【作者】 杨慧；

【导师】 赵龙凤；

【作者基本信息】 山西医科大学 ， 内科学， 2012， 博士

【摘要】 肝纤维化是各种慢性肝病的共同病理学基础,是形成肝硬化的必经病理阶段,也是临床治疗慢性肝病的关键环节。其组织学特征表现为以胶原为主的细胞外基质(extracellular matrix, ECM)成分合成增多、降解相对不足而在肝内过量沉积。肝星状细胞(hepatic stellate cell, HSC)是产生ECM的主要来源,HSC的增殖活化是肝纤维化发生的中心事件。通过抑制HSC增殖、诱导HSC凋亡来阻断甚至逆转肝纤维化的发展,是抗肝纤维化治疗的重要策略。血红素加氧酶-1(Heme Oxygenase-1, HO-1)是人类和哺乳动物组织中广泛存在的一种加氧酶,是催化血红素的起始酶和限速酶。HO-1及其催化产物一氧化碳(Carbon Monoxide, CO)、胆红素及转铁蛋白在体内有显著的抗炎、抗氧化、调控细胞凋亡等重要作用。有研究显示,HO-1在肝移植、急性肝损伤和缺血再灌注损伤等多种肝脏损害中,对肝细胞均具有保护作用。在慢性肝病进展过程中诱导HO-1表达,可以减少Ⅰ型胶原的分泌,有效阻止肝纤维化的进展。过氧化物酶体增生因子激活受体(peroxisome proliferator-activated receptor, PPAR)是一种配体激活的转录因子,其亚型PPARγ主要分布于HSC。在肝纤维化进展过程中,上调PPARγ表达可以抑制HSC激活、诱导HSC凋亡,进而减轻肝纤维化的程度。核转录因子-κB(nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB)是具有转录激活功能的蛋白质,活化的NF-κB可促进HSCs增殖及胶原的产生、诱导TNF-a、IL-6及TGF-β等相关细胞因子与纤维化因子的释放、并减少HSCs凋亡,在促进在肝纤维化进程中同样发挥重要作用。研究发现,PPARγ可以直接与NF-κB的亚基p50/p65结合,或通过竞争结合协同活化因子p300和CBP来抑制NF-κB DNA合成、转录与表达,诱导HSC的凋亡。对肝纤维化发生、发展起重要的负调控作用。已有其他领域的研究显示,HO-1同PPARγ之间存在相互调控,HO-1启动子区的基因多态性影响PPARγ的转录活性；HO-1的启动子区域还有NF-κB结合位点,诱导HO-1表达可以抑制NF-κB、TNF-α及IL-6等相关细胞因子的分泌。有关HO-1对肝脏保护机制的研究目前主要集中于HO-1的分解产物如胆绿素、一氧化碳(CO)和自由铁的抗氧化及抗炎作用等方面,而HO-1对肝纤维化相关信号分子调控方面的研究尚未见报道。为此,本课题从在体动物实验和体外细胞实验两方面入手,观察HO-1的表达对大鼠肝纤维化及HSC-T6增殖、活化与凋亡的影响,同时研究HO-1表达对HSC-T6中PPARγ、NF-κB及其下游炎性信号分子与凋亡相关因子表达的影响,以进一步探讨HO-1调控HSC-T6增殖、活化及凋亡,进而阻止肝纤维化进展的信号分子机制。本课题包括以下三部分：第一部分血红素加氧酶-1对大鼠肝纤维化及相关信号分子表达的影响第二部分血红素加氧酶-1对大鼠肝星状细胞增殖、活化及凋亡的影响第三部分血红素加氧酶-1对大鼠肝星状细胞调控作用的信号分子机制第一部分血红素加氧酶-1对大鼠肝纤维化及相关信号分子表达的影响目的：研究在构建实验性肝纤维化大鼠模型的过程中抑制/诱导HO-1的表达对大鼠肝纤维化进程及相关信号分子表达的影响。方法：采用复合因素构建肝纤维化大鼠模型。实验分组：正常对照组,4周模型组,6周模型组,ZnPP-Ⅸ干预组,Hemin干预组。在造模第4～6周分别给予HO-1的诱导剂Hemin及抑制剂Znpp-Ⅸ隔口腹腔注射进行干预。HE、Masson染色观察肝脏病理组织学变化；免疫组织化学染色观察肝组织中HO-1及α-SMA的分布与表达；全自动生化分析仪检测血清各项生化指标；酶联免疫吸附试验(ELISA)检测血清中透明质酸(HA)及Ⅲ型前胶原(PⅢP)水平；分光光度法检测组织中羟脯氨酸(Hyp)含量;采用Real-time PCR技术检测肝组织中α-SMA、HO-1、PPARγ及NF-κB的mRNA表达;采用Western blot技术检测肝组织中HO-1、PPARγ及NF-κBp65的蛋白表达。结果：1、HE及Masson染色结果表明肝纤维化模型构建成功。Hemin干预组胶原纤维的增生、肝小叶的破坏及假小叶的形成程度明显低于同期模型组及Znpp-Ⅸ干预组(P<0.05)：2、免疫组织化学染色及Real-time PCR、Western blot检测各实验组大鼠肝脏HO-1表达的结果显示：随着造模时间延长,纤维化肝组织中HO-1表达范围扩大且.强度增加(P<0.01)：Hemin干预组HO-1表达范围及强度较对照组及模型组增加更加明显(P<0.01),而Znpp-Ⅸ干预组HO-1表达强度则减弱(P<0.01);3、免疫组织化学染色及Real-time PCR检测大鼠肝脏α-SMA表达的结果显示：肝纤维化进展过程中肝组织中α-SMA表达逐渐增加,Hemin的干预使α-SMA的表达明显下降(P<0.01),Znpp-Ⅸ干预组α-SMA的表达则增加(P<0.05)；4、各实验组大鼠肝脏生化指标的变化：大鼠肝功能的损害程度随肝纤维化的进展而逐渐加重(P<0.05)；Hemin干预组ALT、AST、ALB及TBIL各项生化指标较同期模型组及Znpp-Ⅸ干预组则明显恢复(P<0.05);5、各实验组大鼠肝脏胶原代谢的变化：大鼠血清HA、PⅢP及肝组织中Hyp含量随着肝纤维化程度的加重而逐渐增加(P<0.05),Hemin的干预使HA、PⅢP及Hyp含量较同期模型组.及Znpp-Ⅸ干预组明显下降(P<0.05)；6、Real-time PCR及Western blot检测各实验组大鼠肝组织中PPARy mRNA及蛋白表达显示：随着肝纤维化程度的加重,肝组织内PPARy的mRNA表达逐渐下降(P<0.01),使用Znpp-IX的干预组同6周模型组相比虽无统计学意义,但仍有下降趋势(P=0.195)；而Hemin干预组PPARy的mRNA表达则较6周模型组明显增加(P<0.05)。PPARy蛋白表达与mRNA变化趋势一致；7、Real-time PCR及Western blot检测各实验组大鼠肝组织中NF-κB mRNA及蛋白表达显示：随着肝纤维化程度的加重,肝组织中NF-κB的mRNA表达逐渐增加(P<0.01)；Znpp-IX十预组较6周模型组增加更为明显(P<0.05),而Hemin十预组中的NF-κB mRNA表达则显著下降(P<0.05)。NF-κB蛋白表达与mRNA变化趋势一致。结论：HO-1的诱导表达可以明显减轻肝纤维化大鼠肝脏的炎症反应程度、降低肝组织a-SMA及胶原纤维的表达,减轻肝纤维化；而HO-1阻止肝纤维化进展的肝脏保护作用可能是通过对肝组织中PPARy及NF-κB表达的调控实现的。第二部分血红素加氧酶-1对大鼠肝星状细胞增殖、活化及凋亡的影响目的：研究抑制／诱导HO-1的表达对HSC-T6增殖、活化及凋亡的影响。方法：以活化的HSC-T6为研究对象。不同浓度的Hemin (10μmol/l、20μmol/l、40μmol/l)及Znpp-IX (5μmol/l、10μmol/l、20μmol/l)作用于HSC-T6不同的时间(12h,24h,48h),通过MTT比色法检测HSC-T6的增殖情况、台盼蓝拒染实验观察细胞存活率及乳酸脱氢酶(LDH)释放实验观察药物对细胞的毒性作用,以此筛选出Hemin或Znpp-IX发挥诱导／抑制HO-1表达效应的最适浓度及作用时间用于后续实验。实验分组：空白对照组、Hemin干预组、Znpp-IX干预组及Hemin+Znpp干预组。采用MTT法检测各实验组HSC-T6的增殖情况；ELISA方法检测HSC-T6上清液中HA及PⅢP含量；免疫细胞化学技术检测HSC-T6中HO-1及α-SMA的分布与表达；Real-time PCR检测HSC-T6中HO-1及α-SMA的mRNA表达；Western blot检测HSC-T6中HO-1及α-SMA蛋白表达;TUNEL法及AnnexinV-FITC/PI联合标记流式细胞术检测HSC-T6的凋亡；Real-time PCR检测HSC-T6中抗凋亡蛋白Bcl-2及Caspase-3的mRNA表达。结果：1、HO-1的诱导剂Hemin对HSC-T6增殖发挥抑制作用且无明显细胞毒性的最适浓度为20μmol/l (13μg/ml)。抑制剂ZnPP-IX对HSC-T6增殖发挥促进作用且无明显细胞毒性的最适浓度为10μmol/l (3μg/ml),作用时间选择24h；2、免疫细胞化学技术、Real-time PCR及Western blot对各组HSC-T6中HO-1表达的检测显示：Hemin干预组的HO-1表达范围及强度较正常对照及其他干预组均显著升高(P<0.01)：Znpp-Ⅸ干预组同对照组相比虽无显著差异,但也呈下降趋势；Hemin+Znpp-Ⅸ共同干预组中HO-1的表达范围及强度较单纯Hemin干预组显著下降(P<0.05)；3、免疫细胞化学技术、Real-time PCR及Western blot对各组HSC-T6中α-SMA表达的检测显示：Hemin干预组可见α-SMA表达范围及强度较对照组显著下降(P<0.01),Znpp-Ⅸ干预组则升高(P<0.01)；Hemin+Znpp-Ⅸ共同干预组α-SMA表达范围及强度较单纯Hemin干预组增加(P<0.01)；4、各实验组HSC-T6增殖情况：Hemin干预组HSC-T6的增殖活性(MTT值0.867±0.023)较对照组下降16.15%(P<0.01)；Znpp-Ⅸ组(MTT值为1.161±0.015)较对照组升高12.28%(P<0.01)；Hemin+Znpp-Ⅸ组则比Hemin单独处理组HSC-T6增殖活性升高(P<0.05)；5、各实验组HSC-T6胶原代谢的变化：Hemin干预组细胞上清液中HA及PIIIP含量较对照组显著下降(P<0.01),Znpp-Ⅸ十预组二者均显著升高(P<0.01),而Hemin+Znpp-Ⅸ干预组细胞上清液中HA及PⅢP含量较单纯Hemin干预组升高(P<0.01)；6、TUNEL及AnnexinV-FITC/PI流式细胞术检测各组HSC-T6的凋亡：TUNEL结果显示：Hemin干预组HSC-T6凋亡指数(23.5%±2.02%)较对照组(3.25%±0.63%)增加了6.23倍(P<0.01)；Znpp-Ⅸ干预组(4.00%±0.82%)同对照组相比无显著差异(P=0.574)；而Hemin+Znpp-Ⅸ组凋亡指数(16.25%±1.38%)则较单纯Hemin干预组下降(P<0.01)。流式细胞术检测结果同上述趋势一致；7、Real-time PCR检测凋亡相关蛋白Bcl-2及Caspase-3的mRNA表达显示：同对照组相比较Hemin干预组Bcl-2mRNA表达显著下降(P<0.05),Caspase-3mRNA表达升高(P<0.01);Znpp-Ⅸ干预组Bcl-2mRNA升高(P<0.05),而Caspase-3mRNA表达下降；Hemin及Znpp-Ⅸ共同干预组中Bcl-2mRNA表达量较单纯Hemin干预组又有所回升(P<0.05),而Caspase-3mRNA的表达则有所下降(P<0.05)。结论：HO-1的诱导表达能够抑制HSC-T6的增殖活性及胶原蛋白的代谢能力；HO-1还能够通过影响凋亡相关蛋白如Bcl-2及Caspase-3的表达来调控HSC-T6的凋亡。第三部分血红素加氧酶-1对大鼠肝星状细胞调控作用的信号分子机制目的：研究抑制/诱导HO-1表达对HSC-T6内PPARγ、NF-κB及下游炎性细胞因子表达的调控作用。方法：实验分组为空白对照组、Hemin干预组、Hemin+GW9662组、Znpp-Ⅸ干预组、Znpp-Ⅸ+罗格列酮组。采用免疫细胞化学技术检测PPARy在HSC-T6中的表达;免疫荧光技术检测NF-κB p65在HSC-T6中的表达；Real time-PCR技术检测HSC-T6中HO-1、PPARy及NF-κB的mRNA表达；Western blot检测HSC-T6中HO-1、PPARy及NF-κBp65的蛋白表达;采用ELISA方法检测各实验组HSC-T6培养上清液中TGF-β1及IL-6的含量。结果：1、Real-time PCR及Western blot检测各组HSC-T6中HO-1的mRNA及蛋白表达：Hemin干预组HO-1表达较对照组明显增加(P<0.01)；Hemin+GW9662组HO-1的mRNA表达较单纯Hemin干预组下降19.96%(P<0.05),蛋白表达下降16.05%(P<0.05)；Znpp-Ⅸ干预组HO-1表达较对照组明显下降(P<0.01)；而Znpp-Ⅸ+罗格列酮组,HO-1的mRNA表达较单纯Znpp-Ⅸ干预组升高18.32%(P<0.05),蛋白表达升高13.7%(P<0.05)；2、Real-time PCR及Western blot检测各组HSC-T6中PPARγ、NF-κBp65的mRNA及蛋白表达：Western blot结果显示：Hemin干预组PPARy表达较对照组升高(P<0.01),NF-κBp65表达则下降(P<0.01)；Znpp-IX干预组PPARy表达较对照组下降(P<0.01),而NF-κBp65表达升高(P<0.01)。Hemin+GW9662同单纯Hemin组比较PPARy表达下降17.78%、NF-κB的表达则升高28.94%(P<0.05);Znpp-Ⅸ+罗格列酮组同单纯Znpp-Ⅸ组比较PPARy表达升高18.6%、NF-κB蛋白表达则下降23.16%(P<0.01)。Real-time PCR的mRNA检测同上述趋势基本一致；3、免疫细胞化学及免疫荧光检测HSC-T6中PPARγ、NF-κBp65的表达：PPARy及NF-κBp65主要定位于HSC-T6细胞核。对照组细胞核PPARy表达微弱；Hemin干预后细胞核PPARy表达明显增强,而应用GW9662预孵育的实验组PPARy表达则较单纯Hemin干预组减弱；Znpp-Ⅸ干预组HSC-T6细胞核中PPARy表达较对照组下降,使用诱导剂罗格列酮预孵育的实验组则可看到PPARy表达较单纯Znpp-Ⅸ干预组略有增加。而NF-κBp65的免疫荧光检测结果则同PPARy变化趋势相反；4、各实验组HSC-T6培养上清液中TGF-β1及IL-6的含量：ELISA检测结果显示,TGF-β1及IL-6在各组的变化趋势一致。Hemin干预组细胞上清液中TGF-β1及IL-6的释放量较对照组明显下降(P<0.01),Hemin+GW9662组则观察到二者较单纯Hemin组均有所增加(P<0.05)；Znpp-IX干预组细胞上清液中TGF-β1及IL-6的释放量较对照组显著升高(P<0.01),Znpp-Ⅸ+罗格列酮组二者的释放量较单纯Znpp-Ⅸ干预组则下降。结论：HO-1抑制活化HSC-T6增殖、诱导活化HSC-T6凋亡的作用同其对PPARγ、 NF-κB表达及下游炎性细胞因子释放的调控有关。更多还原

【Abstract】 Liver fibrosis is the mechanism of compensation and reparation after chronic hepatic injury, which is a necessary pathologic stage from chronic hepatitis to cirrhosis. The main pathological characteristic of liver fibrosis is the increased concentration and deposition of extracellular matrix (ECM). Hepatic stellate cells (HSC) are increasingly being recognized as the key players in liver fibrosis which is the main source of ECM. The activation and proliferation of HSC play a important role in fibrosis process. So inhibiting the proliferation or inducing apoptosis of activated HSC play a key role in reversing the liver fibrosis.Heme Oxygenase-1(HO-1) is a microsomal enzyme and rate-limiting enzyme for heme degradation in a wide range of human and mammalian tissues. HO-1can catalyze the heme degrading into biliverdin, iron atoms and carbon monoxide (CO) which play vital physiological roles in anti-inflammation, anti-oxidation and regulation of apoptosis. Many reseachers recently confirmed that HO-1had protective effects on liver cells, such as liver transplantation, acute liver injury, ischemia/reperfusion injury and so forth. In chronic liver disease, induction of HO-1is important to prevent the development of liver fibrosis effectively. Peroxisome Proliferator-Activated Receptor (PPAR) is a ligand activated transcription factors. PPARy is one of the subtypes which is mainly expressed in HSC. Up-regulation of PPARy resulted in a significant reduction of HSC activation, and reversed the development of liver fibrosis. Nuclear Factor-kappa B (NF-κB) is an important nuclear transcription factor. Up-regulating the activation of NF-κB would promote the proliferation of HSC, the releasing of cytokines and decrease HSC apoptosis which play important role in liver fibrosis. Studies have found that PPARy can inhibit the transcription and DNA synthesis of NF-κB by binding p50/p65subunits to form transcriptional repressor complexes directly or by binding p300and CBP co-activating factors to inhibit the transcription and expression of NF-κB competitively. Studies in other areas have shown that co-regulation existed between HO-1and PPARy, and there was an NF-κB binding site in the HO-1promoter region. Up-regulation of HO-1could decrease NF-κB activity drastically and inhibiting the serum levels of TNF-α and IL-6significantly.Reseach about the defense mechanism of HO-1againsting many kinds of liver injuries concentrate on the role of anti-inflammation, anti-oxidation and apoptosis regulation induced by its degrading production such as biliverdin, iron atoms and CO. But no reports have been found about the regulation of liver signals and molecules by HO-1. So, in this study we design animal and cells research to explored the effects of HO-1on rat liver fibrosis and HSC proliferation or apoptosis, and the expression of PPARγ、NF-κB and the downstream signals or inflammation cytokines which are aim to explore the signals and molecules mechanism of HO-1on the proliferation and apoptosis of activated HSC and the prevention of liver fibrosis.Reseach about the defense mechanism of HO-1againsting many kinds of liver injuries concentrate on the role of anti-inflammation, anti-oxidation and regulation of apoptosis induced by degrading production such as biliverdin, iron atoms and CO. But no reports have been found about the regulation of liver signals and molecules by HO-1. So, in this study we designed animal and cells research to explore the effects of HO-1on rat liver fibrosis and HSC proliferation or apoptosis, and the expression of PPARγ、NF-κB and the downstream signals or inflammation cytokines which were aim to explore the signals and molecules mechanism of HO-1on the proliferation and apoptosis of activated HSC and the prevention of liver fibrosis.The project contain three parts as below:Part1:Effects of HO-1induction or inhibition on liver fibrosis and the potential mechanismsPart2:Effects of HO-1induction or inhibition on the proliferation and apoptosis of activated HSCPart3:The signaling and molecular mechanisms of HO-1on HSC proliferation and apoptosisPart1:Effects of HO-1on liver fibrosis and the potential mechanismObjective:To explore the impacts of HO-1induction or inhibition on liver fibrosis and the potential mechanism by using liver fibrosis modelsMethods:Liver fibrosis models in rats was constructed by using composite factors which were randomly divided into five groups:①Normal group,②4weeks model,③6weeks model,④Znpp-IX treated model,⑤Hemin treated model. Two opposite reagents were used to induce or inhibite the expression of HO-1such as hemin (20μmol/kg) and ZnPP-IX (30μimol/kg) which were peritoneal injected to rats every other day from week4to week6. Histopatho logical changes were evaluated by hematoxylin and eosin (HE) and Masson staining. The expressions of HO-1and a-SMA in hepatic tissues were measured by immunohistochemical staining. Liver function including ALT, AST, ALB and TBIL were detected by using an automated biochemistry clinical analyzer. Levels of serum hyaluronic acid (HA) and Type Ⅲ procollagen (PⅢP) were determined by Enzyme-linked immunoadsorbent assay (ELISA). The content of hydroxyproline (Hyp) in the liver specimens were determined by using spectrophotometric method. The mRNA expression of a-SMA、HO-1、PPARy and NF-κB were determined by Real-time PCR. The protein expression of HO-1、PPARy and NF-κB were determined by Western blot.Results:(1) HE and Masson’s staining showed rat modes of hepatic fibrosis were established successfully by using composite factors mainly with CC14. The degree of collagen proliferation, hepatic lobule destruction and the pseudolobuli formation in livers of Hemin treated model was significantly lower than the6week model control and Znpp-IX treated model (P<0.05).(2) The expression of HO-1mRNA and protein increased gradually during the process of liver fibrosis by using immunohistochemistry, Western blot and Real-time PCR (P<0.01). Hemin promoted the expression of HO-1compared with model control(P<0.01), but Znpp-IX diminished it (P<0.01).(3) The expression of a-SMA with immunohistochemistry showed that in hepatic tissues of normal rats, there was weakly expression of a-SMA in the smooth muscle cells of the vessel wall. With the development of hepatic fibrosis the a-SMA positive cells in hepatic tissues of rats increased significantly. The expression of a-SMA could be decreased by Hemin (P<0.01), but promoted by Znpp-IX (P<0.01). The relative expression of HO-1mRNA was consistent with the results above.(4) The degree of hepatic injury was increased gradually during the process of liver fibrosis as evidenced by significantly higher serum concentrations of ALT, AST, TBIL and lower concentration of ALB. Hemin treatment could change the injury of liver function by ameliorating the increased of ALT, AST and TBIL, and the decrease of ALB compared with model control (P<0.05). But the results in Znpp-IX treated group was opposite (P<0.05).(5) Results about the changes of collagen metabolism in livers of each group showed that the levels of HA、PⅢP in serum and Hyp in liver tissues increased in the process on liver fibrosis (P<0.05). Hemin treatment decreased the levels of HA、PⅢP and Hyp compared with6week model control and Znpp-IX treated group (P <0.05).(6) The expression of PPARy mRNA and protein decreased gradually during the process of liver fibrosis by using Real-time PCR and Western blot (P<0.01). Especially in Znpp-Ⅸ treated group PPARy decreased more evidently compared with6week model group, although no statistically significant (P=0.195).But the expression of PPARy was promoted significantly in Hemin treated group (P<0.05).(7) The expression of NF-κB mRNA and protein increased gradually during the process of liver fibrosis by using Real-time PCR and Western blot (P<0.01). NF-κB increased more significantly compared with6week model group especially in Znpp-IX treated group (P<0.05),but decreased apparently in Hemin treated group (P<0.05)Conclusions:The induction of HO-1in liver of fibrosis rats model could decrease the a-SMA expression, collagen synthesis, liver injury and the degree of fibrosis, thus prevent liver fibrosis progression. Then, our data demonstrated a potential mechanism that the liver protective effect of HO-1could be achieved by enhancing the expression of PPARγ and decreasing the expression of NF-κB in liver tissues.Part2:Effects of HO-1on the proliferation and apoptosis of activated HSCObjective:To explore the effects of HO-1induction or inhibition on activated HSC proliferation and apoptosisMethods:Activated HSC-T6were used as object of study which were incubated with Hemin (10μmol/l,20μmol/l,40μmol/l) or Znpp-Ⅸ (5μmol/l,10μmol/l,20μmol/l) for12h,24h and48h. MTT assay was used to determine cell proliferation, trypan blue staining was used to observe the survival rate of HSC-T6and lactate dehydrogenase (LDH) release assay was used to observe the toxicity of Hemin and Znpp-Ⅸ to HSC-T6. Through above the best concentration and action time about Hemin and Znpp-Ⅸ were chosen for the following experiments. Cells were grouped as follows:①Normal control group,②Hemin treated group,③Znpp-Ⅸ treated group,④Hemin and Znpp-Ⅸ co-treated group. MTT assay was used to determine cell proliferation. ELISA was used to detect the levels of HA and PⅢP in cell cultured supernatant. The expressions of HO-1and α-SMA in HSC-T6were measured by immunocytochemical staining, Real-time PCR and Western blot. TUNEL assay and AnnexinV-FITC/propidium iodide (PI) co-labed flow cytometry (FCM) were used to detect cell apoptosis. Changes of Bcl-2and Caspase-3mRNA in HSC-T6were monitored by Real-time PCR.Results:(1) The best concentration of Hemin inducting HO-1expression was20μmol/l (13μg/ml), and ZnPP-IX inhibiting HO-1expression was10μmol/l (3μg/ml) which had been proved to be effective and no toxicity to cultured HSC-T6. The best action time was24h.(2) The expression of HO-1in Hemin treated group was more extensively and strong than normal control and other treated group by using immunocytochemical staining. Results from Western blot and Real-time PCR also showed that HO-1expression was significantly higher in Hemin treated group compared with normal control (P<0.01), but lower in Znpp-IX treated group although no significant difference with normal control. HO-1expression in Hemin and Znpp-Ⅸ co-treated group was lower than merely Hemin treated group (P<0.05).(3) The expression of α-SMA about extent and intensity was lower in Hemin treated group than normal control and other treated group by using immunocytochemical staining. The mRNA and protein expression about α-SMA were lower in Hemin treated group (P<0.01), but higher in Znpp-Ⅸ treated group than normal control (P<0.01) by using Real-time PCR and Western blot. In Hemin and Znpp-Ⅸ co-treated group, a-SMA expression was higher than merely Hemin treated group (P<0.05).(4)The proliferation of HSC-T6decreased16.15%in Hemin treated group (MTT value was0.867±0.023), but increased12.28%in Znpp-Ⅸ treated group (MTT value was 1.161±0.015) compared with normal control (P<0.01). The proliferation of HSC-T6in Hemin and Znpp-Ⅸ co-treated group was higher than merely Hemin treated group (P<0.05).(5) Results about the changes of collagen metabolism in cultured HSC-T6supernatant of different groups showed that the levels of HA、PⅢP decreased significantly in Hemin treated group (P <0.01), but increased in Znpp-Ⅸ treated group compared with normal control (P<0.01) Levels of HA and PⅢP in Hemin and Znpp-Ⅸ co-treated group were higher than merely Hemin treated group (P<0.01).(6) TUNEL assay showed that the apoptotic rates of HSC-T6in Hemin treated group (23.5%±2.02%) increased6.23folds compared with normal control (3.25%±0.63%)(P<0.01). There was no significant difference between Znpp-Ⅸ treated group (4.00%±0.82%) and normal control (P=0.574). But the apoptotic rate in Hemin and Znpp-Ⅸ co-treated group (16.25%±1.38%) was lower than merely Hemin treated group (P <0.01). Results about the apoptotic rates of HSC analyzed by AnnexinV-FITC/PI co-labed FCM were coincidence with TUNEL.(7) Real-time PCR analysis showed that the expression of Bcl-2mRNA was decreased significantly in Hemin treated group compared with normal control (P<0.05), but increased in Znpp-Ⅸ treated group (P<0.05). Bcl-2mRNA in Hemin and Znpp-Ⅸ co-treated group was higher than merely Hemin treated group (P<0.05). In contrast, the expressions of Caspase-3mRNA was increased in Hemin treated group (P<0.01) but decreased in Znpp-Ⅸ treated group compared with normal control. Caspase-3mRNA in Hemin and Znpp-Ⅸ co-treated group was lower than merely Hemin treated group (P<0.05)Conclusions:The induction of HO-1could inhibit HSC-T6proliferation and collagen metabolism, and induce HSC-T6apoptosis by regulating the expression of apoptosis correlated protein such as Bcl-2and Caspase-3.Part3:The signaling and molecular mechanisms of HO-1on HSC proliferation and apoptosisObjective:To investigate the signaling and molecular mechanisms of HO-1on the proliferation and apoptosis of activated HSC-T6.Methods:Cells were grouped as follows:①Normal control group,②Hemin treated group,③Hemin+GW9662treated group,④Znpp-Ⅸ treated group,⑤Znpp-Ⅸ+Rosiglitazone treated group. The expression of HO-1in HSC-T6was detected by Real-time PCR and Western blot. The expression of PPARγ and NF-κB in HSC-T6were measured by immunocytochemical staining or immunofluorescence, Real-time PCR and Western blot. The contents of TGF-β1and IL-6in the supernatants of HSC-T6were detected by ELISA.Results:(1) Western blot and Real-time PCR demonstrated that the expression of HO-1was higher in Hemin treated group than this in normal control (P<0.01). But in Hemin+GW9662group the expression of HO-1mRNA decreased19.96%and HO-1protein decreased17.28%compared with Hemin treated group (P<0.05). The expression of HO-1was lower in Znpp-IX treated group than this in normal control (P<0.01). But in Znpp-IX+Rosiglitazone group the expression of HO-1mRNA increased18.32%and HO-1protein increased13.7%compared with Znpp-IX treated group (P<0.05).(2) The protein expression of PPARy detected by Western blot was higher in Hemin treated group than normal control (P<0.01), but the protein expression of NF-κB p65was lower than normal control (P <0.01). In contrast, in Znpp-IX treated group the expression of PPARy was lower but NF-κB p65was higher than normal group (P<0.01). In Hemin+GW9662treated group, PPARy decreased17.78%but NF-κB p65increased28.94%compared with merely Hemin treated group (P<0.05). In Znpp-IX+Rosiglitazone treated group, PPARγ increased18.6%but NF-κB p65decreased23.16%compared with merely Hemin treated group (P<0.01). The mRNA expression of PPARγ and NF-κB detected by Real-time PCR were coincidenced with the protein expression above.(3) Results about the expression of PPARγ from immunocytochemical staining showed that they all located in nucleus of HSC-T6. PPARγ expressed weakly in normal control, but strengthen in Hemin treated group. When GW9662pretreated with Hemin, the expression of PPARy was lower than Hemin merely treated group. PPARy expressed more weaker in Znpp-IX treated group than normal group. But in Rosiglitazone pretreated group, the expression of PPARy was higher than Znpp-IX merely treated group. Results about the expression of NF-κB p65from immunofluorescence were opposite to PPARy.(4) The changes of TGF-β1and IL-6in HSC-T6supernatant were same. Levels of TGF-β1and IL-6were lower in Hemin treated group (P<0.01), but significantly higher in Znpp-IX treated group (P<0.01) than normal control. In Hemin+GW9662treated group, levels of TGF-β1and IL-6all increased compared with Hemin treated group (P<0.05). In Znpp-Ⅸ+Rosiglitazone treated group, levels of TGF-β1and IL-6all decreased compared with Znpp-IX treated group.Conclusions:The induction of HO-1could up-regulate the expression of PPARy and negatively regulate the expression of NF-κB at same time in activated HSC-T6, and then impact the release of inflammatory cytokines such as TGF-β1and IL-6, and thus inhibit the proliferation and apoptosis of activated HSC-T6.更多还原