【作者】 王徐毅；

【导师】 薛博瑜；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2013， 博士

【摘要】 背景：慢性乙型病毒性肝炎感染乙型肝炎病毒(HBV)后,机体体液及细胞免疫引起的以肝脏损伤为主的全身性疾病。本病流行范围之广、传播途径之复杂、发病率之高、危害性之大,居各种传染病之首,是严重危害人类生命健康的传染病。慢性乙型肝炎在全球范围内分布,全世界有20多亿人被感染,约3.5亿人成为慢性HBV感染者。慢性乙型肝炎患者中约50%-75%有活跃的病毒复制和肝脏炎症改变,其中10%-20%可发展为肝硬化,1%-5%可演变为肝癌。慢性乙型肝炎是WHO公布的人类疾病死亡原因中第9位的疾病。现代医学主要治疗手段包括保肝、降酶、抗病毒、免疫调节等,有效的抗病毒治疗是根治慢性病毒性肝炎的关键。目标是清除或持续抑制HBV,从而阻止或减轻肝脏的炎症、坏死。大量的临床研究证实抗病毒较为理想的药物是干扰素和核苷类似物。但干扰素适应面窄,副作用明显,远期疗效不理想,价格昂贵。核苷类似物易于产生病毒变异等,其临床应用受到限制。大量的临床和实验研究证实,中药复方在保护肝细胞、调节免疫、抗肝纤维化,进而通过综合作用抑制病毒等方面显示了优越性。但在中医药研究中尚存在不少问题,如迄今中医对慢性乙型肝炎的病因、病机、辨证诊断等方面的认识不统一,与目前国际上普遍采用的以肝组织病理学分级分期、临床表现分型的慢性乙型肝炎诊断标准不相适应,难以准确反应慢性乙型肝炎的病变程度,临床缺乏有效、科学、系统的治疗方法,不能满足慢性乙型病毒性肝炎中医临床的要求。目的：通过实验研究,探索芪珠方治疗慢性乙型病毒性肝炎作用机理,明确芪珠方药物组成特点,为临床寻找有效的治疗本病的中医药方法奠定基础。方法：芪珠方是由叶下珠、虎杖、丹参、黄芪,赤芍、白花蛇舌草、土茯苓七味药组成,为保证疗效,最大限度的提出有效成分,本研究选择加热回流法进行提取,通过RP-HPLC对复方中的化学成分进行分析,选择四种不同浓度的乙醇进行提取,分析不同溶剂提取的样品中化学成分的变化。建立肝纤维化模型动物的基础上,中药高、中剂量组分别以16.20g/kg、9.72g/kg(芪珠方生药含量/大鼠体重)灌胃,每日1次,首次造模次日开始给药,连续8周直至动物处死前一天；秋水仙碱组(将秋水仙碱药片磨碎,制成水溶液,每1mL水中含0.1mg秋水仙碱)5.0mL/kg(秋水仙碱含量/大鼠体重)灌胃,每日11次,首次造模次日开始给药,连续8周直至动物处死前一天；模型组用等同于芪珠方大剂量组灌胃体积的生理盐水灌胃,肝组织内TNF-a,IL-4,IL-8,IL-10,TIMP-1,MMP-1,MMP-2,MMP-13表达水平,血清内TIMP-1,MMP-1, MMP-2,MMP-13表达。结果：不同溶剂提取的芪珠方(70%醇提进样为80μL,其余均为10μL),所得样品出峰数不同,水提及50%、70%、95%提取的复方在25min之前基本上没有出峰,实验过程中,通过加大进样量比较,70%醇提芪珠方出峰最多,且峰形大小和形状较好。肝纤维化模型动物模型组大鼠肝组织中TNF-α,IL-4,IL-8的表达相对空白组明显增加,IL-10的表达则显著减少肝组织中TNF-α,IL-4,IL-8的表达显著减少(P<0.01),IL-10的表达显著增加(P<0.01)；芪珠方高剂量组TNF-α, IL-4,IL-8的表达低于中剂量组,IL-10的表达高于中剂量组；芪珠方水提组IL-4, IL-8, TNF-α的表达低于同剂量乙醇提取组,IL-10的表达高于同剂量乙醇提取组(P<0.01)。与秋水仙碱组相比,芪珠方治疗组的IL-4的表达均高于秋水仙碱组且有显著差异(P<0.01)；芪珠方治疗组IL-8的表达显著高于秋水仙碱组(P<0.01)；芪珠方治疗组IL-10的表达均低于秋水仙碱组且有显著差异(P<0.01)；芪珠方治疗组TNF-α表达均高于秋水仙碱组(P<0.01)。模型组TIMP-1, MMP-2的表达相对高于正常组,MMP-1, MMP-13的表达相对低于正常组,两组相比差异有显著差异(P<0.01)。所有治疗组TIMP-1,MMP-2的表达低于模型组,MMP-1,MMP-13的表达高于模型组,均有显著差异(P<0.01)。芪珠方高剂量组TIMP-1,MMP-2的表达低于中剂量组,MMP-1,MMP-13的表达高于中剂量组,有明显的剂量依赖性(P<0.01)。芪珠方水提组TIMP-1, MMP-2的表达高于同剂量乙醇提取组(P<0.01), MMP-1, MMP-13的表达均低于同剂量乙醇提取组(P<0.01)。与秋水仙碱组相比,芪珠方4组治疗组的TIMP-1的表达均高于秋水仙碱组且有显著差异(P<0.01)；大剂量组的MMP-1表达显著高于秋水仙碱组(P<0.01),中剂量组的表达低于秋水仙碱组,其中醇提中剂量组有差异(P<0.05),水提中剂量组有显著差异(P<0.01)；4组治疗组MMP-2的表达均低于秋水仙碱组,其中复方大剂量组与醇提组大、中剂量3组有显著差异(P<0.01),水提中剂量组无统计学意义(P>0.05)；芪珠方大剂量组MMP-13表达高于秋水仙碱组(P<0.01),中剂量组表达低于秋水仙碱组(P<0.01)。模型组血清TIMP-1, MMP-2的表达相对高于正常组,MMP-1, MMP-13的表达相对低于正常组,两组相比差异有显著差异(P<0.01)。所有治疗组血清TIMP-1,MMP-2的表达低于模型组,MMP-1,MMP-13的表达高于模型组,均有显著差异(P<0.01)。芪珠方高剂量组血清TIMP-1,MMP-2的表达低于中剂量组,MMP-1,MMP-13的表达高于中剂量组,有明显的剂量依赖性(P<0.01)。芪珠方水提组中剂量组血清TIMP-1的表达高于同剂量醇提取中剂量组(P<0.01),高剂量组间无统计学意义(P>0.05)；水提组MMP-1的表达均低于同剂量醇提组(P<0.01)；水提组MMP-2的表达高于同剂量醇提组(P<0.01);MMP-13的表达均低于同剂量乙醇提取组(P<0.01)。与秋水仙碱组相比,芪珠方4组治疗组的TIMP-1的表达均高于秋水仙碱组,水提大、中剂量组和醇提中剂量组有显著差异(P<0.01),醇提大剂量组无统计学意义(P>0.05)；大剂量组的MMP-1表达高于秋水仙碱组(P<0.01),中剂量组的表达低于秋水仙碱组(P<0.01)；4组治疗组MMP-2的表达均低于秋水仙碱组,其中复方大剂量组与醇提组大、中剂量3组有显著差异(P<0.01),水提中剂量组无统计学意义(P>0.05)；芪珠方大剂量组MMP-13表达高于秋水仙碱组(P<0.01),中剂量组表达低于秋水仙碱组(P<0.01)。结论：芪珠方醇提能够治疗慢性乙型病毒性肝炎及肝纤维化,可能是通过调节肝脏及血清的TNF-a,IL-4,IL-8,IL-10,TIMP-1,MMP-1,MMP-2,MMP-13等发挥作用。更多还原

【Abstract】 Background:Chronic hepatitis B, hepatitis B virus (HBV) infection, liver damage caused by the humoral and cellular immune systemic disease. The wide range of the epidemic spread of the complexity of ways, the high incidence of the dangers of large, ranking the first of a variety of infectious diseases, and infectious diseases of serious harm to human life and health. Chronic hepatitis B worldwide distribution, more than20million people are infected worldwide, about350million people with chronic HBV infection. About50%-75%of the chronic hepatitis B patients with active viral replication and liver inflammation change, of which10%to20%may develop cirrhosis, and1%to5%may develop into liver cancer. Chronic hepatitis B is a human disease, the cause of death in the WHO announced nine diseases. Modern medical treatment, including the liver, reducing enzyme, antiviral, immunomodulatory, and effective anti-viral therapy cure for chronic viral hepatitis. The goal is to clear or sustained suppression of HBV to prevent or reduce liver inflammation, necrosis. A large number of clinical studies confirmed the ideal antiviral drugs interferon and nucleoside analogues. But interferon adapt narrow obvious side effects, long-term effect is not satisfactory, the price is expensive. Nucleoside analogues are prone to virus mutation, its clinical application is limited. A large number of clinical and experimental studies confirmed that the traditional Chinese medicine to protect liver cells, immune regulation, anti-fibrotic, and then by the combined effects of inhibition of viruses and other shows superiority. Not uniform, but the understanding of the Traditional Chinese Medicine Research surviving many problems, such as the so far Chinese medicine for chronic hepatitis B etiology and pathogenesis, syndrome diagnosis liver histopathological grading and commonly used in the international stage, typing clinical manifestations of chronic hepatitis B diagnostic criteria are incompatible, chronic hepatitis B is difficult to accurately reflect the severity of the clinical treatment of the lack of effective, science, systems can not meet the chronic hepatitis B in Chinese medicine clinical requirements.Objective:explore stilbene beads side through experimental research, the treatment of chronic hepatitis B mechanism, clear the stilbene beads prescription drug composition characteristics, looking for clinical lay the foundation for effective medicine in the treatment of this disease.Qizhu decoction side by Phyllanthus, Polygonum cuspidatum, Radix, TPG, diffusa, soil Fuling Qiwei drug composition, the maximum in order to ensure the efficacy of active ingredients, chosen for this study heating reflux extraction by RP-HPLC analysis of the chemical composition of the compound, and choose one of four different concentrations of ethanol extraction and analysis of the changes in the chemical composition of samples of different solvent extraction. Establishment of animal models of liver fibrosis based on traditional Chinese medicine, the middle dose group were16.20g/kg,9.72g/kg (Qi Zhu Fang Sheng the medicine content/rat body weight) orally, once a day, for the first time modeling the administration began the next day, for eight consecutive weeks until the animals were killed the day before; colchicine group (ground, made of an aqueous solution of colchicine tablets per1mL of water containing0.1mg colchicine)5.0mL/kg (colchicine alkali content/rat body weight) orally, once daily, the model for the first time the next day, the administration began eight consecutive weeks until the animals were killed the day before; model group, the same as the the stilbene beads Fangda dose group was given volume of physiological saline gavage, liver tissue TNF-a, IL-4, IL-8, IL-10, TIMP-1, MMP-1, MMP-2, MMP-13expression levels, serum TIMP-1, MMP-1, MMP-2, MMP-13expression.Results:different solvent extraction stilbene beads side (70%alcohol into the sample to80μL, the rest are10μL) sample obtained a number of peaks, mentioned by50%of water,70%,95%compound extracted basic25min before does not have a peak, during the experiment, by increasing the injection volume comparison,70%ethanolic the stilbene beads side peaks up to the size and shape of the peak shape better. Liver fibrosis model in animal models of rats liver tissue TNF-a, IL-4, IL-8expression relative blank group significantly increased IL-10expression significantly reduced liver tissue TNF-a, IL-4, the expression of IL-8was significantly (P<0.01) reduction in the expression of IL-10’s significantly with increased (P<0.01); stilbene beads side high dose group of TNF-a, of IL-4, of IL-8’s expression lower than the middle dose group IL-10expression is higher than the middle dose group; the stilbene beads to water extraction group, IL-4, IL-8, TNF-a expression is lower than the same dose of ethanol extract group, IL-10expression is higher than the same dose of ethanol extract group (P<0.01). Expression of IL-4compared to colchicine group, stilbene the beads side treatment group were higher than the colchicine group and there is a significant difference (P<0.01); stilbene beads to treatment group of IL-8expression was significantly higher than in autumn Narcissus alkali group (P<0.01); the stilbene beads to treatment group the expression of IL-10were lower than the colchicine group and a significant difference (P<0.01); the stilbene beads to treatment group were higher than the expression of TNF-a colchicine alkali group (P<0.01). Model group, TIMP-1, MMP-2expression is relatively higher than the normal group, MMP-1, MMP-13expression is relatively lower than the normal group, the two groups differ significant differences (P<0.01). All treatment groups TIMP-1, MMP-2expression is lower than the model group, MMP-1, MMP-13expression is higher than the model group, there were significant differences (P<0.01). Stilbene the beads side high dose group of TIMP-1, MMP-2expression is lower than the middle dose group, MMP-1, MMP-13expression is higher than the middle dose group, significant dose-dependent manner (P<0.01). The stilbene beads to water extraction group, TIMP-1, MMP-2expression is higher than the same dose of ethanol extract group (P<0.01), MMP-1, MMP-13expression were lower than the same dose of ethanol extract group (P<0.01). TIMP-1expression stilbene beads side4treatment groups were higher than the colchicine group compared with the colchicine group, and there is a significant difference (P<0.01); expression of MMP-1in the high-dose group was significantly higher than in autumn Narcissus alkali group (P <0.01), the expression of the middle dose group below the colchicine group in alcohol extract dose group difference (P<0.05) were significantly different (P<0.01), water extraction in the dose group; expression of MMP-2treatment groups were lower than the colchicine group, compound high-dose group and the alcohol extract group, middle dose group, a significant difference (P<0.01), no significant water extraction in the dose group (P>0.05); the stilbene beads Fangda dose group MMP-13expression above the colchicine group (P<0.01), the middle dose group was lower than the colchicine group (P<0.01). Model group, serum of TIMP-1, MMP-2expression is relatively higher than the normal group, MMP-1, MMP-13expression is relatively lower than the normal group, the two groups differ significant differences (P<0.01). All treatment groups serum TIMP-1, MMP-2expression is lower than the model group, MMP-1, MMP-13expression is higher than the model group, there were significant differences (P<0.01). Stilbene the beads side high dose of serum TIMP-1, MMP-2expression is lower than the middle dose group, MMP-1, MMP-13expression is higher than the middle dose group, significant dose-dependent manner (P<0.01). Stilbene beads to water extraction group dose serum TIMP-1expression extract the middle dose group (P<0.01) higher than the same dose of alcohol, the high-dose group was not statistically significant (P>0.05); MMP-1water extract group expression were lower than the same dose of alcohol extract group (P<0.01) higher than the same dose of alcohol extract group (P<0.01); aqueous extract group, the expression of MMP-2; MMP-13expression were lower than the same dose of ethanol extract group (P<0.01). TIMP-1expression compared with colchicine, stilbene beads side4treatment groups were higher than the colchicine group, large water extraction, the middle dose group and the alcohol extract middle dose group were significantly different (P<0.01) alcohol extract high-dose group was not statistically significant (P>0.05); MMP-1expression in the high-dose group than colchicine group (P<0.01), the middle dose group was lower than the colchicine group (P<0.01); expression of MMP-2treatment group were lower than the colchicine group, compound high-dose group and the alcohol extract group, middle dose group, a significant difference (P <0.01), the water was no mention in dose statistically significant (P>0.05); the stilbene beads Fangda dose group MMP-13expression is higher than the colchicine group (P<0.01), the middle dose group was lower than the colchicine group (P<0.01).Conclusion:Qizhu decoction party alcohol extract to treat chronic hepatitis B and liver fibrosis by regulating the liver and serum TNF-a, IL-4, IL-8, IL-10, TIMP-1, MMP-1, MMP-2, MMP-13.更多还原