人纤维化肝组织中结缔组织生长因子的表达及其意义

目的观察结缔组织生长因子（connective tissue growth factor, CTGF）在人纤维化肝组织中的表达，探讨CTGF及其介导的生物学信号在肝纤维化发生中的作用机制。方法44例人肝组织（其中包括12例正常肝组织、32例慢性病毒性肝炎和肝硬化组织）来自诊断明确的病理存档标本。采用免疫组化方法对肝组织中CTGF的表达及分布进行检测。结果免疫组化显示，CTGF主要表达于人肝星状细胞胞质中。在人纤维化肝组织中，CTGF表达水平显著高于正常人肝组织（P〈0．01）。结论CTGF作为一种促纤维化因子，其过度表达可促进肝星状细胞的增殖活化，促进细胞外基质的形成，从而促进肝纤维化的发生、发展。     

龙血素A对大鼠肝星状细胞增殖及Frizzled-4受体蛋白表达的影响

［摘要］目的　研究WNT信号通路中卷曲蛋白-低密度脂蛋白相关蛋白复合受体（Frizzled/LRP）在体外培养活化大鼠肝星状细胞（aHSCs）中的表达及龙血素A（Loureirin A）干预对大鼠肝星状细胞（aHSC）增殖及α-平滑肌肌动蛋白、转化生长因子β1分泌的影响,探讨龙血素A抗肝纤维化的细胞分子机制．方法　分离SD大鼠肝星状细胞,体外传代培养诱导细胞活化,倒置相差显微镜下观察肝星状细胞活化前后形态学变化,用不同浓度龙血素A处理活化的大鼠肝星状细胞（aHSCs）．MTT法测定龙血素A对肝星状细胞的生长抑制率;Western Blot方法从蛋白水平检测肝星状细胞中Frizzled-4受体蛋白;Elisa测定药物干预后细胞培养上清液中α-SMA和TGFβ1的含量．结果　与对照组相比,龙血素A抑制肝星状细胞增殖并有明显的时间-剂量效应关系,IC50=0.30 μg/μL;龙血素A在蛋白水平显著下调受体蛋白Frizzled-4表达,同时抑制肝星状细胞分泌α-SMA、TGFβ1（P＜0.05）．结论　Frizzled-4受体蛋白在活化的大鼠肝星状细胞（aHSCs）中高表达,经龙血素A干预后,Frizzled-4表达量明显降低,细胞增殖受到抑制,α-SMA、TGFβ1合成和分泌量下降,这提示龙血素A可能与WNT信号通路调节肝纤维发生和血管新生的分子机制相关．     

胰岛素样生长因子与肝纤维化的研究进展

肝星状细胞(hepatic stellate cells,HSCs)的激活和增殖是肝纤维化的中心环节,胰岛素样生长因子(insulin-like growth factors,IGFs)是一类重要的促细胞活化、分裂的生长因子.IGFs与其受体结合后通过Ras/细胞外信号调节激酶途径、PI3-K途径、影响Na /H 交换以及抑制肝星状细胞凋亡等方式促进肝星状细胞增殖,从而加速肝纤维化.     

PICK1在肝纤维化中的表达变化及功能研究

目的：研究蛋白激酶 C 结合蛋白1（PICK1）在小鼠肝纤维及活化的肝星状细胞中的表达变化,并探讨其对人肝星状细胞株（LX-2）活化的影响。方法建立四氯化碳（CCl4）诱导的小鼠肝纤维化模型,观察 PICK1在肝纤维化过程中的表达变化;转化生长因子β1（TGF-β1）刺激 LX-2活化,West-ern blot 测定 PICK1的蛋白表达情况;转染 PICK1过表达质粒至 LX-2中,再以 TGF-β1诱导活化,Western blot 检测PICK1、α平滑肌肌动蛋白（α-SMA）、I 型胶原（Col1a1）和Smad2、3及其磷酸化水平的蛋白表达情况。结果正常组小鼠肝脏中 PICK1表达较高,随着肝纤维化的加重,PICK1的表达逐渐递减。TGF-β1诱导活化的 LX-2细胞中 PICK1表达降低。转染 PICK1过表达质粒后,TGF-β1诱导的α-SMA、Colla1的表达明显减少,且 Smad2、3磷酸化水平显著下降。结论 PICK1在纤维化肝组织及活化的 HSC 中表达下调。过表达 PICK1可抑制 TGF-β1诱导的 LX-2活化,可能是通过抑制 TGF-β／Smad 通路而发挥作用,为肝纤维化的防治研究提供了新的思路和靶点。     

Smad蛋白与肝纤维化关系的研究进展

肝星形细胞(HSC)是肝纤维化发生发展的关键细胞,转化生长因子β1(TGF-β1)是促进HSC活化、细胞外基质合成的主要因子。在肝纤维化形成的过程中,TGF-β1/Smad信号转导对HSC的作用非常重要。深入研究TGF-β1/Smad信号转导通路可进一步阐述肝纤维化的发病机制,为肝纤维化的防治提供新的有效途径。     

以瘦素为靶点中药单体成分防治肝纤维化研究策略及进展

近年来大量研究表明瘦素在肝纤维化发生发展中发挥着重要作用。瘦素能够通过血小板衍化生长因子（PDGF）、血管生成因子（VEGF）、转化生长因子-β（TGF-β）等相关细胞因子和JAK/STAT3、MAPK/ERK1/2、PI3K/AKT等信号通路激活肝星状细胞。中药单体成分通过抑制细胞因子的释放和阻断信号通路来防治肝纤维化的机制研究已越来越深入。通过对与瘦素相关的肝星状细胞激活通路和细胞因子进行概括总结,并对中药单体成分通过瘦素靶点抗肝纤维化作用进行展望。     

靶向结缔组织生长因子的siRNA对于肝星状细胞介导肝纤维化的研究进展

肝纤维化是多种慢性因素导致肝内细胞发生持续、反复的坏死或炎症刺激,机体发生的修复反应,以肝内细胞外基质（extracellular matrix,ECM）过度沉积和肝星状细胞（hepatic stellate cells,HSC）活化为特征。它不是一个独立的疾病,而是许多慢性肝病的共同病理过程,持续进展可导致肝内正常组织结构改建形成肝硬化并出现多种危及生命的并发症,预后极差。由于体内存在纤维降解过程,故肝纤维化逆转可以避免病情进展至肝硬化。研究表明靶向结缔组织生长因子（connective tissue growth factor,CTGF）小干扰RNA（small interfering RNA,siRNA）可能抑制HSC活化、增殖及ECM的合成,使肝纤维化进程受阻。siRNA介导的CTGF基因沉默为肝纤维化治疗开辟了新的途径。     

细胞因子在肝星状细胞中的信号转导机制

肝星状细胞是产生肝脏细胞外基质的主要细胞.其活化、增殖是肝纤维化发生的中心环节.肝星状细胞的活化受许多生长因子及细胞因子如转化生长因子、血小板衍生生长因子、白介素-1、白介素-10等的调节.近十年来有关细胞因子在肝星状细胞中的信号转导通路逐渐被阐明.现就细胞因子在肝星状细胞中的信号转导机制进行综述.     

转化生长因子β1/Smads信号转导途径及其与肝纤维化的关系

目前认为,在肝纤维化进程中,转化生长因子β1(transforming growth factor bata,TGF-β1)是最强的致纤维化细胞因子[1],它有很强的促肝星状细胞(HSC)激活和分泌细胞外基质(ECM)的作用,与肝纤维化进程密切相关.     

酒精性肝纤维化中肝星状细胞活化的机制研究进展

肝星状细胞（HSC）活化是各种病因所致肝纤维化过程中的关键环节。在酒精性肝病的发展过程中，乙醇和代谢产物乙醛可改变机体氧化还原状态，活化枯否细胞，释放大量细胞因子，诱导HSC活化、增殖和胶原合成，形成肝纤维化。本文将对酒精性肝纤维化中HSC活化的机制和相关靶点加以综述，为开发抗肝纤维化药物和临床治疗提供新的思路。     

Research Progress in Astragalus membranaceus and Its Active Components on Immune Responses in Liver Fibrosis

The interaction between immune cells and hepatic stellate cells (HSCs) can modulate the development of hepatic fibrosis. It can also regulate hepatic fibrosis and liver cirrhosis caused by excessive deposition of extracellular matrix (ECM). This article reviews the action mechanism of immune cells on liver fibrosis and the effect of Astragalus membranaeus and its active components on immune cells. In-depth study of interaction between immune cells and HSCs on the pathogenesis of liver fibrosis, and the regulatory effect of Astragalus membranaeus and its active components on immune mechanism will provide new insights in the treatment of liver fibrosis.     

自然杀伤细胞与肝纤维化

 肝淋巴细胞中含有丰富的自然杀伤细胞（natural killer cells,NK）,后者通过影响天然免疫抵御肝内病毒感染及肿瘤转移。而活化的肝星状细胞（hepatic stellate cells,HSCs）在肝纤维化发生发展中起着关键作用。近来一些研究表明NK细胞可通过产生具有抗纤维化作用的IFN-γ以及直接杀死活化HSCs,起到明显的抗肝纤维化作用。因此,刺激NK细胞活化极可能成为治疗肝纤维化的新途径,但其有效性及安全性需进一步通过临床研究来证明。     

肝星形细胞中细胞凋亡和存活的信号调控

肝星状细胞(hepatic stellate cells,HSCs)在肝脏纤维化发生过程中起着关键作用.当正常肝脏受到损伤时,HSCs由静息状态转分化为类肌成纤维细胞,并保持这种处于激活状态的表型,它们接收到的凋亡和存活的生物信号将决定激活态HSCs的最终细胞寿命.HSCs凋亡的发生与一系列复杂而又相互关联的生物信号传导和调控有关,HSCs凋亡信号来自于细胞膜受体,如死亡受体、神经生长因子受体和外周型苯甲二氮卓受体(peripheral-type benzodiazepine receptor);以及胞浆蛋白,如Bcl-2家族蛋白和细胞周期蛋白等.HSCs存活信号受到多种激酶和细胞因子的诱导,如金属蛋白酶组织抑制剂-1(tissue jnhibitors of metalloproteinase-1)、Rho/Rho激酶、血小板源生长因子(platelet-derived growth factor)、转化生长因子-β1(transforming growth factor-β1)和胰岛素样生长因子(insulin-like growth factor-1)等.特异性地诱导HSCs发生凋亡是治疗肝脏纤维化的直接和有效手段,虽然目前对HSCs由激活态到静息状态的转归尚需进一步研究,但诱导HSCs凋亡将是治疗肝脏纤维化和肝硬化的研究热点和主要发展方向.     

An adipocentric view of liver fibrosis and cirrhosis

Liver fibrosis is the consequence of chronic or repeated liver injury caused by hepatotoxic agents like alcohol and viruses, as well as immune and congenital metabolic disorders. Nonalcoholic fatty liver disease (NAFLD), caused by obesity and abnormal lipid metabolism, may be the latest known cause of liver fibrosis and cirrhosis. Furthermore, NAFLD with obesity can provide a terrain in which alcoholic and viral liver diseases, such as chronic hepatitis C, are prone to cause liver cirrhosis. Insulin, insulin-like growth factor (IGF)-1, peroxisome proliferator-activated receptors (PPARs), leptin, adiponectin, and preadipocyte factor-1/delta-like1 (Pref-1/dlk1) are hormones, growth factors, nuclear receptors, and cytokines that are actively involved in lipid metabolism. They share common target cells important in liver fibrosis, i.e., hepatic stellate cells (HSCs). Activation of HSCs is known to initiate and perpetuate liver fibrosis. Insulin and IGF-1 stimulate HSC activation and collagen production in vitro. However, IGF-1 alleviates liver fibrosis in vivo. Ligands of PPARy inhibit HSC activation and collagen synthesis in vivo and in vitro, and are helpful in decreasing liver fibrosis. But ligands of PPARbeta enhance proliferation of HSCs. Leptin is profibrogenic, and liver fibrosis is decreased in leptin- or leptin receptor-deficient mice. Adiponectin is, on the contrary, anti-fibrogenic. Extensive liver fibrosis may develop in adiponectin-knockout mice and is alleviated by administration of recombinant adiponectin. Pref-1/dlkl is implicated in fibrogenesis of the liver through its modulation of HSCs. The use of such biologically active molecules in lipid metabolism as ligands of PPARgamma and adiponectin might not help slim down a patient on the whole, but can potentially be used to halt the progression of liver fibrosis. Weight reduction, a strategy for controlling obesity and metabolic syndromes, may also be a tool for decreasing NAFLD and alleviating liver cirrhosis.     

肝纤维化过程中肝星状细胞的移行机制

目的探讨肝纤维化过程中肝星状细胞（HSC）移行的机制和肝纤维化病变过程中新的病理生理机制。方法运用改良的Boyden腔系统,在体外条件下模拟体内正常Disse间隙的微环境及肝纤维化时的相关改变,以HSC为研究对象,通过细胞迁移实验、明胶酶谱和凝胶免疫印迹等实验方法,研究肝纤维化时致纤维化生长因子和细胞外基质促进HSC移行的机制。结果肝纤维化时增高的血小板衍化生长因子BB（PDGF—BB）、转化生长因子β1（TGF—β1）以及上皮细胞生长因子（EGF）均可以刺激活化的HSC分泌基质金属蛋白酶2（MMP-2）,而MMP-2通过降解胶原又可以促进HSC的移行（4．9倍）;HSC的移行是由其表面的整合素α1和以所α2,不同致纤维化生长因子诱导HSC移行时依赖着不同的整合素的介导;由HSC分泌的细胞外基质对HSC自身的行为有反馈调节作用,间质类基质可促进HSC的移行（3．2倍）,而基底膜样的基质则可抑制HSC的移行（1．2倍）。结论肝纤维化时Disse间隙微环境的改变导致了HSC的移行,其机制与促进HSC高表达MMP-2相关;肝纤维化时HSC的移行由整合素α1和以所α2;不同的细胞外基质对HSC的移行行为有着不同的调节作用。     

过氧化物酶增殖物激活受体γ抑制大鼠肝星状细胞中转化生长因子-β1诱导的结缔组织生长因子表达

目的 探讨过氧化物酶增殖物激活受体γ(PPARγ)对大鼠肝星状细胞(HSC)中转化生长因子-β1(TGF-β1)诱导的结缔组织生长因子(CTGF)表达的抑制作用.方法 常规培养大鼠HSC,预先给予或不给予PPARγ特异性拮抗剂GW9662处理,再经系列浓度的PPARγ天然配体(15-d-PGJ2)或合成配体(GW7845)及TGF-β1序贯作用后,通过半定量RT-PCR及Western-blotting分析CTGF表达状况,透射电镜观察HSC形态学变化.结果 15-d-PGJ2和GW7845可显著抑制HSC中TGF-β1诱导的CTGF表达(同时在转录和转录后水平),且PPARγ配体对CTGF表达的抑制作用可被GW9662部分或完全逆转,说明此种抑制效应确由PPARγ所介导;电镜观察亦显示HSC由活化态向静息态的形态学转变.结论 PPARγ活化可显著抑制HSC中TGF-β1诱导的CTGF表达,提示PPARγ可作为逆转肝纤维化治疗的新的有效靶点.

Abstract：

Objective To investigate the effect of peroxisome proliferator-activated receptor gamma(PPARγ)activation on transforming growth factor betal(TGF-β1)-induced connective tissue growth factor(CTGF)expression in rat hepatic stellate cells(HSCs).Methods Cultured HSCs with or without PPARγ-specific antagonist GW9662 treatment prior to the addition of an increasing amount of PPARγ natural ligand(15-d-PGJ2)or synthetic ligand(GW7845)were stimulated with TGF-β1.The mRNA and protein levels of CTGF expression were detected by semi-quantitative RT-PCR and Western blotting,respectively.The morphological changes of the HSC were obgerved by electron microscope.Results 15-d-PGJ2 and GW7845 significantly inhibited TGF-β1-induced CTGF expression at both mRNA and protein levels in HSCs, and the inhibitory effect was dramatically,if not completely,abolished by pretreatment with GW9662,suggesting that the inhibition was mediated by PPARγ. Morphological observation revealed that PPARγactivation caused obvious changes of HSCs from activated to quiescent phenotypes.Conclusion PPARγ ligand shows potent inhibitory effect on TGF-β1-induced CTGF expression in rat HSCs,suggesting its potential as a candidate agent for treatment and prevention of hepatic fibrosis.     

活化型肝星状细胞清除机制及其相关抗肝纤维化策略

已知肝星状细胞(hepatic stellate cells, HSCs)活化是肝纤维化的核心环节。越来越多的基础和临床研究证实,肝纤维化可以逆转。而肝纤维化消退的主要表现之一是活化型HSCs数量不断减少。因此,如何清除活化型HSCs并使之成为抗肝纤维化治疗新策略正逐渐受到关注。现有两种可能机制用于解释活化型HSCs的清除现象,即活化型HSCs的表型逆转和凋亡。本文旨在对基于活化型HSCs清除机制的抗肝纤维化策略进展做一综述。     

Progress of Targeting Transforming Growth Factor-β1 Small Interfering RNA in Liver Fibrosis

Liver fibrosis is a common pathological consequence of a variety of chronic stimuli, including viral, autoimmune, drug-induced, cholestatic and metabolic diseases. Fibrosis is driven by a dynamic process involving increased synthesis of matrix components and a failure of physiological mechanisms of matrix turnover. Activation of hepatic stellate cells （HSCs） remains a central event in fibrosis. HSCs are the main source of extracellular matrix （ECM）. Transforming growth factor-beta （TGF-β）, which is the fibrogenic master cytokine, can induce the activation of HSCs to produce a large amount of ECM, and is capable of inducing apoptosis of liver cells. RNA interference （RNAi） is a novel gene disruption technology. Studies have shown that small interfering RNA （siRNA） targeting TGF-β1 may inhibit the activation and proliferation of HSCs, suppress ECM synthesis and block liver fibrosis. TGF-β1 siRNA-mediated gene silencing therapy provides a new avenue for liver fibrosis. This review summarizes recent progresses in research on HSCs, TGF-β1 and TGF-β1 siRNA in liver fibrosis.     

18beta-glycyrrhetinic acid induces ROS-mediated apoptosis to ameliorate hepatic fibrosis by targeting PRDX1/2 in activated HSCs

Hepatic stellate cells (HSCs) are essential drivers of fibrogenesis. Inducing activated-HSC apoptosis is a promising strategy for treating hepatic fibrosis. 18beta-glycyrrhetinic acid (18β-GA) is a natural compound that exists widely in herbal medicines, such as Glycyrrhiza uralensis Fisch, which is used for treating multiple liver diseases, especially in Asia. In the present study, we demonstrated that 18β-GA decreased hepatic fibrosis by inducing the apoptosis in activated HSCs. 18β-GA inhibited the expression of α-smooth muscle actin and collagen type I alpha-1. Using a chemoproteomic approach derived from activity-based protein profiling, together with cellular thermal shift assay and surface plasmon resonance, we found that 18β-GA covalently targeted peroxiredoxin 1 (PRDX1) and peroxiredoxin 2 (PRDX2) proteins via binding to active cysteine residues and thereby inhibited their enzymatic activities. 18β-GA induced the elevation of reactive oxygen species (ROS), resulting in the apoptosis of activated HSCs. PRDX1 knockdown also led to ROS-mediated apoptosis in activated HSCs. Collectively, our findings revealed the target proteins and molecular mechanisms of 18β-GA in ameliorating hepatic fibrosis, highlighting the future development of 18β-GA as a novel therapeutic drug for hepatic fibrosis.