【作者】 唐晓明；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2009， 博士

【摘要】 肝脏由于其特殊的生理位置和血液供应,易于受到多种外界因素的刺激,进而发生肝脏损伤。肝脏长时期处于损伤-恢复的状态,往往会导致肝纤维化的发生。肝纤维化(hepatic fibrosis)是慢性肝病长期发展后的必然过程。其疾病特征是以胶原为主的细胞外基质(extracellular matrix,ECM)在肝内沉积,造成肝小叶结构改建、假小叶和结节形成,严重的肝纤维化可以发展为肝硬化,乃至肝功能衰竭。近来研究认为,肝纤维化属可逆性病变。如果能够去除损伤因素,随着时间的变化,肝纤维化是可以自然恢复的,表现为沉积的胶原降解,肝脏结构和肝脏功能的恢复。文献报道,在肝纤维化形成及恢复阶段,Kupffer细胞发挥了双刃剑的作用。在肝纤维化进展期可促进HSC的激活,促进肝纤维化的形成;在肝纤维化恢复期能促进活化的HSC失活、凋亡,促进ECM的降解,从而抑制肝纤维化的形成。然而,Kupffer细胞在肝纤维化恢复期诱导HSC凋亡的研究较少,其作用的细胞因子、细胞信号传导通路目前尚不清楚。为了进一步揭示肝纤维化恢复过程中Kupffer细胞的作用,我们拟通过分离肝纤维化恢复模型大鼠的Kupffer细胞来进行相关研究。Lefluomide是一种新的抗炎免疫药,目前主要应用在抗类风湿性关节炎的病人上。本实验室率先开展了lefluomide对实验性肝纤维化作用的研究。A771726且可显著抑制HSC的增殖,同时,A771726能够抑制Leptin对HSC的活化,降低胶原的产生。而且,在整体动物模型上,A771726对实验性肝纤维化动物模型具有防治作用,能够降低肝脏纤维化的严重程度。继续研究lefluomide对HSC的作用将有利于探讨lefluomide对肝纤维化防治作用的分子机制,为临床肝纤维化治疗药物的选择及肝纤维化治疗药物的开发与筛选提供新的思路。本研究制备了大鼠肝纤维化恢复模型,观察到恢复期的肝组织中胶原的含量明显下降,同时胶原纤维向组织内延伸的程度也明显降低。进一步采用原位灌注、密度梯度离心的方法分离得到Kupffer细胞和HSC,并收集了Kupffer细胞条件性培养基(KCCM)。BrdU细胞增殖实验显示,恢复期KCCM抑制HSC的增殖;细胞凋亡实验发现恢复期KCCM能够促进HSC的凋亡。使用A771726预处理HSC,能够增强恢复期KCCM抑制HSC的作用。为检测恢复期KCCM诱导HSC是否依赖caspase活化,本文采用Western blot检测了恢复期KCCM处理的HSC的caspase活化和PARP剪切水平,结果显示与对照组相比,恢复期KCCM处理组HSC的caspase-3活性亚基明显增多,PARP被剪切成典型的肽段,caspase-9前体的水平明显降低;A771726预处理能够明显增强这种作用。结果提示caspase信号途径参与了恢复期KCCM诱导的HSC凋亡。为了进一步证明caspase信号途径在这一过程中的作用,采用caspase广谱抑制剂Z-VAD-FMK , caspase-3抑制剂Z-DEVD-FMK,caspase-8抑制剂Z-IETD-FMK,或caspase-9抑制剂Z-LEHD-FMK分别预处理HSC,结果显示均能明显抑制恢复期KCCM诱导的HSC凋亡。这些结果表明恢复期KCCM诱导HSC凋亡依赖caspase途径的活化。实时定量PCR(Q-PCR)检测结果显示,与正常Kupffer细胞相比,恢复期Kupffer细胞的FasL和TNF-α表达水平没有明显改变;而恢复期Kupffer细胞的TRAIL表达水平明显升高。利用RNA干涉实验进一步证明TRAIL在恢复期KCCM诱导HSC凋亡中的作用。荧光报告基因实验显示,恢复期KCCM能够诱导HSC中NF-κB的活化;用A771726预处理HSC能明显地抑制恢复期KCCM诱导的NF-κB活化,且呈剂量依赖性。结果提示A771726通过抑制HSC的NF-κB活化,发挥对恢复期KCCM诱导HSC凋亡的促进作用。综上,我们的结果显示,在肝纤维化恢复期,肝脏的Kupffer细胞能够通过分泌TRAIL来诱导活化的HSC发生凋亡,同时,A771726能够通过抑制NF-κB的转录活性,促进HSC的凋亡。为了进一步研究lefluomide在肝纤维化中应用的可能性,我们利用人的星型细胞系LX-2作为细胞模型,以重组的人TRAIL为凋亡诱导因子,在体外检测lefluomide对人星型细胞系LX-2的影响及其对TRAIL诱导HSC凋亡的作用和相应的分子机制,为评价lefluomide可能用于治疗肝纤维化提供新的实验证据。细胞凋亡实验发现,TRAIL能够诱导人星型细胞系LX-2发生凋亡,lefluomide的预处理会明显加速这种凋亡现象。Caspase-3活性检测、PARP剪切水平检测和caspase抑制剂预处理的细胞凋亡实验结果表明,lefluomide促进TRAIL诱导LX-2细胞凋亡依赖caspase途径的活化。Q-PCR实验结果显示,lefluomide并没有上调DR4、DR5的表达水平。TRAIL与其受体相互作用后,可以引发JNK和NF-κB的活化。在LX-2细胞中,TRAIL能够诱导JNK和c-Jun发生磷酸化增强;lefluomide的预处理能够抑制LX-2细胞中JNK和c-Jun的磷酸化。同时,lefluomide能够抑制IκB的磷酸化和p65亚基的核转运,这会导致NF-κB的转录活性的降低。双荧光素酶报告基因实验显示,lefluomide在人的星型细胞系LX-2中同样能够抑制NF-κB的转录活性。利用JNK或NF-κB信号通路的抑制剂进行细胞凋亡实验,结果表明lefluomide通过抑制TRAIL诱导的JNK和NF-κB通路活化来发挥促进TRAIL诱导LX-2细胞凋亡的作用。综上,本实验证明了在大鼠肝纤维化恢复期kupffer细胞促进HSC凋亡,探讨其主要发挥作用的细胞因子和对HSC内可能的信号转导通路;证明了TRAIL能够诱导人肝星型细胞系LX-2细胞发生凋亡,lefluomide能够促进这种作用,并初步阐明了lefluomide这种功能的分子机制,为lefluomide应用于肝纤维化治疗提供了坚实的体外实验基础,为评价、筛选肝纤维化治疗药物提供了新的思路。更多还原

【Abstract】 Hepatic fibrosis is a common consequence of chronic liver injury of any etiology, and hepatic stellate cells (HSCs) are the major source of increased extracellular matrix (ECM) proteins in chronic liver diseases. If hepatic injury persists, HSCs proliferate and undergo dramatic transdifferentiation from quiescent vitamin A-storing cells to activated myofibroblast-like cells. These activated HSCs secrete large amounts of ECM proteins, which is a seminal event in hepatic fibrogenesis.The factors that modulate HSC activation influence the progression of liver fibrosis. Kupffer cells, the resident macrophages in the liver, act as a double-edged sword in the progression of and recovery from hepatic fibrosis. It has been widely reported that Kupffer cells can promote HSC activation during the progression of fibrosis. On the other hand, Kupffer cells can negatively regulate activated HSCs during recovery from liver fibrosis associated with enhancing matrix degradation. The mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis, however, is still far from being fully understood. Leflunomide, an isoxazole derivative, is a unique immunomodulatory agent, capable of treating rheumatoid arthritis (RA), allograft and xenograft rejection,and systemic lupus erythematosus. After administration, it is metabolized to its active form, A771726, which leads to the immunosuppressive activity. It has also been reported to block cell cycle progression in the G0/G1 phase. Recent evidence has suggested that A771726 can significantly inhibit the proliferation and activation of HSCs by inhibiting the Janus kinase (JAK) and protein kinase B (Akt) pathways. In vivo, A771726 showed an inhibitory effect on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats. Based on these results, the aim of this study was to elucidate the mechanism by which Kupffer cells regulate HSC biology during regression of hepatic fibrosis and the effect of leflunomide on this process.Following stimulation with recovery KCCM, HSCs showed a decrease in proliferation and an increase in apoptosis by a caspase-dependent mechanism. Furthermore, pretreatment with A771726 markedly enhanced these effects. Real-time quantitative PCR (Q-PCR) analysis showed increased expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in Kupffer cells during the spontaneous recovery phase. The pro-apoptotic function of KCCM prepared from TRAIL siRNA-treated Kupffer cells was obviously decreased, suggesting that TRAIL played an important role in recovery from hepatic fibrosis. Moreover, A771726 enhanced recovery KCCM-induced apoptosis of HSCs by a mechanism involving the inhibition of nuclear factor-kappa B (NF-κB) activation. Our results showed the role of TRAIL in recovery Kupffer cell-induced apoptosis of activated HSCs and provided insights into the resolution of fibrosis and the mechanisms by which leflunomide might act upon liver fibrosis.During the resolution phase of hepatic fibrosis, a crucial mechanism is the apoptosis of activated hepatic stellate cells (HSCs). It is necessary to find more anti-fibrosis drugs that would modulate HSCs to be more susceptible to apoptotic stimuli. Here we showed that A771726, the active metabolite of leflunomide, markedly enhanced tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in the human hepatic stellate cell line LX-2. A771726 could increase caspase activity in LX-2 cells in a dose-dependent manner. A771726 did not increase the expression of TRAIL receptors in LX-2 cells but could inhibit activation of the c-Jun NH2-terminal kinase (JNK) pathway through decreasing TRAIL-induced JNK and c-Jun phosphorylation. Moreover, A771726 could accelerate TRAIL-induced apoptosis via inhibiting nuclear factor-kappaB (NF-κB) activation in LX-2 cells. In conclusion, our results indicated leflunomide could enhance the sensitivity of LX-2 cells to TRAIL-induced apoptosis via inhibiting the survival pathways and provided a promising approach to anti-fibrotic therapy with leflunomide.更多还原