【作者】 张纨；

【导师】 李佃贵；

【作者基本信息】 河北医科大学 ， 中西医结合临床， 2010， 博士

【摘要】 肝纤维化(hepatic fibrosis,HF)是指肝细胞发生坏死或炎症刺激时,肝脏内纤维结缔组织增生与分解失衡,从而在肝内异常沉积的病理过程,是多种慢性肝病的重要病理特征和发展至肝硬化的必经阶段。2006年美国第三届肝纤维化专题会议明确了肝纤维化是可逆的。Hans Popper教授说:“谁能阻止或延缓肝纤维化,谁将能医治大多数慢性肝病”。肝纤维化发生的关键是肝星状细胞(hepatic stellate cell,HSC)的激活及其激活后细胞外基质(extracellular matrixc,ECM)异常表达。从细胞因子及其信号转导角度研究药物的干预机制,有利于促进HF治疗学研究。转化生长因子-β1(transforming growth factor-beta1-1,TGF-β1)是最重要的促肝纤维化细胞因子之一,具有抑制肝细胞增殖、诱导肝细胞凋亡,激活HSC、促进其产生ECM等作用,TGFβ/Smad信号转导通路是TGFβ发挥这些生物效应重要的信号转导通路。目前临床仍缺乏高效、无明显毒副作用的治疗HF的西药。中医药通过多途径、多环节、多靶点的综合药理学作用在抗HF方面展现出优势,显示了良好的前景。李佃贵教授经过多年的临床实践和基础研究,结合当今疾病模式及疾病谱的变化,提出以“浊毒”立论治疗肝纤维化的理论,并应用化浊解毒调肝法进行治疗,取得较好疗效。依据此理论确立的肝复健方能显著改善HF、慢性肝炎患者临床症状,促进HF指标的恢复;肝复健方能明显改善HF模型大鼠肝功能、抑制肝纤维化大鼠血清HA、LN、Ⅳ-C、PCIII水平,抑制胶原纤维增生等作用。本课题以肝纤维化大鼠模型,以秋水仙碱为对照,观察肝复健方对HF大鼠肝脏TGF-β1及其受体、Smad2/3、Smad4、Smda7的表达情况,探讨其对TGF-β/Smad信号传导通路的影响,旨在对本方抗HF的药效和可能的作用机制作进一步的研究,为其临床应用提供实验依据。本实验分为以下五部分:第一部分浊毒理论与肝纤维化目的:以浊毒理论为依据探讨其在肝纤维化治疗中的地位,进而提出新的观点与治疗方法。方法:温习、回顾了中医理论中浊毒源流,提出浊毒相关为害是肝纤维化的主病机,系统总结了肝纤维化浊毒特性的临床表现、基本治法、用药规律及兼夹证的治疗。结果:梳理了浊毒之源流,探讨了肝纤维化与浊毒的关系,以及化浊解毒调肝法提出的理论依据,从临床论证了化浊解毒调肝方药治疗肝纤维化的可行性。结论:浊毒是肝纤维化形成和进展过程中重要的始动因素和关键环节,临床上应用化浊解毒法不仅能改善其症状,还可恢复肝脏生理特性,截断或逆转肝脏病理性改变。第二部分肝复健方对肝纤维化大鼠肝脏病理组织学的影响目的:将肝复健方应用于造模成功的HF大鼠,观察肝复健方对HF大鼠一般情况和病理组织学的影响。方法:健康雄性SD大鼠60只,适应性喂养一周,随机分为正常组(Normal)10只,造模组50只。除正常组外,造模组采用腹腔注射未灭活猪血清法造模,每只大鼠每次注射猪血清0.5 ml,每周2次,共8周。造模成功大鼠分为:模型组,肝复健方小剂量组(以下简称GFJL),肝复健方中剂量组(以下简称GFJM),肝复健方大剂量组(以下简称GFJH),秋水仙碱组,每组均10只。GFJL给予含1.5 g生药/ml水煎剂2ml灌胃;GFJM给予含2.25 g生药/ml水煎剂2ml灌胃;GFJH给予含3.0 g生药/ml水煎剂2ml灌胃;秋水仙碱组给予0.154 g/kg·d灌胃;空白组、模型组均给予等量生理盐水灌胃。各组均灌胃至12周末。造模用药期间观察大鼠一般情况。于12周结束时,断颈处死各组大鼠,留取大鼠肝组织,采用苏木精-伊红染色(HE染色)和Masson染色(胶原纤维特殊染色法)观察肝组织病理形态学改变并进行大鼠肝纤维化病变半定量分析。结果:1一般情况观察肝复健方可明显改善HF大鼠的精神状态、饮食、皮毛色泽,增加其体重。2肝脏大体形态观察正常组大鼠肝脏质地柔软,表面光滑。模型组大鼠肝脏质地坚韧,表面粗糙,颜色晦暗,有些可见颗粒状的小结节。秋水仙碱组及肝复健方药物治疗组大鼠肝表面较模型组光滑,质地较柔软。3肝复健方对肝纤维化大鼠肝组织病理的影响正常组肝小叶结构完整,肝细胞围绕中央静脉呈放射状排列,无变性坏死,无纤维组织增生。模型组肝小叶结构严重破坏,肝细胞广泛坏死伴炎性细胞浸润,纤维组织大量增生并形成纤维间隔。与模型组比较,各药物治疗组肝小叶结构破坏明显减轻,肝脏胶原纤维增生亦明显减轻,纤维条索疏松变窄。肝细胞水肿好转,变性情况明显改善,炎细胞浸润减少。其中肝复健方高、中剂量组肝脏结构改善较为明显。4大鼠肝纤维化病变半定量分析按肝纤维化病变半定量分析判断标准的计分标准,正常组大鼠纤维化程度为0,模型组大鼠纤维化程度为(15.76±0.33),秋水仙碱组大鼠纤维化程度为(5.46±0.20),GFJL大鼠纤维化程度为(11.54±0.36),GFJM大鼠纤维化程度为(9.6±0.16),GFJH大鼠纤维化程度为(5.2±0.12)。与模型组比较,秋水仙碱组、GFJL、GFJM、GFJH大鼠纤维化程度均明显减轻,差异有统计学意义(P<0.05)。结论:肝复健方能改善HF大鼠一般状况,能显著减轻大鼠肝组织炎症活动度及纤维化程度,体现出较强的保肝、抗HF作用。第三部分肝复健方对肝纤维化大鼠肝组织TGF-β1的影响目的:探讨肝复健方对HF大鼠TGF-β1的影响。方法:免疫组织化学法和RT-PCR法分别检测HF大鼠肝组织TGF-β1的表达。结果:1肝复健方对大鼠肝组织TGF-β1蛋白表达的影响正常肝组织中TGF-β1蛋白微弱表达,显色极淡。模型组较正常组TGF-β1阳性信号明显增强;不同剂量肝复健方组TGF-β1蛋白的表达较模型组明显减少(p<0.05),GFJH作用最为明显;秋水仙碱组TGF-β1蛋白的表达较模型组降低(p<0.05)。2肝复健方对大鼠肝组织TGF-β1mRNA表达的影响与正常对照组相比,模型组、秋水仙碱组和肝复健方大、中、小剂量组TGF-β1mRNA的表达均明显增强,差异有统计学意义(p<0.05)。与模型对照组相比,秋水仙碱组和肝复健方大、中、小剂量组TGF-β1mRNA表达明显减弱,差异有统计学意义(p<0.05)。且肝复健方大、中剂量组优于秋水仙碱组(p<0.05)。结论:肝复健方可能通过降低肝组织TGF-β1蛋白及基因的表达而达到抗纤维化的作用。第四部分肝复健方对肝纤维化大鼠肝组织TβRI、TβRII的影响目的:探讨肝复健方对HF大鼠TβRI、TβRII表达的影响。方法:免疫组织化学法和RT-PCR法分别检测HF大鼠肝组织TβRI、TβRII的表达。结果:1肝复健方对大鼠肝组织TβRI、TβRII蛋白表达的影响正常对照组肝细胞和间质细胞可见TβRI和TβRII阳性染色,模型组TβRI和TβRII阳性染色显著增强(P<0.05),尤以纤维增生组织和炎性细胞周围多见。与正常组相比,模型组和各治疗组表达明显升高(P<0.05),而与模型组比较,秋水仙碱组和肝复健方各剂量组大鼠肝组织TβRI和TβRII表达则明显降低(P<0.05);各治疗组比较,肝复健方大、中剂量组优于秋水仙碱组(P<0.05)。2肝复健方对大鼠肝组织TβRI、TβRII mRNA表达的影响与正常对照组相比,模型对照组、秋水仙碱组和肝复健方大、中、小剂量组TβRI和TβRIImRNA的表达均明显增强,差异有统计学意义(p<0.05)。与模型对照组相比,秋水仙碱组和肝复健方大、中、小剂量组TβRI和TβRIImRNA表达明显减弱,差异有统计学意义(p<0.05)。且肝复健方大、中剂量组优于秋水仙碱组(p<0.05)。结论:肝复健方能够显著抑制HF大鼠肝组织TβRI、TβRII型受体蛋白及基因的表达,从而抑制HF的发生、发展。第五部分肝复健方对肝纤维化大鼠肝组织Smads表达的影响目的:探讨肝复健方对Smad2/3,Smad4,Smad7蛋白表达的影响,结合二、三、四部分,观察其对TGF-β/samd信号通路的影响。方法:Western blot检测HF大鼠肝组织Smad2/3,Smad4,Smad7的表达。结果:1肝复健方对大鼠肝组织Smad2/3、Smad4蛋白表达的影响(1)所有实验组大鼠肝组织中Smad2/3、Smad4的表达量均高于正常对照组(P<0.05);(2)肝复健方各剂量组及秋水仙碱组与模型组相比Smad2/3、Smad4的表达量有显著性降低(P<0.05);(3) GFJL与秋水仙碱组相比Smad2/3、Smad4表达量均无显著性差异(P>0.05);肝复健方中、大剂量组分别与秋水仙碱组相比Smad2/3、Smad4的表达量有显著性降低(P<0.05);(4)GFJH与GFJM相比Smad2/3、Smad4表达量有下降趋势,但无显著性差异(P>0.05)。2肝复健方对大鼠肝组织Smad7蛋白表达的影响(1)所有实验组大鼠肝组织中Smad7的表达量均低于正常对照组(P<0.05)。(2)肝复健方各剂量组及秋水仙碱组与模型组相比Smad7的表达量显著升高(P<0.05)。(3)GFJL与秋水仙碱组相比Smad7表达量均无显著性差异(P>0.05);肝复健方中、大剂量组分别与秋水仙碱组相比Smad7的表达量显著升高(P<0.05)。(4)GFJH与GFJM相比Smad7表达量有上升趋势,但无显著性差异(P>0.05)。结论:肝复健方能够抑制受体活化性蛋白smad2和smad3的表达,下调通用性蛋白Smad4的表达,促进抑制性蛋白Smad7的表达,抑制HF大鼠肝脏中TGF-β/smad信号传导通路的激活,从而抑制肝纤维化的发生和发展。更多还原

【Abstract】 Hepatic fibrosis (HF)is a pathologic process that every kind of disease cause liver injure and inflammation. Its essense is excessive deposition of extracellular matrix(ECM) components in liver,which occurs due to an imbalance between the production and degradation of matrix. It is the hallmark of most chronic liver diseases. The United States 3th liver fibrosis conferences in 2006 indicated that HF is reversible. Professor Hans Popper says:”Who can prevent or delay liver fibrosis, who will be able to cure most chronic liver disease.”The activation and proliferation of hepatic stellate cell (HSC), which are the major source of ECM, are the crucial event to liver fibrosis. It is advantageous in HF treatment from the cell factor and its signal pathways to study mechanism of drug intervention.Transforming growth factor-beta1-1 (TGF-β1)is one of the most important Hepatic Fibrosis cell factors, can inhibition of hepatocyte proliferation, induce hepatocyte apoptosis, activate HSC,promote formation of ECM.TGF-β1 exerts biological effect mainly through TGF-β/samd signal pathway.At present clinical still lackes highly effective and no obvious poisonous side effect medicine to treat HF.Traditional Chinese Medicine has seen significant advancement against liver fibrosis. After many years clinical practices and experiment research, combining with current disease pattern and disease spectrum changes,professor Li dian-gui advances the zhuodu theory , applies this theory to treat Hepatic Fibrosis patients ,which obtaining the good curative effect.Ganfujian formula based on zhuodu theory can improve patients’clinical findings, inhibiting inflammatory reaction and fibroplasias to promote the reversion of hepatic fibrosis. The experimental study indicated that,it can obviously reduce HF rat blood serum HA、LN、Ⅳ-C、PCⅢ,reduces the liver inflammation and the fibrosis degree.We still used Hepatic Fibrosis rat’s model, taking the colchicine as the comparison, to determine if Ganfujian formula could inhibit the expression of TGFβ1,TβRI,TβRII,smad2,smad3 and promote the expression of smad7 and then intercepts TGF-β/Samd signal pathway,.This provides scientific evidence for clinic to apply Ganfujian formula in treating cirrhosis. This experiment was separated into five parts:Part one ZhuoDu theory and Hepatic FibrosisObjective: The aim was to study the effect of The ZhuoDu theory in the position of therapy for Hepatic Fibrosis in order to proposes the new viewpoint and the treatment method for HF.Methods: Reviewed zhuodu’s source and course in the Chinese medicine theory,put forward that the main pathogenesis of Hepatic Fibrosis was zhuodu,and made a systematic summarization on the characteristics of Hepatic Fibrosis,symptoms,general therapies and the treatment of combined syndromes.Results: We compiled the history of zhuodu ,discussed the relations of Hepatic Fibrosis and zhuodu, searched the treatment of huozhuo jiedu tiaogan theoretical basis,put forward that Hepatic Fibrosis’s main pathogenesis was zhuodu. Clinical curative effect proved that zhuodu theory has a positive position in treatment methods of Hepatic Fibrosis.Conclusion: Zhuodu closely links with Hepatic Fibrosis in etiology and pathogenesis, also is it’s main pathogenesis.The therapy of huazhuo jiedu used in clinic can not only relieve the symptoms,but also restore the liver physiology characteristic and reversal liver pathology changes.Part two Effect of Ganfujian formula on liver histopathology in HF ratsObjective: The aim was to observe Ganfujian formula’s effect on rats’ordinary circumstances and change of liver histopathology in HF rats.Methods:Sixty male Sprague-Dauley rats were selected in this experiment.The animals were randomly divided into normal group(10 rats)and HF model group(50 rats).Besides the normal group, pig’s serum(0.5ml per mouse,twice a week)was established in rats by intraperitoneal injection for eight weeks. The rats of HF model were randomly divided into five groups: the model group,colchicines group,Ganfujian formula low dose group(GFJL), Ganfujian formula of medium dose group(GFJM) and Ganfujian formula of high dose group(GFJH)with 10 rats for each group. After HF models were made successfully,the drugs started be given to the rats according to adult dosage 5,15,20 times,respectively,once daily.The rats were respectively treated with Ganfujian low dose(1.5g crude-drug/ml),Ganfujian middle dose(2.25g crude-drug/ml),Ganfujian high dose(3.0g crude-drug/ml) and colchicine(0.154g/kg·d) intragastrically,the normal group and the model group were given physiological saline by gavage.Each group was given the medicine for 12 weeks. Observed rats’ordinary circumstances.All animals were killed ,the specimens were taken and the degree of hepatic fibrosis was judged by routine haematoxylin-eosin staining and Masson staining. Fibrosis score was calculated by semi-quantitative analysis of liver histopathology.Results:1 Rat ordinary circumstances observationThe rat ordinary circumstances observation demonstrated that,The strong 1iver fries by the stasis may obviously improve the HF rat’s state of mind,the diet, the superficial knowledge luster,increases its body weight.2 Gross findingsLivers of control group were tender and smooth.The ones of model group were hard,coarse and tarnish,with some observable small nodules, whereas the ones of drug groups were smooth and tender.3 Effect of Ganfujian formula to pathomorphology in hepatic fibrosis ratsThe structure of hepatic lobule were integrated hepatic cord radially ranged as the center of navel vein. There was not degeneration and necrosis in liver tissue.There was not collagen proliferation.Compared with normal group,the model control rats had obvious liver inflammation including degeneration,necrosis,fibrosis and collagen proliferation.Pseudolobule proliferation could be seen in individual animal. Compared with model group,in various drug groups,the destructions were lightened,proliferatio ns of collagen fibers were also lightened,fiber cords were loosened and narrowed.Swelling of liver cells and degeneration alleviated,infiltrating cells decreased.These improvements were more significant in high and medium-dose GFJ groups.4 Semi-quantitative analysis of liver histopathology in HF ratsAccording to HF semi-quantitative analysis standard,fibrosis score was evaluated on the liver tissue sections performing with HE and Reticulation’s stain.The fibrosis score of normal group was 0,model group was 15.76±0.33, colchicines group was 5.46±0.20, GFJL was 11.54±0.36, GFJM was 9.6±0.16, GFJH was 5.2±0.12. Compared with model group, the fibrosis score all decreased in colchicines group, GFJL, GFJM and GFJH(p<0.05).Conclusion:Ganfujian formula can improve the HF rat’s ordinary circumstances and obviously reduce the rat liver organization inflammation activity and the fibrosis degree,presents the good local constable liver,the anti-HF function.Part three Effect of Ganfujian formula on TGF-β1 in HF ratsObjective: The aim was to explore Ganfujian formula’s effect on expression of TGF-β1 in HF rats.Methods: TGFβ1 protein and mRNA expression were determined by using immunohistochemical technique and RT-PCR.Results:1 Experimental study on Ganfujian Formula effect on TGF-β1 protein of the hepatic fibrosis ratsLess positive expression of TGF-β1 cell can be seen in the liver tissue of rats form normal group.The expression of TGF-β1 was strongly positive in model group. The cell proportion of TGF-β1 cell expression in the liver tissue of rats form model group were significantly higer than that of the normal group,which were manifested by the unsymmetric brown yellow particulates.The protein level of TGF-β1 of hepatic fibrosis rats could be decreased from Ganfujian group and colchicines group significantly.The effect of GFJH group was better than that others group. 2 Experimental study on Ganfujian formula effect on TGF-β1 mRNA of the hepatic fibrosis ratsCompared with normal group, the level of TGF-β1 mRNA all increased in model group ,Ganfujian group and colchicines group(p<0.05).But the level of TGF-β1 mRNA decreased in Ganfujian group and colchicines group as compared with that in model group(p<0.05),in which,the reduction in medium dose group and high dose group were more than colchicines group(p<0.05).Conclusion: Ganfujian formula possibly reduce or prevent hepatic fibrosis through decreasing the expression of TGF-β1 protein and mRNA. Part four Effect of Ganfujian formula on TβRI,TβRII in HF ratsObjective: The aim was to explore Ganfujian formula’s effect on expression of TβRI,TβRII in HF rats.Methods: TβRI,TβRII protein and mRNA expression were determined by using immunohistochemical technique and RT-PCR, respectively.Results:1 Expression of TβRI,TβRII protein in liver tissueTβRI,TβRII protein expressed in hepatocellular and interstitial cells. The expression of TβRI,TβRII protein in model group was stronger than which in normal group(P<0.05), TβRI,TβRII protein especially expressed around fibre hyperplasia and inflammatory cells.Compared with normal group,the expression of TβRI,TβRII protein in other groups was stronger(P<0.05).Compared with model group,the expression of TβRI,TβRII protein in Ganfujian groups and colchicines group was significantly lighter(P<0.05).The expression of TβRI,TβRII protein in GFJH group and GFJM group were lighter than that of colchicines group, there were significantly difference among those groups(P<0.05).2 Expression of TβRI,TβRII mRNA in liver tissueCompared with normal group,the expression of TβRI,TβRII mRNA in other groups was stronger(P<0.05).Compared with model group,the expression of TβRI,TβRII mRNA in Ganfujian groups and colchicines group was significantly lighter(P<0.05).The expression of TβRI,TβRII mRNA in GFJH group and GFJM group were lighter than that of colchicines group, there were significantly difference among those groups(P<0.05).Conclusion: Ganfujian formula can significantly inhibits the expression of TGFβ1 receptors’protein and mRNA.Part five Effect of Ganfujian formula on Smads in HF ratsObjective: The aim was to explore Ganfujian formula’s effect on expression of Smads’protein in HF rats.Combined with part two,three and four, Our study is to determine if Ganfujian formula intercepts TGF-β/Samd signal pathway.Methods: Western blotting was used for detecting the protein expression level of Smad2/3,Smad4,Smad7, respectively.Results:1 Expression of Smad2/3, Smad4 protein in liver tissueCompared with normal group,the expression of Smad2/3, Smad4 in other groups was stronger(P<0.05).Compared with model group,the expression of Smad2/3, Smad4 in Ganfujian groups and colchicines group was significantly lighter(P<0.05).Compared with colchicines group, the expression of Smad2/3, Smad4 in GFJM group and GFJH group was significantly lighter(P<0.05),and there was no difference between colchicines group and GFJL group(P>0.05). The expression of Smad2/3, Smad4 in GFJH group was lighter than that of GFJM group,but there was no difference between two groups(P>0.05).2 Expression of Smad7 protein in liver tissueCompared with normal group,the expression of Smad7 in other groups was lower(P<0.05).Compared with model group,the expression of Smad7 in Ganfujian groups and colchicines group was significantly stronger(P<0.05).Compared with colchicines group, the expression of Smad7 in GFJM group and GFJH group was significantly stronger(P<0.05),and there was no difference between colchicines group and GFJL group(P>0.05). The expression of Smad7 in GFJH group was stronger than that of GFJM group,but there was no difference between two groups(P>0.05).Conclusion: Ganfujian could inhibit the expression of smad2,smad3 and promote the expression of smad7 which could intercepts TGF-β/Samd signal pathway, then delay the process of HF and become an effective therapy for HF.更多还原