【作者】 黄晓菡；

【导师】 朱科伦；

【作者基本信息】 广州医学院 ， 传染病学， 2009， 硕士

【摘要】 肝损伤是指在一系列理化因素的作用下,肝细胞发生不同程度的肿胀、变性、坏死和凋亡,是各种肝病发生发展的最基本的病理状态。在我国,以病毒性肝炎引起的肝损伤最为常见。我国是HBV感染的高发区,根据2008年全国病毒性肝炎血清流行病学调查,1至59岁人群HBsAg携带率达7.18%,约9300万人。其中部分病毒性肝炎最终会发展为肝硬化,甚至肝细胞癌,危害极大。目前病毒性肝炎没有特效治疗,以综合治疗为主,包括合理的休息和营养,心理辅导,改善和恢复肝功能,调节机体免疫,抗病毒,抗纤维化等治疗。近年来抗病毒治疗虽然取得了很大的进展,但其价格昂贵,停药后易复发,副作用较多,近期疗效较低,且与患者应答反应性有关,具有一定的局限性。而我国中医药资源丰富,多种中草药也被证实有改善和恢复肝功能,调节免疫,抗病毒和抗肝纤维化的作用。故此探索中药对肝脏的保护作用以及抗炎的机制对开发新的抗肝炎、保护肝脏药物有重要意义。丹芪活肝胶囊主要由黄芪、黄芩、菊花、山楂、丹参、灵芝孢子粉组方,临床药效主要是活血化淤、清热解毒、抗肝纤维化。作为院方制剂已经广泛应用于临床,治疗慢性肝炎及肝硬化,在长期的临床应用中已证实在抗肝炎、肝纤维化方面有较好的疗效,但相关的基础研究极少。本研究旨在通过建立肝损伤模型进行药效学试验和进行治疗机理研究,推动其在临床上更为广泛的应用。对肝炎病毒易感的动物很局限,且造模复杂、困难。而急性四氯化碳（CCl₄）肝损伤动物模型和氨基半乳糖（D-GalN）肝损伤模型中的病理状态、生化指标以及组织变化都与人的肝炎有一定的相似之处,比较经典且制模方法已经比较成熟。特别是D-GalN肝损伤模型,是目前公认的比较好的研究病毒性肝炎的发病机制及有效治疗药物的实验动物模型。故此,本研究通过制备这两个动物模型,并检测各种指标,从肝脏功能、脂质过氧化、细胞因子的表达及病理变化来探讨丹芪活肝胶囊对肝脏的保护作用及其对炎症干预作用的相关机制。研究目的研究丹芪活肝胶囊对CCl₄致小鼠急性肝损伤模型转氨酶的影响,探讨丹芪活肝胶囊对肝细胞膜通透性的影响。探讨丹芪活肝胶囊对D-GalN致大鼠肝损伤模型大鼠的肝功能、肝脏的病理形态学改变及脂质过氧化损伤的影响。研究方法1丹芪活肝胶囊对CCl₄致小鼠急性肝损伤的保护作用采用药物灌胃,四氯化碳腹腔注射方法,观察丹芪活肝胶囊对CCl₄致肝损伤模型小鼠血清中谷丙转氨酶（ALT）及谷草转氨酶（AST）含量的影响。2丹芪活肝胶囊对D-GalN致大鼠急性肝损伤的保护作用采用药物灌胃,氨基半乳糖腹腔注射方法,观察丹芪活肝胶囊对模型大鼠血清中ALT、AST、TP、ALB、TBIL、SOD、GSH含量的影响,肝匀浆中SOD、MDA含量的影响。并做HE染色,观察肝脏组织细胞形态结构的变化。用E1isa试剂盒检测血清中炎性细胞因子TNF-α浓度的变化。研究结果1丹芪活肝胶囊对CCl₄致小鼠的急性肝损伤的保护作用与空白组比,模型组的小鼠血清ALT、AST水平显著增高。丹芪活肝胶囊高剂量组及低剂量组能明显降低受损伤小鼠血清中的ALT含量;丹芪活肝胶囊高、中、低剂量组均能明显降低受损伤小鼠血清中的AST含量。其与模型组相比,差异有显著性（P<0.01或P<0.05）。2丹芪活肝胶囊对D-GalN致大鼠急性肝损伤的保护作用模型组大鼠血清中的ALT、AST、TBIL明显升高,TP、ALB明显降低,肝匀浆中SOD、GSH的含量明显降低,血清中TNF-α浓度明显升高,肝脏组织内大量慢性炎细胞浸润,肝细胞大片坏死,坏死区域累及肝小叶大部分或相连的几个肝小叶,小叶结构不清,残存的肝细胞轻度水肿,肝窦受压。丹芪活肝胶囊高剂量组能明显降低大鼠血清中ALT水平,使血清中TP、ALB、SOD、GSH含量升高,肝匀浆中SOD含量升高,MDA含量降低,病理显示其对受损的肝脏组织有明显改善作用。丹芪活肝胶囊中剂量组能使大鼠血清中TP、ALB、SOD含量升高,肝匀浆中SOD含量升高,并对受损的肝脏组织有明显改善作用。丹芪活肝胶囊低剂量组能使大鼠血清中TP、GSH含量升高,肝匀浆中SOD含量升高,MDA含量降低,血清中的TNF-α浓度明显降低。结论1丹芪活肝胶囊对CCl₄所造成的肝脏损伤有一定的保护作用。2丹芪活肝胶囊能显著减轻D-GalN所致大鼠急性肝损伤过程中的肝脏损伤,增加肝脏的蛋白质合成,减轻脂质过氧化损伤,减轻炎症反应,改善病理状态。更多还原

【Abstract】 Liver injury, the most fundamental pathological status of liver diseases, is the swelling, degeneration, necrosis and apoptosis to various degrees of liver cells induced by a series of chemicophysical factors. The most common type of liver injury in China is induced by viral hepatitis. China is a high prevalence area of viral hepatitis. According to a nationwide epidemiological investigation taken in 2008 on viral hepatitis serum samples, HBsAg carriers account for 7.18% of the population aged 1-59, i.e. approximately 93 million people. In some cases, viral hepatitis can lead to hepatic cirrhosis or even hepatocellular carcinoma（HCC）, which is fatal. There is no specific treatment for viral hepatitis at present except for combined therapy, including sufficient repose and appropriate nutrition, psychological counseling, improvement and recovery of hepatic functions,adjustment of the immune system, anti-virus treatment, anti-fibrosis treatment as the routine treatment for viral hepatitis patients. Anti-virus treatment has seen a great progress in recent years but its application is limited due to its high price, frequent relapse, various side effects, low short-term efficacy and its pertinence to patient reaction. As an alternative, a wide variety of Chinese herbal medicine has been proven to be effective in improving and rehabilitating liver functions, in adjusting immunity and resisting virus and fibrosis. With the ample resources of Chinese medicine in this country, therefore, the study on the protective effect and anti-inflammatory mechanism of Chinese medicine on liver has great significance in developing new anti-hepatitis and liver-protecting medication. The major Clinical efficacy of Danqi Huogan Capsule（DHC） includes "Huoxuehuayu" （activating blood flow and removing blood stasis）, "Qingrejiedu"（clearing away heat and toxic materials）, and anti-fibrosis effect, with its major ingredients being Radix Astragali, Scutellaria, Chrysanthemi Flos, Fructus Crataegi, Salvia Miltiorrhiza and Ganoderma lucidum sporus powder. As a hospital prescription, DHC has had wide clinical application in curing chronic hepatitis and hepatic cirrhosis and long proven to have had good anti-hepatitis and anti-fibrosis effects. Relevant basic research is restricted, however. This study hereby aims to promote broader clinical application of DHC by establishing experimental models of hepatotoxicity for pharmacodynamic study and mechanism of treatment.As is known, only a limited variety of animals are susceptible to hepatitis viruses. Nonetheless, the pathological status, biochemical indices and change of tissue in animal models with acute hepatotoxicity caused respectively by CCl₄ and D-GalN are similar to human hepatitis conditions. These are classical experimental animal models and the modeling methods are relatively mature, especially the D-GalN model, which is recognized to be a good model thus far to study the pathogenesis and effective cure for viral hepatitis. Therefore, by establishing these two models and testing various indices, this study dwells on the hepatic protection of DHC and its relevant machanism on inflammation interference in terms of liver functions, lipid peroxidation, expression of cytokine and pathological change.ObjectiveTo investigate the influence of DHC on 1)Transaminase and hepatic cell membrane permeability modeling on mice with acute hepatotoxicity induced by CCl₄; 2)Liver function, pathomorphism change and lipid peroxidation damage modeling on rats with acute hepatotoxicity induced by D-GalN.Methods1. Protective effect of DHC on mice with acute hepatotoxicity induced by CCl₄ The animal model is established by intragastric administration （ig） of DHC and intraperitoneal injection （ip） of CCl₄ on mice. The influence of DHC on mice with acute hepatotoxicity is then observed in terms of the alanine aminotransferase （ALT） and aspartate aminotransferase （AST） content in serum.2. Protective effect of DHC on rats with acute hepatotoxicity induced by D-GalNThe animal model is established by intragastric administration （ig） of DHC and intraperitoneal injection （ip） of D-GalN on rats. The influence of DHC on rats with acute hepatotoxicity in terms of the content of ALT, AST, TP, ALB, TBIL, SOD, GSH in serum as well as the content of SOD, MDA in Liver homogenate is then observed. HE staining （hematoxylin and eosin stain） is also applied to examine the change of cell morphology and structure in liver tissues. E1isa Kit is used to detect the change of TNF-αconcentration of inflammatory cytokines in serum.Results1. Protective effect of DHC on mice with acute hepatotoxicity induced by CCl₄ Compared with the blank group, ALT and AST level in model groups are significantly higer. ALT content in groups with both high and low dosage of DHC is significantly decreased. AST content in all groups administered with DHC is significantly decreased.2. Protective effect of DHC on rats with acute hepatotoxicity induced by D-GalNIn model groups, ALT, AST, TBIL levels are significantly increased while TP, ALB levels significantly decreased; SOD, GSH levels in liver homogenate see a conspicuous decrease; TNF-αconcentration in serum witnesses a conspicuous rise. The following is detected: Inflammatory cell infiltration in liver tissue to a large extent, liver cells necrosis in large area, covering most part of the hepatic lobule or ajacent hepatic lobules with unclear lobular structure, slight edema in remnant liver cells, liver sinusoids pressured.In the model group of rats administered with high dosage of DHC, ALT level is significantly lowered, TP, ALB, SOD, GSH levels in serum increased, SOD level increased while MDA level decreased in liver homogenate. The pathological section indicates significant improvement in the damaged live tissues.In the model group of rats administered with medium dosage of DHC, TP, ALB, SOD levels in serum as well as SOD level in liver homogenate are increased. The Significant improvement has been detected in the damaged live tissues.In the model group of rats administered with low dosage of DHC,TP, GSH levels in serum are increased; SOD level in liver homogenate are increased while MDA level decreased. TNF-αconcentration in serum is significantly lowered.Conclusion1. DHC has certain protective effect from hepatotoxicity induced by CCl₄.2. DHC can significantly reduce the damage on rats with acute hepatotoxicity induced by D-GalN by increasing hepatic protein synthesis, lessening lipid peroxidation damage, abating inflammatory reaction and improving pathological status.更多还原