研究背景与目的：随着分子生物学和细胞生物学的不断进展，对肝纤维化的发生机制有了进一步认识。肝纤维化是各种慢性肝病发展为肝硬化的共同病理过程，是肝脏内纤维组织过度沉积、肝脏细胞外基质(ECM)的合成与降解失衡的结果，其中心环节是肝星状细胞(HSC)的活化。它是肝硬化的早期阶段和必经阶段，在一定情况下可被逆转，若病因持续存在，肝纤维化逐渐加重，即发展为不可逆转的肝硬化。20世纪50年代，沙利度胺(thalidomide，反应停)在欧洲曾广泛应用于怀孕相关的晨吐，因其有强烈的致畸作用而退出世界市场。后来，因其对一系列炎性或免疫相关性疾病有一定疗效，因此，沙利度胺越来越多的用于免疫功能异常所致的器官损伤。细胞粘附分子-1 (ICAM-1)是免疫球蛋白超家族成员之一，作为免疫分子对介导T1淋巴细胞对肝细胞的损伤及肝纤维化的发病起着关键作用。有报道沙利度胺可以显著降低人T白血病细胞和人脐静脉内皮细胞粘附分子ICAM-1和淋巴细胞功能相关抗原-1 (LFA-1)的表达和细胞之间的粘附。据报道，沙利度胺可抑制TNF-A和IL-1的生成，故其被应用于治疗实验性肝纤维化疗效是令人乐观的。沙利度胺可以不同的方式、不同的细胞水平上影响白细胞、内皮细胞等靶细胞，改变粘附分子的浓度导致炎症组织的白细胞外渗及抑制炎症反应。这种对粘附分子下调机制能否用于对肝纤维化的治疗目前仍罕见。NF-κB是一种具有转录激活功能的蛋白质，对多种细胞因子有重要调控作用。最常见的是由P65和P50形成的异源二聚体，NF-κB已被证实其在炎症反应时对多种细胞因子在转录水平进行调控。有研究表明，NF-κB活性增强时，P65明显增加，KC分泌IL-1、IL-6等炎症介质也增多，通过炎症反应放大，诱导纤维化因子等基因表达，激活HSC介导肝纤维化。TNF-α还可诱导中性粒细胞和巨噬细胞大量浸润，增强KC作用。因此TNF-α、IL-1与NF-κB之间造成细胞因子网络中的恶性循环。抑制纤维化肝组织NF-κB/IκB信号道路时沙利度胺的作用靶点之一。T辅助细胞(Th细胞)是肝内细胞因子的又一来源，不同的Th细胞通过产生不同细胞因子影响肝纤维化进程，在肝纤维化的发病机制中Th细胞的失衡发挥了重要作用。又报道Th2反应有利于形成肝纤维化，Th1反应则相反。但目前尚唯有Th细胞失衡参与沙利度胺治疗肝纤维化有关机制的报道。本研究的目的是观察沙利度胺对猪血清诱导的大鼠肝纤维化的影响和分析其同ICAM-1、IL-1、TNF-α，NF-κB和Th细胞免疫调节表达的影响。 方法：雄性Wistar大鼠80只随机分为正常对照组、模型组、沙利度胺小剂量治疗组和沙利度胺大剂量治疗组(每组20只)。模型组和治疗组大鼠以猪血清腹腔注射诱导大鼠肝纤维化模型，正常对照组大鼠则给与等量花生油腹腔注射。自第一周起，治疗组大鼠给予沙利度胺灌胃，小剂量治疗组用量为每日20㎎/㎏，大剂量治疗组用量为每日100㎎/㎏，共7周；正常对照组和模型对照组大鼠给予等量生理盐水灌胃，共7周。所有大鼠于试验7周末处死，收集血清和肝脏备用。 HE染色和Masson三色染色检测肝组织病理形态学改变。生化法检测血清丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、前白蛋白(PA)、白蛋白(ALB)、球蛋白(GLB)；放免法检测血清透明质酶(HA)、层粘连蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型胶原(CⅣ)、Ⅰ型前胶原(PC Ⅰ)；免疫组化法检测肝组织ICAM-1、 NF-κBp65、TNF-α、IL-1、IL-10； IL-2、IFN-γ；Westen blot 法检测肝组织ICAM-1、TNF-α ，IL-1、IL-10、IL-2，IFN-γ 细胞浆蛋白和 NF-κBP65 核蛋白表达；逆转录-聚合酶链反应(RT-PCR)方法测定肝组织ICAM-1、TNF-α、NF-κ BP65、IL-1mRNA 的表达。 结果：大剂量沙利度胺治疗组与模型组相比：可以显著减轻肝细胞坏死和纤维增生，使纤维间隔变薄和减轻炎症细胞浸润，使肝小叶结构显著改善；显著降低肝组织 Knodell积分(P＜0.01)；显著降低血清 ALT、AST、HA、LN、PCⅢ、C Ⅳ、PC Ⅰ水平，显著升高PA和ALB水平(P＜0.01)；显著降低肝组织ICAM-1、 TNF-α、NF-κBP65、IL-1 mRNA(P＜0.01)；显著降低肝组织 ICAM-1、NF-κBP65、 IL-1、TNF-α、IL-10 蛋白表达，升高IL-2、IFN-γ 蛋白表达(P＜0.01)； NF-κ BP65mRNA表达与ICAM-1、TNF-α、IL-1mRNA表达之间存在较强的正相关关系(VS ICAM-1，r=0.78，P＜0.01； VS TNF-α，r=0.72 ，P＜0.01； VS IL-1，r=0.82， P＜0.01)。 结论：沙利度胺可以有效地抑制肝纤维化的发展，通过阻断NF-κB信号通路从而抑制粘附分子、IL-1、TNF-α的表达，以及可能与机体免疫调节功能有关。 关键词：沙利度胺 肝纤维化 核因子-Kappa B 粘附分子 Th细胞

 Background and aims: With the development of molecular biology and cell biology, the understanding on the mechanism of how liver fibrosis happens. Liver fibrosis is a common pathology process during the course which various kinds of chronic liver disease develops into liver cirrhosis, it's also the consequence caused by over-deposit of the fibrous tissue in liver, and the synthesis and dagradation disequilibrium of extracellular matrix (ECM) of the liver cell. Liver fibrosis is the early but inevitable stage in liver cirrhosis, and can be reversed under certain condition. However, if the etiological factor continues to exist, the liver fibrosis gradually aggravates and eventually develops into irreversible liver cirrhosis. In 1950s, thalidomide was widely used in morning sickness caused by pregnance in Europe.but it was withdrawn from the world market for its well-known tragic teratogenic effects. Afterwards, thalidomide was more and more widely used in organ damage caused by immune disfunction because it was found to have curative effect in a series of inflammation and immunity relevant disease. Being one of the members of immunoglobulin superfamily, Intercellular adhesion molecular-1 (ICAM-1) plays a key role in mediating the injury of the T1 lymphcyte to liver cells as well as in the pathogenesis of liver fibrosis. It has been reported that thalidomide can significantly reduce cell adhesion molecule expression such as ICAM-1 and LFA-1 in human T leukemic cells and human umbilical vein endothelial cells and cell adhesion between them. According to the report, thalidomide can inhibit the production of TNF-α and IL-1, so thalidomide can be applied to treat the experimental liver fibrosis and its curative effect is optimistic. Thalidomide targets leucocytes and endothelial cells, affecting them in different manners and at different cellular levels, and induces the changes in the density of adhesion molecules, which alter leucocyte extravasation and the inflammatory response in the tissue involved. Whether the down-regulation of adhesion molecules by thalidomide can be used in the treatment of liver fibrosis is still rare yet. Nuclear factorκB (NF-κB) is a functional protein depending on activation of transcription, which plays a regulative function in many types of cytokine. The heterodimer NF-αB, composed of p65 and p50, has been proved to regulate many types of cytokine in transcription level in inflammtory action. Some research indicated that when NF-αB activity enhances,p65 increases manifestly, mediators of inflammation such as IL-1、IL-6 secreted by KC also increases, and this will induce gene expression of fibrosis factor,and activate HSC to mediate liver fibrosis. TNF-α can also induce massive infiltration of neutrophilic granulocyte and macrophages, which enhances the function of KC. So TNF-α、IL-1 and NF-κB result in a infernal circle among the cytokine network. One of the function targets of thalidomide is the suppression of NF-κB/IkB signaling pathway in fibrous hepatic tissue. Th cell is another source for liver cytokine, different types of Th cells affect liver fibrosis process by generating different types of cytokine, and the disequilibrium of Th cell plays a key role in the pathogenesis of liver fibrosis. It has been reported that Th2 reaction is advantageous to liver fibrosis, while Th1 reaction on the contrary. There aren't any reports yet on the mechanism how the disequilibrium of Th cell plays its role in thalidomide when it cures liver fibrosis. The aim of this study is to inverstigate the effect of thalidomide on pig blood serum induced rat liver fibrosis and to analyze its association with the expressions of ICAM-1、IL-1、TNF-α、NF-κB and Th cell immunomodulation. Methods: Seven male Wistar rats were randomly divided into four groups (each group, n=20) as follows: normal control group, model group, group treated with low dose of thalidomide, group treated with high dose of thalidomide. The rats in model group and therapeutic groups were intragastric injected with pig blood serum to induce liver fibrosis model, and the rats in nomal control group received peanut oil. From the first week,the rats in the therapeutic groups were given low dose (20㎎/㎏) or high dose (100㎎/㎏) of thalidomide by intragastric administration respectively, meanwhile the rats in the normal control group and model group were given normal saline by intragastric administration. All rats were sacrificed at the end of the seven week, and their blood and liver were collected for analysis. The liver histopathology was examined by haematoxylin-eosin staining and Masson trichrome staining. Serum alanine aminotransferase(ALT), aspartate aminotransferase(AST), prealbumin(PA), albumin(ALB), globumin(GLO) were detected by biochemical analysis and hyaluronic acid(HA), laminin(LN), procollagen type Ⅲ(PCⅢ), procollagen type Ⅰ (PC Ⅰ ), type Ⅳ collagen(CIV) were detected by radioimmunoassay in all the rats.ICAM-1, NF-κBp65, TNF-α; IL-1; IL-10; IL-2; IFN-γ in the liver was detected by immunohistochemistry. ICAM-1、TNF-α、IL-1、IL-10、IL-2、IFN-γ protein in cytoplasm and NF-κBp65 protein in nucleus were detected by western blot. ICAM-1, TNF-α, NF-κBP65, IL-1 mRNA levels in the liver were studied by using reverse transcriptase polymerase chain reaction (RT-PCR). Results: Compared with the model group, the severity of hepatocellular necrosis and fibroplasis were significantly decreased in the liver of rats treated with high dose of thalidomide, meanwhile there were thin fibro-septal and decreased inflammatory cell infiltration in the liver, which resulted in the markedly improved architecture of hepatic lobule. Its total Knodell's score was significantly lower than that in the model group (P＜0.01) .Serum ALT, AST, HA, LN, PCⅢ、 PC Ⅰ and CⅣ were significantly decreased in this group, meanwhile PA and ALB were significantly elevated (P＜0.01) .The expressions of ICAM-1, TNF-α, NF-κBP65, IL-1 mRNA in the liver were significantly decreased in this group (P＜0.01) . The expressions of ICAM-1, NF-κBP65, IL-1, TNF-α, IL-10 protein decreased in the livers of rats treated with high dose of thalidomide (P＜0.01) ,but IL-2, IFN-γ protein was elevated (P＜0.01) .There was a close positive correlation between the expression of NF-KBp65 mRNA and that of ICAM-1, TNF-α, IL-1 mRNA respectively (VS ICAM-1,r=0.78, P＜0.01; VS TNF-α, r=0.72, P＜0.01; VS IL-1, r=0.82, P＜0.01) . Conclusion: Thalidomide might exert its effect on the inhibiton of adhesion molecules, IL-1 , TNF-α and the organism immunoregulation function via down-regulation of NF-κB signaling pathway to prevent the progression of liver fibrosis. Key words: thalidomide, liver fibrosis, nuclear factor-κB, adhesion molecule, Th cell