【作者】 熊振芳；

【导师】 张赤志；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2010， 博士

【摘要】 研究背景与目的：肝纤维化是肝脏对不同病因所致的慢性损害产生的创伤愈合反应,表现为肝脏细胞外基质各种成分的过度沉积及分布异常,是纤维增生和纤维降解不平衡的结果,是多种慢性肝病向肝硬化发展的中间病理阶段,目前的研究认为肝纤维化病变经过适当的积极治疗有希望延缓甚至逆转,而发展到肝硬化阶段则很难逆转。中医药治疗肝纤维化具有多靶点、多环节、多途径复合作用的特点,针对肝纤维化发病的复杂机制,体现了中医药的传统优势。蕲艾是湖北省的道地药材,临床研究发现蕲艾有明显的抗肝纤维化的作用,本课题通过建立免疫性肝纤维化动物模型,初步了解蕲艾提取液抑制大鼠肝纤维化的作用,并通过其对大鼠肝组织中TGFβ-Smad信号转导通路中关键的信号传导分子TGFβ1、Smad 3、Smad 4、Smad 7表达水平的变化,以及对抗凋亡基因Bcl-2和细胞周期蛋白的影响,探讨蕲艾提取液抗肝纤维化的分子机理。方法：1.采用猪血清诱导肝纤维化模型,将72只Wistar大鼠适应喂养1周后,随机分为正常对照组、肝纤维化模型组、蕲艾提取液低剂量、中剂量和高剂量组及丹参治疗组,每组12只。除正常对照组外,其余各组腹腔注射无菌猪血清0.3ml,每周2次,共10周。蕲艾提取液高、中和低剂量治疗组分别给予6ml/只、3ml/只和1.5ml/只的蕲艾提取液灌胃,每日1次；丹参治疗组给予丹参注射液6ml/只灌胃,每日1次；模型组给予生理盐水6ml/只灌胃,每日1次；正常对照组大鼠按100g体重给予生理盐水0.3ml腹腔注射,每周2次,共10周,并给予生理盐水6ml/只灌胃,每日1次。于10周末最后一次注射猪血清后48h,断头处死各组动物,眼球取血留取足够的血,分离血清,并取一部分肝脏组织,4%福尔马林固定,常规石蜡包埋切片。一部分新鲜肝组织梯度置于-70℃保存,用于实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)。2.血清学检测：各组实验大鼠血清分别检测肝纤维化血清学指标透明质酸(HA)、层粘蛋白(LN)、Ⅲ型前胶原(PCⅢ)、Ⅳ型前胶原(IV-C)的含量,以及脂质过氧化指标超氧化物歧化酶(SOD)、丙二醛(MDA)O ELISA法检测血清中TGFβ1含量。3.肝组织学检测：采用HE染色和Masson三重胶原染色法观察大鼠肝组织病理学变化。4.免疫组织化学检测：采用免疫组化SABC法,结合显微镜,观察大鼠肝组织TGFβ1、Smad3、Smad4、Smad7以及Bcl-2、Cyclin D1的表达变化。5.采用实时荧光定量反转录聚合酶链式反应(RT-PCR)法及蛋白印迹实验(Western blot)分别检测TGFβ1、Smad3、Smad4、Smad7 mRNA和蛋白及Bcl-2、cyclinDl mRNA和蛋白的表达变化。结果1.蕲艾提取液抑制大鼠肝纤维化作用的实验研究与正常对照组比较,模型组中肝纤维化血清学指标HA、PCⅢ、LN、IV-C含量均明显升高(P<0.01),血清TGF-β1水平亦明显升高(P<0.05),血清MDA含量上升、SOD活性显著降低(P<0.01)；与模型组比较,蕲艾提取液治疗各组HA、PCⅢ、LN、IV-C含量均显著降低(P<0.05),血清TGF-β1含量亦明显下降(P<0.05),血清脂质过氧化指标MDA含量显著降低(P＜0.05), SOD活性显著升高(P<0.05),且蕲艾提取液治疗组与正常对照组无显著性差异(P>0.05)。光镜下观察：模型组大鼠可见肝组织结构部分破坏,肝细胞发生广泛的变性水肿,水样变性甚至气球样变性,还可见程度、范围不等的肝细胞坏死；肝板排列紊乱,部分肝小叶失去正常结构；汇管区纤维隔内有大量嗜酸性细胞、单核淋巴细胞及成纤维细胞浸润,大量胶原沉积,有较明显纤维组织增生,胶原纤维沿汇管区及中央静脉周围向肝小叶内延伸,可见相互连接的纤维分割肝小叶,甚至有部分区域有早期肝硬化表现。蕲艾提取液及丹参治疗各组大鼠肝脏在色泽、质地、大小、表面光滑度方面较模型组有明显改善,细胞变性和坏死程度显著减轻,肝小叶结构破坏不明显,炎性细胞浸润和纤维组织增生减少,中央静脉和汇管区有少量纤维组织延伸,不同剂量蕲艾提取液治疗组间无显著性差异。2.蕲艾提取液对大鼠肝组织中TGFβ-Smad信号转导通路蛋白表达的影响免疫组织化学结果显示,TGFβ1、Smad3、Smad4在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见于间质细胞、炎性细胞、损伤的肝细胞,阳性物质主要集中在细胞膜上,胞质内有少量表达；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。RT-PCR及Western blot检测结果显示,TGFβ1、Smad3、Smad4 mRNA及其蛋白,在正常对照组中表达明显低下,而Smad7 mRNA及其蛋白表达则明显升高；蕲艾提取液高、中、低剂量组TGFβ1、Smad 3、Smad4 mRNA及其蛋白与模型组比较表达逐渐减少,Smad7 mRNA及其蛋白表达与模型组比较则逐渐升高,其中中、高剂量组TGFβ1、Smad3、Smad7 mRNA及其蛋白与模型组比较有显著性差异(P<0.05,P<0.01),而与丹参组比较无显著性差异(P>0.05)。3.蕲艾提取液对抗凋亡基因Bcl-2及细胞周期蛋白Cyclin D1表达的影响免疫组织化学结果显示,Bcl-2及Cyclin D1蛋白在正常对照组肝细胞内无阳性细胞表达,模型组肝内表达明显增强,主要见汇管区、肝细胞、间质细胞膜上及细胞浆内,阳性物质主要集中在细胞膜上；蕲艾提取液各剂量组及丹参组阳性细胞数目明显减少,阳性染色程度明显减轻。RT-PCR及Western blot检测结果显示,正常对照组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组低下,蕲艾提取液各剂量组Bcl-2、Cyclin D1 mRNA及蛋白表达较模型组逐渐减少,其中高剂量组与模型组比较有显著性差异(P<0.05),与正常对照组和丹参组比较无显著性差异(P>0.05)。结论1.蕲艾提取液可以明显改善猪血清诱导的大鼠免疫性肝纤维化的肝组织病理学损害,减轻纤维组织增生,明显减少胶原纤维间隔、肝细胞坏死、变性以及炎性细胞的浸润,并能有效降低肝纤维化血清学指标,其抗纤维化的机理可能与抗脂质过氧化有关。2.蕲艾提取液能够显著抑制大鼠肝组织中TGFβ1、Smad3、Smad4 mRNA及其蛋白表达水平,上调Smad7 mRNA及其蛋白表达水平,表明其可能通过有效调控TGFβ1-Smad信号转导通路,从而抑制大鼠肝纤维化。3.蕲艾提取液能够降低大鼠肝组织中抗凋亡基因Bcl-2 mRNA及其蛋白表达水平,提示其可能通过有效诱导大鼠HSC凋亡,从而达到抗肝纤维化的作用。4.蕲艾提取液可能通过下调大鼠肝组织中细胞周期蛋白Cyclin D1 mRNA及其蛋白表达水平,发挥对细胞周期的调控,抑制细胞增殖,而达到抗肝肝纤维化的作用。更多还原

【Abstract】 Background and Objective:Hepatic fibrosis is regarded as a pathologically repair reac-tion followed various kinds of chronic liver diseases. Fibrosis mainly presents the imbalance of the ECM compound production and degradation, which makes the ECM deposit pervadingly in liver. Hepatic fibrosis is a common stage of most chronic liver diseases regardless of the etiology, and its progression may lead to hepatic cirrhosis or hepatocelluar carcinoma (HCC). Although hepatic fibro-sis is thought to be a reversible pathological state, there is no established effective therapy for hepatic fibrosis yet.Because of its special feature of poly-target, poly-lint, poly-channel, and its compound effects, the Chinese medicine can aim to the complicated mechanisms of hepatic fibrosis accordingly, which makes it have traditional and elearly prevalence against the western medicine. The topic is through the establishment of immune animal model of hepatic fibrosis, to get a preliminary understanding the inhibition of Artemisia argyi extracting solution on rat hepatic fibrosis, and through its rat liver tissue TGFβ-Smad signal transduction pathway in the key signal transduction molecules TGFβ1, Smad 3, Smad 4, Smad 7 expression level changes, as well as against the apoptosis gene bcl-2 and cell cycle proteins, to explore Artemisia argyi extracting solution the molecular mechanism of anti-fibrosis.Methods:1. Use of pig serum-induced liver fibrosis model,72 Wistar rats were fed 1 week after adaptation,were randomly divided into normal control group, liver fibrosis model group, high dose, middle dose and low-dose treatment of Artemisia argyi extracting solution grou-ps,12 rats in each. In addition to the normal control group, the rest of the rats by intraperitoneal injection of sterile body weight 100g pig serum 0.3ml,2 times per week, a total of 10 weeks,20 times. Artemisia argyi extracting solution large, medium and low-dose treatment group were given 6ml/only,3ml/only and 1.5ml/only with Artemisia argyi extracting solution fed to pray daily 1; model group were given 6ml/only normal saline gavage, every day 1 times; the normal control group was given intraperitoneal injection of saline,2 times per week,10 weeks a total of 20 times, and to give 6ml/only normal saline gavage, day 1 times. At 10 over the weekend after the last injection of pig serum 48h, animals in each group were decapitated, eyes, enough to take blood specimens from blood, separa-tion of serum, and take part of the liver,4% formalin-fixed, conven-tional paraffin-embedded sections.Part of the gradient of fresh liver tissue placed in -70℃to save, for real-time fluorescent quantitative reverse transcription-polymerase chain reaction(RT-PCR) method and protein blot (Western blot).2. Serological test:The rats in each group were detected in serum indicators of liver fibrosis serum hyaluronic acid (HA),laminin (LN),Ⅲprocollagen (PCⅢ),Ⅳprocollagen (Ⅳ-C)content, as well as indicators of lipid peroxidation superoxide dismutase(SOD), malondialdehyde (MDA). ELISA, serum TGFβ1 levels。3. Liver histology tests:using HE staining and Masson staining of collagen triple-pathological changes in rat liver.4.Immunohistochemistry:using of immunohistochemistry SABC method, combined with microscope to observe the rat liver tissue TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl of expression.5. Using real-time fluorescent quantitative reverse transcription-poly mer-ase chain reaction (RT-PCR) method and Western blot test (Western blot) were measured TGFβ1, Smad3, Smad4, Smad7 and Bcl-2, cyclinDl mRNA and protein expression.Results:1. Artemisia argyi extract solution inhibited rat liver fibrosis in rats.Compared with the normal control group, the serum markers of liver fibrosis HA,PCⅢ,LN,Ⅳ-C levels of model group,were signi-ficantly higher(P＜0.01), serum TGF-β1 levels are significantly higher (P＜0.05), increase in serum MDA concentration, SOD activity was significantly decreased(P＜0.01);with the model group,Artemi-sia argyi extracting solution treatment for all groups HA, PCIII, LN,Ⅳ-C levels were significantly lower (P＜0.05), serum TGF-β1 content also decreased significantly(P＜0.05), serum indicators of lipid peroxidation MDA contents were significantly lower(P＜0.05), SOD activity was significantly increased (P＜0.05), and Artemisia argyi extracting solution treatment group and normal control group had no significant difference (P＞0.05).Optical microscope observation:model group can be seen part of the organizational structure of liver damage, liver cells a wide range of variability of edema, water balloon-like degeneration or degeneration, but also shows the extent, scope, ranging from liver cell necrosis; liver disorder board arrangement, some of loss of normal structure of hepatic lobule; periportal fibrous septum in a large number of eosinophils, monon-uclear cell infiltration of lymphocytes and fibroblasts, a large number of collagen deposition, there are more obvious fibrous tissue hyperplasia, collagen fibers along the periportal areas and around the central vein to the hepatic lobule within the extension,we can see the connection between fiber split hepatic lobule, and even some regions the performance of early liver cirrhosis. Artemisia argyi extracting solution therapy in liver of rats in each group in the color, textu-re, size, surface smoothness in a significant improvement compared with model group, cell degeneration and necrosis significantly reduced hepatic lobule was not obvious structural damage, inflame-matory cell infiltration and fibrous tissue reduced proliferation, the central veins and periportal extension of a small amount of fibrous tissue, different doses of Artemisia argyi extracting solution between the treatment group no significant difference.2. Artemisia argyi extracting solution expect rat liver tissue TGFβ-Smad signal transduction pathway protein expression.Immunohistochemistry results showed that, TGFβ1, smad3, smad4 liver cells in the normal group no positive cells, liver expression was significantly increased in model group, mainly seen in interstitial cells, inflammatory cells, injury of liver cells, the positive substances mainly concentrated on the membrane, there is a little expression in the cytoplasm; Artemisia argyi extracting solution each dose group significantly reduced the number of positive cells, positive staining is reduced significantly.RT-PCR and Western blot test results showed, TGFβ1, smad3, smad4 mRNA and protein expression in the normal control group, significantly lower, while the smad7 mRNA and protein expression was significantly increased; Artemisia argyi extracting solution high, medium and low dose group TGFβ1, smad3, smad4 mRNA and protein expression compared with model group gradually reduced, smad7 mRNA and protein expression compared with model group were increased gradually, in which middle and high dose group TGFβ1, smad3, smad7 mRNA and protein and model group and there was significant difference (P＜0.05, P＜0.01), compared with the SM group was no significant difference (P＞0.05). 3. Artemisia argyi extracting solution against apoptosis gene Bcl-2 and cell cycle protein expression of cyclinDl.Immunohistochemistry results showed that, Bcl-2 and cyclinDl protein in normal liver cells there is no positive cells, liver expression was significantly increased in model group, mainly seen in portal area,hepatic cells and stromal cells’membrance and cytoplasm, positive substance is mainly concentrated in the membr-ance; Artemisia argyi extracting solution each dose group signifi-cantly reduced the number of positive cells, positive staining is reduced significantly.RT-PCR and Western blot test results showed that the normal control group, Bcl-2, CyclinDl mRNA and protein expression compared with model group, low, Artemisia argyi extracting solution every dose group Bcl-2, CyclinD1 mRNA and protein expression compared with model group gradually reduced, which high-dose group compared with the model group were significantly different (P＜0.05), with the normal group and the SM group showed no significant difference (P＞0.05).Conelusions:1. Artemisia argyi extracting solution can significantly improve the rat liver fibrosis histopathological damage induced by immune pig serum, reduce fibrous tissue proliferation and decreased collagen fibers interval,liver cell necrosis,degeneration,and inflammatory cell infiltration,and can effectively reduce serum markers of liver fibrosis and its mechanism may be anti-fibrosis and anti-lipid peroxidation, reducing the concentration of serum TGF-β1.2. Artemisia argyi extracting solution can significantly inhibit rat liver tissue TGFβ1, Smad3, Smad4 protein and mRNA expression levels, increase the level of Smad7 protein and mRNA expression, indicating that it can effectively regulate TGFβ-Smad signal transduction pathway, thereby suppressing rat liver fibrosis.3. Artemisia argyi extracting solution can reduce the rat liver tissue of anti-apoptotic gene Bcl-2 protein and mRNA expression levels, suggesting that it can effectively induce apoptosis in rat HSC to achieve the anti-fibrosis.4. Artemisia argyi extracting solution could be reduced in rat liver tissue of cyclin CyclinDl protein and mRNA expression levels to play on cell cycle regulation, so that stagnation in the G0/G1 phase cell replication, inhibit cell proliferation.更多还原