【作者】 洪汝涛；

【导师】 许建明；

【作者基本信息】 安徽医科大学 ， 老年医学， 2009， 博士

【摘要】 背景/目的肝纤维化是慢性肝病共有的病理特征,其过程涉及到多细胞、多分子事件,最终导致胶原和细胞外基质(extracellular matrix, ECM).在狄氏间隙沉积,并能发展为不可逆性肝硬化。近年来的研究证实,肝纤维化在未进入肝硬化之前,尚有逆转可能。在此阶段,如果处理得当,肝硬化有可能避免。然而,目前尚无理想的治疗肝纤维化药物。因此,探讨肝纤维化的机理,开发有效和毒副作用小的治疗肝纤维化的新药是目前研究的重要方向。目前有证据提示,氧化应激在肝纤维化的发生和发展中起重要作用。褪黑素(N-acetyl-5-metyoxytryptamine, melatonin)是松果体分泌的一种激素,为强大的内源性抗氧化剂,能清除氧自由基和中间活性体。体内和体外实验显示,在由黄樟素、脂多糖(LPS)、四氯化碳(CCl4)、缺血再灌注、淀粉状蛋白质、电离辐射等诱导的氧化损伤中,褪黑素具有保护细胞、组织和器官的作用。另外,褪黑素还通过提高谷胱甘肽过氧化物酶(GPx)、超氧化物歧化酶(SOD)、谷胱甘肽(GSH)这些潜在性的抗氧化剂的水平而间接发挥抗氧化作用。近年研究显示,褪黑素具有减少纤维母细胞增殖、抑制胶原合成作用。但褪黑素对肝纤维化的保护作用研究尚少。核转录因子кB(Nuclear factor-kappaB ,NF-кB)是由二聚体构成的转录因子家族,具有调控炎症、免疫、创伤愈合、细胞生存、细胞功能等作用。生理应激、氧化应激、暴露于某些化学品均可导致NF-кB激活,进而发挥其关键性的应激反应调节作用。NF-кB过度激活与许多疾病有关。近来NF-кB在肝脏疾病中的功能和调控受到关注,肝脏的损伤和纤维增生涉及到NF-кB的活化。NF-кB在肝纤维化中的确切作用尚不甚清楚,褪黑素对纤维化肝脏中NF-кB活性的影响及其后果未见系统研究。本课题在建立大鼠CCl4诱导的化学性肝纤维化模型基础上,探讨MEL对肝纤维化的抑制作用和可能机制。同时,探讨NF-кB在CCl4诱导的化学性肝纤维化中的作用,以及MEL是否通过抑制NF-кB活性而发挥抗纤维化作用。方法采用四氯化碳(carbon tetrachloride ,CCl4)制备SD大鼠化学性肝纤维化模型。所有大鼠随机分为6组:正常对照组(normal control group,normal组,11只)、模型对照组(model control group, model组,20只)、CCl4+N-乙酰-L-半胱氨酸组(NAC组,为阳性药对照,20只)、CCl4+低剂量褪黑素组(MEL1组,20只)、CCl4+中剂量褪黑素组(MEL2组,20只)、CCl4+高剂量褪黑素组(MEL3组,20只)。将CCl4与精制花生油按2 :3比例配成40%CCl4油剂溶液,除正常对照组外,余按3ml/kg给大鼠皮下注射,每周两次,连续12周,正常对照组注射等容量花生油作为对照。自动物皮下注射CCl4之日起,开始给药, NAC 100 mg/kg, i.p.,每天1次;MEL低、中、高剂量组分别给予MEL 2.5 mg/kg、5 mg/kg、10 mg/kg,均为腹腔注射。12周实验结束后,所有动物予以麻醉,腹主动脉取血,取出肝脏并称湿重。HE染色和Van Gieson (VG)胶原纤维染色观察肝脏病理改变;全自动生化分析仪检测血清谷丙转氨酶(alanine aminotransferase, ALT)、谷草转氨酶(aspartate aminotransferase ,AST)、白蛋白(albumin, A)、球蛋白(globulin, G )水平;生化法测定肝脏羟脯氨酸(hydroxyproline ,Hyp)、丙二醛(malondialdehyde ,MDA)含量和谷胱甘肽过氧化物酶活性(glutathione peroxidase ,GPx);放射免疫法测定血清透明质酸(hyaluronic acid,HA)、层粘连蛋白(laminin,LN)、三型前胶原氨端肽(procollagenⅢN-terminal peptide,PⅢN P)含量;免疫组化法测定肝组织中NF-кB P50、NF-кB P65、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)的表达;RT-PCR法测定肝组织中Ⅰ型前胶原(procollagen typeⅠ)mRNA表达;电泳迁移率改变分析(electrophoretic mobility shift assay ,EMSA)测定肝组织中NF-кB DNA结合活性;同时,用原位灌注加密度梯度离心法分离正常SD大鼠肝脏星状细胞,以免疫组化法鉴定(用desmin抗体),在培养的肝星状细胞中加入脂多糖(LPS)进行刺激,用MTT法测定不同浓度的MEL对肝星状细胞增殖的抑制作用。结果1. MEL对大鼠化学性肝纤维化的保护作用通过12周皮下注射CCl4,成功建立了大鼠肝纤维化模型。病理分级显示:正常对照组和CCl4处理各组之间差异显著(P < 0.01),和模型组相比,MEL(10 mg/kg)组肝纤维化程度较轻,有统计学意义(P < 0.05),表明MEL(10 mg/kg)有抑制肝纤维化作用。与正常对照组比较,CCl4处理各组血清ALT、AST水平显著升高(P < 0.01),与模型组比较,MEL(5, 10 mg/kg)能显著降低升高的的ALT、AST水平(P < 0.05,P < 0.01),NAC也能显著降低升高的ALT、AST水平(P < 0.05),其作用强度和MEL相近。同样,与模型组比较,MEL(10 mg/kg)能降低肝脏Hyp含量和LN、HA水平,表明MEL对纤维化标志物的改善。另外,MEL对氧化应激指标也有明显影响,和模型组比较,MEL(5, 10 mg/kg)能显著降低肝匀浆MDA含量(P < 0.05),升高GPx活性(P < 0.05,P < 0.01),NAC也有同样作用,其作用强度和MEL(10 mg/kg)相近。2. MEL对LPS介导的肝星状细胞增殖的影响通过原位灌注和密度梯度离心法成功分离并纯化大鼠肝星状细胞,刚分离的星状细胞纯化至70-80%,存活率在90 %以上,纯化的星状细胞得率2×107/肝。传第一代的细胞,S-P法作免疫细胞化学鉴定,Desmin表达阳性细胞为90%以上。与对照组(无LPS刺激)相比,培养的肝星状细胞经LPS(10ng/ml)刺激后,增殖明显加快,与LPS组相比, MEL(0.1mmol/L)能显著抑制由LPS刺激的肝星状细胞增殖。3. MEL对纤维化大鼠肝组织I型前胶原mRNA表达的影响正常对照组大鼠肝组织I型前胶原mRNA表达较少,和CCl4处理各组相比,差异显著(P<0.05,P<0.01)。和模型组相比,MEL(10 mg/kg)组、NAC组表达明显减少(P<0.05),MEL(2.5 mg/kg, 5 mg/kg)表达有下降趋势,但无统计学意义。这些结果表明MEL有抑制肝脏纤维化大鼠I型前胶原mRNA表达作用。4. NF-кB在CCl4诱导的大鼠肝纤维化中的作用以及MEL对NF-кB信号通路的影响NF-кB P65、NF-кB P50在正常对照组大鼠肝组织中仅有微量表达,表现为细胞浆中淡黄色颗粒,较之正常对照组,CCl4处理各组表达明显增强,表现为棕黄色和/或棕褐色颗粒,且细胞浆和细胞核中均有表达,和模型组相比,MEL(5, 10 mg/kg)、NAC均能显著降低NF-кB P65、NF-кB P50的表达。进一步, NF-кB DNA结合活性被测定,结果显示,正常对照组NF-кB结合活性较弱,模型组NF-кB结合活性较其它各组明显增强,和模型组相比,MEL能明显抑制NF-кB的结合活性。TNF-α、ICAM-1在正常对照组大鼠肝组织中仅很微量表达,表现为细胞浆中淡黄色颗粒,较之正常对照组,CCl4处理各组表达明显增强,表现为棕黄色和/或棕褐色颗粒,以血管周围,汇管区炎性细胞较多的区域最为明显,其表达强度弱于NF-кB P65、NF-кB P50,和模型组相比,MEL(10 mg/kg)、NAC均能显著降低TNF-α、ICAM-1的表达。结论1.连续12周CCl4皮下注射成功复制出大鼠肝纤维化模型,该模型具有典型肝纤维化病理表现,同时符合有关血液、肝组织生化检查改变,适合药物防治肝纤维化的实验研究。2.MEL具有保护肝细胞、抑制肝纤维化作用,这种作用考虑与其抗氧化有关。3.NF-кB与肝纤维化关系密切,其表达和DNA结合活性与肝纤维化程度一致,在肝纤维化的发展中起重要作用。4.MEL保护肝细胞、抑制肝纤维化作用可能部分地与其抑制NF-кB活化,下调NF-кB信号通路有关。以上结果将为开发MEL作为防治肝纤维化药物提供实验基础。更多还原

【Abstract】 BACKGROUND/AIMS Hepatic fibrosis, a common pathological process of chronic hepatic disease, can lead to irreversible cirrhosis, and involves multiple cellular and molecular events that ultimately result in accumulation of collagen and extra cellular matrix protein in space of Disse. Hepatic fibrosis generally considered reversible conditions. If treated properly at fibrosis stage, cirrhosis can be prevented. However, no effective antifibrosis drugs are available at present. Therefore , one of important trends focuses on investigation the mechanisms of hepatic fibrosis and exploration new effective drugs without ill effects. Several lines of evidence suggest that oxidative stress plays an important role in the etiopathogenesis of hepatic fibrosis.Melatonin (N-acetyl-5-metyoxytryptamine), a secretory product of the pineal gland, is a powerful endogenous antioxidant, regulates circadian rhythms, sleep and immune system activity, behaves as a free radical scavenger, eliminates oxygen free radicals and reactive intermediates. Both in vitro and in vivo experiments have shown that melatonin can protect cells, tissues, and organs against oxidative damage induced by a variety of free-radical-generating agents and processes, such as safrole, lipopolysaccharide (LPS), carbon tetrachloride (CCl4), ischemia-reperfusion, amyloid-protein, and ionizing radiation. In addition, melatonin also has an indirect antioxidant effect by enhancing the levels of potential antioxidants such as glutathione peroxidase (GPx), superoxide dismutase (SOD), and glutathione (GSH). Recent studies showed that melatonin reduces fibroblast proliferation and collagen synthesis.Nuclear factor-kappaB (NF-кB) is a family of dimeric transcription factors that regulate inflammation, innate and adaptive immunity, wound healing respones, and cell fate and function. Physical stress , oxidative stress ,and exposure to certain chemicals can lead to activation of NF-кB , which may function as a critical mediator of stress responses. Excessive activation of NF-кB relates to many diseases.Emerging attention has been focused on the regulation and function of NF-кB during liver injury. NF-кB activation have been implicated as mediators of liver injury and fibrogenesis. The present study was designed to investigate the protective effects of melatonin on carbon tetrachloride (CCl4)-induced hepatic fibrosis in experimental rats and its possible mechanisms. Meanwhile, we evaluated the role of NF-кB on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and investigated whether treatment with melatonin was able to modulate NF-kappaB activation.METHODS All rats were randomly divided into normal control group, model control group treated with CCl4 for 12 wk, CCl4 + NAC group treated with CCl4 + NAC (100 mg/kg, i.p.) for 12 wk, CCl4 + MEL-1 group treated with CCl4 + melatonin (2.5 mg/kg) for 12 wk, CCl4 + MEL-2 group treated with CCl4 + melatonin (5.0 mg/kg) for 12 wk, and CCl4 + MEL-3 group treated with CCl4 + melatonin (10 mg/kg) for 12 wk. Rats in the treatment groups were injected subcutaneously with sterile CCl4 (3 mL/kg, body weight) in a ratio of 2:3 with arachis oil twice a week. Rats in normal control group received hypodermic injection of arachis oil at the same dose and frequency as those in treatment groups. At the end of experiment, rats in each group were anesthetized and sacrificed. Hematoxylin and eosin (HE) staining and Van Gieson staining were used to examine changes in liver pathology. Serum activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and protein concentration were measured with routine laboratory methods using an autoanalyzer. Hydroxyproline (Hyp) content in liver and malondialdehyde (MDA) and glutathione peroxidase (GPx) levels in liver homogenates were assayed by spectrophotometry. Serum hyaluronic acid (HA), laminin (LN), and procollagenⅢN-terminal peptide (PⅢNP) were determined by radioimmunoassay. Expression of NF-кB P50 ,NF-кB P65, TNF-αand ICAM-1 in liver tissue was detected by immunohistochemistry and expression of procollagen typeⅠmRNA in liver tissue was detected by semiquantified RT-PCR. NF-kappaB binding activity in liver tissue was determined by electrophoretic mobility shift assay (EMSA). HSCs are isolated from the liver of SD rat through in situ perfusion with pronase and collagenas and they were identified by immunohistochemistry stain with desmin antibody. With LPS and various concentrations of MEL, the inhibition of HSC was determined by MTT method.RESULTS1. Protective effects of MEL on chemical hepatic fibrosis induced by CCl4 in ratThe hepatic fibrosis models in rats were successfully induced by CCl4. Pathologic grading showed that the fibrogenesis was much less severe in CCl4 + MEL3 group than in model control group (u = 2.172, P < 0.05), indicating that melatonin (10 mg/kg) can significantly ameliorate CCl4-induced hepatic fibrotic changes. The serum levels of ALT and AST were markedly lower in CCl4 + MEL treatment groups (5, 10 mg/kg) than in model control group. Similarly, melatonin (10 mg/kg) not only decreased hydroxyproline (Hyp) contents in liver , but also decreased the elevated level of laminin (LN) and hyaluronic acid (HA) in serum, which indicate that melatonin decrease the ECM production of hepatic fibrosis in rats. Moreover, melatonin also ameliorated the oxidative stress state of hepatic fibrosis rats. Treatment with melatonin (5, 10 mg/kg) significantly reduced the MDA content and increased the GPx activity in liver homogenates compared with model control group.2. Effects of MEL on LPS-mediated HSC proliferationHSCs are successfully isolated from the liver of SD rat through in situ perfusion with pronase and collagenase, crude cell suspensions were purified to 70-80% homogeneity by density gradient centrifugation. The cell viability was more than 90 %. The harvest rate of HSCs was 2×107cells per liver. HSC was identified by immunohistochemistry stain with desmin antibody. The desmin postive cell rate was more than 90 % in subculture.Compared with the control group, LPS (10ng/ml) significantly promoted the proliferation of rat HSCs. Compared with the LPS group, MEL obviously inhibited the proliferation of rat HSCs at the concentration of 0.1mmol/ml.3. Effect of MEL on hepatic mRNA expressions of procollagen typeⅠCompared with normal control group, the expression of procollagen typeⅠm RNA in liver tissue in CCl4-treated groups was significantly increased. Compared with model control group , the expression of procollagen typeⅠmRNA in liver tissue in CCl4 + MEL group(10 mg/kg) and CCl4+NAC group was significantly decreased .4. The role of NF-кB on hepatic fibrosis induced by CCl4 in rats and the effect of MEL on the NF-кB signaling pathwayImmunohistochemical analysis of liver tissue sections revealed that there were few positive expressions of NF-кB P65, NF-кB P50, TNF-αand ICAM-1 in the normal rat liver. NF-кB P65 and NF-кB P50 expression, as brown or yellow granulae in cytoplasm and nuleus, was apparently increased in response to 12-week subcutaneous injection of CCl4. MEL(5, 10 mg/kg) and NAC significantly reduced NF-кB P65 and NF-кB P50 expression. Meanwhile, expression of TNF-αand ICAM-1 whose transcription are regulated by NF-кB was also apparently increased in response to 12-week subcutaneous injection of CCl4. MEL(10 mg/kg)and NAC significantly reduced TNF-αand ICAM-1 expression. Furthermore, the NF-kappaB-DNA binding activity was confirmed by EMSA. The activation of NF-кB in the normal control group was weak. It was elevated significantly in the model control group. Treatment with melatonin significantly suppressed the NF-кB binding activity in a dose-dependent manner to some degree.CONCLUSIONS1. In this study, hepatic fibrosis was successfully induced by subcutaneous injection of sterile CCl4 twice weekly for 12 wk. Through this hepatic fibrosis model, the effects of melatonin on hepatic fibrosis induced by CCl4 in rats were examined.2. Melatonin may have beneficial effects on hepatic fibrosis induced by CCl4 in rats. The protective effect of melatonin on hepatic fibrosis may be related to its antioxidant activities.3. NF-кB activation are implicated as mediator of fibrogenesis. NF-кB plays an important role on hepatic fibrosis induced by CCl4 in rats.4. Melatonin may exert an effect on the inhibition of oxidation stress via downregulation of NF-кB signaling pathway to prevent the progression of hepatic fibrosis.All above-mentioned research results may provide the experimental basis for melatonin being developed to new drug treating hepatic fibrosis.更多还原