.
[39]Sanchez-Fueyo A, Tian J, Picatella D, et al. Tim-3 inhibits T helper type 1-mediated auto-and allo-immune responses and promotes immunological tolerance. Nat ImmunOl.2003,4(11):1093-1101.
[40]Kobayashi, N., P. Karisola, V. Pena-Cruz, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity.2007,27:927-940.
[41]Miyanishi, M.,K. Tada, M. Koike, et al. Nagata. Identification of Tim4 as a phosphatidylserine receptor. Nature.2007,450:435-439.
[42]续力云,祁建妮,梁晓红,等.稳定高表达小鼠Tim-4巨噬细胞系RAW264.7-T4的建立[J].山东大学学报(医学版),2008,46(6)：551-555.
[43]钱晖,彭秀娟,许晓蒙,等.Tim4基因定量检测方法的建立及在消化道肿瘤中的初步应用[J].江苏大学学报(医学版),2009,19(6)：465-468.
[44]何金洋,张小虎,郭兴伯.中药治疗乙肝的机制研究进展[J].中医药学报, 2002,3(4)：65-66.
[45]杨鉴英等.中药抗乙型肝炎病毒的实验研究[J].中西医结合杂志,1989,9(8)：494.
[46]陈压西,郭树华,齐珍元,等.叶下珠有效部位抗鸭乙型肝炎病毒及体外抗HBV活性的研究[J].中药药理与临床,1996：15(6)：16-18.
[47]黄正昌,邓学龙,朱宇同,等.台湾细叶柚柑体外抗乙肝病毒作用研究[J].广州中医药大学学报,2000,17(3)：260-263.
[48]张晓刚,吕志平,谭秦湘等.白背叶根抗乙型肝炎病毒的体外实验研究[J].时珍国医国药,2006,17(8)：1437-1438.
[49]郭姗姗,高英杰,时宇静,等.一枝蒿总黄酮体外抗乙肝病毒作用机理的实验研究[J].中国实验方剂学杂志,2009,15(10)：72-74.
[50]范惠敏,肖会泉,高小玲.肝舒胶囊抗鸭乙型肝炎病毒体内实验[J].中国中西医结合杂志,2002,11(3)：207-208.
[51]王岭,张均倡,罗上武,等.叶下珠复方肝丹体外抗HBV作用特点的实验研究[J].中国中医基础医学杂志,1999,5(7)29-33.
[52]刘中景,熊曼琪,李塞美,等.小柴胡汤体外抗HBV作用及其组方机理的实验研究[J].中国中医基础医学杂志,2001,7(6)：17-19.
[53]吴国庆.赤珠清毒汤治疗慢性乙型肝炎94例疗效观察[J].中医药通报,2003,2(2)：119-121.
[54]范惠敏,肖会泉,吴婉芬等.肝舒胶囊对慢性肝炎患者免疫功能的影响[J].中国中西医结合脾胃杂志,1998,6(4)：210-211.
[55]徐德先.肝苏颗粒治疗慢性乙型肝炎128例的疗效观察[J].中国临床医药研究杂志,2000,31(12)：4926.
[56]李承栋.一种治疗慢性乙型肝炎的新药及制备方法[P].中国专利,CN1281701,2001-01-31.
[57]蔡永江,张秀婷,王立静.芪蓝扶正解毒剂对于HBsAg阳性转阴的临床观 察[J].中医现代医生,2007,45(22)：77-78.
[58]汪锐.中药治疗乙肝患者前后血清中ALT、AST及AST／ALT的变化[J].临床和实验医学杂志,2010,9(15)：1176-1177.
[59]钱涯邻.乙肝II号治疗慢性乙型病毒性肝炎临床研究[J].中国中医药信息杂志,2009,16(8)：68-69.
[60]钱涯邻,陈维铭,张燕.中药乙肝II号联合a-干扰素治疗慢性乙型肝炎临床观察[J].中国中医药信息杂志,2010,17(11)：70-71.
[61]刘厚佳,胡晋红,蔡溱,等.中药及其有效成分抗乙型肝炎病毒的研究进展[J].解放军药学学报,2000,11(1)：20.
[62]沈洪,张蓓.急性肝损伤研究进展[J].现代中西医结合杂志,2009,18(18)：2211-2213.
[63]Dey A, Cederbsum AI. Alcohol and oxidative liver injury. Hepatology.2006, 43(2 Suppl1):863-874.
[64]朱润芝,李京敬,谢超.过氧化作用与肝脏疾病[J].世界华人消化杂志,2010,18：1134-1140.
[65]Sakaguchi S. Furusawa S. Oxidative stress and septic shock:metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia. FEMS Immunol Med Microbiol,2006.47:167-177.
[66]Grattagliano I, Bonfrate L, Diogo CV, et al. Biochemical mechanisms in drug induced liver injury:certainties and doubts. World J Gastroenterol,2009,15: 4865-4876.
[67]Katagiri H, Ito Y, Ito S, et al.TNF-a induces thromboxane receptor signaling-dependent microcirculatory dysfunction in mouse liver. Shock,2008, 30(4):463-467.
[68]宋润波,马建亭.IL-10对大鼠重症胰腺炎炎症相关细胞因子的调节作用[J].河北医药,2009,31：1451-1452
[69]Mits A, Hashikura Y, Tagawa Y, et al. Expression of Fas ligand by hepatic macrophages in patients with fulminant hepatic failure. Am J Gastroenteml, 2005,100:2551-2559.
[70]Kimura K, Sekiguchi S, Hayashi S, et al. Role of interleukin-18 intrahepatic inflammatory cell recruitment in acute liver injury. J Leukoe Biol, 2011,89(3): 433-42.
[71]胡骁骅,张会英,葛艳玲,等.乌司他丁对严重烧伤后多脏器功能的保护作用[J].中华医学杂志,2005,85：2889-2894.
[72]黄吕州,张宗明,裘法祖.缺血再灌注损伤埘大鼠肝细胞钙池操纵的钙通道电流的影响及药物拮抗[J].世界华人消化杂志,2005,13：739-742.
[73]刘亮明,罗杰,张吉翔.内毒素诱导D-半乳糖胺致敏大鼠急性肝衰竭的研究[J].中华医学杂志,2006,86：2122-2126.
[74]Webb c, Twedt D. Oxidative stress and liver disease. Vet Clin North Am Small Anita Pract,2008,38:125-135.
[75]钟卫卫,林世德.内质网应激与肝损伤研究进展[J].世界华人消化杂志,2010,18：1021-1025.
[76]伏建峰,史清海,路西春,等.大鼠慢性酒精诱导肝损伤中肝细胞凋亡与Fas表达[J].西北国防医学杂志,2006,27：362-364.
[77]Nanji AA,Jokehinen K,Rahemtulla A,et al. Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat[J]. Hepatology.1999,30(4):934-943.
[78]Suetsugu H, Iimuro Y, Uehara T, et al. Nuclear factor{kappa} B in activation in the rat liver ameliorates short term total warm ischaemia/reperfusion injury[J]. Gut,2005,54(6):835-842.
[79]Ding WX, Tin XM. Dissection of the multiple mechanisms of TNF induced apoptosis in liver injury[J]. J Cell Mol Med,2004,8(4):445-454.
[80]李跃华,朱丽.哈团柱,等.刀豆素A诱导小鼠肝损伤增加肝脏NF-K B活性[J].中华肝脏病杂志,2003,11(2)：115
[81]赵宗豪,高人焘.核因子-κB与肝纤维化的研究进展[J].实用肝脏病杂志,2004,7(2)：119-121.
[82]Tiegs G, Hemschd J, Wendel A. A T cell dependent experimental liver injury in mice induced by concanavalin A[J]. J Clin Invest.1992,90:196-203.
[83]Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta [J]. Nature, 2000,403:98-103.
[84]Gong W, Pecci A, Roth S, et al. Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand [J]. Hepatology,1998,28(2):492-502.
[85]黄才国.李医明,贺祥.玄参中苯丙素苷对大鼠肝损伤细胞凋亡的影响[J].中西医结合肝病杂志,2004,14(3)：160-161.
[86]杨小红,袁江,周远明,等.白首乌粗多糖对酒精性肝损伤的保护作用研究[J].时珍国医国药,2009,20(11)：2704-2705.
[87]陈旭,杜正彩.桃金娘多糖对大鼠急性肝损伤保护作用的研究[J].安徽农业科学,2010,38(11)：5663-5664.
[88]Xu H, Guo T, Guo Y, et al. Characterization and protection on acute liver injury of a polysaccharide MP-I from Mytilus coruscus[J]. Glycobiology,2008, 18(1):97-103
[89]宋淑亮,肖增平,吉爱.绞股蓝多糖对HepG2细胞酒精性损伤的保护作用[J].中国生化药物杂志,2008,29(5)：302-305.
[90]张波,徐永杰.牛蒡菊糖对亚慢性酒精肝损伤作用的研究[J].食品工业科技.2010,31(8)：329-331.
[91]吕雄文,李俊,金涌,等.豹皮樟总黄酮对小鼠急性酒精性肝损伤的保护 作用[J].中成药,2010,3(32)：489-491.
[92]林冠宇,姚楠,何蓉蓉,等.淡竹叶总黄酮对拘束负荷所致小鼠肝损伤的保护作用[J].中国实验方剂学杂志,2010,16(7)：177-179.
[93]李中华,涂燕云,赵晓芳.半枝莲总黄酮对肝纤维化大鼠血清白介素-10表达的影响[J].中药药理与临床,2010,26(2)：44-46.
[94]潘莹,张林丽.大果山楂黄酮提取物对四氯化碳致大鼠慢性肝损伤的保护作用[J.].时珍国医国药,2008,19(2)：318-319.
[95]韩刚,万红,翟冠钰,等.姜黄素固体分散体对四氯化碳致小鼠急性肝损伤的保护作用[J].时珍国医国药,2009,6(20)：1321-1322.
[96]喻松仁,程绍民,周步高,等.葛根总黄酮对大鼠慢性肝损伤的保护作用[J].江西中医学院学报,2010,22(2)：70-72.
[97]陈立波,郝六平,王宏,等.注射用盐酸苦参碱对卡介苗加脂多糖致小鼠免疫性肝损伤的保护作用[J].现代预防医学,2008,35(21)：4305-4306.
[98]洪振丰,李天骄,赵锦燕,等.粗叶悬钩子总生物碱对急性肝损伤大鼠肝组织CYP2E1与CYP3A1酶mRNA表达的影响[J].中国中西医结合杂志,2009,29(8)：711-715.
[99]毕明刚,周娟,许扬,等.岩黄连总碱提取物对小鼠免疫性肝损伤的改善作用[J].中国药理学与毒理学杂志,2009,23(1)：39-44.
[100]周劲光.岩黄连总碱对慢性肝损伤大鼠的肝保护作用观察[J].山东医药,2010,50(27)：11.
[101]王晓丽,郑立运,张艳,等.盐酸千金藤碱对急性肝损伤小鼠保护作用的研究[J].中华中医药杂志,2009,24,(10)：1384-1387.
[102]陈象青,王钦茂,方华武,等.白芍总苷与当归提取物合用对实验性肝炎的保护作用[J].安徽中医学院学报,2002,2(3)：44.
[103]李瑞麟,马勇,魏伟,等.白芍总苷治疗四氯化碳致大鼠肝纤维化的作用与其影响肝星状细胞功能的关系[J].中国新药杂志,2007,16(9)：685-689.
[104]邱彦,陈敏,茆玉国,等.注射用西黄总苷对四氯化碳致大鼠慢性肝损伤的保护作用研究[J].药学实践杂志,2008,26(3)：188-190.
[105]樊文研,罗力元.三七总皂苷对异烟肼和利福平合用所致小鼠肝损伤的保护作用[J].中国医药导报,2009,6(35)：7-8.
[106]卢春凤,陈廷玉,赵锦程,等.槲皮素对INH诱导的大鼠肝损伤及肝组织Bcl-2和Bax表达的影响[J].解剖学研究,2010,32(1)：28-31.
[107]荣黎,戴立里,曾维政,等.红景天甙对拟高原缺氧大鼠肝损伤的保护作用[J].中国组织工程研究与临床康复,2010,14(31)：5813-5817.
[108]张国良,黄川锋.芦荟苷对急性肝损伤小鼠NO和IFN-γ的影响[J].中药药理与临床,2010,26(1)：27-29.
[109]罗景慧,杨迎暴.白藜芦醇对酒精性肝损伤大鼠血清中TNF-α、IL-6、ICAM-1水平的影响及机制[J].中药药理与临床,2010,26(4)：14-16.
[110]林兴,黄权芳,张士军,等.六月青总皂苷对四氯化碳诱导小鼠肝损伤的脂质过氧化反应的影响[J].中成药,2009,12(31)：1937-1939.
[111]刘莉,管小琴,李芬,等.茶多酚对酒精性肝病大鼠核因子-K B及环氧合酶2表达的影响[J].中国实验动物学报,2008,3(16)：209-214.
[112]沈洪,朱路佳,薛洁,等.生姜油不同部位对小鼠急性肝损伤保护作用的比较[J].中成药,2008,9(30)： 8-10.
[113]冯文文,谭勇,李国玉,等.紫草提取物对小鼠急性肝损伤保护作用的研究[J].农垦医学,2010,32(2)：108-111.
[114]田立.决明子的不同提取物及炮制品对四氯化碳致小鼠急性肝损伤的保护作用[J].时珍国医国药,2008,8(19)：2005-2006.
[115]谢丽琼,李海芳,迪里努尔.小鼠中甘草毛状根、甘草切片总抗氧化能力和对CC14致肝损伤保护的比较研究[J].食品科学,2009,30(23)：432-435.
[116]周春晖,苏烨,李凤芝.金银花提取物对小鼠化学性肝损伤的保护作用初步研究[J].现代食品科技,2010,26(4)：351-353.
[117]苏晓聆,李福安,魏全嘉,等.秦艽水煎液对小鼠急性肝损伤肿瘤坏死因子-α和白细胞介素-10表达的影响[J].时珍国医国药,2010,21(4)：827-828.
[118]陈世林,洪汝涛,刁磊,等.水飞蓟素对大鼠急性酒精性肝损伤的保护作用[J].安徽医科大学学报,2010,45(2)：209-212.
[119]伍义行,郝冰洁,胡少青,等.六棱菊提取物对醋氨酚致小鼠肝损伤的影响[J].世界华人消化杂志,2010,18(7)：711-715.
[120]刘协,顾呈华,胡启之,等.甘草甜素脂质体对小鼠急性酒精性肝损伤的保护作用[J].中国临床康复,2004,8(36)：8260-8261.
[121]陈梁,朱锦,任建平.柴胡疏肝散对四氯化碳所致大鼠急性肝损伤的保护作用[J].中西医结合肝病杂志,2004,14(1)：42-43.
[122]李海燕,董礼阳,杨景云.四君子汤对急性肝损伤伴发菌群失调及内毒素血症的保护作用[J].中国微生态学杂志,2005,17(2)：100-101.
[123]苏晓聆,李福安,魏全嘉,等.秦艽水煎液对小鼠急性肝损伤肿瘤坏死因子-a和白细胞介素-10表达的影响[J].时珍国医国药,2010,21(4)：827-828.
[124]马丽,高玉杰,钱月慧,等.丹葛解醒汤对大鼠酒精性肝损伤的保护作用研究[J].时珍国医国药,2010,21(7)：1596-1597.
[125]薛晓伟,丰平,董坤,等.槲芪散对小鼠酒精性肝损伤的干预作用[J].实验动物科学,2010,27(4)：27-32.
[126]Tungyuan Lai, Yijiun Weng, Weiwen, Kuo, et al. Taohe Chengqi Tang ameliorates acute liver injury induced by carbon tetrachloride in rats[J]. Journal of Chinese Integrative Medicine,2010,8(1):49-55.
[127]张引强,唐旭东,王凤云,等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-456.
[128]孔祥才,张方信,陈嘉屿,等.大黄甘草汤对大鼠重症急性胰腺炎并发肝损伤时核因子-K B及细胞因子的影响[J].中国中西医结合消化杂志,2010, 18(2)：82-85.
[129]林旭楷,张奕,许森源.胃舒散对五氯硝基苯致肝损伤的保护作用[J].广州中医药大学学报,2009,26(4)：371-373.
[130]蒋洁云,徐强.四逆散及其各组成中药对实验性肝损伤的影响[J].中国天然药物,2004,2(1)：45-49.
[131]刘思纯,马博.甘草酸二铵治疗急性药物性肝损伤的疗效观察[J].中国处方药,2006,10(55)：62-64.
[132]Lino Set al.Hepatal Res 2001,19(1):31-40.
[133]吉孝祥,夏仲芳.黄芪对有机磷农药中毒患者肝脏的保护作用研究[J].现代中西医结合杂志,2003,12(20)：2158.
[134]刘成,薛惠明,徐列明,等.人工冬虫夏草苗丝制剂治疗肝炎后肝硬化22例[J].上海中医药杂志,1986(6)：30-31.
[135]周良楣,续月明,朱庆义,等.冬虫夏草菌丝治疗慢性乙型病毒性肝炎的近期疗效观察[J].中国中药杂志,,1990,15(1)：53.
[136]徐列明,刘成,刘平.桃仁提取物合虫草菌丝治疗肝炎后肝硬化的病理免疫组化研究[J].中西医结合肝病杂志,1994,(4)：9.
[137]巫善明.参三七注射液治疗27例久治难愈的血淤型慢性肝炎疗效观察[J].中成药研究,1978,3(4)：41.
[138]高寒.红参、三七、激素等联用治疗重型肝炎70例报告[J].中华内科杂志,1980,19(4)：313.
[139]张火昆.三七粉降低谷丙转氨酶及改善血浆蛋白的观察[J].中医杂志,1980,21(5)：25.
[140]路彬,刘晓丹,高墉发,等.苦参碱注射液治疗肝功能异常40例疗效分析[J].现代中西医结合杂志,2005,14(4)：495.
[141]吴福全.苦参碱治疗慢性乙型肝炎疗效观察[J].实用医学杂志,2002,18(11)：1232-1234.
[142]卢宝玲,钟丽华.苦参素治疗慢性乙型肝炎疗效观察[J].黑龙江医学,2002,26(1)：46-48.
[143]江远,熊丽.莪术治疗肝病的研究进展[J].中西医结合肝病杂志,2005,15(2)：127-129.
[144]李宝红,梁念慈.莪术油制剂的临床应用及实验研究进展[J].中药材,2003,26(1)：78-71.
[145]韩铭钧,任克.油性中药抗癌剂OCC肝动脉栓塞治疗肝癌84例3年随访报告[J].临床放射学杂志,1998,17(2)：112-114.
[146]陈春永,徐凯.莪术油肝动脉灌注栓塞治疗继发性肝癌28例疗效观察[J].新中医,2003,35(3)：23-24.
[147]陈斌,孙克伟,谢凤瑛,等.莪术治疗慢性肝损伤时的肝毒作用研究[J].中西医结合肝病杂志,2003,13(1)：20.
[148]江远,熊丽.莪术治疗肝病的研究进展[J].中西医结合肝病杂志,2005,15(2)：127-129.
[149]高尚社.35年茵陈蒿汤治疗传染性黄疸型肝炎的研究进展[J].云南中医杂志,1985,(5)：54-57.
[150]张志勇.加味茵陈蒿汤治疗黄疸疗效观察[J].湖北中医杂志,2007,29(9)：33.
[151]石定,艾宏伟,王文菊.乌鸡白凤丸与马洛替酯联合治疗肝炎后肝硬化低白蛋白血症40例[J].中国中西医结合脾胃杂志,2000,8(4)： 247.
[152]刘洪明,毕青,张俊富.乌鸡白凤丸对肝硬化白蛋白的影响[J].天津中医药,2003,20(1)：29.
[153]方焱明.鳖甲煎丸合乌鸡白凤丸治疗慢性血吸虫病肝纤维化46例[J].时珍国医国药,2004,15(12)：846.
[154]欧阳云.发泡疗法合乌鸡白凤丸治疗慢性乙型肝炎9例[J].中国民间疗法,2003,11(11)： 9.
[155]何炳福,黄丹文.小柴胡汤对乙肝病毒携带者治疗疗效观察[J].浙江中西医结合杂志,2007,17(1)：34-35.
[156]王彦刚,周琰,杨金国,等.痛泻要方合小柴胡汤加减治疗脂肪肝疗效观察[J].河北中医药学报,2007,22(1)： 6-8.
[157]梁炳根,莫尤美.芍药甘草汤治疗148例病毒性肝炎的临床观察[J].上海中医药杂志,1989(6)：4-6.
[158]马列清.逍遥散加减对乙型病毒性肝炎肝纤维化指标的影响[J].北京中医,2002,21(5)：285-286.
[159]芦连菊.参麦注射液对化疗后肝功能损伤的临床疗效观察[J].中草药,2000,31(12)：92.
[1]吕志平.红背叶根在制备治疗慢性肝炎药物中的应用：[P]中国专利：200610 122307
[2]Sarent JM, Tlaylor CG. Appeaisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukemia. Br J Cancer,1989.60(2):206-210.
[3]蒋美娟,李玉虎.中药单方乙醇提取液抗乙肝病毒的实验研究[J].医学信息,2011,24(8)：4910-4912
[4]郑浩杰.利用2.2.15细胞株筛选抗乙肝病毒中草药的体外实验研究进展[J].陕西中医学院学报,2003,26(2)：61-63
[5]Sells M A, Chen ML,Aes G. Production of hepatitis B virus particles in Hep-G2 cells transfected with cloned hepatitis B virus DNA[J]. ProcNad Acad Sci USA,1987,84:1005
[6]中华人民共和国卫生部药政管理局.中药新药研究指南[S].北京：1994.82
[7]宋晓凯.天然药物化学[M].北京：化学工业出版社,2004：21-22
[8]张燕明,刘妮,朱宇同,等.诃子醇提物抗HBV的体外实验研究[J].中医药学刊,2003,21(3)：383-4
[9]刘妮,朱辫弦,黄正昌,等.大黄醇提液对2.2.15细胞分泌HBsAg、HBeAg的抑制作用[J].中药材,2004,27(6)：419-421
[10]周长征.生物碱类成分抗病毒研究进展[J].健康必读,2012,11(5)：5
[11]杨志伟,周娅,曹秀琴.苦豆子生物碱体外抗柯萨奇B3病毒的作用[J].四川中医,2003,21(3)：14-16
[12]黄洁春,吕志平,刘强,等.红背叶根提取物对四氯化碳致小鼠肝损伤的作用研究[J].陕西中医,2011,32(1)：113-5
[13]金奇.医学分子病毒学[M].北京：科技出版社,2001：338
[1]Sells MA, Mei Ling Chen, George Acsl. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Cell Biol,1987:1005-1009.
[2]罗红雨,杨旭,邹文,等.2.2.15细胞上清HBVDNA定量检测方法的比较P[J].中国现代医学杂志,2005,1(3):383-387.
[3]任建林,焦兴元.现代消化病诊疗学[M].第一版.人民军医出版社.2007：366-367.
[1]MASON WS, SEAL G, SUMMERS J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus[J]. J Virol,1980,36: 829-836.
[2]胡权,杨东亮.鸭乙型肝炎病毒感染模型的研究进展[J].实验肝脏病杂志,2007,10(3)：201-204.
[3]杨书兰,张奉学,朱宇同,等.用PCR法筛选先天感染鸭乙肝模型及与后天感染模型的比较[J].广州中医药大学学报,2001,18(3)：256-259.
[4]Ganem D, Schneider RJ. Hepadnaviridae:The viruses and their replication. In: Knipe DM, Howley PM, eds. Fields Virology.Volume 2. Philadelphia: Lippincott, Williams & Wilkins,2001; 2923-2969.
[5]Anneke Funk, Mouna Mhamdi, Hans Will.Avian hepatitis B viruses:Molecular and cellular biology,phylogenesis, and host tropism.World J Gastroenterol 2007; 13(1):91-103.
[6]MASON WS, CULLEN J, SAPUTELLI J, et al. Characterization of the antiviral effects of 2carbodeoxyguanosine in ducks chronically infected with duck hepatitis B virus [J]. Hepatology,1994,19(2):398-411.
[7]OFFENSPERGER WB,OFFENSPERGER S, KEPPLERHAFKEMEYER A, et al. Antiviral activities of Denciclovirand famciclovir on duck hepatitis B virus in vitro and in ViVo[J]. Antivir Ther,1996,1(3):141-146.
[8]LIN E, LUSCOMBE C, COLLEDGE D, et al. Long term therapy with the guanine nucleoside analog penciclovir controls chronic duck hepatitis B virus infection in vivo [J]. Antimicrob Agents Chemother,1998,42(8):2132-2137.
[1]唐云安,刘玉清,王国钦.肝损伤动物模型研究进展[J].卫生毒理学杂志,2002；16(4)：236-238.
[2]宋雨鸿,刘强,陈育尧,等.红背叶根对小鼠免疫性肝损伤的保护作用[J].南方医科大学学报,2008,28(3)：494-496.
[3]黄洁春,吕志平,刘强,等.红背叶根提取物对四氯化碳致小鼠肝损伤的作用研究[J].陕西中医,2011,32(1)：113-115
[4]闫冰,蔡秀江,姚卫峰等.二至丸保肝活性成分群对CCl4致小鼠急性肝损伤保护作用研究[J].中国中药杂志,2012,37(9)：1303-1305.
[5]广东中药志编辑委员会.广东中药志(第二卷)[M].广东科技出版社出版,1996：365-367.
[6]吕小燕,刘强,吕志平.红背叶根拮抗酒精性肝纤维化大鼠模型的作用机理研究[J].南方医科大学学报,2007,27(2)：153-155.
[7]吕志平.红背叶根在制备治疗慢性肝炎药物中的应用：中国,200610122307.4
[8]Tiegs G, Hentschel J, Wendel A. T Cell-dependent experimental liver injury in mice inducible by concanavalin A[J]. J Clin Invest,1992,90(1):196-203.
[9]Ganmer F, Leiat M, Lohse AW, et al. Concanavalin A induced T Cell-mediated hepatic injury in mice:The role of tumor necrosis factor[J]. Hepatology,1995,21(1):1901-98.
[10]曲建慧,张修礼.小鼠Con-A性肝损伤模型[J].世界华人消化杂志,2001,9(5)：571-574.
[11]邓利娟,李湛军,罗楹等.Caspase抑制剂F1013对刀豆蛋白A诱导小鼠急性肝损伤的治疗作用[J].中国药理学通报,2012,28(1)：136-139.
[12]吕小燕,吕志平,孙学刚,等.红背叶根对肾问质纤维化大鼠血流变及微循环的影响[J].北京中医药大学学报,2010,33(6)：398-401
[1]刘丹卓,赵新广.肝损伤病因病理机制及中医药治疗研究近况[J].国医论坛,2006,21(6)：53-55
[2]张定凤.乙型肝炎的发病机理及临床[M].重庆出版社,1992：1.
[3]Y H Zou,Y Yang, J Li, WPLi, Q Wu. Prevention of hepatic mjury by a
[4]traditional Chinese formulation, BJ-JN, in mice treated with Bacille-Calmette-Gu&in and lipopolysaccharide[J]. J Ethnopharrnacol,2006; 107:442-448.
[5]肖碧跃,赵国荣,贺又舜,等.理脾护肝调脂丸对脂肪肝大鼠肝脏MDA,SOD影响的研究[J].中国实验方剂学杂志,2011,17(11)：230.
[6]王君明,崔瑛,季莉莉,等.超氧化物歧化酶参与肝损伤的研究进展[J].中国实验方剂学杂志,2011,17(4)：265.
[7]周艳丽,张磊,刘维.白芍总苷对雷公藤多昔片所致小鼠急性肝损伤保护作用的实验研究[J].天津中医药,2007,24(1)：61.
[8]许金鹏,张慧慧,李朝品,等.原卟啉钠对四氯化碳致小鼠急性肝损伤的保护作用及其机制[J].中国实验方剂学杂志,2011,17(5)：168.
[9]Masayo Hatano, Soichiro Sasaki, Shinsuke Ohata, et al. Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis. Cellular Immunology. 2008,251:25-30.
[10]Schumann J,Wolf D, Pahl A,et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol.2000,157:1671-1683.
[11]Y. Tagawa, K. Sekikawa, Y. lwakura, Suppression of concanavalin A-induced hepatitis in IFN-gamma (_/_)mice. but not in TNF-alpha(_/_)mice:role of IFN-gamma in activating apoptosis of hepatocytes. J. Immunol.1997,159: 1418-1428.
[12]S. Kusters, G. Tiegs, L. Alexopoulou, et al. In vivo evidence for a functional role of both tumor necrosis factor(TNF) receptors and transmembrane TNF in experimental hepatitis. Eur. J. Immunol.1997,27:2870-2875.
[13]G. Kunstle, H. Hentze, P. G. Germann, et al. Concanavalin A hepatocytotoxicity in mice:tumor necrosis factor-mediated organ failure independent of caspase-3-like protease activation. Hepatology.1999,30: 1241-1251.
[14]Kremer M, Hines IN, Milton RJ, et al. Favored T helper 1 response in a mouse model of hepatosteatosis is associated with enhanced T cell-mediated hepatitis[J]. Hepatology,2006,44(1):216-227.
[15]Tiegs C, Hentschel J, Wendel A. T Cell-dependent experimental liver injury in mice inducible by Concanavalin A[J]. J Clin Invest,1992,90(1); 196-203.
[16]Gantner F, Leist M, Lohse AW,et al. Coneanavalin A induced r-Cell-mediated hepatic injury in mice:The role of tumornecrosis factor[J]。Hepatology,1995, 21(1):190-198.
[17]Trautwein CR, Akemann T, Brenner DA. Concanavalin A induced liver cell damage:activation of intracellular pathways triggered lby tumor necrosis factor in mice[J]. Gastroenterology,1998,114(5):1035-1045.
[18]Mizuhara H, Uno M, Seki Y, et al. Critical involvement of interferon gamma in the pathogenesis of T-cell activation-associated hepatitis and regulatory mechanisms of hepatitis [J]. Hepatology,1996,23(6):1608-1615.
[19]Mizuhara H, Uno M, Seki N, et al. Critical involvement of interferon-y in the pathogenesis of T-cell activation associated hepatitis and regulatory mechanisms of interleukin-6 for the manifestations of hepatitis. Hepatology.1996; 23:1608-1615.
[20]Yeo W,Chun SH. Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy [J]. Hepatology,2007,45(6):1382-1389.
[21]李昌明,肖青.细胞介素-6的临床应用进展[J].中国误诊学杂志,2007,7(1)：30-32.
[22]符正远,李勇忠,邓英,等.重型肝炎患者血清细胞因子的变化及其临床意义[J].中华感染控制杂志,2006,5(4)：306-308.
[23]Kuchroo V K, Dardalhon V, Xiao S, et al. New roles for TIM family members in immune regulation [J].Nat Rev Immunol,2008,8:577-580.
[24]祁建妮.Tim-4对小鼠免疫性肝炎的抑制作用研究[D].山东：山东大学,2009.
[25]Kobayashi N, Karisola P, Pena-Cruz V, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells[J], Immunity.2007,27(6):927-940.
[26]Litvack ML. Palaniyar N. Review:soluble innate immune pattern-recognitionproteins for clearing dying ceils and cellular components: implications on exacerbating or resolving inflammation[J]. Innate hnmun, 2010,16(3):191-200.
[27]Wang Q, Imamura R, Motani K, et al. Pyroptotic cells externalize eat-me and release find-me signals and a%e efficiently engulfed by macrophages[J]. Int Immunol,2013,25(6):363-372.
[2]陈圻,邓存良.TLR4在肝脏损伤中的作用研究进展[J].医学综述,2008,14(19)：2897-2899.
[3]Beutler B. The toll-like receptors:analysis by forward genetic methods[J]. Immunogenetics,2005,57(6):385-392.
[4]Katsargyris A, Klonaris C, Alexandrou A, et al. Toll-like receptors in liver ischemia reperfusion injury:a novel target for therapeutic modulation?[J]. Expert Opin Ther Targets,2009,13(4):427-442.
[5]Akira S, Uematsu S, Takeuchi O. Pathogen lecognition and innate immunity[J]. Cell,2006,124(4):783-801.
[6]Liew FY, Xu D, Brint EK, et al. Negative regulation of toll-like preceptor-mediated immune responses[J]. Nat Rev Immunol,2005,5(6):446-458.
[7]Janeway CA Jr, Medzhitov R. Innate immune recognition. Annu Rev Immunol,2002,20:197-216.
[8]Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell,2006,124:783-801.
[9]KURT-JONES E A, POPOVA L,KWINN L, et al. Pattem recognition receptors TLR4 and CD14 mediate response to respiratory syncytial vinIs[J]. Nat Immunol,2000,1(5):398-401.
[10]Yin Mo Bradford BU, Wheeler MD, et al. Reduced early alcohol-induced liver injury in CD14-deficient mice. J Immunol,2001,166:473-4742.
[11]Uesugi T, Froh M, Arteel GE, et al. Toll-like receptor 4 is involved in the mechanism of early alcohol-induced liver injury in mice. Hepatology,2001, 34:101-108.
[12]Masanori Isogawa, Michael D. Robek, Yoshihiro Furuichi,et al. Toll-Like Receptor Signaling Inhibits Hepatitis B Virus Replication In Vivo. J Virol.2005 Jun;79(11):7269-7272.
[13]Evans J T, Cluff C W, Johnson D A,et al. Enhancement of antigen-specific immunity via the TLR4 ligans MPL adjuvant and Ribi.529[J]. Expert Rev.Vaccines,2003,2(2):219-229.
[14]Dupont J, Altclas J, Lepetic A,et al. A controlled clinical trial comparing the safety and immunogenicity of a new adjuvanted hepatitis B vaccine with a standard hepatitis B vaccine[J]. Vaccine,2006,24(49-50):7167-7174.
[15]O'Neill L A,Bryant C E, Doyle S L. Therapeautic targeting Toll-like receptors for infection and inflammatory diseases and cancer[J]. Pharm.Rev.,2009,61(2): 177-197.
[16]Magness ST, Bataller R, Yang L, et al. A dual reporter gene trans-genic mouse demonstrates heterogeneity in hepatic fibrogenic cell populations[J]. Hepatology,2004,40(5):1151-1159.
[17]Seki E, De Minicis S, Osterricher CH, et al. TLR4 enhances TGF-β signaling and hepatic fibrosis[J]. Nat Med,2007,13(11):1324-1332.
[18]McIntire JJ, Umetsu SE, Akbari O, et al. Identification of Tapr (an airway hyperreactivity regulatory locus) and the linked Tim gene family. Nat Immunol.2001,2(12):1109-1116.
[19]Monney L, Sabatos C A, Gaglia J L, et al. Thl-specific cell surface protein TIM-3 regulates macrophage activation and severity of an autoimmune disease[J]. Nature,2002,415:536-541.
[20]Chan R C, Chan A, Jeon M, Wu T F, Pasqualone D, Rougvie A E, et al. Chromosome cohesion is regulated by a clock gene paralogue TIM-1[J]. Nature,2003,423:1002-1009.
[21]Meyer P, Saez L, Young M W. PER-TIM interactions in living Drosophila cells: an interval timer for the circadian clock[J]. Science,2006,311:226-229.
[22]Kuchroo V K, Meyers J H, Umetsu D T, DeKruyff R H. TIM family of genes in immunity and tolerance[J]. Adv Immunol,2006,91:227-249.
[23]Kuchroo V K, Umetsu D T, DeKruyff R H, Freeman G J. The TIM gene family: emerging roles in immunity and disease[J]. Nat Rev Immunol,2003,3:454-462.
[24]MEYERS J H, CHAKAVARTI S, SCHLESINGER D, et al. TIM-4 is the ligand For TIM-1,and the TIM-1-TIM-4 interaction regulates T cell proliferation[J]. Nat Immunol,2005,6:455-464.
[25]Umetsu S E, Lee W L, McIntire J J, et al. TIM-1 induces T cell activation and inhibits the development of peripheral tolerance[J]. Nat Immunol,2005,6: 447-454.
[26]Nakae S, Iwakura Y, Suto H, Galli S J. Phenotypic differences between Thl and Th17 cells and negative regulation of Thl cell differentiation by IL-17[J]. J Leukoc Biol,2007,81:1258-1268.
[27]Binn L L, Scott M L, Rennert P D. Human TIM-1 associates with the TCR complex and up-regulates T cell activation signals[J]. J Immunol,2007, 178:4342-4350.
[28]Chakravarti S, Sabatos C A, Xiao S, et al. Tim-2 regulates T helper type 2 responses and autoimmunity[J]. J Exp Med,2005,202:437-444.
[29]Koguchi K, Anderson D E, Yang L, et al. Dysregulated T cell expression of TIM3 in multiple sclerosis[J]. J Exp Med,2006,203:1413-1418.
[30]Zhao J,Lei Z,Liu Y,Li B,Zhang L,Fang H, et al. Human pregnancy up-regulates Tim 3 in innate immune cells for systemic immunity [J]. J Immunol,2009,182: 6618-6624.
[31]Mizui M, Shikina T, Arase H, Suzuki K, Yasui T, Rennert P D, et al. Bimodal regulation of T cell-mediated immune responses by TIM-4[J]. Int Immunol, 2008,20:695-708.
[32]Khademi M, Illes Z Alexander W, et al. T cell Ig-and mucin-domain-containing molecule-3(TIM-3)and TIM-1 molecules are differentially expressed on human Thl and Th2 cells and in cerebrospinal fluid-derived mononuclear cells in multiple sclerosis[J]. J Immunol,2004,172(11):7169-7176.
[33]Cai PC, Wu Qw, Wang L, et al. Distribution characteristics and linkage disequilibrium of TIM4 promoter polymorphisms in asthma patients of Chinese Han population[J]. J Huazhong Univ Sci Teehnolog Med Sci,2008,28(4): 447-450.
[34]夏义平,崔天盆,吴健民.TIM4基因多态性与湖北汉族变应性哮喘的相关性研究[J].中华医学遗传学杂志,2007,24(2)：213-216.
[35]Monney L, et al. Thl-specific cell surface protein Tim-3 regulates macrophage activation and severity of an autoimmune disease. Nature 2002,415:536-541.
[36]Sanchez-Fueyo A, et al. Tim-3 inhibits T helper type 1-mediated auto-and alloimmune responses and promotes immunological tolerance. Nat Immunol 2003,4:1093-1101.
[37]Jones RB, et al. Tim-3 expression defines a novel population of dysfunctional T cells with highly elevated frequencies in progressive HIV-1 infection. J Exp Med 2008;205:2763-2779.
[38]Golden-Mason L,et al. Negative immune regulator Tim-3 is overexpressed on T cells in hepatitis C virus infection and its blockade rescues dysfunctional CD4+ and CD8+T cells. J Virol 2009;83:9122-9130.
[39]Sanchez-Fueyo A, Tian J, Picatella D, et al. Tim-3 inhibits T helper type 1-mediated auto-and allo-immune responses and promotes immunological tolerance. Nat ImmunOl.2003,4(11):1093-1101.
[40]Kobayashi, N., P. Karisola, V. Pena-Cruz, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells. Immunity.2007,27:927-940.
[41]Miyanishi, M.,K. Tada, M. Koike, et al. Nagata. Identification of Tim4 as a phosphatidylserine receptor. Nature.2007,450:435-439.
[42]续力云,祁建妮,梁晓红,等.稳定高表达小鼠Tim-4巨噬细胞系RAW264.7-T4的建立[J].山东大学学报(医学版),2008,46(6)：551-555.
[43]钱晖,彭秀娟,许晓蒙,等.Tim4基因定量检测方法的建立及在消化道肿瘤中的初步应用[J].江苏大学学报(医学版),2009,19(6)：465-468.
[44]何金洋,张小虎,郭兴伯.中药治疗乙肝的机制研究进展[J].中医药学报, 2002,3(4)：65-66.
[45]杨鉴英等.中药抗乙型肝炎病毒的实验研究[J].中西医结合杂志,1989,9(8)：494.
[46]陈压西,郭树华,齐珍元,等.叶下珠有效部位抗鸭乙型肝炎病毒及体外抗HBV活性的研究[J].中药药理与临床,1996：15(6)：16-18.
[47]黄正昌,邓学龙,朱宇同,等.台湾细叶柚柑体外抗乙肝病毒作用研究[J].广州中医药大学学报,2000,17(3)：260-263.
[48]张晓刚,吕志平,谭秦湘等.白背叶根抗乙型肝炎病毒的体外实验研究[J].时珍国医国药,2006,17(8)：1437-1438.
[49]郭姗姗,高英杰,时宇静,等.一枝蒿总黄酮体外抗乙肝病毒作用机理的实验研究[J].中国实验方剂学杂志,2009,15(10)：72-74.
[50]范惠敏,肖会泉,高小玲.肝舒胶囊抗鸭乙型肝炎病毒体内实验[J].中国中西医结合杂志,2002,11(3)：207-208.
[51]王岭,张均倡,罗上武,等.叶下珠复方肝丹体外抗HBV作用特点的实验研究[J].中国中医基础医学杂志,1999,5(7)29-33.
[52]刘中景,熊曼琪,李塞美,等.小柴胡汤体外抗HBV作用及其组方机理的实验研究[J].中国中医基础医学杂志,2001,7(6)：17-19.
[53]吴国庆.赤珠清毒汤治疗慢性乙型肝炎94例疗效观察[J].中医药通报,2003,2(2)：119-121.
[54]范惠敏,肖会泉,吴婉芬等.肝舒胶囊对慢性肝炎患者免疫功能的影响[J].中国中西医结合脾胃杂志,1998,6(4)：210-211.
[55]徐德先.肝苏颗粒治疗慢性乙型肝炎128例的疗效观察[J].中国临床医药研究杂志,2000,31(12)：4926.
[56]李承栋.一种治疗慢性乙型肝炎的新药及制备方法[P].中国专利,CN1281701,2001-01-31.
[57]蔡永江,张秀婷,王立静.芪蓝扶正解毒剂对于HBsAg阳性转阴的临床观 察[J].中医现代医生,2007,45(22)：77-78.
[58]汪锐.中药治疗乙肝患者前后血清中ALT、AST及AST／ALT的变化[J].临床和实验医学杂志,2010,9(15)：1176-1177.
[59]钱涯邻.乙肝II号治疗慢性乙型病毒性肝炎临床研究[J].中国中医药信息杂志,2009,16(8)：68-69.
[60]钱涯邻,陈维铭,张燕.中药乙肝II号联合a-干扰素治疗慢性乙型肝炎临床观察[J].中国中医药信息杂志,2010,17(11)：70-71.
[61]刘厚佳,胡晋红,蔡溱,等.中药及其有效成分抗乙型肝炎病毒的研究进展[J].解放军药学学报,2000,11(1)：20.
[62]沈洪,张蓓.急性肝损伤研究进展[J].现代中西医结合杂志,2009,18(18)：2211-2213.
[63]Dey A, Cederbsum AI. Alcohol and oxidative liver injury. Hepatology.2006, 43(2 Suppl1):863-874.
[64]朱润芝,李京敬,谢超.过氧化作用与肝脏疾病[J].世界华人消化杂志,2010,18：1134-1140.
[65]Sakaguchi S. Furusawa S. Oxidative stress and septic shock:metabolic aspects of oxygen-derived free radicals generated in the liver during endotoxemia. FEMS Immunol Med Microbiol,2006.47:167-177.
[66]Grattagliano I, Bonfrate L, Diogo CV, et al. Biochemical mechanisms in drug induced liver injury:certainties and doubts. World J Gastroenterol,2009,15: 4865-4876.
[67]Katagiri H, Ito Y, Ito S, et al.TNF-a induces thromboxane receptor signaling-dependent microcirculatory dysfunction in mouse liver. Shock,2008, 30(4):463-467.
[68]宋润波,马建亭.IL-10对大鼠重症胰腺炎炎症相关细胞因子的调节作用[J].河北医药,2009,31：1451-1452
[69]Mits A, Hashikura Y, Tagawa Y, et al. Expression of Fas ligand by hepatic macrophages in patients with fulminant hepatic failure. Am J Gastroenteml, 2005,100:2551-2559.
[70]Kimura K, Sekiguchi S, Hayashi S, et al. Role of interleukin-18 intrahepatic inflammatory cell recruitment in acute liver injury. J Leukoe Biol, 2011,89(3): 433-42.
[71]胡骁骅,张会英,葛艳玲,等.乌司他丁对严重烧伤后多脏器功能的保护作用[J].中华医学杂志,2005,85：2889-2894.
[72]黄吕州,张宗明,裘法祖.缺血再灌注损伤埘大鼠肝细胞钙池操纵的钙通道电流的影响及药物拮抗[J].世界华人消化杂志,2005,13：739-742.
[73]刘亮明,罗杰,张吉翔.内毒素诱导D-半乳糖胺致敏大鼠急性肝衰竭的研究[J].中华医学杂志,2006,86：2122-2126.
[74]Webb c, Twedt D. Oxidative stress and liver disease. Vet Clin North Am Small Anita Pract,2008,38:125-135.
[75]钟卫卫,林世德.内质网应激与肝损伤研究进展[J].世界华人消化杂志,2010,18：1021-1025.
[76]伏建峰,史清海,路西春,等.大鼠慢性酒精诱导肝损伤中肝细胞凋亡与Fas表达[J].西北国防医学杂志,2006,27：362-364.
[77]Nanji AA,Jokehinen K,Rahemtulla A,et al. Activation of nuclear factor kappa B and cytokine imbalance in experimental alcoholic liver disease in the rat[J]. Hepatology.1999,30(4):934-943.
[78]Suetsugu H, Iimuro Y, Uehara T, et al. Nuclear factor{kappa} B in activation in the rat liver ameliorates short term total warm ischaemia/reperfusion injury[J]. Gut,2005,54(6):835-842.
[79]Ding WX, Tin XM. Dissection of the multiple mechanisms of TNF induced apoptosis in liver injury[J]. J Cell Mol Med,2004,8(4):445-454.
[80]李跃华,朱丽.哈团柱,等.刀豆素A诱导小鼠肝损伤增加肝脏NF-K B活性[J].中华肝脏病杂志,2003,11(2)：115
[81]赵宗豪,高人焘.核因子-κB与肝纤维化的研究进展[J].实用肝脏病杂志,2004,7(2)：119-121.
[82]Tiegs G, Hemschd J, Wendel A. A T cell dependent experimental liver injury in mice induced by concanavalin A[J]. J Clin Invest.1992,90:196-203.
[83]Nakagawa T, Zhu H, Morishima N, et al. Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta [J]. Nature, 2000,403:98-103.
[84]Gong W, Pecci A, Roth S, et al. Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand [J]. Hepatology,1998,28(2):492-502.
[85]黄才国.李医明,贺祥.玄参中苯丙素苷对大鼠肝损伤细胞凋亡的影响[J].中西医结合肝病杂志,2004,14(3)：160-161.
[86]杨小红,袁江,周远明,等.白首乌粗多糖对酒精性肝损伤的保护作用研究[J].时珍国医国药,2009,20(11)：2704-2705.
[87]陈旭,杜正彩.桃金娘多糖对大鼠急性肝损伤保护作用的研究[J].安徽农业科学,2010,38(11)：5663-5664.
[88]Xu H, Guo T, Guo Y, et al. Characterization and protection on acute liver injury of a polysaccharide MP-I from Mytilus coruscus[J]. Glycobiology,2008, 18(1):97-103
[89]宋淑亮,肖增平,吉爱.绞股蓝多糖对HepG2细胞酒精性损伤的保护作用[J].中国生化药物杂志,2008,29(5)：302-305.
[90]张波,徐永杰.牛蒡菊糖对亚慢性酒精肝损伤作用的研究[J].食品工业科技.2010,31(8)：329-331.
[91]吕雄文,李俊,金涌,等.豹皮樟总黄酮对小鼠急性酒精性肝损伤的保护 作用[J].中成药,2010,3(32)：489-491.
[92]林冠宇,姚楠,何蓉蓉,等.淡竹叶总黄酮对拘束负荷所致小鼠肝损伤的保护作用[J].中国实验方剂学杂志,2010,16(7)：177-179.
[93]李中华,涂燕云,赵晓芳.半枝莲总黄酮对肝纤维化大鼠血清白介素-10表达的影响[J].中药药理与临床,2010,26(2)：44-46.
[94]潘莹,张林丽.大果山楂黄酮提取物对四氯化碳致大鼠慢性肝损伤的保护作用[J.].时珍国医国药,2008,19(2)：318-319.
[95]韩刚,万红,翟冠钰,等.姜黄素固体分散体对四氯化碳致小鼠急性肝损伤的保护作用[J].时珍国医国药,2009,6(20)：1321-1322.
[96]喻松仁,程绍民,周步高,等.葛根总黄酮对大鼠慢性肝损伤的保护作用[J].江西中医学院学报,2010,22(2)：70-72.
[97]陈立波,郝六平,王宏,等.注射用盐酸苦参碱对卡介苗加脂多糖致小鼠免疫性肝损伤的保护作用[J].现代预防医学,2008,35(21)：4305-4306.
[98]洪振丰,李天骄,赵锦燕,等.粗叶悬钩子总生物碱对急性肝损伤大鼠肝组织CYP2E1与CYP3A1酶mRNA表达的影响[J].中国中西医结合杂志,2009,29(8)：711-715.
[99]毕明刚,周娟,许扬,等.岩黄连总碱提取物对小鼠免疫性肝损伤的改善作用[J].中国药理学与毒理学杂志,2009,23(1)：39-44.
[100]周劲光.岩黄连总碱对慢性肝损伤大鼠的肝保护作用观察[J].山东医药,2010,50(27)：11.
[101]王晓丽,郑立运,张艳,等.盐酸千金藤碱对急性肝损伤小鼠保护作用的研究[J].中华中医药杂志,2009,24,(10)：1384-1387.
[102]陈象青,王钦茂,方华武,等.白芍总苷与当归提取物合用对实验性肝炎的保护作用[J].安徽中医学院学报,2002,2(3)：44.
[103]李瑞麟,马勇,魏伟,等.白芍总苷治疗四氯化碳致大鼠肝纤维化的作用与其影响肝星状细胞功能的关系[J].中国新药杂志,2007,16(9)：685-689.
[104]邱彦,陈敏,茆玉国,等.注射用西黄总苷对四氯化碳致大鼠慢性肝损伤的保护作用研究[J].药学实践杂志,2008,26(3)：188-190.
[105]樊文研,罗力元.三七总皂苷对异烟肼和利福平合用所致小鼠肝损伤的保护作用[J].中国医药导报,2009,6(35)：7-8.
[106]卢春凤,陈廷玉,赵锦程,等.槲皮素对INH诱导的大鼠肝损伤及肝组织Bcl-2和Bax表达的影响[J].解剖学研究,2010,32(1)：28-31.
[107]荣黎,戴立里,曾维政,等.红景天甙对拟高原缺氧大鼠肝损伤的保护作用[J].中国组织工程研究与临床康复,2010,14(31)：5813-5817.
[108]张国良,黄川锋.芦荟苷对急性肝损伤小鼠NO和IFN-γ的影响[J].中药药理与临床,2010,26(1)：27-29.
[109]罗景慧,杨迎暴.白藜芦醇对酒精性肝损伤大鼠血清中TNF-α、IL-6、ICAM-1水平的影响及机制[J].中药药理与临床,2010,26(4)：14-16.
[110]林兴,黄权芳,张士军,等.六月青总皂苷对四氯化碳诱导小鼠肝损伤的脂质过氧化反应的影响[J].中成药,2009,12(31)：1937-1939.
[111]刘莉,管小琴,李芬,等.茶多酚对酒精性肝病大鼠核因子-K B及环氧合酶2表达的影响[J].中国实验动物学报,2008,3(16)：209-214.
[112]沈洪,朱路佳,薛洁,等.生姜油不同部位对小鼠急性肝损伤保护作用的比较[J].中成药,2008,9(30)： 8-10.
[113]冯文文,谭勇,李国玉,等.紫草提取物对小鼠急性肝损伤保护作用的研究[J].农垦医学,2010,32(2)：108-111.
[114]田立.决明子的不同提取物及炮制品对四氯化碳致小鼠急性肝损伤的保护作用[J].时珍国医国药,2008,8(19)：2005-2006.
[115]谢丽琼,李海芳,迪里努尔.小鼠中甘草毛状根、甘草切片总抗氧化能力和对CC14致肝损伤保护的比较研究[J].食品科学,2009,30(23)：432-435.
[116]周春晖,苏烨,李凤芝.金银花提取物对小鼠化学性肝损伤的保护作用初步研究[J].现代食品科技,2010,26(4)：351-353.
[117]苏晓聆,李福安,魏全嘉,等.秦艽水煎液对小鼠急性肝损伤肿瘤坏死因子-α和白细胞介素-10表达的影响[J].时珍国医国药,2010,21(4)：827-828.
[118]陈世林,洪汝涛,刁磊,等.水飞蓟素对大鼠急性酒精性肝损伤的保护作用[J].安徽医科大学学报,2010,45(2)：209-212.
[119]伍义行,郝冰洁,胡少青,等.六棱菊提取物对醋氨酚致小鼠肝损伤的影响[J].世界华人消化杂志,2010,18(7)：711-715.
[120]刘协,顾呈华,胡启之,等.甘草甜素脂质体对小鼠急性酒精性肝损伤的保护作用[J].中国临床康复,2004,8(36)：8260-8261.
[121]陈梁,朱锦,任建平.柴胡疏肝散对四氯化碳所致大鼠急性肝损伤的保护作用[J].中西医结合肝病杂志,2004,14(1)：42-43.
[122]李海燕,董礼阳,杨景云.四君子汤对急性肝损伤伴发菌群失调及内毒素血症的保护作用[J].中国微生态学杂志,2005,17(2)：100-101.
[123]苏晓聆,李福安,魏全嘉,等.秦艽水煎液对小鼠急性肝损伤肿瘤坏死因子-a和白细胞介素-10表达的影响[J].时珍国医国药,2010,21(4)：827-828.
[124]马丽,高玉杰,钱月慧,等.丹葛解醒汤对大鼠酒精性肝损伤的保护作用研究[J].时珍国医国药,2010,21(7)：1596-1597.
[125]薛晓伟,丰平,董坤,等.槲芪散对小鼠酒精性肝损伤的干预作用[J].实验动物科学,2010,27(4)：27-32.
[126]Tungyuan Lai, Yijiun Weng, Weiwen, Kuo, et al. Taohe Chengqi Tang ameliorates acute liver injury induced by carbon tetrachloride in rats[J]. Journal of Chinese Integrative Medicine,2010,8(1):49-55.
[127]张引强,唐旭东,王凤云,等.荣肝合剂对刀豆蛋白A诱导急性免疫性肝损伤小鼠的保护作用[J].中医杂志,2010,51(5)：453-456.
[128]孔祥才,张方信,陈嘉屿,等.大黄甘草汤对大鼠重症急性胰腺炎并发肝损伤时核因子-K B及细胞因子的影响[J].中国中西医结合消化杂志,2010, 18(2)：82-85.
[129]林旭楷,张奕,许森源.胃舒散对五氯硝基苯致肝损伤的保护作用[J].广州中医药大学学报,2009,26(4)：371-373.
[130]蒋洁云,徐强.四逆散及其各组成中药对实验性肝损伤的影响[J].中国天然药物,2004,2(1)：45-49.
[131]刘思纯,马博.甘草酸二铵治疗急性药物性肝损伤的疗效观察[J].中国处方药,2006,10(55)：62-64.
[132]Lino Set al.Hepatal Res 2001,19(1):31-40.
[133]吉孝祥,夏仲芳.黄芪对有机磷农药中毒患者肝脏的保护作用研究[J].现代中西医结合杂志,2003,12(20)：2158.
[134]刘成,薛惠明,徐列明,等.人工冬虫夏草苗丝制剂治疗肝炎后肝硬化22例[J].上海中医药杂志,1986(6)：30-31.
[135]周良楣,续月明,朱庆义,等.冬虫夏草菌丝治疗慢性乙型病毒性肝炎的近期疗效观察[J].中国中药杂志,,1990,15(1)：53.
[136]徐列明,刘成,刘平.桃仁提取物合虫草菌丝治疗肝炎后肝硬化的病理免疫组化研究[J].中西医结合肝病杂志,1994,(4)：9.
[137]巫善明.参三七注射液治疗27例久治难愈的血淤型慢性肝炎疗效观察[J].中成药研究,1978,3(4)：41.
[138]高寒.红参、三七、激素等联用治疗重型肝炎70例报告[J].中华内科杂志,1980,19(4)：313.
[139]张火昆.三七粉降低谷丙转氨酶及改善血浆蛋白的观察[J].中医杂志,1980,21(5)：25.
[140]路彬,刘晓丹,高墉发,等.苦参碱注射液治疗肝功能异常40例疗效分析[J].现代中西医结合杂志,2005,14(4)：495.
[141]吴福全.苦参碱治疗慢性乙型肝炎疗效观察[J].实用医学杂志,2002,18(11)：1232-1234.
[142]卢宝玲,钟丽华.苦参素治疗慢性乙型肝炎疗效观察[J].黑龙江医学,2002,26(1)：46-48.
[143]江远,熊丽.莪术治疗肝病的研究进展[J].中西医结合肝病杂志,2005,15(2)：127-129.
[144]李宝红,梁念慈.莪术油制剂的临床应用及实验研究进展[J].中药材,2003,26(1)：78-71.
[145]韩铭钧,任克.油性中药抗癌剂OCC肝动脉栓塞治疗肝癌84例3年随访报告[J].临床放射学杂志,1998,17(2)：112-114.
[146]陈春永,徐凯.莪术油肝动脉灌注栓塞治疗继发性肝癌28例疗效观察[J].新中医,2003,35(3)：23-24.
[147]陈斌,孙克伟,谢凤瑛,等.莪术治疗慢性肝损伤时的肝毒作用研究[J].中西医结合肝病杂志,2003,13(1)：20.
[148]江远,熊丽.莪术治疗肝病的研究进展[J].中西医结合肝病杂志,2005,15(2)：127-129.
[149]高尚社.35年茵陈蒿汤治疗传染性黄疸型肝炎的研究进展[J].云南中医杂志,1985,(5)：54-57.
[150]张志勇.加味茵陈蒿汤治疗黄疸疗效观察[J].湖北中医杂志,2007,29(9)：33.
[151]石定,艾宏伟,王文菊.乌鸡白凤丸与马洛替酯联合治疗肝炎后肝硬化低白蛋白血症40例[J].中国中西医结合脾胃杂志,2000,8(4)： 247.
[152]刘洪明,毕青,张俊富.乌鸡白凤丸对肝硬化白蛋白的影响[J].天津中医药,2003,20(1)：29.
[153]方焱明.鳖甲煎丸合乌鸡白凤丸治疗慢性血吸虫病肝纤维化46例[J].时珍国医国药,2004,15(12)：846.
[154]欧阳云.发泡疗法合乌鸡白凤丸治疗慢性乙型肝炎9例[J].中国民间疗法,2003,11(11)： 9.
[155]何炳福,黄丹文.小柴胡汤对乙肝病毒携带者治疗疗效观察[J].浙江中西医结合杂志,2007,17(1)：34-35.
[156]王彦刚,周琰,杨金国,等.痛泻要方合小柴胡汤加减治疗脂肪肝疗效观察[J].河北中医药学报,2007,22(1)： 6-8.
[157]梁炳根,莫尤美.芍药甘草汤治疗148例病毒性肝炎的临床观察[J].上海中医药杂志,1989(6)：4-6.
[158]马列清.逍遥散加减对乙型病毒性肝炎肝纤维化指标的影响[J].北京中医,2002,21(5)：285-286.
[159]芦连菊.参麦注射液对化疗后肝功能损伤的临床疗效观察[J].中草药,2000,31(12)：92.
[1]吕志平.红背叶根在制备治疗慢性肝炎药物中的应用：[P]中国专利：200610 122307
[2]Sarent JM, Tlaylor CG. Appeaisal of the MTT assay as a rapid test of chemosensitivity in acute myeloid leukemia. Br J Cancer,1989.60(2):206-210.
[3]蒋美娟,李玉虎.中药单方乙醇提取液抗乙肝病毒的实验研究[J].医学信息,2011,24(8)：4910-4912
[4]郑浩杰.利用2.2.15细胞株筛选抗乙肝病毒中草药的体外实验研究进展[J].陕西中医学院学报,2003,26(2)：61-63
[5]Sells M A, Chen ML,Aes G. Production of hepatitis B virus particles in Hep-G2 cells transfected with cloned hepatitis B virus DNA[J]. ProcNad Acad Sci USA,1987,84:1005
[6]中华人民共和国卫生部药政管理局.中药新药研究指南[S].北京：1994.82
[7]宋晓凯.天然药物化学[M].北京：化学工业出版社,2004：21-22
[8]张燕明,刘妮,朱宇同,等.诃子醇提物抗HBV的体外实验研究[J].中医药学刊,2003,21(3)：383-4
[9]刘妮,朱辫弦,黄正昌,等.大黄醇提液对2.2.15细胞分泌HBsAg、HBeAg的抑制作用[J].中药材,2004,27(6)：419-421
[10]周长征.生物碱类成分抗病毒研究进展[J].健康必读,2012,11(5)：5
[11]杨志伟,周娅,曹秀琴.苦豆子生物碱体外抗柯萨奇B3病毒的作用[J].四川中医,2003,21(3)：14-16
[12]黄洁春,吕志平,刘强,等.红背叶根提取物对四氯化碳致小鼠肝损伤的作用研究[J].陕西中医,2011,32(1)：113-5
[13]金奇.医学分子病毒学[M].北京：科技出版社,2001：338
[1]Sells MA, Mei Ling Chen, George Acsl. Production of hepatitis B virus particles in HepG2 cells transfected with cloned hepatitis B virus DNA. Cell Biol,1987:1005-1009.
[2]罗红雨,杨旭,邹文,等.2.2.15细胞上清HBVDNA定量检测方法的比较P[J].中国现代医学杂志,2005,1(3):383-387.
[3]任建林,焦兴元.现代消化病诊疗学[M].第一版.人民军医出版社.2007：366-367.
[1]MASON WS, SEAL G, SUMMERS J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus[J]. J Virol,1980,36: 829-836.
[2]胡权,杨东亮.鸭乙型肝炎病毒感染模型的研究进展[J].实验肝脏病杂志,2007,10(3)：201-204.
[3]杨书兰,张奉学,朱宇同,等.用PCR法筛选先天感染鸭乙肝模型及与后天感染模型的比较[J].广州中医药大学学报,2001,18(3)：256-259.
[4]Ganem D, Schneider RJ. Hepadnaviridae:The viruses and their replication. In: Knipe DM, Howley PM, eds. Fields Virology.Volume 2. Philadelphia: Lippincott, Williams & Wilkins,2001; 2923-2969.
[5]Anneke Funk, Mouna Mhamdi, Hans Will.Avian hepatitis B viruses:Molecular and cellular biology,phylogenesis, and host tropism.World J Gastroenterol 2007; 13(1):91-103.
[6]MASON WS, CULLEN J, SAPUTELLI J, et al. Characterization of the antiviral effects of 2carbodeoxyguanosine in ducks chronically infected with duck hepatitis B virus [J]. Hepatology,1994,19(2):398-411.
[7]OFFENSPERGER WB,OFFENSPERGER S, KEPPLERHAFKEMEYER A, et al. Antiviral activities of Denciclovirand famciclovir on duck hepatitis B virus in vitro and in ViVo[J]. Antivir Ther,1996,1(3):141-146.
[8]LIN E, LUSCOMBE C, COLLEDGE D, et al. Long term therapy with the guanine nucleoside analog penciclovir controls chronic duck hepatitis B virus infection in vivo [J]. Antimicrob Agents Chemother,1998,42(8):2132-2137.
[1]唐云安,刘玉清,王国钦.肝损伤动物模型研究进展[J].卫生毒理学杂志,2002；16(4)：236-238.
[2]宋雨鸿,刘强,陈育尧,等.红背叶根对小鼠免疫性肝损伤的保护作用[J].南方医科大学学报,2008,28(3)：494-496.
[3]黄洁春,吕志平,刘强,等.红背叶根提取物对四氯化碳致小鼠肝损伤的作用研究[J].陕西中医,2011,32(1)：113-115
[4]闫冰,蔡秀江,姚卫峰等.二至丸保肝活性成分群对CCl4致小鼠急性肝损伤保护作用研究[J].中国中药杂志,2012,37(9)：1303-1305.
[5]广东中药志编辑委员会.广东中药志(第二卷)[M].广东科技出版社出版,1996：365-367.
[6]吕小燕,刘强,吕志平.红背叶根拮抗酒精性肝纤维化大鼠模型的作用机理研究[J].南方医科大学学报,2007,27(2)：153-155.
[7]吕志平.红背叶根在制备治疗慢性肝炎药物中的应用：中国,200610122307.4
[8]Tiegs G, Hentschel J, Wendel A. T Cell-dependent experimental liver injury in mice inducible by concanavalin A[J]. J Clin Invest,1992,90(1):196-203.
[9]Ganmer F, Leiat M, Lohse AW, et al. Concanavalin A induced T Cell-mediated hepatic injury in mice:The role of tumor necrosis factor[J]. Hepatology,1995,21(1):1901-98.
[10]曲建慧,张修礼.小鼠Con-A性肝损伤模型[J].世界华人消化杂志,2001,9(5)：571-574.
[11]邓利娟,李湛军,罗楹等.Caspase抑制剂F1013对刀豆蛋白A诱导小鼠急性肝损伤的治疗作用[J].中国药理学通报,2012,28(1)：136-139.
[12]吕小燕,吕志平,孙学刚,等.红背叶根对肾问质纤维化大鼠血流变及微循环的影响[J].北京中医药大学学报,2010,33(6)：398-401
[1]刘丹卓,赵新广.肝损伤病因病理机制及中医药治疗研究近况[J].国医论坛,2006,21(6)：53-55
[2]张定凤.乙型肝炎的发病机理及临床[M].重庆出版社,1992：1.
[3]Y H Zou,Y Yang, J Li, WPLi, Q Wu. Prevention of hepatic mjury by a
[4]traditional Chinese formulation, BJ-JN, in mice treated with Bacille-Calmette-Gu&in and lipopolysaccharide[J]. J Ethnopharrnacol,2006; 107:442-448.
[5]肖碧跃,赵国荣,贺又舜,等.理脾护肝调脂丸对脂肪肝大鼠肝脏MDA,SOD影响的研究[J].中国实验方剂学杂志,2011,17(11)：230.
[6]王君明,崔瑛,季莉莉,等.超氧化物歧化酶参与肝损伤的研究进展[J].中国实验方剂学杂志,2011,17(4)：265.
[7]周艳丽,张磊,刘维.白芍总苷对雷公藤多昔片所致小鼠急性肝损伤保护作用的实验研究[J].天津中医药,2007,24(1)：61.
[8]许金鹏,张慧慧,李朝品,等.原卟啉钠对四氯化碳致小鼠急性肝损伤的保护作用及其机制[J].中国实验方剂学杂志,2011,17(5)：168.
[9]Masayo Hatano, Soichiro Sasaki, Shinsuke Ohata, et al. Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis. Cellular Immunology. 2008,251:25-30.
[10]Schumann J,Wolf D, Pahl A,et al. Importance of Kupffer cells for T-cell-dependent liver injury in mice. Am J Pathol.2000,157:1671-1683.
[11]Y. Tagawa, K. Sekikawa, Y. lwakura, Suppression of concanavalin A-induced hepatitis in IFN-gamma (_/_)mice. but not in TNF-alpha(_/_)mice:role of IFN-gamma in activating apoptosis of hepatocytes. J. Immunol.1997,159: 1418-1428.
[12]S. Kusters, G. Tiegs, L. Alexopoulou, et al. In vivo evidence for a functional role of both tumor necrosis factor(TNF) receptors and transmembrane TNF in experimental hepatitis. Eur. J. Immunol.1997,27:2870-2875.
[13]G. Kunstle, H. Hentze, P. G. Germann, et al. Concanavalin A hepatocytotoxicity in mice:tumor necrosis factor-mediated organ failure independent of caspase-3-like protease activation. Hepatology.1999,30: 1241-1251.
[14]Kremer M, Hines IN, Milton RJ, et al. Favored T helper 1 response in a mouse model of hepatosteatosis is associated with enhanced T cell-mediated hepatitis[J]. Hepatology,2006,44(1):216-227.
[15]Tiegs C, Hentschel J, Wendel A. T Cell-dependent experimental liver injury in mice inducible by Concanavalin A[J]. J Clin Invest,1992,90(1); 196-203.
[16]Gantner F, Leist M, Lohse AW,et al. Coneanavalin A induced r-Cell-mediated hepatic injury in mice:The role of tumornecrosis factor[J]。Hepatology,1995, 21(1):190-198.
[17]Trautwein CR, Akemann T, Brenner DA. Concanavalin A induced liver cell damage:activation of intracellular pathways triggered lby tumor necrosis factor in mice[J]. Gastroenterology,1998,114(5):1035-1045.
[18]Mizuhara H, Uno M, Seki Y, et al. Critical involvement of interferon gamma in the pathogenesis of T-cell activation-associated hepatitis and regulatory mechanisms of hepatitis [J]. Hepatology,1996,23(6):1608-1615.
[19]Mizuhara H, Uno M, Seki N, et al. Critical involvement of interferon-y in the pathogenesis of T-cell activation associated hepatitis and regulatory mechanisms of interleukin-6 for the manifestations of hepatitis. Hepatology.1996; 23:1608-1615.
[20]Yeo W,Chun SH. Hepatitis B viral load predicts survival of HCC patients undergoing systemic chemotherapy [J]. Hepatology,2007,45(6):1382-1389.
[21]李昌明,肖青.细胞介素-6的临床应用进展[J].中国误诊学杂志,2007,7(1)：30-32.
[22]符正远,李勇忠,邓英,等.重型肝炎患者血清细胞因子的变化及其临床意义[J].中华感染控制杂志,2006,5(4)：306-308.
[23]Kuchroo V K, Dardalhon V, Xiao S, et al. New roles for TIM family members in immune regulation [J].Nat Rev Immunol,2008,8:577-580.
[24]祁建妮.Tim-4对小鼠免疫性肝炎的抑制作用研究[D].山东：山东大学,2009.
[25]Kobayashi N, Karisola P, Pena-Cruz V, et al. TIM-1 and TIM-4 glycoproteins bind phosphatidylserine and mediate uptake of apoptotic cells[J], Immunity.2007,27(6):927-940.
[26]Litvack ML. Palaniyar N. Review:soluble innate immune pattern-recognitionproteins for clearing dying ceils and cellular components: implications on exacerbating or resolving inflammation[J]. Innate hnmun, 2010,16(3):191-200.
[27]Wang Q, Imamura R, Motani K, et al. Pyroptotic cells externalize eat-me and release find-me signals and a%e efficiently engulfed by macrophages[J]. Int Immunol,2013,25(6):363-372.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |