【作者】 王根生；

【导师】 刘耕陶；

【作者基本信息】 中国协和医科大学 ， 生化药理， 1994， 博士

【摘要】 肝炎的治疗仍是一个巨大难题。虽然用于治疗肝炎的药物品种繁多，但有效者则廖廖无几，既往对保肝药的研究是建立在对化学性肝损伤的保护基础之上。化学性肝损伤与人体肝炎在发病机制上相差甚远。所以，经常出现这样的情况即有些对化学性肝损伤有效的药物在临床治疗肝炎时却未见明显疗效。为开辟新的研究途径、建立新的研究方法，我们建立了卡介苗(BCG)加脂多糖(LPS)免疫性肝损伤模型。近年研究表明，肝炎的发病与积聚在肝脏中的炎症细胞有密切关系，一氧化氮(NO)和肿瘤坏死因子(TNF)是炎症细胞释放的主要介质。关于NO在肝损伤中的作用国内尚未有人涉及。本文首次对)NO在BCG+LPS肝损伤中的作用进行探讨，并对肿瘤坏死因子在免疫性肝损伤中的作用进行研究，以期为寻找保肝新药开辟新途径。Sy-801是我所研制的继DDB之后第二代保肝新药。Sy-640也是我所研制的另一保肝新药。既往实验表明，这两种药对化学性肝损伤具有显著保护作用，且效果优于DDB。本文旨在探讨NO、TNF与肝细胞损伤的关系以及抗肝炎新药Sy-8O1和Sy-640对此的影响及其作用机理。结果如下：1．BCG+LPS可诱导小鼠血浆一氧化氮(NO)剧烈升高及严重的肝损伤。抑制NO的合成则可加重肝损伤的程度，而促进NO的合成则可减轻肝损伤的程度。四氯化碳、扑热息痛及D-半乳糖氨虽使小鼠血浆转氨酶剧烈升高，但并诱导血浆NO的显著变化。2．BCG十LPS可诱导小鼠血浆肿瘤坏死因子的显著升高。脾切除对BCG+LPS诱导小鼠血浆NO和TNF升高及肝损伤的程度均无显著影响。3．激活枯否氏细胞，导致BCG+LPS诱导的小鼠血浆NO及TNF更趋升高，并伴有肝损伤程度的增加；而抑制枯否氏细胞功能，则降低BCG+LPS诱导的小鼠血浆NO及TNF的升高，并减轻肝损伤。4．预先给小鼠口服Sy-801及Sy-640均使BCG+LPS诱导小鼠血浆NO进一步升高，TNF显著降低，肝损伤程度减轻。药物的作用呈剂量依赖关系。5．Sy-801及Sy-640显著降低BCG+LPS诱导小鼠腹腔巨噬细胞释放NO及TNF；药物的作用呈剂量依赖关系。6．Sy-801及Sy-640均不同程度地抑制BCG+LPS诱导小鼠腹腔巨噬细胞TNF mRNA水平的升高。7．将小鼠肝细胞与BCG+LPS激活的小鼠腹腔巨噬细胞共同培养，可导致培养体系中NO和瓜氨酸含量显著升高及肝细胞表面膜结构的损伤。Sy-801及Sy-640均可降低上述系统中NO和瓜氨酸的水平，并对肝细胞表面膜结构的损伤具有显著保护作用。以上结果表明：NO参与了BCG+LPS诱导的肝损伤且呈现一定的保护作用。NO在化学性肝损伤中所起的作用极微。看来，NO在免疫性肝损伤中呈现双向作用。来源于吞噬细胞的NO具有损伤作用，而其它来源的NO则似有保护肝细胞损伤的作用。TNF水平的升高与BCG+LPS诱导的肝损伤程度呈平行关系。脾脏在BCG+LPS诱导小鼠肝损伤中所起的作用极微。枯否氏细胞通过释放NO及TNF从而介导肝损伤。抗肝炎新药Sy-801及Sy-640的保肝机理与它们升高血浆NO及降低TNF基因表达有关。本文首次发现NO参与了BCG+LPS诱导的肝损伤，为研究肝损伤的产生机制和保肝药的作用机理，提供了新的途径和方法，具有重大的理论意义和实际意义。更多还原

【Abstract】 The therapy of viral hepatitis is still a big difficulty, although various kinds of drugs have been used in the clinic. In the past decades, the investigation of anti-hepatitis drugs was based on the protection against chemical-induced liver injury. It is well known that chemical-induced liver injury by certain chemicals differs far from human hepatitis in the aspect of pathogenesis. Some drugs are effective in chemical-induced liver injury but showed no remarkable effects in human hepatitis. In order to explore a new therapeutic path and new method for study the mechanism underlying the liver injury and the mechanism of protective action of drugs against liver damage, we have established an immunological liver injury model by injection of micro lipopolysaccha-ride(LPS) into BCG(bacilli Calmette Guérin)-primed mice. The pathogenesis of BCG+LPS induced hepatitis was reported to be closely related to the infiltration of inflammatory cells into the liver.Nitric oxide(NO) and tumor necrosis factor(TNF) are two main inflammatory mediators released from phagocytes. The role of NO in liver injury induced by BCG+LPS has not been studied both domestically and aborad. The present studies were to evaluate the role of NO and TNF in liver damage in order to lay a base for exploring a new approach for searching hepato- prectants.Sy-801, the second generation of DDB, and Sy-640 are two new hepatoprectants developed by our institute. Both Sy-801 and Sy-640 showed significantly protective action against chemical-induced liver injury. The potency of the two hepatoprectants are greater than DDB. The present paper reports the role of NO and TNF in immunological liver injury induced by BCG+LPS in mice as well as the mechanisms of protective action of both Sy-801 and Sy-640 on this immunological liver injury model. The results are described as follows:Remarkable plasma NO elevation and severe liver necrosis were induced by i. v. injection of LPS into BCG-primed mice. The inhibition of NO synthesis by N~G- monomethyl-L-arginine, an inhibitor of NO synthase, resulted in exacerbation of liver injury. However, promotion of NO synthesis induced alleviation of liver damage.TNF was also involved in liver damage induced by BCG plus LPS. Splenectomy neither showed influence on BCG+LPS induced increases of plasma NO and TNF levels, nor alter the degree of liver necrosis.Activation of Kupffer cells by large doses of retinol induced further increase of plasma NO and TNF levels, and was associated with increment of liver necrosis induced by BCG+LPS. In contrast, suppression of Kupffer cells by silica or carbon resulted in reduction of plasma NO and TNF levels, and was related to the alleviation of liver necrosis.Pretreatment of mice with Sy-801 and Sy-640, the two new anti-hepatitis drugs, induced further increase of plasma NO level and reduction of TNF level induced by BCG+LPS, and amelioration of the liver damage.Sy-801 and Sy-640 exhibited significant inhibiting effect on TNF and NO production by BCG-elicited murine macrophages when stimulated with LPS.Sy-801 and Sy-640 significantly inhibited BCG+LPS induced elevation of TNF mRNA level in murine peritoneal macrophages.Marked increases of NO and citrulline levels and damage of hepatocyte surface architecture were noticed when the mouse hepatocytes were cocultured with BCG- primed murine peritoneal macrophages in case of challenge with LPS. Sy-801 and Sy-640 significantly inhibited the production of NO and citrulline and ameliorated of the damage of the hepatocyte surface membrane.It may be deduced from the above results that NO plays a dual action on liver damage, i. e. damaging and protective. TNF released by macrophages is also one of the key factors leading to the damage of liver cells. Kupffer cells were involved in the pathogenesis of BCG+LPS induced liver injury by releasing NO and TNF. The spleen plays a minimal role in BCG+LPS induced liver injury in mice. The mechanism(s) by which Sy-801 and Sy-640 protect BCG+LPS induced damage may be the results of stimulating NO production by certain cells and inhibition of TNF expression by macrophages.更多还原