【作者】 刘鸣昊；

【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】 背景肝纤维化是多种慢性肝病发展至肝硬化的必经阶段。研究证实肝纤维化是可逆的,但如果不加以治疗,肝纤维化可能会转变为肝硬化。晚期肝硬化病人3年的存活率仅仅为16%,全世界直接死于肝硬化的人数位居人类总死亡数的第6位。肝纤维化的形成原因是由于肝脏受到损害与炎症反应后,肝组织内细胞外基质(ECM)过度增生与异常沉积,导致肝脏内胶原组织的增多,使肝脏结构和肝功能产生异常病变。转化生长因子-β1是肝纤维化的重要靶点,它可以激活肝星状细胞、调节细胞外基质合成和降解导致肝纤维化发生,TGF-β1促纤维化作用主要是通过TGF-β1/Smad信号转导通路来实现的。西医对肝纤维化的治疗多为阻断肝纤维化诱因的针对治疗,临床上尚无理想的、公认的抗纤维化特效药物。中医药在治疗肝纤维化方面具有多成分、多环节、多靶点的优势,并已经取得了较好的疗效,拥有广阔的开发和应用前景。芪珠方有清热解毒、益气活血等功效,前期研究表明芪珠方在慢性乙型肝炎的治疗和临床研究中有一定的保肝降酶、抗纤维化作用；对急慢性肝损伤和肝纤维化模型大鼠有一定的保护作用。本研究拟运用分子生物学和细胞学技术,观察芪珠方对CC14肝纤维化模型大鼠肝组织TGF-β1/Smad信号通路相关因子及TIMPs/MMPs, ILs等细胞因子的影响,部分阐明抗肝纤维化机制；利用HPLC法检测芪珠方中的有效成分；检测芪珠方有效成分对外源性TGF-β1刺激后的HSC-T6细胞增殖抑制、细胞凋亡、细胞周期以及TGF-β1/Smad信号传导通路的影响,进一步探明芪珠方抗肝纤维化的作用机制。方法1.体内实验SD大鼠70只,随机分为空白组、模型组、芪珠方水提大、中剂量组、芪珠方醇提大、中剂量组,秋水仙碱组,组间大鼠体重差异无显著性。除正常组外,其余各组大鼠皮下注射40%四氯化碳(cC14)橄榄油造模,首次5mL每100g体重,以后每次3mL每100g体重,每周3次,共8周。于造模开始次日给药,芪珠方高、低剂量组16.2,9.72g每100g体重,秋水仙碱组0.5mg每100g体重灌胃,每日1次,连续8周。空白组不做任何处理,模型组大鼠给予同大剂量组灌胃剂量生理盐水灌胃。造模结束后所有大鼠处死,肝脏组织学观察,测定肝脾指数,酶联免疫吸附试验(ELISA)技术检测TNF-a、IL-4、IL-8、IL-10、TIMP-1、MMP-1、MMP-2、MMP-13在各组大鼠肝组织内的表达,TIMP-1、MMP-1、 MMP-2、MMP-13在各组大鼠血清内表达。蛋白免疫印迹法(Western blott)检测Smad2、 Smad3、Smad4、Smad7、TGF-β1蛋白表达：实时荧光定量Real-time PCR)检测Smad2、 Smad3、Smad4、Smad7、TGF-β1基因表达。2.芪珠方HPLC实验采用高效液相色谱法,色谱条件为Agilent C18柱(250mm×4.6mm,5μm);流动相：A为乙腈,B为水溶液,梯度洗脱,流速0.8mL/min,柱温30℃,检测波长230nm,检测芪珠方与含药血清中有效成分,采用中药色谱指纹图谱相似度评价系统(A版)进行相似度评价。3.体外实验HSC-T6细胞,DMEM培养基+10%FBS培养(37℃,5%C02),青链霉素作为双抗,0.25%胰蛋白酶消化,细胞生长至75%传代。分组：空白组、叶下珠次素组、虎杖苷组、芍药苷组、黄芪甲苷组,取对数生长期的HSC-T6细胞,每组设6个复孔,加入浓度为5ng/mL外源性TGF-β1作为模型组,空白组与模型组分别加入叶下珠次素、虎杖苷、芍药苷、黄芪甲苷,观察12h、24h、48h。检测各组HSC-T6细胞增殖情况,流式细胞仪检测各组24h细胞周期变化：蛋白免疫印迹法(western blot)检测Bcl-2、Bax、CyclinD1、Smad2、Smad3、 Smad4、Smad7、TGF-pβ,24h蛋白表达；实时荧光定量(real-time PCR)检测Bcl-2、Bax、 CyclinD1、Smad2、Smad3、Smad4、Smad7、TGF-β1,24h基因表达。所有结果以均数±标准误差形式,利用SPSS18.0软件进行统计学分析,组间比较使用t检验或方差分析(ANOVA),当p<0.05时,认为处理组和对照组间有统计学差异。结果第一部分体内实验1.与正常组相比,模型组大鼠体质量下降(P<0.01),肝、脾指数上升(P<0.01)；模型组与治疗组相比,各治疗组大鼠体重有增加,大剂量组有显著改变(P<0.01)：脾脏指数降低(P<0.05)；大鼠肝脏大体标本肉眼观察,各治疗组外观接近正常对照组；模型组HE切片可见肝细胞肿胀,广泛气球样变与大量纤维增生,芪珠方组HE切片大鼠肝组织炎性病变程度减轻,肝细胞变性或坏死减少,纤维组织增生减少或逆转。提示肝纤维化模型复制建立成功,芪珠方对肝纤维化有一定的预防作用。2.相比空白组,模型组大鼠肝组织内IL4、IL8、TNF-a、TIMP-1、MMP-2表达上调,IL10、MMP-1、MMP-13表达下调(P<0.01)；芪珠方能降低肝纤维化大鼠肝组织BL4, IL-8, TNF-a的表达,提高IL-10的表达(P<0.01)；同时能降低肝组织TIMP-1、MMP-2的表达,提高MMP-1、MMP-13的表达(P<0.01),血清内的TIMPs/MMPs的表达与肝组织表达一致(P<0.01)。3.相比空白组,模型组大鼠肝组织内TGF-β1、Smad2、Smad3、Smad4蛋白和mRNA表达上调,Smad7蛋白和mRNA表达下调(P<0.05)；芪珠方干预后,芪珠方治疗组大鼠肝组织内TGF-β1、Smad2、Smad3、Smad4蛋白和mRNA表达不同程度下调(P<0.05),Smad7蛋白和mRNA表达不同程度上调(P<0.05)。第二部分芪珠方的HPLC检测HPLC结果显示芍药苷、虎杖苷、叶下珠次素是芪珠方中的主要成分；芪珠方醇提法比水提法能更有效的提取方中药材的有效成分。第三部分体外实验1.叶下珠次素、虎杖苷各剂量组以及芍药苷、黄芪甲苷中剂量组在12h、24h、48h3个时间段对HSC-T6细胞分别有一定的增殖抑制作用,在24h时间段抑制作用最为明显和稳定。2.叶下珠次素、虎杖苷、黄芪甲苷组能上调空白组HSC-T6细胞与模型组HSC-T6细胞BaxmRNA和蛋白表达(P<0.05),下调Bcl-2mRNA和蛋白表达(P<0.05),芍药苷能上调空白组HSC-T6细胞与模型组HSC-T6细胞BaxmRNA和蛋白表达(P<0.05),下调Bcl-2蛋白表达(P<0.05)。3.叶下珠次素有通过下调模型组HSC-T6细胞周期相关因子CyclinD1蛋白和mRNA表达,该组HSC-T6细胞与模型组相比停留在G1期的数量更多(P<0.05)。4.相比空白组,模型组HSC-T6细胞内源性TGF-β1、Smad2,Smad3的蛋白和mRNA表达高于空白组(P<0.05), Smad7的表达低于空白组(P<0.05)；叶下珠次素可以下调模型组HSC-T6细胞Smad2蛋白表达、Smad3mRNA表达,上调Smad7蛋白与mRNA表达,下调内源性TGF-β1mRNA与蛋白的表达(P<0.05)；虎杖苷可以下调Smad2mRNA与蛋白的表达,下调TGF-β1mRNA与蛋白的表达(P<0.05)；芍药苷可以下调Smad2,Smad3蛋白表达,下调Smad3mRNA表达,上调Smad7蛋白与mRNA表达,下调TGF-β1mRNA与蛋白的表达(P<0.05)；黄芪甲苷可以上调Smad7蛋白与mRNA表达,下调TGF-β1蛋白的表达(P<0.05)。结论1.芪珠方能提高肝纤维化模型大鼠体重,减轻肝脏肿大及肝脾湿重,改善肝组织内的纤维化程度,提示芪珠方有保护肝纤维化大鼠和抗肝纤维化作用。2.芪珠方能降低肝纤维化大鼠肝组织IL4、IL-8、TNF-a、TIMP-1、MMP-2的表达,提高IL-10、MMP-1、MMP-13的表达,提示芪珠方抗肝纤维化的作用与调节TIMPs/MMPs,ILs和TNF-a,从而减少细胞外基质的分泌,平衡肝内胶原的分泌与降解有关。3.芪珠方能通过下调肝纤维化大鼠肝组织内Smad2、Smad3、Smad4mRNA和蛋白的表达,上调Smad7mRNA和蛋白的表达,芪珠方能调节TGF-β1/Smad信号传导通路,从而抑制TGF-β1mRNA和蛋白的表达、抑制肝星状细胞的激活,减少细胞外基质的分泌,降低肝纤维化程度。4.叶下珠次素、虎杖苷、芍药苷是芪珠方的有效成分,醇提法在提取上述单体的效果好于水提法,提示芪珠方醇提物调节TIMPs/MMPs、TGF-β1/Smad信号传导通路的作用优于水提物与可以更有效的提取中药中的有效成分有关。5.叶下珠次素、虎杖苷各剂量组以及芍药苷、黄芪甲苷中剂量组在12h、24h、48h三个时间段对HSC-T6细胞和外源性TGF-β1刺激模型组HSC-T6细胞有一定的增殖抑制作用,提示芪珠方的抗肝纤维化作用与其有效成分可以抑制肝星状细胞增殖有关。6.叶下珠次素、芍药苷、黄芪甲苷、虎杖苷均能调节Bcl-2和Bax的表达从而促进HSC-T6细胞,其抑制HSC-T6细胞增殖作用可能是通过上调Bax和下调Bc1-2表达,促进HSC-T6细胞凋亡而形成的,芪珠方抗肝纤维化的作用可能与其有效成分能促进肝星状细胞凋亡有关。7.叶下珠次素有通过下调CyclinD1mRNA与蛋白表达,从而调控细胞周期,使HSC-T6细胞停留在G1期,叶下珠次素抑制HSC-T6细胞增殖作用可能与能调控HSC-T6细胞周期有关。8.芪珠方抗肝纤维化的作用与芪珠方中的有效成分叶下珠次素、虎杖苷、芍药苷、黄芪甲苷可以分别调控、共同作用于TGF-β1/Smad信号传导通路,抑制TGF-β1表达,从而抑制肝星状细胞的激活,减少肝组织内ECM沉积有关。更多还原

【Abstract】 BackgroundHepatic fibrosis is a necessary stage in many chronic liver diseases developing to liver cirrhosis and this process is reversible proved by research. But if Hepatic fibrosis is left untreated, it may be transformed into liver cirrhosis. The survival rate of end-stage liver cirrhosis is only16%in3years, directly death in liver cirrhosis is on the sixth place in human total death of the whole world.The mainly formation mechanism of liver fibrosis is that:after the liver damage by inflammation, there are excessive proliferation and abnormal deposition of Extracellular matrix (ECM), leading an abnormal changes of liver structure and function. Transforming growth factor-β1(TGF-β1) is the main reason of Hepatic fibrosis by activate hepatic stellate cells, regulate synthesis and degradation of extracellular matrix, and it is the important target for liver fibrosis. The effect of TGF-β1is mainly realized by TGF-β1/Smad signal transduction pathway. The treatment for Hepatic fibrosis on Modern Medicine is blocking the inducement and there is no specific medicine for Hepatic fibrosis on clinic. In contrast, Traditional Chinese Medicine has various advantages and achieved effect on the treatment of Hepatic fibrosis such as multiple components, multiple links, and multiple targets, has broad prospects for development and application. Compound Qizhu Tablet has many functions such as clearing heat and removing toxicity, benefiting qi for activating blood circulation and it has proved its effect in the treatment of chronic hepatitis B as protection of liver, dropping enzyme and anti-liver fibrosis. Our previous studies have shown that Compound Qizhu Tablet has a protective effect on acute and chronic liver injury and hepatic fibrosis rat model.The experiment aims to observe the systematic effect of Compound Qizhu Tablet on Hepatic fibrosis, and make further research on the mechanism. The conclusion of this experiment, providing exploratory research to develop the treatment of Hepatic fibrosis drugs from Traditional Chinese Medicine.Objective: This study aims to research the effect of TGF-β1/Smad signaling pathway and cell factor as TIMPs/MMPs and ILs influenced by Compound Qizhu Tablet on CC14rats hepatic fibrosis model and verify the mechanism partly on molecular biology and cell biology; To find out the effective components of Compound Qizhu Tablet by HPLC; To observe the influence of effective components of Compound Qizhu Tablet on HSC-T6cell effected by exogenous TGF-β1, by testing cell apoptosis, cell cycle and TGF-β1/Smad signal transduction pathway effects, to provide further experimental evidence for the treatment of liver fibrosis in Traditional Chinese Medicine.Methods:Experiment1:Treatment of liver fibrosis of Compound Qizhu Tablet in vivo experimentsSeventy SD rats were randomly divided into normal group, model group, Qizhu high-dose group (water preparation), Qizhu low dose group (water preparation), Qizhu high-dose group (ethanol preparation), Qizhu low dose group (ethanol preparation), and colchicine group. All the rats except normal group received40%CCl4subcutaneous injection twice a week for8weeks, rats was firstly given40%CCl4(5mL per1OOg body weight) and3mL per100g body weight secondly. Rats in both Qizhu high-dose group and small dose group were given16.2,9.72g per100g body weight by ig. Qizhu decoction respectively while rats in colchicine group were fed with0.5mg per100g body weight colchicine. Liver histology and liver and spleen index were observed after the model is done. The expression of TNF-a, IL-4,IL-8,IL-10,TIMP-1,MMP-1,MMP-2,MMP-13in hepatic tissue was detected by enzyme linked immune sorbent assay (ELISA),and as same as the expression of TIMP-1, MMP-1, MMP-2,MMP-13in the blood serum. To detect the protein expression levels of Smad2, Smad3, Smad4, Smad7, and TGF-β1by Western-blot, the mRNA expression levels of Smad2, Smad3, Smad4, Smad7, and TGF-β1by real time quantitative RT-PCR.2. Experiment2:HPLC for Compound Qizhu TabletHPLC Method was applied to establish the Compound Qizhu Tablet. The separation was performed on a Agilent C18column (250mm×4.6mm,5μm) with a gradient elution composed of acetonitrile(A) and water (B). The column temperature was set at30℃and the flow rate was0.8Ml/min.The detective wave length was at280nm. HPLC fingerprint was performed on the Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (A)Experiment3:Treatment of liver fibrosis of the effective components of Compound Qizhu Tablet in vitro experimentsHSC-T6cells were cultured in DMEM medium supplemented with10%fetal bovine serum,50U penicillin/mL and50μg streptomycin/mL. For sub culturing purposes, cells were detached by treatment with0.25%trypsin/0.02%EDTA at37℃. Cultures were used at75%confluence. Cultures were divided into control group, Polydatin group, Hypophyllanthin group,Astragaloside group, Peoniflorin group, and model group is add exogenous TGF-β1. ALL groups are measured for proliferation and cell cycle change at24h by flow cytometry; The mRNA and protein expression levels of Bcl-2、Bax、CyclinD1、Smad2、Smad3、Smad4、Smad7、TGF-β1were detected by real time quantitative RT-PCR and Western-blot respectively at24h.Data are expressed as Mean±SD. The Student’s t-test was used to determine the statistical significance of the experimental data.ResultsExperiment1:Treatment of liver fibrosis of Compound Qizhu Tablet in vivo experiments1.Compared with normal group, body weight of rats in model group decreased (P<0.01), liver index, spleen index increased (P<0.01); Compared with the model group, body weight of rats in treatment group increased, the high dose group were improved significantly (P<0.01); spleen index decreased (P<0.05); On the rat gross anatomy, the treatment group appearance similar to the normal group; According to the pathological condition, the rats in model group were observed that the liver cells swelling, widely balloon degeneration and fibrosis, the rats in treatment group showed Compound Qizhu Tablet can improve liver inflammatory lesions, reduce liver cell degeneration or necrosis, stop or reverse the hyperplasia of fibrous tissue. It showed that liver fibrosis model replication is established successfully, and Compound Qizhu Tablet has a preventive effect on liver fibrosis.2. Compared with normal group, the expression of IL4,IL8,TNF-a,TIMP-1,MMP-2in liver fibrosis rats increased, the expression of IL-10,MMP-1,MMP-13decreased (P<0.01);Compound Qizhu Tablet can reduce the expression of IL4, IL-8, TNF-a in liver fibrosis rats, increase the expression of IL-10(P<0.01); At the same time, Compound Qizhu Tablet can reduce the expression of reduce the expression of TIMP-1,MMP-2in liver fibrosis rats, increase the expression of MMP-1,MMP-13(P<0.01), it showed the same TIMPs/MMPs expression in the serum of liver fibrosis rats (P<0.01).3.Compared with normal group, protein and gene expression of TGF-β1, Smad2, Smad3, Smad4in model group increased, protein and gene expression of Smad7decreased (P<0.05); Compared with model group, protein and gene expression of TGF-β1, Smad2, Smad3,Smad4in treatment group decreased,and expression of Smad7increased (P<0.05) Experiment2:HPLC for Compound Qizhu TabletResult of HPLC in Compound Qizhu Tablet showed that Polydatin, Hypophyllanthin, and Peoniflorin is the effective components of Compound Qizhu Table; And ethanol preparation is more effective than water preparation.Experiment3:Treatment of liver fibrosis of the effective components of Compound Qizhu Tablet in vitro experiments1. Hypophyllanthin,Polydatin in each dosage group and dose group of Paeoniflorin, Astragaloside showed effect of proliferation inhibition on HSC-T6cells in12h,24h,48h,and the most obvious performance in on24h.2. Hypophyllanthin, Polydatin, Astragaloside can increase protein and gene expression of Bax in normal group and model group HSC-T6cells, decrease the expression of Bcl-2; Paeoniflorin can increase protein and gene expression of Bax in normal group and model group HSC-T6cells, decrease the protein expression of Bcl-2.3. Hypophyllanthin can hold the HSC-T6cells of model group in G1phase by decrease the protein and gene expression of cell cycle related factors CyclinD1.4. Compared with normal group, HSC-T6in model group protein and gene expression of TGF-β1, Smad2,Smad3increased, protein and gene expression of Smad7decreased; Hypophyllanthin can decrease protein expression of Smad2, gene expression of Smad3, increase protein and gene expression of Smad7and decrease protein and gene expression of endogenous TGF-β1; Polydatin can decrease protein and gene expression of Smad2, decrease protein and gene expression of endogenous TGF-β1; Paeoniflorin can decrease protein and gene expression of Smad3, protein expression of Smad2, increase protein and gene expression of Smad7, and decrease protein and gene expression of endogenous TGF-β1; Astragaloside can increase protein and gene expression of Smad7and decrease protein expression of endogenous TGF-β1.Conclusions1. Compound Qizhu Tablet can improve the weight of the rats of liver fibrosis model, reduce the enlargement of the liver and spleen wet weigh, and restore the damage of fibrosis in liver tissue.It shows Compound Qizhu Tablet can protect liver fibrosis rats and treat liver fibrosis.2.Compound Qizhu Tablet can reduce the expression of IL4, IL-8, TIMP-1, MMP-2,TNF-a in liver fibrosis rats, increase the expression of IL-10, MMP-1, MMP-13.It shows Compound Qizhu Tablet treat hepatic fibrosis though regulate TTMPs/MMPs, ILs and TNF-a in the liver, reduce the expression of extracellular matrix in liver and balance liver collogen.3.Compound Qizhu Tablet can decrease protein and gene expression of Smad2, Smad3, Smad4,and increase expression of Smad7, regulat the TGF-β1/Smad Signal Transduction Pathway to decrease the expression of TGF-β1and Inhibiting activation of hepatic stellate cells, reduce the secretion of extracellular matrix, reduce the degree of liver fibrosis.4. According to HPLC on Compound Qizhu Tablet, Hypophyllanthin, Phyllanthus, polydatin are the effective component of Compound Qizhu Tablet, ethanol preparation is better than water preparation in the extraction of the monomer, it showed ethanol preparation regulate TIMPs/MMPs, TGF-β1/Smad signal transduction pathway more effective than water preparation With the relevant of in the extraction of the effective component.5.Hypophyllanthin,Polydatin in each dosage group and dose group of Paeoniflorin, Astragaloside showed effect of proliferation inhibition on HSC-T6cells in12h,24h,48h,and the most obvious performance in on24h.It showed that treatment of Compound Qizhu Tablet on Hepatic fibrosis is relative to its effective component’s effect of proliferation inhibition on HSC-T6cell.6. Hypophyllanthin, Polydatin, Astragaloside and Paeoniflorin increase protein and gene expression of Bax in normal group and model group HSC-T6cells; decrease the expression of Bcl-2. Compound Qizhu Tablet’s effect on Hepatic fibrosis is relative to proliferation inhibition on HSC-T6cells, as well as the effective component’s effect on Promoting HSC-T6apoptosis.7. Hypophyllanthin can hold the HSC-T6cells of model group in G1phase by decrease the protein and gene expression of cell cycle related factors CyclinD1and the proliferation inhibition on HSC-T6cells is relative to the control of cell cycle.8. Effect on hepatic fibrosis of Compound Qizhu Tablet is relative to its effective component such as Hypophyllanthin, Polydatin, Astragaloside and Paeoniflorin, which can respectively control the TGF-β1/Smad signal transduction pathway, inhibition TGF-β1expression and activation of hepatic stellate cells, reduced ECM deposition in liver tissue.更多还原