【作者】 郝瑞春；

【导师】 门九章；

【作者基本信息】 湖北中医药大学 ， 中医内科学， 2011， 博士

【摘要】 肝纤维化(hepatic fibrosis, HF)主要是一种组织病理学概念。HF指肝组织内细胞外基质成分过度增生与异常沉积,导致肝结构或(和)功能异常的病理变化。多数慢性肝炎均可发生肝纤维化,部分肝纤维化进一步发展可形成肝硬化和肝癌,严重影响人类健康和生命。而目前临床上尚无理想的抗HF药物,因此能否延缓、终止HF的进展,甚或逆转HF至正常,具有重大意义。HF的形成涉及诸多因素和环节,单纯应用针对某一环节作用的药物可能难以逆转纤维化进程,研究表明,中药复方有多途径,多靶点抗HF的综合药理作用,着眼于肝纤维化发生发展的多个环节,发掘出疗效较好的抗肝纤维化中药复方是中医药工作者的重要使命。雄芍汤乃导师门九章教授治疗肝纤维化的有效验方,本论文中我们一方面进行了雄芍汤抗大鼠免疫性肝纤维化的作用和机理实验研究,另一方面进行了雄芍汤含药血清对体外培养的人肝星状细胞影响的实验研究,简述如下：目的：从症状体征、肝功、肝纤四项、病理形态学等方面综合评价雄芍汤防治免疫性肝纤维化的疗效,并从肝纤维化发病机制中起关键作用的环节入手明确雄芍汤抗肝纤维化的机理。方法：1.动物实验采用猪血清腹腔注射法制造大鼠肝纤维化模型。设立正常对照组、模型组对照组、扶正化瘀胶囊对照组、雄芍汤预防组和雄芍汤高、低剂量组(以下分别简称正常组、模型组、扶正组、雄预组、雄高组、雄低组)。观察大鼠毛色泽、体重等一般情况；肉眼观察大鼠新鲜肝脏的外观；计算肝重与体重的比值,脾重与体重的比值；常规方法检测大鼠血清肝功能指标：丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、白蛋白(ALB)、球蛋白(GLB)、白蛋白与球蛋白比值(A/G)；同位素放射免疫分析法检测肝纤维化血清标志物：透明质酸(HA)、层粘连蛋白(LN)、Ⅲ型前胶原氨端肽(PCⅢ)、Ⅳ型胶原(CIV)的含量；黄嘌呤氧化酶法检测血清SOD活力,TBA法测定血清MDA含量；ELISA法检测大鼠血清MMP-9、IgE、C3的含量；消化法检测肝组织羟脯氨酸含量；HE染色和Masson染色法观察肝组织病理形态学改变；免疫组织化学染色法检测大鼠肝组织Ⅰ型胶原、Ⅲ型胶原、α-SMA、TGF-β1表达；实时荧光定量逆转录聚合酶链反应(RT-PCR)技术检测肝组织中TIMP-1mRNA的表达。2.细胞实验采用MTT比色法测定大鼠雄芍汤药物血清对体外培养的人肝星状细胞LX-2增殖的影响；免疫组织化学染色法测LX-2中Ⅰ型胶原、Ⅲ型胶原、α-SMA、TGF-β1的表达。结果：1.给大鼠腹腔注射猪血清15周后,肝纤维化模型复制成功。2.雄芍汤可以改善免疫性肝纤维化大鼠毛色、进食量和体重等一般情况。3.肝功能指标检测结果：模型组血清AST、ALT、GLB均显著高于正常组(P<0.01),ALB、A/G均显著低于正常组(P<0.01)；除了雄低组AST与模型组差别不显著外,雄芍汤各治疗组血清AST、ALT、GLB均显著低于模型组(P<0.05或P<0.01),ALB、A/G均显著高于模型组(P<0.01),雄预组血清GLB显著低于扶正组(P<0.05),雄预组和雄高组A/G显著高于扶正组(P<0.05)。4.血清肝纤维化标志物检测结果：模型组血清HA、LN、PCⅢ、CIV均显著高于正常组(P<0.01),各治疗组血清HA、PCⅢ、LN均显著低于模型组(P<0.05或P<0.01),其中雄高组血清HA和LN显著低于雄预组,且雄高组血清LN显著低于扶正化瘀(P<0.05),雄预组血清CIV显著低于模型组(P<0.05,),而其他治疗组血清CIV与模型组差别不明显。5.肝重指数、脾重指数结果：所有实验组肝重指数均无显著差别；模型组脾重指数明显大于正常组(P<0.01),雄预组和雄低组脾重指数均明显小于模型组(P<0.01),雄高组和扶正组脾重指数均与模型组差别不明显。6.肝组织病理观察结果：模型组大鼠肝组织炎症活动度、纤维化半定量计分均显著高于正常组(P<0.01,)；除雄低组外,各治疗组均显著低于模型组(P<0.05或P<0.01),显著高于正常组(P<0.05或P<0.01)。7.肝组织Hyp检测结果：模型组肝组织Hyp含量显著高于正常组(P<0.01),各治疗组均显著低于模型组(P<0.05或P<0.01),雄预组和雄高组均低于扶正组(P<0.05)。8.大鼠血清SOD和MDA含量检测结果：模型组大鼠血清SOD含量显著低于正常组(P<0.01),MDA含量显著高于正常组(P<0.01),除扶正组SOD含量与模型组差别不显著外,各治疗组SOD含量均显著高于模型组(P<0.05或P<0.01),MDA含量均显著低于模型组(P<0.01),其中雄预组SOD含量显著高于雄低组(P<0.05),雄低组显著高于雄高组,各治疗组间MDA含量差别不显著。9.大鼠血清IgE、C3的含量检测结果：模型组大鼠血清IgE、补体C3均与正常组差别不显著,雄预组IgE显著高于其他所有组(P<0.05或P<0.01),雄预组补体C3显著低于正常组和模型组。10.大鼠肝组织Ⅰ型胶原、Ⅲ型胶原、α-SMA、TGF-β1表达免疫组织化学染色结果：模型组大鼠肝组织Ⅰ型胶原、Ⅲ型胶原、α-SMA、TGF-β1含量显著高于正常组(P<0.01),各治疗组均显著低于模型组(P<0.01),各治疗组中雄预组Ⅰ型胶原、Ⅲ型胶原和α-SMA含量显著低于扶正组(P<0.05或P<0.01),雄高组Ⅲ型胶原含量也显著低于扶正组(P<0.01),雄高组和雄预组Ⅲ型胶原、α-SMA含量均显著低于雄低组(P<0.01)雄低组TGF-β1表达显著低于雄高组(P<0.01)。11.大鼠血清MMP-9含量和肝组织TIMP-1mRNA表达检测结果：模型组大鼠血清MMP-9含量显著低于正常组(P<0.05),肝组织TIMP-1mRNA表达显著高于正常组(P<0.01)；雄预组血清MMP-9含量显著高于模型组(P<0.01),其他用药组与模型组差别不显著；雄高组肝组织TIMP-1mRNA表达显著低于模型组(P<0.01),与正常组差别不显著。12.雄芍汤含药血清对LX-2增殖的抑制率结果：20%和10%空白血清组抑制率均低于相应浓度的扶正组和雄芍汤组(P<0.01),5%空白血清组抑制率与相应浓度的扶正组和雄芍汤组差别不显著；雄芍汤各浓度含药血清组对LX-2增殖的抑制率显著高于相应浓度的扶正含药血清组(P<0.05或P<0.01)。雄芍汤各浓度含药血清组中,20%组和10%组对LX-2增殖的抑制率差别不显著,但均显著高于雄芍汤5%组(P<0.01)。13.含药血清对LX-2中Ⅰ型胶原、Ⅲ型胶原、α-SMA、TGF-β1的表达影响的免疫组织化学染色结果：雄芍血清组和扶正血清组LX-2中Ⅰ型胶原、Ⅲ型胶原、α-SMA和TGF-β1含量显著低于空白血清组(P<0.01),雄芍血清组和扶正血清组Ⅰ型胶原差别不显著,扶正血清组Ⅲ型胶原、α-SMA含量显著低于雄芍血清组(P<0.05),雄芍血清组TGF-β1含量显著低于扶正血清组(P<0.01)。结论：1.雄芍汤有确切的抗免疫性肝纤维化疗效,早期预防用药综合疗效较好,其抗肝纤维化的机理与扶正化瘀胶囊不完全相同,且在一定条件下某些方面的抗肝纤维化疗效优于扶正化瘀胶囊。2.雄芍汤抗肝纤维化的作用机理主要有如下几方面2.1保护肝细胞,改善肝功能的作用。2.2既抑制胶原等ECM的合成,又促进其降解。2.3提高肝纤维化大鼠清除氧自由基和抑制脂质过氧化能力2.4调节免疫功能2.5促进MMP生成,抑制TIMP-1mRNA表达,从而促进ECM降解2.6抑制HSC活化和增殖,减少促纤维化细胞因子的生成更多还原

【Abstract】 Hepatic fibrosis (HF) is mainly histopathological conception. It refers to the pathological changes of hyperplasia and abnormal deposition of the component of extracellular matrix in liver tissue, which resulting in liver structure or (and) the function abnormal. The majority of chronic hepatitis can cause HF, and Further development of HF can develop cirrhosis and liver cancer, which have serious impact on human health and life. At present there is no ideal drug on clinical which can cure HF.Therefore if or not the development of HF can be delayed, even be stoped or inversed to normal is of great significance.The formation of HF involves many factors and links, in versing the development of HF is possibly difficult by use medicine,which aim only directly at one link.Studies have shown that the pharmacologic actions of anti-HF chinese herbal compound recipe was comprehensive,and was multi-channels,Multi-targeting.Chinese herbal compound recipe was able to focus on Multiple links of the development of HF.Discovering anti-HF chinese herbal compound recipe whose therapeutic effect is better had been our important mission.Xiongshao decoction is a effective proved recipe of anti-HF,by which my advisor Prof. Men Jiuzhang treat HF.In this theses, on one hand we had an empirical study on the effects of Xiongshao decoction on Immune HF in rats and its mechanism,on the other hand,we had done an empirical study on the effects of Xiongshao decoction drugs blood serum on human hepatic stellate cell(HSC),which were cultivated in vitro.ObjectiveSynthetically evaluate the curative effect of Xiongshao decoction on HF by the symptoms and signs,assaying liver function and serumal marker of HF,observing the change of pathomorphology;Identify the anti-HF mechanism of Xiongshao decoction beginning with pathogenetic capital link of HF.Methods(1)In animal experiment,the HF model were induced by injecting porcine serum to the rats’abdominal cavity. We set up the normal control group,the model group,FZHY capsule group,the Xiongshao decoction preservation group, the Xiongshao decoction high-dose group and low-dose group.We had observed the general state of health of rats, including coat color, body weight and so on; observed the appearance of rats’fresh liver by naked eye; calculated the ratio of liver weight and body weight, the ratio of spleen weight and body weight;By routine methods detected the rats’serumal hepatic function parameter:ALT,AST,ALB, GLB and A/G; Using isotopes radio immunoassay detected the rats’ serumal marker of HF:hyaluronic acid(HA),laminin(LN),ProcollagenⅢN-terminal peptide(PCⅢ) and collogen IV(CIV);Using xanthine oxidase detected the vitality of SOD in rats’serum,and using thiobarbituric acid(TBA) detected the content of MDA in rats’serum;Using enzyme linked immunosorbent assay(ELISA) detected the content of MMP-9, IgE, and C3 in rats’serum;By digestive method detected the content of Hyp in rats’hepatic tissue;Using HE and Masson staining observed the alteration of pathomorphology in rats’hepatic tissue;Using immunohistostaining measured the expression of collagenⅠ, collagenⅢ,α-SMA and TGF-β1 in rats’hepatic tissue;Using real-time fluorescence quantitative reverse transcription polymerase chain reaction(RT-PCR) detected the expression of TIMP-1mRNA in rats’hepatic tissue;(2) In the cell experiment, we observed the effect of rats’drug serum in which there was Xiongshao decoction on the multiplication of human HSC LX-2 using MTT colorimetry; and measured the expression of collagenⅠ,collagenⅢ,α-SMA and TGF-β1 in LX-2 using immunohistostaining.Resultsl.The HF model in rats was successfully duplicated after injecting pig serum to rats’abdominal cavity for 15 weeks.2.Xiongshao decoction can improve the general status of immunitas HF rat,such as the coat color, the food-intake, the body weight, and so on.3.liver function test results:the content of AST,ALT and GLB in the model group rats’serum are obviously higher than in the normal group rats’serum (P<0.01), and the content of ALB and A/G in the model group rats’serum are obviously lower than in the normal group rats’ serum (P<0.01); Besides the content difference of AST between in Xiongshao decoction low-dose group rats’serum and in model group rats’serum was not remarkable, the content of AST, ALT and GLB in every treatment group of Xiongshao decoction rats’serum were all obviously lower than in the model group (P<0.05 or P<0.01), and the content of ALB and A/G were all obviously higher than in the model group (P<0.01), the content of GLB in Xiongshao decoction preservation group rats’serum was obviously lower than in the FZHY group (P<0.05),the ratios of A/G in the Xiongshao decoction prevention group and high-dose group rats’serum was obviously higher than in the FZHY group (P<0.05)4.serum HF marker test results:the content of HA, LN, PCIII and CIV in model group rats’serum were obviously higher than in the normal group (P<0.01),the content of HA, PCIII and LN in every treatment group rats’serum were obviously lower than in the model group (P<0.05 or P<0.01),the content of HA and LN in Xiongshao decoction high-dose group rats’serum were obviously lower than in the prevention group (P<0.05),and the content of LN in Xiongshao decoction high-dose group rats’serum was obviously lower than in FZHY group (P<0.05), the content of CIV in prevention group rats’serum was obviously lower than in the model group (P<0.05), but the content difference of CIV between in other treatment group rats’serum and the model group were not remarkable.5.Liver heavy index and spleen heavy index result:the difference of all experimental groups liver heavy index were not remarkable;The spleen heavy index of the model group was obviously bigger than the normal group (P< 0.01),the Xiongshao decoction prevention group and low-dose group is smaller than the model group(P<0.01),the difference of spleen heavy index of the Xiongshao decoction high-dose group and the FZHY group were not remarkable.6.Hepatic tissue pathology results:observing through the microscope,every treatment group rats’hepatic inflammation and fibration was milder than the model group. Among every treatment group,Xiongshao decoction low-dose group rats’hepatic inflammation and fibration was the most severe.Analyzing through statistics,the model group rats’hepatic inflammation and fibration semiquantitative scoring were remarkably higher than the model group(P<0.01); Besides Xiongshao decoction low-dose group,each treatment group was obviously lower than the model group (P<0.05 or P<0.01), and was obviously higher than the normal group (P<0.05orP<0.01)7.Hyp in the liver organizes test results:the content of Hyp in the model group rats’hepatic tissue was obviously higher than the normal group (P<0.01), each treatment group was obviously lower than the model group (P<0.05 or P<0.01), both Xiongshao decoction prevention group and Xiongshao decoction high-dose group were lower than the FZHY group (P<0.05)8.SOD and MDA in rats’serum test results:the content of SOD in model group rats’serum was obviously lower than in the normal group (P<0.01),the content of MDA was obviously higher than in the normal group (P<0.01), besides the difference of the content of SOD between in FZHY group and in the model group was not remarkable,SOD in each treatment group was obviously higher than in the model group (P<0.05 or P<0.01),the content of MDA in every treatment group were obviously lower than in the model group (P<0.01),the content of SOD in Xiongshao decoction prevention group was obviously higher than in Xiongshao decoction low-dose group (P<0.05),and in Xiongshao decoction low-dose group was higher than in the Xiongshao decoction high-dose group,the content difference of MDA among treatment groups was not remarkable.9.IgE and C3 in rats’serum test results:the content difference of IgE and C3 in rats’serum between the model group and the normal group was not remarkable,the content of IgE in rats’serum of Xiongshao decoction prevention group was obviously higher than other all groups (P<0.05 or P<0.01),the content of C3 in rats’serum of Xiongshao decoction prevention group was obviously lower than the normal group and the model group.10.Immunohistostaining results ofⅠcollagen,Ⅲollagen,α-SMA,TGF-β1 in rats’hepatic tissue:the contents ofⅠcollagen,Ⅲcollagen,α-SMA,TGF-β1 in the model group rats’hepatic tissue were obviously higher than in the normal group (P<0.01),each treatment group was obviously lower than the model group (P<0.01). Among each treatment group,the contents ofⅠcollagen,Ⅲcollagen and a-SMA in the Xiongshao decoction prevention group rats’hepatic tissue were obviously lower than in the FZHY group (P<0.05 or P<0.01),the content ofⅢcollagen in Xiongshao decoction high-dose group was obviously lower than in the FZHY group (P<0.01),the contents ofⅢcollagen andα-SMA in the Xiongshao decoction high-dose group and Xiongshao decoction prevention group were obviously lower than in the Xiongshao decoction low-dose group (P<0.01).the content of TGF-β1 in the Xiongshao decoction low-dose group was obviously lower than in the Xiongshao decoction high-dose group (P<0.01)11.MMP-9 in rats’ serum and TIMP-1mRNA in rats’ hepatic tissue test results:the content of MMP-9 in the model group rats’ serum was obviously lower than in the normal group (P<0.05),the expression of TIMP-1mRNA in the model group rats’ hepatic tissue was obviously higher than in the normal group (P<0.01);the content of MMP-9 in the Xiongshao decoction prevention group rats’serum was obviously higher than in the model group(P<0.01),the content difference between other treatment groups and the model group was not remarkable;the expression of TIMP-1mRNA in Xiongshao decoction high-dose group rats’ hepatic tissue was obviously lower than in the model group, the content difference between Xiongshao decoction high-dose group and the normal group was not remarkable.12.The inhibition ratio results of drug serum in which there was Xiongshao decoction to LX-2 reduplication:the inhibition ratio of 20% and 10% vacuity serum group was lower than homologue concentration FZHY group and Xiongshao decoction group (P<0.01), the inhibition ratio difference among 5% vacuity serum group,homologue concentration FZHY group and Xiongshao decoction group were not remarkable;the inhibition ratio of each concentration drug serum in which there was Xiongshao decoction was obviously higher than homologue concentration drug serum in which there was FZHY (P<0.05或P<0.01).the inhibition ratio difference between 20% and 10% drug serum in which there was Xiongshao decoction to LX-2 reduplication was not remarkable,but the inhibition ratio of 20% and 10% drug serum in which there was Xiongshao decoction were both higher than 5% drug serum in which there was Xiongshao decoction (P<0.01)13.Immunohistostaining results of the effect of drug serum in which there was Xiongshao decoction on the expression ofⅠcollagen,Ⅲcollagen,a-SMA,TGF-β1 in LX-2:the contents ofⅠcollagen,Ⅲcollagen,a-SMA and TGF-β1 in Xiongshao decoction serum group LX-2 and FZHY serum group LX-2 were obviously lower than in the vacuity serum group (P<0.01),the content difference of I collagen in the Xiongshao decoction serum group LX-2 and FZHY serum group LX-2 was not remarkable,the contents ofⅢcollagen and a-SMA in FZHY serum group LX-2 were obviously lower than in Xiongshao decoction serum group LX-2 (P<0.05),the content of TGF-β1 in Xiongshao decoction serum group LX-2 was obviously lower than in FZHY serum group (P<0.01)Conclusion1. Xiongshao decoction has exact curative effect on immune,the synthesis curative effect was best if Xiongshao decoction were used to prevent HF in prophase,its anti-HF mechanism was not identical with FZHY capsule, and under the controlled condition,its some aspects’ anti-HF curative effect surpassed FZHY capsule.2.anti-HF mechanism of Xiongshao decoction was mainly the following several aspects.2.1 the effect of protecting the hepatic cells and improving hepatic function.2.2 both inhibiting the synthese of ECM,such as collagen and so on, and promoting its degradation.2.3 Sublimating the ability of HF rats eliminating oxygen radicals and suppressing lipid peroxidation. 2.4 adjusting immunologic function.2.5 promoting MMP-9 production and suppressing TIMP-1mRNA expression,thus promoting ECM degradation.2.6 suppressing the activating and multiplication of HSC and reducing the productions of cytokines,which could promote fibrosis.更多还原