【作者】 张万国；

【导师】 胡晋红；

【作者基本信息】 第二军医大学 ， 药理学， 2002， 博士

【摘要】 肝纤维化是伴随肝损伤之后的一种病理修复反应，表现为肝内胶原纤维异常增生，肝细胞变性坏死，肝组织结构和功能遭到破坏。临床研究和动物实验都已证明，肝纤维化是慢性肝病发展至肝硬化的必经途径。肝纤维化具有可逆性，采用适当的措施减轻炎症反应的程度，抑制肝脏内过度表达的胶原合成能力，同时通过提高胶原酶的活性，促进已沉积胶原的消除，可以阻止肝纤维化状况的进一步恶化，并引导其向正常方向发展直至完全恢复。因此抗肝纤维化对于治疗慢性肝病和预防肝硬化具有十分重要的意义，倍受国内外肝病学者们的重视。由于肝纤维化形成机制极其复杂，临床治疗中难以取得满意的效果，所以寻找疗效更为确切的药物，成为药学工作者致力开拓的重要课题之一。本文根据多孔真菌科植物桑黄的药理特点，进行抗实验性肝纤维化的研究，并从多方面探讨其作用机理。 以CCl4诱发大鼠肝损伤，同时给予桑黄灌胃进行预防，结果预防组肝细胞变性减轻，肝内胶原纤维增生减少；血清HA、LN、Ⅳ-C含量与正常组接近（P>0.05），PCⅢ含量也降至模型组一半以下（P<0.01）。肝功能血清酶谱测定结果显示，预防组较模型组肝脏蛋白合成能力增强，TBIL下降明显（P<0.05），氨基转移酶ALT、AST水平显著下降（P<0.01）。这些结果表明桑黄对肝脏具有保护作用，能明显延缓肝纤维化的进程，预防慢性肝损伤的发生与发展。 采用高、中、低三个剂量对已建立起肝纤维化的大鼠进行治疗，比较各剂量对肝脏病变程度、血清酶谱以及胶原成分的影响。结果发现高剂量桑黄（1.0g/100g体重）表现出非常好的治疗效果，血清胶原成分较模型对照组显著下降（P<0.01），表明肝脏内纤维增生减轻，这一点与病理观察结果一致；血清酶谱各指标水平已恢复至接近正常值（P>0.05），肝功能明显改善，病理组织观察显示肝细胞变性坏死明显减轻。中剂量组（0.5g/100g体重）也显示出较好的治疗效果，虽然各项指标改善程度不及高剂量组。低剂量组治疗效果不明显，各项指标与正常组比较差异明显（P<0.01，P<0.05），但与模型组相比，各项指标仍有一定幅度的下降。桑黄对肝纤维<WP=6>化的治疗作用，预示着桑黄具有逆转肝纤维化的潜力，这种潜力与药物剂量呈正相依赖性关系。 为了阐明桑黄抗肝纤维化的机理，考虑到中药成分的多样性和药理作用的广泛性，本文从不同侧面来分析桑黄对肝纤维化的影响： 1. 血液流变学参数显示，CCl4诱导大鼠肝纤维化与血液流变学性质的改变密切相关，血液粘度增高，红细胞流变性降低，影响肝区微循环，肝细胞得不到充足的营养供给，又进一步加重了肝细胞损伤。与模型组比较，桑黄能明显改善肝纤维化大鼠的血液流变学参数，尤其是全血粘度显著下降（P<0.01），红细胞刚性下降（P<0.01），红细胞压积减小（P<0.05），反映红细胞对血液粘度影响的参数全血还原粘度也明显降低（P<0.05）。肝组织ATPase和ChE活性升高，从另一侧面反映了肝细胞活性增强。这就提示桑黄可通过改善血液流变学性能，促进肝区微循环，增加肝细胞营养，从而表现出保肝作用。 2. 脂质过氧化在CCl4致大鼠肝损伤中扮演着重要的角色，桑黄在治疗大鼠肝纤维化过程中，能显著降低血清中活性氧成分，而它们是致肝细胞损伤的直接因子。与模型组相比，桑黄治疗组大鼠肝组织SOD活性提高（P<0.10），脂质过氧化产物MDA减少（P<0.20）。由于体外抗氧化试验未能证明桑黄对自由基的抑制作用，因此我们推测桑黄可能通过提高SOD活性和机体清除自由基的能力而发挥抗脂质过氧化作用。 3. IL-4是肝纤维化过程中的炎症因子，而IFN-γ则是肝纤维化的抑制因子。本文选择在肝纤维化中功能相反的两种细胞因子，考察桑黄在治疗肝纤维化时对细胞因子的调节作用。结果治疗组血清IL-4水平较模型组 下降，分别为8.93±2.77 pg/ml和10.65±2.89 pg/ml （P>0.05）。相反治疗组IFN-γ水平较模型组显著升高，分别为97.38±14.95 pg/ml和41.67±6.83 pg/ml （P<0.01）。体外细胞试验进一步表明，桑黄能增强人外周血单个核细胞（PMNC）产生IFN-γ，PMNC与100μg/ml PHA单独培养时，上清中IFN-γ含量为209.89±4.80 pg/ml；当与100μg/ml PHA和200μg/ml 桑黄共同培养时，上清中IFN-γ含量升高到361.19±14.06 pg/ml。IFN-γ在抗肝纤维化方面的作用早已被证实，其作用机理是多方面的，主要有抑制<WP=7>HSC细胞的活化与增殖，抑制胶原基因mRNA表达，并有抗病毒、抗炎作用等。本研究提示，桑黄可促进淋巴细胞分泌IFN-γ，进而调节机体免疫反应，间接发挥抗肝纤维化作用。 4. 以HSC-T6和NIH-3T3细胞作为肝纤维化的体外细胞模型，研究桑黄对胶原产生的影响，结果显示桑黄对两种细胞的增殖及胶原合成能力都有明显的抑制作用，呈浓度依赖性特征。2000μg/ml的桑黄对HSC-T6和NIH-3T3细胞的3H-TdR掺入抑制率分别为45.03%和27.93%，对两者的3H-Proline掺入抑制率更高达68.40%和40.99%。另外桑黄对BRL肝细胞增殖呈现一定的促进作用，这将有利于机体内肝细胞的再生。 5. 应用流式细胞分析技术检测桑黄对HSC-T6细胞内受体PDGFR-β表达的影响，结合HSC-T6?更多还原

【Abstract】 Hepatic fibrosis is a process of repair response to the liver injury, and involves the abnormal accumulation of collagen, degeneration and necrosis of hepatocytes, and damage to liver architecture and function. It has been proved by clinical research and experimental work on animals that hepatic fibrosis is the key process in the conversion of chronic liver disease into liver cirrhosis. Hepatic fibrosis is a reversible process, and can be resorted to health through various ways such as suppressing the inflammation reaction, inhibiting the excessive synthesis of collagen, and accelerating the degradation of collagen by enhancing the activities of collagenase. Today much attention has been paid to anti-fibrosis procedure in the treatment of chronic liver disease or prevention of liver cirrhosis, but seldom can a satisfying result be achieved in clinical practice due to the extremely complicated formation of hepatic fibrosis. Therefore finding eutherapeutic medicines has become an important task for pharmaceutical researchers to fulfil. We study the inhibitory effects of Phellinus igniarius (PI), a polyporus fungal medicine, on the experimental hepatic fibrosis, and further reveal the polyergic mechanism of its action. Hepatic injury was induced by CCl4 in rats. Histomorphology study showed that PI could prevent the degeneration of hepatocytes and proliferation of collagen in the liver. Serum levels of HA, LN and Ⅳ-C from prophylactic group were reduced close to normal group (P>0.05), while the PCⅢ level was reduced to the value half of the model group (P<0.01). Levels of TBIL, ALT and AST were also declined significantly (P<0.05, P<0.01). The results showed that PI had an protective effect on the liver. It could also retard the progress of hepatic fibrosis and prevent the occurring and developing of chronic liver injury. The curative effect of PI on hepatic fibrosis was studied after administration of PI in high, median and low dosage to rats. High dosage (1.0g/100g body weight) of PI was very effective to cure hepatic fibrosis. It could significantly reduce the serum levels of collagen and various enzymes and relieve the<WP=9>symptom of collagen accumulation in liver. Median dosage (0.5g/100g body weight) could also cure hepatic fibrosis though it was not as effective as high dosage. Low dosage (0.25g/100g body weight) had little effect on hepatic fibrosis and could only slightly reduce the serum levels of collagen and enzymes. The curative effect of PI suggested that PI had the potentiality to reverse hepatic fibrosis, and there existed a positive correlation between the efficacy and PI dosage. The mechanisms of PI inhibiting hepatic fibrosis were revealed on multiaspect because Chinese traditional medicine had many ingredients and involved in various pharmacological activities. 1. Hemorheological studies in rats displayed an intimated connection between hepatic fibrosis and hemorheological changes. When hepatic fibrosis was induced by CCl4, blood viscosity became high and erythrorheological ability became low, thus led to microcirculatory disturbance, which would aggravate hepatic injury. Compared with model group, the whole blood viscosity, reduced blood viscosity, hematocrit and red blood cell (RBC) rigidity in PI group were decreased obviously (P<0.01, P<0.05). The levels of ATPase and ChE in liver tissue were increased in PI group. Therefore it could be conclude that PI could protect the liver through meliorating hemorheological properties, improving blood circulation in liver and supplying sufficient nutrition for hepatocytes. 2. Lipid peroxidation plays an important role in the process of CCl4 inducing liver injury. As compared with model group, serum level of active oxygen in PI group significantly decreased (P<0.01), tissue level of SOD activity was elevated (P<0.10) and MDA content was decreased (P<0.20). However, the experiment in vitro on anti-photooxidation didn’t provide any positive evidence of PI inhibiting oxygen free radicals. We infered that PI might prevent lipid更多还原