大鼠肝纤维化恢复期肝星状细胞凋亡的分子机制及来氟米特的调节作用
|
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。
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.
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。
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.
引文
1. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology, 2008; 134(6): 1655-1669
2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005; 115(2): 209-218
3. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. MedGenMed, 2002; 4(3): 27
4. Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Semin Liver Dis, 2001; 21(3): 427-436
5. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology, 1991; 13(6): 1193-1202
6. Arthur MJ, Iredale JP, Mann DA. Tissue inhibitors of metalloproteinases: role in liver fibrosis and alcoholic liver disease. Alcohol Clin Exp Res, 1999; 23(5): 940-943
7.申月明,朱萱.肝星状细胞激活和信号转导.世界华人消化杂志,2007; 15(8): 873-878
8. Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H, Dianzani MU, Laffi G, Abboud HE, Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology, 1997; 112(4): 1297-1306
9. Mallat A, Gallois C, Tao J, Habib A, Maclouf J, Mavier P, Préaux AM, Lotersztajn S. Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation. Role of a prostaglandin/cyclic AMP pathway and cross-talk with endothelin receptors. J. Biol. Chem., 1998; 273: 27300-27305
10. Pinzani M, Gesualdo L, Sabbah GM, Abboud HE. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured ratliver fat-storing cells. J Clin Invest, 1989; 84(6): 1786-1793
11. Castilla A, Prieto J, Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease. Effects of interferon alfa therapy. N Engl J Med, 1991; 324: 933 -940
12. Garcia-Trevijano ER, Iraburu MJ, Fontana L, Dominguez-Rosales JA, Auster A, Covarrubias-Pinedo A, Rojkind M. Transforming growth factor beta1 induces the expression of alpha1(I) procollagen mRNA by a hydrogen peroxide-C/EBPbeta-dependent mechanism in rat hepatic stellate cells. Hepatology, 1999; 29(3): 960-970
13 Watanabe T, Niioka M, Hozawa S, Kameyama K, Hayashi T, Arai M, Ishikawa A, Maruyama K, Okazaki I. Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride. J Hepatol, 2000; 33(2): 224-235
14. Shiratori Y, Geerts A, Ichida T, Kawase T, Wisse E. Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells. J Hepatol, 1986; 3(3): 294-303
15. Friedman SL. Mac the knife? Macrophages- the double-edged sword of hepatic fibrosis. J Clin Invest, 2005; 115(1): 29-32
16. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest, 2005; 115(1): 56-65
17. Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, Kirschbaum BJ. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol, 1999; 93(3): 198-208
18. Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry, 1996; 35(4): 1270-1273
19. Yao HW, Li J, Chen JQ, Xu SY. Inhibitory effect of leflunomide on hepatic fibrosisinduced by CCl4 in rats. Acta Pharmacol Sin, 2004; 25(7): 915-920
20. Si HF, Li J, LüXW, Jin Y. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation. Experimental Biology and Medicine (Maywood, N.J.), 2007; 232(3): 427-436
21. Li D, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol, 1999; 14(7): 618-633
22. Issa R, Zhou X, Trim N, Millward-Sadler H, Krane S, Benyon C, Iredale J. Mutation in collagen-I that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J, 2003; 17(1): 47-49
23. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998; 102(3): 538-549
24. Guo J, Friedman SL. Hepatic fibrogenesis. Semin Liver Dis, 2007; 27(4): 413-426
25. Isoda K, Kagaya N, Akamatsu S, Hayashi S, Tamesada M, Watanabe A, Kobayashi M, Tagawa Y, Kondoh M, Kawase M, Yagi K. Hepatoprotective effect of vitamin B12 on dimethylnitrosamine-induced liver injury. Biol Pharm Bull, 2008; 31(2): 309-311
26. Kusunose M, Qiu B, Cui T, Hamada A, Yoshioka S, Ono M, Miyamura M, Kyotani S, Nishioka Y. Effect of Sho-saiko-to extract on hepatic inflammation and fibrosis in dimethylnitrosamine induced liver injury rats. Biol Pharm Bull, 2002; 25(11): 1417-1421
27. Wu J, Norton PA. Animal models of liver fibrosis. Scand J Gastroenterol, 1996; 31(12): 1137-1143
28.许建明,徐叔云,张运芳,梅俏,吴继锋.四氯化碳诱导小鼠肝纤维化模型的建立.中国药理学通报, 2000; 16(3): 339-341
29. Panduro A, Shalaby F, Biempica L, Shafritz DA. Changes in albumin,alpha-fetoprotein and collagen gene transcription in CCl4-induced hepatic fibrosis. Hepatology, 1988; 8(2): 259-266
30. George J, Rao KR, Stern R, Chandrakasan G. Dimethylnitrosamine-induced liver injury in rats: the early deposition of collagen. Toxicology, 2001; 156(2-3): 129-138
31. Rubin E, Lieber CS. Fatty liver, alcoholic hepatitis and cirrhosis produced by alcohol in primates. N Engl J Med, 1974; 290(3): 128-135
32. Prakash S, Nanji AA, Robbins PW. Fibrosin: A novel lymphokine in alcohol-induced fibrosis. Exp Mol Pathol, 1999; 67(1): 40-49
33. Symeonidis A, Trams EG. Morphologic and functional changes in the livers of rats after ligation or excision of the common bile duct. Am J Pathol, 1957; 33(1): 13-27
34. Lee SS, Girod C, Braillon A, Hadengue A, Lebrec D. Hemodynamic characterization of chronic bile duct-ligated rats: effect of pentobarbital sodium. Am J Physiol, 1986; 251: G176-180
35. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem, 1987; 161(1): 207-218
36. Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest, 1989; 84(6): 1780-1785
37. Harwood SM, Yaqoob MM, Allen DA. Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis. Ann Clin Biochem, 2005; 42: 415- 431
38. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296
39. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs theATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 2000; 101(1): 25-33
40. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and
22-nucleotide RNAs. Genes Dev, 2001; 15(2): 188-200
41. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of
21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001; 411(6836): 494-498
42. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409(6818): 363-366
43. Sontheimer EJ. Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol, 2005; 6(2): 127-138
44. Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM. The Receptor for the Cytotoxic Ligand TRAIL. Science, 1997; 276(5309): 111-113
45. Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA, Goodwin RG, Rauch CT. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J, 1997; 16(17): 5386-5397
46. Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity, 1997; 7(6): 821-830
47. Schneider P, Tschopp J. Apoptosis induced by death receptors. Pharm Acta Helv, 2000; 74(2-3): 281-286
48. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science, 1997; 277(5327): 815-818
49. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A.Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science, 1997; 277(5327): 818-821
50. Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, Yuan J, Gurney A, Goddard AD, Godowski P, Ashkenazi A. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol, 1997; 7(12): 1003-1006
51. Marsters SA, Pitti RA, Sheridan JP, Ashkenazi A. Control of apoptosis signaling by Apo2 ligand. Recent Prog Horm Res, 1999; 54: 225-234
52. Leverkus M, Neumann M, Mengling T, Rauch CT, Br?cker EB, Peter H. Krammer PH, Walczak H. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res, 2000; 60(3): 553-559
53. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med, 1999; 5(2): 157-163
54. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, Blenis J, Tschopp J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol, 2000; 2(4): 241-243
55. Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology, 2004; 126(7): 1795-1808
56. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998; 102(3): 538-549
57. Oakley F, Meso M, Iredale JP, Green K, Marek CJ, Zhou X, May MJ,Millward-Sadler H, Wright MC, Mann DA. Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis. Gastroenterology, 2005; 128(1): 108-120
58. Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, Arthur MJ, Iredale JP, Mann DA. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology, 2001; 121(3): 685-698
59. Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis, 2001; 21(3): 437-451
60. Chawla-Sarkar M, Bauer JA, Lupica JA, Morrison BH, Tang Z, Oates RK, Almasan A, DiDonato JA, Borden EC, Lindner DJ. Suppression of NF- B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL. J Biol Chem, 2003; 278: 39461-39469
61. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gelinas C, Fuchs EJ, Bedi A. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nat Cell Biol, 2001; 3(4): 409-416
62. Afford SC, Adams DH. Following the TRAIL from hepatitis C virus and alcohol to fatty liver. Gut, 2005; 54(11): 1518-1520
63. Lan L, Gorke S, Rau SJ, Zeisel MB, Hildt E, Himmelsbach K, Carvajal-Yepes M, Huber R, Wakita T, Schmitt-Graeff A, Royer C, Blum HE, Fischer R, Baumert TF. Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner. J Immunol, 2008; 181: 4926-4935
64. Finnberg N, Klein-Szanto AJ, El-Deiry WS. TRAIL-R deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis. J Clin Invest, 2008; 118(1): 111-123
65. Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediatedapoptosis. Hepatology, 2003; 37(1): 87-95
66. Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J, 2004; 18(13): 1612-1614
67. Imose M, Nagaki M, Kimura K, Takai S, Imao M, Naiki T, Osawa Y, Asano T, Hayashi H, Moriwaki H. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB. Hepatology, 2004; 40(5): 1160-1169
68. Vrenken TE, Buist-Homan M, Kalsbeek AJ, Faber KN, Moshage H. The active metabolite of leflunomide, A771726, protects rat hepatocytes against bile acid-induced apoptosis. J Hepatol, 2008; 49(5): 799-809
69. Leger DY, Liagre B, Beneytout JL. Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents. Apoptosis, 2006; 11(10): 1747-1760
70. Migita K, Miyashita T, Maeda Y, Nakamura M, Yatsuhashi H, Ishibashi H, Eguchi K. An active metabolite of leflunomide, A771726, inhibits the production of serum amyloid A protein in human hepatocytes. Rheumatology, 2005; 44(4): 443-448
71. Manna SK, Aggarwal BB. Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-κB activation and gene expression. J Immunol, 1999; 162(4): 2095-2102
72. Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. J Immunol, 2000; 165(10): 5962-5969
73. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002; 298: 1911-1912
74. Schnabl B, Bradham CA, Bennett BL, Manning AM, Stefanovic B, Brenner DA. TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. Hepatology, 2001;34(5): 953-963
75. Yoshida K, Matsuzaki K, Mori S, Tahashi Y, Yamagata H, Furukawa F, Seki T, Nishizawa M, Fujisawa J, Okazaki K. Transforming growth factor-? and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury. Am J Pathol, 2005; 166: 1029-1039
76. Chen A, Davis BH. UV irradiation activates JNK and increases I(I) collagen gene expression in rat hepatic stellate cells. J Biol Chem, 1999; 274: 158-164
77. Burger D, Begué-Pastor N, Benavent S, Gruaz L, Kaufmann MT, Chicheportiche R, Dayer JM. The active metabolite of leflunomide, A771726, inhibits the production of prostaglandin E2, matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes. Rheumatology, 2003; 42(1): 89-96
78. Alexander D, Friedrich B, Abruzzese T, Gondolph-Zink B, Wulker N, Aicher WK. The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression. Cell Physiol Biochem, 2006; 17(1-2): 69-78
1. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology, 2008; 134(6): 1655-1669
2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005; 115(2): 209-218
3. Boigk G,Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology, 1997; 26(3): 643-649
4. Vitamin E therapy in patients with NASH. Yoneda M, Hasegawa T, Nakamura K, Tamano M, Kono T, Terano A. Hepatology, 2004; 39(2): 568
5. Zou WL, Yang Z, Zang YJ, Li DJ, Liang ZP, Shen ZY. Inhibitory effects of prostaglandin E1 on activation of hepatic stellate cells in rabbits with schistosomiasis. Hepatobiliary Pancreat Dis Int, 2007; 6(2): 176-181
6. Hui AY, Dannenberg AJ, Sung JJ, Subbaramaiah K, Du B, Olinga P, Friedman SL. Prostaglandin E2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha 1(I) in hepatic stellate cells. J Hepatol, 2004 ;41(2): 251-258
7. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. MedGenMed, 2002; 4(3): 27
8. Rockey DC, Maher JJ, Jarnagin WR, Gabbiani G, Friedman SL. Inhibition of rathepatic lipocyte activation in culture by interferon-gamma. Hepatology, 1992; 16(3): 776-784
9. Wei HS, Li DG, Lu HM, Zhan YT, Wang ZR, Huang X, Zhang J, Cheng JL, Xu QF. Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl(4). World J Gastroenterol, 2000; 6(4): 540-545
10. Wei YH, Jun L, Qiang CJ. Effect of losartan, an angiotensin II antagonist, on hepatic fibrosis induced by CCl4 in rats. Dig Dis Sci, 2004; 49(10): 1589-1594
11. Ramalho LN, Ramalho FS, Zucoloto S, Castro-e-Silva Junior O, Correa FM, Elias Junior J, Magalhaes JF. Effect of losartan, an angiotensin II antagonist, on secondary biliary cirrhosis. Hepatogastroenterology, 2002; 49(48): 1499-1502
12. Jonsson JR, Clouston AD, Ando Y, Kelemen LI, Horn MJ, Adamson MD, Purdie DM, Powell EE. Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis. Gastroenterology, 2001; 121(1): 148-155
13. Ueno T, Hashimoto O, Kimura R, Torimura T, Kawaguchi T, Nakamura T, Sakata R, Koga H, Sata M. Relation of type II transforming growth factor-beta receptor to hepatic fibrosis and hepatocellular carcinoma. Int J Oncol, 2001; 18(1): 49-55
14. Iwamoto H, Nakamuta M, Tada S, Sugimoto R, Enjoji M, Nawata H. Platelet-derived growth factor receptor tyrosine kinase inhibitor AG1295 attenuates rat hepatic stellate cell growth. J Lab Clin Med, 2000; 135(5): 406-412
15. Kweon YO, Paik YH, Schnabl B, Qian T, Lemasters JJ, Brenner DA. Gliotoxin-mediated apoptosis of activated human hepatic stellate cells. J Hepatol. 2003;39(1):38-46
16. Siegmund SV, Qian T, de Minicis S, Harvey-White J, Kunos G, Vinod KY, Hungund B, Schwabe RF. The endocannabinoid 2-arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species. FASEB J, 2007; 21(11): 2798 - 2806
17. Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellatecells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis. Hepatology, 2003; 37(1): 87-95
18. Horani A, Muhanna N, Pappo O, Melhem A, Alvarez CE, Doron S, Wehbi W, Dimitrios K, Friedman SL, Safadi R. Beneficial effect of glatiramer acetate (Copaxone) on immune modulation of experimental hepatic fibrosis. Am J Physiol Gastrointest Liver Physiol, 2007; 292(2): G628-638
19. Guo YT, Leng XS, Li T, Peng JR, Song SH, Xiong LF, Qin ZZ. Effect of ligand of peroxisome proliferator-activated receptor gamma on the biological characters of hepatic stellate cells. World J Gastroenterol, 2005; 11(30): 4735-4739
20. Ikeda H, Nagashima K, Yanase M, Tomiya T, Arai M, Inoue Y, Tejima K, Nishikawa T, Omata M, Kimura S, Fujiwara K. Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2003; 285(5): G880-886
21. Sakaida I, Matsumura Y, Kubota M, Kayano K, Takenaka K, Okita K. The prolyl 4-hydroxylase inhibitor HOE 077 prevents activation of Ito cells, reducing procollagen gene expression in rat liver fibrosis induced by choline-deficient L-amino acid-defined diet. Hepatology, 1996; 23(4): 755-763
22. Matsumura Y, Sakaida I, Uchida K, Kimura T, Ishihara T, Okita K. Prolyl 4-hydroxylase inhibitor (HOE 077) inhibits pig serum-induced rat liver fibrosis by preventing stellate cell activation. J Hepatol, 1997; 27(1): 185-192
23. Pines M , Nagler A. Halofuginone: a novel antifibrotic therapy. Gen Pharmacol, 1998; 30(4): 445-450
2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005; 115(2): 209-218
3. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. MedGenMed, 2002; 4(3): 27
4. Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Semin Liver Dis, 2001; 21(3): 427-436
5. Geerts A, Lazou JM, De Bleser P, Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology, 1991; 13(6): 1193-1202
6. Arthur MJ, Iredale JP, Mann DA. Tissue inhibitors of metalloproteinases: role in liver fibrosis and alcoholic liver disease. Alcohol Clin Exp Res, 1999; 23(5): 940-943
7.申月明,朱萱.肝星状细胞激活和信号转导.世界华人消化杂志,2007; 15(8): 873-878
8. Marra F, Gentilini A, Pinzani M, Choudhury GG, Parola M, Herbst H, Dianzani MU, Laffi G, Abboud HE, Gentilini P. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology, 1997; 112(4): 1297-1306
9. Mallat A, Gallois C, Tao J, Habib A, Maclouf J, Mavier P, Préaux AM, Lotersztajn S. Platelet-derived growth factor-BB and thrombin generate positive and negative signals for human hepatic stellate cell proliferation. Role of a prostaglandin/cyclic AMP pathway and cross-talk with endothelin receptors. J. Biol. Chem., 1998; 273: 27300-27305
10. Pinzani M, Gesualdo L, Sabbah GM, Abboud HE. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured ratliver fat-storing cells. J Clin Invest, 1989; 84(6): 1786-1793
11. Castilla A, Prieto J, Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease. Effects of interferon alfa therapy. N Engl J Med, 1991; 324: 933 -940
12. Garcia-Trevijano ER, Iraburu MJ, Fontana L, Dominguez-Rosales JA, Auster A, Covarrubias-Pinedo A, Rojkind M. Transforming growth factor beta1 induces the expression of alpha1(I) procollagen mRNA by a hydrogen peroxide-C/EBPbeta-dependent mechanism in rat hepatic stellate cells. Hepatology, 1999; 29(3): 960-970
13 Watanabe T, Niioka M, Hozawa S, Kameyama K, Hayashi T, Arai M, Ishikawa A, Maruyama K, Okazaki I. Gene expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by carbon tetrachloride. J Hepatol, 2000; 33(2): 224-235
14. Shiratori Y, Geerts A, Ichida T, Kawase T, Wisse E. Kupffer cells from CCl4-induced fibrotic livers stimulate proliferation of fat-storing cells. J Hepatol, 1986; 3(3): 294-303
15. Friedman SL. Mac the knife? Macrophages- the double-edged sword of hepatic fibrosis. J Clin Invest, 2005; 115(1): 29-32
16. Duffield JS, Forbes SJ, Constandinou CM, Clay S, Partolina M, Vuthoori S, Wu S, Lang R, Iredale JP. Selective depletion of macrophages reveals distinct, opposing roles during liver injury and repair. J Clin Invest, 2005; 115(1): 56-65
17. Fox RI, Herrmann ML, Frangou CG, Wahl GM, Morris RE, Strand V, Kirschbaum BJ. Mechanism of action for leflunomide in rheumatoid arthritis. Clin Immunol, 1999; 93(3): 198-208
18. Davis JP, Cain GA, Pitts WJ, Magolda RL, Copeland RA. The immunosuppressive metabolite of leflunomide is a potent inhibitor of human dihydroorotate dehydrogenase. Biochemistry, 1996; 35(4): 1270-1273
19. Yao HW, Li J, Chen JQ, Xu SY. Inhibitory effect of leflunomide on hepatic fibrosisinduced by CCl4 in rats. Acta Pharmacol Sin, 2004; 25(7): 915-920
20. Si HF, Li J, LüXW, Jin Y. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation. Experimental Biology and Medicine (Maywood, N.J.), 2007; 232(3): 427-436
21. Li D, Friedman SL. Liver fibrogenesis and the role of hepatic stellate cells: new insights and prospects for therapy. J Gastroenterol Hepatol, 1999; 14(7): 618-633
22. Issa R, Zhou X, Trim N, Millward-Sadler H, Krane S, Benyon C, Iredale J. Mutation in collagen-I that confers resistance to the action of collagenase results in failure of recovery from CCl4-induced liver fibrosis, persistence of activated hepatic stellate cells, and diminished hepatocyte regeneration. FASEB J, 2003; 17(1): 47-49
23. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998; 102(3): 538-549
24. Guo J, Friedman SL. Hepatic fibrogenesis. Semin Liver Dis, 2007; 27(4): 413-426
25. Isoda K, Kagaya N, Akamatsu S, Hayashi S, Tamesada M, Watanabe A, Kobayashi M, Tagawa Y, Kondoh M, Kawase M, Yagi K. Hepatoprotective effect of vitamin B12 on dimethylnitrosamine-induced liver injury. Biol Pharm Bull, 2008; 31(2): 309-311
26. Kusunose M, Qiu B, Cui T, Hamada A, Yoshioka S, Ono M, Miyamura M, Kyotani S, Nishioka Y. Effect of Sho-saiko-to extract on hepatic inflammation and fibrosis in dimethylnitrosamine induced liver injury rats. Biol Pharm Bull, 2002; 25(11): 1417-1421
27. Wu J, Norton PA. Animal models of liver fibrosis. Scand J Gastroenterol, 1996; 31(12): 1137-1143
28.许建明,徐叔云,张运芳,梅俏,吴继锋.四氯化碳诱导小鼠肝纤维化模型的建立.中国药理学通报, 2000; 16(3): 339-341
29. Panduro A, Shalaby F, Biempica L, Shafritz DA. Changes in albumin,alpha-fetoprotein and collagen gene transcription in CCl4-induced hepatic fibrosis. Hepatology, 1988; 8(2): 259-266
30. George J, Rao KR, Stern R, Chandrakasan G. Dimethylnitrosamine-induced liver injury in rats: the early deposition of collagen. Toxicology, 2001; 156(2-3): 129-138
31. Rubin E, Lieber CS. Fatty liver, alcoholic hepatitis and cirrhosis produced by alcohol in primates. N Engl J Med, 1974; 290(3): 128-135
32. Prakash S, Nanji AA, Robbins PW. Fibrosin: A novel lymphokine in alcohol-induced fibrosis. Exp Mol Pathol, 1999; 67(1): 40-49
33. Symeonidis A, Trams EG. Morphologic and functional changes in the livers of rats after ligation or excision of the common bile duct. Am J Pathol, 1957; 33(1): 13-27
34. Lee SS, Girod C, Braillon A, Hadengue A, Lebrec D. Hemodynamic characterization of chronic bile duct-ligated rats: effect of pentobarbital sodium. Am J Physiol, 1986; 251: G176-180
35. Friedman SL, Roll FJ. Isolation and culture of hepatic lipocytes, Kupffer cells, and sinusoidal endothelial cells by density gradient centrifugation with Stractan. Anal Biochem, 1987; 161(1): 207-218
36. Friedman SL, Arthur MJ. Activation of cultured rat hepatic lipocytes by Kupffer cell conditioned medium. Direct enhancement of matrix synthesis and stimulation of cell proliferation via induction of platelet-derived growth factor receptors. J Clin Invest, 1989; 84(6): 1780-1785
37. Harwood SM, Yaqoob MM, Allen DA. Caspase and calpain function in cell death: bridging the gap between apoptosis and necrosis. Ann Clin Biochem, 2005; 42: 415- 431
38. Hammond SM, Bernstein E, Beach D, Hannon GJ. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000; 404(6775): 293-296
39. Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: double-stranded RNA directs theATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell, 2000; 101(1): 25-33
40. Elbashir SM, Lendeckel W, Tuschl T. RNA interference is mediated by 21- and
22-nucleotide RNAs. Genes Dev, 2001; 15(2): 188-200
41. Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T. Duplexes of
21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001; 411(6836): 494-498
42. Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 2001; 409(6818): 363-366
43. Sontheimer EJ. Assembly and function of RNA silencing complexes. Nat Rev Mol Cell Biol, 2005; 6(2): 127-138
44. Pan G, O'Rourke K, Chinnaiyan AM, Gentz R, Ebner R, Ni J, Dixit VM. The Receptor for the Cytotoxic Ligand TRAIL. Science, 1997; 276(5309): 111-113
45. Walczak H, Degli-Esposti MA, Johnson RS, Smolak PJ, Waugh JY, Boiani N, Timour MS, Gerhart MJ, Schooley KA, Smith CA, Goodwin RG, Rauch CT. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J, 1997; 16(17): 5386-5397
46. Chaudhary PM, Eby M, Jasmin A, Bookwalter A, Murray J, Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-kappaB pathway. Immunity, 1997; 7(6): 821-830
47. Schneider P, Tschopp J. Apoptosis induced by death receptors. Pharm Acta Helv, 2000; 74(2-3): 281-286
48. Pan G, Ni J, Wei YF, Yu G, Gentz R, Dixit VM. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science, 1997; 277(5327): 815-818
49. Sheridan JP, Marsters SA, Pitti RM, Gurney A, Skubatch M, Baldwin D, Ramakrishnan L, Gray CL, Baker K, Wood WI, Goddard AD, Godowski P, Ashkenazi A.Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science, 1997; 277(5327): 818-821
50. Marsters SA, Sheridan JP, Pitti RM, Huang A, Skubatch M, Baldwin D, Yuan J, Gurney A, Goddard AD, Godowski P, Ashkenazi A. A novel receptor for Apo2L/TRAIL contains a truncated death domain. Curr Biol, 1997; 7(12): 1003-1006
51. Marsters SA, Pitti RA, Sheridan JP, Ashkenazi A. Control of apoptosis signaling by Apo2 ligand. Recent Prog Horm Res, 1999; 54: 225-234
52. Leverkus M, Neumann M, Mengling T, Rauch CT, Br?cker EB, Peter H. Krammer PH, Walczak H. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes. Cancer Res, 2000; 60(3): 553-559
53. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M, Chin W, Jones J, Woodward A, Le T, Smith C, Smolak P, Goodwin RG, Rauch CT, Schuh JC, Lynch DH. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med, 1999; 5(2): 157-163
54. Bodmer JL, Holler N, Reynard S, Vinciguerra P, Schneider P, Juo P, Blenis J, Tschopp J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8. Nat Cell Biol, 2000; 2(4): 241-243
55. Issa R, Zhou X, Constandinou CM, Fallowfield J, Millward-Sadler H, Gaca MD, Sands E, Suliman I, Trim N, Knorr A, Arthur MJ, Benyon RC, Iredale JP. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking. Gastroenterology, 2004; 126(7): 1795-1808
56. Iredale JP, Benyon RC, Pickering J, McCullen M, Northrop M, Pawley S, Hovell C, Arthur MJ. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998; 102(3): 538-549
57. Oakley F, Meso M, Iredale JP, Green K, Marek CJ, Zhou X, May MJ,Millward-Sadler H, Wright MC, Mann DA. Inhibition of inhibitor of kappaB kinases stimulates hepatic stellate cell apoptosis and accelerated recovery from rat liver fibrosis. Gastroenterology, 2005; 128(1): 108-120
58. Wright MC, Issa R, Smart DE, Trim N, Murray GI, Primrose JN, Arthur MJ, Iredale JP, Mann DA. Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats. Gastroenterology, 2001; 121(3): 685-698
59. Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis, 2001; 21(3): 437-451
60. Chawla-Sarkar M, Bauer JA, Lupica JA, Morrison BH, Tang Z, Oates RK, Almasan A, DiDonato JA, Borden EC, Lindner DJ. Suppression of NF- B Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL. J Biol Chem, 2003; 278: 39461-39469
61. Ravi R, Bedi GC, Engstrom LW, Zeng Q, Mookerjee B, Gelinas C, Fuchs EJ, Bedi A. Regulation of death receptor expression and TRAIL/Apo2L-induced apoptosis by NF-kappaB. Nat Cell Biol, 2001; 3(4): 409-416
62. Afford SC, Adams DH. Following the TRAIL from hepatitis C virus and alcohol to fatty liver. Gut, 2005; 54(11): 1518-1520
63. Lan L, Gorke S, Rau SJ, Zeisel MB, Hildt E, Himmelsbach K, Carvajal-Yepes M, Huber R, Wakita T, Schmitt-Graeff A, Royer C, Blum HE, Fischer R, Baumert TF. Hepatitis C virus infection sensitizes human hepatocytes to TRAIL-induced apoptosis in a caspase 9-dependent manner. J Immunol, 2008; 181: 4926-4935
64. Finnberg N, Klein-Szanto AJ, El-Deiry WS. TRAIL-R deficiency in mice promotes susceptibility to chronic inflammation and tumorigenesis. J Clin Invest, 2008; 118(1): 111-123
65. Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellate cells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediatedapoptosis. Hepatology, 2003; 37(1): 87-95
66. Saxena NK, Titus MA, Ding X, Floyd J, Srinivasan S, Sitaraman SV, Anania FA. Leptin as a novel profibrogenic cytokine in hepatic stellate cells: mitogenesis and inhibition of apoptosis mediated by extracellular regulated kinase (Erk) and Akt phosphorylation. FASEB J, 2004; 18(13): 1612-1614
67. Imose M, Nagaki M, Kimura K, Takai S, Imao M, Naiki T, Osawa Y, Asano T, Hayashi H, Moriwaki H. Leflunomide protects from T-cell-mediated liver injury in mice through inhibition of nuclear factor kappaB. Hepatology, 2004; 40(5): 1160-1169
68. Vrenken TE, Buist-Homan M, Kalsbeek AJ, Faber KN, Moshage H. The active metabolite of leflunomide, A771726, protects rat hepatocytes against bile acid-induced apoptosis. J Hepatol, 2008; 49(5): 799-809
69. Leger DY, Liagre B, Beneytout JL. Low dose leflunomide activates PI3K/Akt signalling in erythroleukemia cells and reduces apoptosis induced by anticancer agents. Apoptosis, 2006; 11(10): 1747-1760
70. Migita K, Miyashita T, Maeda Y, Nakamura M, Yatsuhashi H, Ishibashi H, Eguchi K. An active metabolite of leflunomide, A771726, inhibits the production of serum amyloid A protein in human hepatocytes. Rheumatology, 2005; 44(4): 443-448
71. Manna SK, Aggarwal BB. Immunosuppressive leflunomide metabolite (A771726) blocks TNF-dependent nuclear factor-κB activation and gene expression. J Immunol, 1999; 162(4): 2095-2102
72. Manna SK, Mukhopadhyay A, Aggarwal BB. Leflunomide suppresses TNF-induced cellular responses: effects on NF-kappa B, activator protein-1, c-Jun N-terminal protein kinase, and apoptosis. J Immunol, 2000; 165(10): 5962-5969
73. Johnson GL, Lapadat R. Mitogen-activated protein kinase pathways mediated by ERK, JNK, and p38 protein kinases. Science, 2002; 298: 1911-1912
74. Schnabl B, Bradham CA, Bennett BL, Manning AM, Stefanovic B, Brenner DA. TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. Hepatology, 2001;34(5): 953-963
75. Yoshida K, Matsuzaki K, Mori S, Tahashi Y, Yamagata H, Furukawa F, Seki T, Nishizawa M, Fujisawa J, Okazaki K. Transforming growth factor-? and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury. Am J Pathol, 2005; 166: 1029-1039
76. Chen A, Davis BH. UV irradiation activates JNK and increases I(I) collagen gene expression in rat hepatic stellate cells. J Biol Chem, 1999; 274: 158-164
77. Burger D, Begué-Pastor N, Benavent S, Gruaz L, Kaufmann MT, Chicheportiche R, Dayer JM. The active metabolite of leflunomide, A771726, inhibits the production of prostaglandin E2, matrix metalloproteinase 1 and interleukin 6 in human fibroblast-like synoviocytes. Rheumatology, 2003; 42(1): 89-96
78. Alexander D, Friedrich B, Abruzzese T, Gondolph-Zink B, Wulker N, Aicher WK. The active form of leflunomide, HMR1726, facilitates TNF-alpha and IL-17 induced MMP-1 and MMP-3 expression. Cell Physiol Biochem, 2006; 17(1-2): 69-78
1. Friedman SL. Mechanisms of hepatic fibrogenesis. Gastroenterology, 2008; 134(6): 1655-1669
2. Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005; 115(2): 209-218
3. Boigk G,Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology, 1997; 26(3): 643-649
4. Vitamin E therapy in patients with NASH. Yoneda M, Hasegawa T, Nakamura K, Tamano M, Kono T, Terano A. Hepatology, 2004; 39(2): 568
5. Zou WL, Yang Z, Zang YJ, Li DJ, Liang ZP, Shen ZY. Inhibitory effects of prostaglandin E1 on activation of hepatic stellate cells in rabbits with schistosomiasis. Hepatobiliary Pancreat Dis Int, 2007; 6(2): 176-181
6. Hui AY, Dannenberg AJ, Sung JJ, Subbaramaiah K, Du B, Olinga P, Friedman SL. Prostaglandin E2 inhibits transforming growth factor beta 1-mediated induction of collagen alpha 1(I) in hepatic stellate cells. J Hepatol, 2004 ;41(2): 251-258
7. Safadi R, Friedman SL. Hepatic fibrosis-role of hepatic stellate cell activation. MedGenMed, 2002; 4(3): 27
8. Rockey DC, Maher JJ, Jarnagin WR, Gabbiani G, Friedman SL. Inhibition of rathepatic lipocyte activation in culture by interferon-gamma. Hepatology, 1992; 16(3): 776-784
9. Wei HS, Li DG, Lu HM, Zhan YT, Wang ZR, Huang X, Zhang J, Cheng JL, Xu QF. Effects of AT1 receptor antagonist, losartan, on rat hepatic fibrosis induced by CCl(4). World J Gastroenterol, 2000; 6(4): 540-545
10. Wei YH, Jun L, Qiang CJ. Effect of losartan, an angiotensin II antagonist, on hepatic fibrosis induced by CCl4 in rats. Dig Dis Sci, 2004; 49(10): 1589-1594
11. Ramalho LN, Ramalho FS, Zucoloto S, Castro-e-Silva Junior O, Correa FM, Elias Junior J, Magalhaes JF. Effect of losartan, an angiotensin II antagonist, on secondary biliary cirrhosis. Hepatogastroenterology, 2002; 49(48): 1499-1502
12. Jonsson JR, Clouston AD, Ando Y, Kelemen LI, Horn MJ, Adamson MD, Purdie DM, Powell EE. Angiotensin-converting enzyme inhibition attenuates the progression of rat hepatic fibrosis. Gastroenterology, 2001; 121(1): 148-155
13. Ueno T, Hashimoto O, Kimura R, Torimura T, Kawaguchi T, Nakamura T, Sakata R, Koga H, Sata M. Relation of type II transforming growth factor-beta receptor to hepatic fibrosis and hepatocellular carcinoma. Int J Oncol, 2001; 18(1): 49-55
14. Iwamoto H, Nakamuta M, Tada S, Sugimoto R, Enjoji M, Nawata H. Platelet-derived growth factor receptor tyrosine kinase inhibitor AG1295 attenuates rat hepatic stellate cell growth. J Lab Clin Med, 2000; 135(5): 406-412
15. Kweon YO, Paik YH, Schnabl B, Qian T, Lemasters JJ, Brenner DA. Gliotoxin-mediated apoptosis of activated human hepatic stellate cells. J Hepatol. 2003;39(1):38-46
16. Siegmund SV, Qian T, de Minicis S, Harvey-White J, Kunos G, Vinod KY, Hungund B, Schwabe RF. The endocannabinoid 2-arachidonoyl glycerol induces death of hepatic stellate cells via mitochondrial reactive oxygen species. FASEB J, 2007; 21(11): 2798 - 2806
17. Taimr P, Higuchi H, Kocova E, Rippe RA, Friedman S, Gores GJ. Activated stellatecells express the TRAIL receptor-2/death receptor-5 and undergo TRAIL-mediated apoptosis. Hepatology, 2003; 37(1): 87-95
18. Horani A, Muhanna N, Pappo O, Melhem A, Alvarez CE, Doron S, Wehbi W, Dimitrios K, Friedman SL, Safadi R. Beneficial effect of glatiramer acetate (Copaxone) on immune modulation of experimental hepatic fibrosis. Am J Physiol Gastrointest Liver Physiol, 2007; 292(2): G628-638
19. Guo YT, Leng XS, Li T, Peng JR, Song SH, Xiong LF, Qin ZZ. Effect of ligand of peroxisome proliferator-activated receptor gamma on the biological characters of hepatic stellate cells. World J Gastroenterol, 2005; 11(30): 4735-4739
20. Ikeda H, Nagashima K, Yanase M, Tomiya T, Arai M, Inoue Y, Tejima K, Nishikawa T, Omata M, Kimura S, Fujiwara K. Involvement of Rho/Rho kinase pathway in regulation of apoptosis in rat hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2003; 285(5): G880-886
21. Sakaida I, Matsumura Y, Kubota M, Kayano K, Takenaka K, Okita K. The prolyl 4-hydroxylase inhibitor HOE 077 prevents activation of Ito cells, reducing procollagen gene expression in rat liver fibrosis induced by choline-deficient L-amino acid-defined diet. Hepatology, 1996; 23(4): 755-763
22. Matsumura Y, Sakaida I, Uchida K, Kimura T, Ishihara T, Okita K. Prolyl 4-hydroxylase inhibitor (HOE 077) inhibits pig serum-induced rat liver fibrosis by preventing stellate cell activation. J Hepatol, 1997; 27(1): 185-192
23. Pines M , Nagler A. Halofuginone: a novel antifibrotic therapy. Gen Pharmacol, 1998; 30(4): 445-450