【作者】 汪敬富；

【导师】 薛博瑜；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2009， 博士

【摘要】 背景:肝硬化是一种以肝组织弥漫性纤维化、假小叶和再生结节形成为特征的慢性肝病。引起肝硬化的病因很多,在我国以病毒性肝炎所致的肝硬化为主,西医认为发病机制主要是在肝脏多种细胞及细胞因子（cytokine,CK）参与下,引起细胞外基质（extracelluar matrix,ECM）的合成与降解失衡,即合成大于降解,导致ECM过度沉积所致。现已证实,肝星形细胞（hepatic stellate cell,HSC）是肝脏产生ECM的主要细胞,HSC活化是肝纤维化发生的中心环节。各种损肝因素导致肝损伤,继而引起肝细胞坏死及炎症刺激,相关细胞分泌多种CK激活HSC,产生大量的ECM。1995年,Sekelsky等在果蝇体内首先发现了Smads蛋白,随后的大量研究证实,Smads蛋白可将TGF-β信号直接由细胞膜激酶受体转导入细胞核内,是目前已知唯一的TGF-β受体（TβR）胞内激酶底物。现已证实,TGFβ-Smad信号转导过程可直接刺激HSC表型转化,诱导HSC凋亡,诱导基质基因表达,抑制基质降解,并且间接影响肝细胞、库普弗细胞、窦内皮细胞多项正常功能的发挥,起到促进肝纤维化发生发展的作用。慢性肝炎肝纤维化的发生率为59.36%,有25%～40%的肝纤维化患者发展成肝硬化,我国以乙型病毒性肝炎为多见。目前西医尚无有效的抗肝硬化的药物,中医药治疗慢性肝病历史悠久,作用明显。目的:本课题以中医理论为指导,根据中医文献中有关“胁痛”“黄疸”“癥积”“臌胀”等的论述,认为肝硬化病位主要在肝脾,久则及肾,古代医家提出“阳虚易治,阴虚难调”,说明肝肾阴虚既是肝硬化失代偿期的一个重要病机,亦是常见而难治的证型。且该证型较其它证型更易引起肝昏迷和大出血等变证。结合现代医学对肝硬化的认识和临床实践体会,病机主要是湿热瘀毒互结,肝肾亏虚所致,确立活血利湿、补益肝肾的治疗大法,治疗采用的药物兰豆护肝颗粒是根据名老中医邹良材教授经验方兰豆枫楮汤研制而成,经临床多年应用观察,疗效确切。本课题临床研究部分方探讨了该方治疗肝硬化的疗效机理。实验研究部分探讨了兰豆护肝颗粒对大鼠肝纤维化的血清学、病理形态学影响,从而为防治肝纤维化的中药新药研究打下基础,并为临床早期诊治肝纤维化提供了依据。方法:按照随机对照原则,本临床研究观察了180例肝炎肝硬化患者,对照组给予西医常规治疗,包括保肝降酶、利尿、退黄、补充白蛋白等基础措施;治疗组在西医一般治疗的基础上,运用兰豆护肝颗粒,主要组成药物有泽兰10g、路路通10g、黑料豆15g、楮实子20g、茵陈15g、牛膝10g、陈葫芦30g,每日3次,每次1包冲服,疗程均为6个月。观察指标包括临床症状体征、肝功能、肝纤指标等血清学检查及影像形态学改变。实验研究运用现代实验技术方法,使用纯中药制剂兰豆护肝颗粒对实验性肝纤维化SD大鼠140只进行干预,方剂由泽兰10g、黑料豆10g、楮实子10g、路路通10g、川牛膝10g、茵陈10g、陈葫芦20g组成,制剂浓度为2.64g/ml。实验分为两个部分,分别为兰豆护肝颗粒的预防用药和治疗用药,选用秋水仙碱作为对照,应用血清生化学检查、常规病理学及免疫病理学检测等技术,观察了兰豆护肝颗粒预防、治疗肝纤维化的疗效,并探讨其作用机制。结果:临床研究表明,兰豆护肝颗粒能够明显降低患者血清转氨酶活性,显著降低γ球蛋白,改善白/球比值,治疗前后两组临床症状体征、肝功能、肝纤指标等血清学检查及形态学均有不同程度改善,治疗后两组比较,疗效差异有统计学意义。治疗组基本治愈24例（27%）,好转49例（54%）,无效17例（19%）,总有效率81.1%。对照组基本治愈19例（21%）,好转34例（38%）,无效37例（41%）,总有效率58.9%。治疗组疗效优于对照组,两组比较经卡方检验,差异有统计学意义（P＜0.05）。同时兰豆护肝颗粒还可降低患者升高的HA、LN、Ⅳ型胶原,与治疗前比有差异有统计学意义（P＜0.05）。实验研究结果表明:预防高剂量组、预防中剂量组、预防低剂量组、预防秋水仙碱组与模型Ⅰ组相比,ALT、AST差异有统计学意义（P＜0.05）,余指标均有不同程度降低,但差异无统计学意义,兰豆护肝颗粒对大鼠急性肝损伤有一定的预防作用,并存在着一定的量效关系。对肝纤维化指标的影响,预防高剂量组、预防中剂量组、预防秋水仙碱组与正常组相比,除LN外,余无显著性差异,预防中剂量组、预防低剂量组纤维化指标均有不同程度的升高;与模型组相比,预防高剂量组、预防中剂量组、预防秋水仙碱组均有不同程度降低,HA（P＜0.01）,PⅢNP（P＜0.05）,差异有统计学意义,LN无明显差异;预防低剂量组HA（P＜0.01）,差异有统计学意义,预防低剂量组LN、PⅢNP降低,但差异无统计学意义。在治疗用药实验部分,治疗高剂量组、治疗中剂量组、治疗低剂量组、治疗秋水仙碱组各组肝功能均有恢复,与模型Ⅱ组相比无显著差异,CCl₄所致实验性大鼠肝损伤模型,停止CCl₄诱导后,肝功能可自我恢复,ALT、AST恢复较快,ALB、TP恢复较慢,大鼠肝脏修复功能比较强大,故各治疗组与模型组之间并差异无统计学意义。肝纤维化指标比较时,HA降低,与正常组比较无明显差异,与模型Ⅰ组比较差异显著（P＜0.01）、LN、PⅢNP较正常组显著升高（P＜0.05）,与模型Ⅰ组比较无显著性差异,提示CCl₄诱导大鼠肝纤维化停止造模用药后有自我恢复状况,实验中模型Ⅱ组较模型Ⅰ组纤维化指标虽有所好转,但总体与正常组差异有统计学意义;治疗各组纤维化指标都有不同程度的降低,与模型Ⅱ组相比,治疗高剂量组、治疗中剂量组、治疗低剂量组、治疗秋水仙碱组各组纤维化指标均有改善,HA（P＜0.01）,治疗高剂量组、治疗中剂量组、治疗秋水仙碱组各组LN（P＜0.05）,治疗高剂量组、治疗秋水仙碱组PⅢNP（P＜0.05）,差异有统计学意义,兰豆护肝颗粒对大鼠肝纤维化有较好的治疗作用,并存在着量效关系。病理形态学观察发现兰豆护肝颗粒对四氯化碳诱导的大鼠肝纤维化模型有较好的预防、治疗作用,能抑制Smad4的表达,减少ECM的沉积。结论:兰豆护肝颗粒能缓解肝炎肝硬化患者的症状体征,保护肝功能,改善肝纤维化指标,实验研究表明兰豆护肝颗粒能显著改善大鼠CCl₄造模肝纤维化模型的肝功能、肝纤维化指标,可能与兰豆护肝颗粒能抑制Smad4的表达,减少ECM的沉积有关。更多还原

【Abstract】 Purpose: From the theory of traditional Chinese medicine, we know that the postion of hepatical cirrhosis lies in the liver, spleen and kidney. Former doctors raise the gist of "it’s easy to cure the yang-deficiency, while hard to yin-deficiency", it is reported that the syndrome of hepaticrenal yin-deficiency is an important pathogenesis, and also is a regular and stubborn syndrome, and this syndrome is easy to become hepaticalencephalopathy and bleeding than others. We are convinced of the pathogenesis of the hepatic cirrhosis is heat interwinding damp, and deficiency of liver and kidney. So activating the blood circulation and supplementing the liver and kidney is established. The compound drugs landou hugan granules is from the professor zouliangcai. After the clinical application, the effect is exact. The clinical part of this project is to declare the way of this compound to cure the hepatical cirrhosis, experimental part discuss the compound’s effect on the hepatic fibrosis and morphologic pathology. And we can make the basis of treating hepatic cirrhosis.Methods: Randomized controlled trial in accordance with the principles of clinical research of the 180 cases of hepatitis observed in patients with liver cirrhosis, the control group given conventional treatment of western medicine, including lowering the liver enzymes, diuretic, dispelling jaundice, supplementary measures such as albumin; treatment group in the general treatment of the basis of Western medicine , the use of Landou Hugan granules, a major component drugs Herba Lycopi , black bean, fructus broussonetiae, fructus liquidambaris, Cyathnla capitata Moq, herba artemisiae, Lagenaria vulgaris Ser, thick fried 100ml, morning and evening service hours, treated for 6 months. Observed clinical signs and symptoms, liver function, liver defibrase indicators serological examination and morphological changes of the image.Experimental study on the use of modern experimental techniques, the use of pure Chinese medicine granules Landou Hugans on experimental hepatic fibrosis in 140 SD rats, prescription from Herba Lycopi , black bean, fructus broussonetiae, fructus liquidambaris, Cyathnla capitata Moq, herba artemisiae, Lagenaria vulgaris Ser, concentration of agents 2.64g/ml. Experiment is divided into two parts, namely, Landou hugan granules prevention and treatment of drug use, selection of colchicine as a control, application inspection serum biochemistry, pathology and immunopathology of conventional detection techniques, observation of the Landou Hugan granules prevention, treatment efficacy of hepatic fibrosis and to explore its mechanism.Result: Clinical studies have shown that Landou Hugan granules can significantly reduced serum transaminase activity, a significant reduction inγ-globulin to improve the A/G ratio, the two groups before and after treatment, the clinical symptoms and signs, liver function, serum indicators of liver defibrase and morphological examination improvement in varying degrees, the two groups after treatment, the difference was statistically significant. Treatment group 24 cases of cure （27%）, improvement in 49 cases （54%）, ineffective in 17 patients （19%）, total effective rate of 81.1%. The control group cured 19 patients （21%）, improvement in 34 cases （38%）, 37 cases of invalid （41%）, the total effective rate 58.9%. Efficacy of the treatment group than the control group, two groups by chi-square test, the difference was statistically significant （P <0.05）. Landou Hugan granules at the same time can reduce the increase in patients with HA, LN, IV collagen, and difference between the time of before and after treatment were statistically significant （P <0.05）.Experimental results show that: the prevention of high-dose group, middle dose group prevention, prevention of low-dose group, the prevention of colchicine group and model group I, ALT, AST were statistically significant differences （P <0.05）, more than indicators are reduced to varying degrees, but the difference was not statistically significant, Landou Hugan granules in rats acute liver injury have some effect in prevention and there is a certain degree of dose-effect relationship. Indicators of the impact of liver fibrosis, prevention of high-dose group, middle dose group prevention, prevention colchicine group compared with the normal group, with the exception of LN, the more was no significant difference in the prevention of middle dose group, low-dose group to prevent fibrosis indicators increased in varying degrees; Compared with model group, the prevention of high-dose group, middle dose group prevention, prevention colchicine group were reduced to varying degrees, HA （P <0.01）, PIIINP （P <0.05）, There were significant differences, LN was no significant difference; the prevention of low-dose group of HA （P <0.01）, the difference was statistically significant, the prevention of low-dose group LN, PIIINP lower, but the difference was not statistically significant.Experimental drugs in the treatment of high-dose treatment groups, treatment dose, treatment of low-dose group, treatment colchicine group had restoration of liver function group, compared with model group II was no significant difference, CCl₄-induced experimental rat liver injury model, after the cessation of CCl₄-induced, self-restoration of liver function, ALT, AST to resume quickly, ALB, TP restoration of slow repair in rat liver was comparatively strong, so the treatment group and model group and the difference is not statistically significant. When compared to hepatic fibrosis, HA compared with the normal group had no significant differences in comparison with the model group I significantly different （P <0.01）, LN, P III NP than the normal group was significantly higher （P <0.05）, with the model group I no significant difference, suggesting that CCl₄-induced rat liver fibrosis model drug after cessation of self-recovery experiment in the model group than in model I II group, despite the improvement in fibrosis index, the overall difference with normal group statistics significance; the treatment of fibrotic markers in each group has a different degree of reduction compared with the model group II, the treatment of high-dose group, the treatment dose, treatment of low-dose group, the treatment of fibrosis colchicine group of indicators in each group were improvement, HA （P <0.01）, treatment of high-dose group, the treatment dose group, treatment group colchicine group LN （P <0.05）, treatment of high-dose group, treatment colchicine group P III NP （P <0.05）, There were significant differences, Portland Hugan bean granules of rat liver fibrosis have a better therapeutic effect and dose-effect relationship exists. Observed that the pathomorphology of Landou Hugan granules on carbon tetrachloride-induced rat liver fibrosis model was better prevention, treatment effect, can inhibit the expression of Smad4 and reduce the deposition of ECM.Conclusion: Landou Hugan granules can improve hepatitis symptoms in patients with liver cirrhosis, catabatic liver function and improve the liver fibrosis, experimental study of Landou Hugans can significantly improve liver function of the model CCM rat model of liver fibrosis, liver fibrosis indicators may be related to Landou Hugan granules inhibiting the expression of Smad4 and reduce the deposition of the ECM.更多还原