多靶点激酶抑制剂sorafenib对肝纤维化大鼠及肝星状细胞胶原代谢的影响及其机制的研究
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摘要
肝纤维化是机体对慢性肝损伤刺激的一种修复反应,以细胞外基质(extracellular matrix, ECM)过度增生与异常沉积为特征,尤其以I型胶原沉积为主。它是肝硬化的早期阶段,在一定情况下可被逆转。但若病因持续存在,肝纤维化逐渐加重,肝脏的正常结构遭到破坏,假小叶形成即发展为肝硬化。肝星状细胞(hepatic stellate cell, HSC)活化、增殖进而合成大量ECM是各种肝脏损伤引起肝纤维化的中心环节。活化的HSC不但是胶原产生的主要细胞,也是调节ECM降解的因子基质金属蛋白酶(matrix metalloproteinases, MMPs)和金属蛋白酶抑制因子(tissue inhibitors of metalloproteinases, TIMPs)的主要来源。HSC活化后表达血小板衍生生长因子受体(platelet-derived growth factor receptor, PDGFR)、血管内皮生长因子受体(vascular endothelial growth factor receptor, VEGFR)、成纤维细胞生长因子受体(fibroblast growth factor receptor, FGFR)、肝细胞生长因子受体(hepatocyte growth factor receptor, HGFR)、神经细胞生长因子受体(nerve growth factor receptor, NGFR)等多种酪氨酸激酶受体,这些受体与相应的细胞因子结合,启动细胞内的信号转导系统,如Ras/Raf/丝裂原活化的蛋白激酶(mitogen-activated protein kinase, MAPK)途径和磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase, PI3K)/Akt/核糖体40S小亚基S6蛋白激酶(70-kDa ribosomal S6 kinase,p70S6K)信号途径,进而从增殖、凋亡、血管形成以及胶原代谢等多方面调节HSC的生物学活性。
多激酶抑制剂是近年来新研发的一类多靶点药物,以PDGFR、VEGFR、干细胞因子受体(c-Kit)和Fms样酪氨酸激酶3(FLT3)等为靶点,抑制Ras/Raf/MAPK和PI-3K/Akt/p70s6k信号通路的激活而发挥抑制细胞增殖和血管生成的双重作用。多激酶抑制剂许多作用靶点在纤维化时是上调的,为分子靶向治疗提供了理论基础。Yoshiji H等发现伊马替尼显著抑制肝纤维化进展,降低肝羟脯氨酸含量,体外实验发现伊马替尼抑制PDGF-BB诱导的HSC增殖、迁移,下调I型胶原α2和α-SMA mRNA表达。Sunitinib是另一个多靶点激酶抑制剂,动物试验发现sunitinib能够减少肝内血管形成、炎症细胞的浸润及肝星状细胞的激活,降低肝脏胶原的表达和门脉压力。Sorafenib为活性更强的多靶点酪氨酸激酶抑制剂,Mejias M等发现sorafenib能够降低肝硬化大鼠的门脉压力,减轻肝组织的损伤、炎症,抑制肝内新生血管的形成。尽管sorafenib在降低门脉高压中的作用得到揭示,但sorafenib对肝纤维化的作用目前仍未明,对HSC的活化、胶原代谢的影响及其机制知之甚少。本实验从体内和体外两个方面研究sorafenib对肝纤维化大鼠和HSC胶原代谢的影响,并初步探讨其对胶原代谢的影响机制。实验内容主要包括以下4部分。
第一部分:Sorafenib对实验性大鼠肝纤维化的影响
目的:研究sorafenib对肝纤维化大鼠肝组织病理、羟脯氨酸含量以及I型胶原表达的影响。
方法:运用胆总管结扎(bile duct ligation, BDL)和二甲基亚硝胺(dimethylnitrosamine, DMN)腹腔注射的方法建立两种不同的肝纤维化动物模型。在造模的第三周和第四周给予sorafenib灌胃治疗,每天一次,共2周。肝组织切片经HE和Masson三色染色检测病理变化,比色法测定肝组织羟脯氨酸含量,Real time Q-PCR和Western blot方法检测I型胶原的表达。
结果:①HE和Masson三色染色结果证明采用胆总管结扎和二甲基亚硝胺腹腔注射两种不同的方法成功建立了肝纤维化动物模型。②Sorafenib治疗后,BDL和DMN大鼠肝内纤维组织增生减轻,炎性细胞浸润减少。肝纤维化计分在BDL大鼠于20mg/kg和40mg/kg治疗组分别为11.92±1.35和8.64±2.13,与溶剂对照组(16.97±2.82)相比显著减低,P<0.01;而在DMN大鼠肝纤维化计分在1mg/kg和5mg/kg治疗组分别为12.41±3.34和9.65±3.02,明显低于溶剂对照组(17.17±3.65),P<0.01。③Sorafenib降低了肝组织中羟脯氨酸含量,BDL大鼠在20 mg/kg和40 mg/kg治疗组肝组织羟脯氨酸含量分别为(247.00±16.05)μg/g和(200.00±50.81)μg/g,低于溶剂对照组(442.50±139.14)μg/g,P<0.05;DMN大鼠在1 mg/kg和5 mg/kg治疗组肝组织羟脯氨酸含量分别为(340.00±19.87)μg/g和(214.40±35.71)μg/g ,在5 mg/kg治疗量组降低明显,P<0.05。④Sorafenib抑制BDL大鼠肝组织Ι型胶原蛋白及mRNA表达,应用Western blot方法发现sorafenib在20 mg/kg和40 mg/kg治疗后,Ι型胶原蛋白表达分别为0.54±0.02和0.51±0.01,表达下调了14.86%和17.89%,P<0.01;Real-time PCR方法发现Ι型胶原α1 mRNA相对表达量分别为1.77±0.33和1.56±0.18,基因表达下调46.69%和53.01%,P<0.01。⑤Sorafenib剂量依赖性地抑制DMN大鼠肝组织Ι型胶原蛋白及mRNA表达,sorafenib在1 mg/kg和5 mg/kg治疗后,Ι型胶原蛋白相对表达量下调了14.92%和32.18%,P<0.05;Real-time PCR方法发现Ι型胶原α1 mRNA相对表达量分别为1.29±0.92和1.14±0.15,mRNA表达下调43.17%和49.78%,P<0.01。而且40 mg/kg治疗组比20 mg/kg治疗组对Ι型胶原抑制效果明显。
结论:胆总管结扎和腹腔注射二甲基亚硝胺诱导肝纤维化形成过程中,肝组织羟脯氨酸含量逐渐增高,I型胶原蛋白和mRNA表达提高;Sorafenib治疗后,肝内炎症和纤维组织增生减轻,纤维化计分和羟脯氨酸含量降低,I型胶原表达下调,肝纤维化程度减轻。表明sorafenib具有抗纤维化作用,提示可能是一种新型抗纤维化药物。
第二部分:Sorafenib对实验性肝纤维化大鼠α-SMA、MMP-13和TIMP-1表达的影响
目的:研究sorafenib对肝纤维化大鼠α-SMA、MMP-13和TIMP-1表达的影响。
方法:运用胆总管结扎和二甲基亚硝胺腹腔注射的方法建立两种不同肝纤维化动物模型。采用不同剂量sorafenib (BDL: 20mg/kg和40 mg/kg; DMN: 1 mg/kg和5 mg/kg)治疗2周。免疫组织化学检测α-SMA、MMP-13和TIMP-1在肝组织的表达情况,Real time Q-PCR和Western blot方法检测MMP-13和TIMP-1的表达。
结果:①随着肝纤维化的进展,α-SMA阳性着色细胞逐渐增多,阳性着色主要分布于汇管区、纤维间隔、肝窦周围及门静脉周围的间质细胞。Sorafenib明显抑制BDL和DMN大鼠肝组织α-SMA表达,sorafenib 20 mg/kg和40 mg/kg组BDL大鼠α-SMA阳性染色平均光密度分别为30.72±1.87%和18.05±4.02%,低于溶剂对照组38.19±2.10%,P<0.01;而DMN大鼠在sorafenib 1 mg/kg和5 mg/kg治疗2周后,α-SMA表达较溶剂对照组明显减弱(23.06±2.20%, 20.63±2.50% vs 32.32±4.40%),P<0.01。②肝纤维化过程中,TIMP-1表达增强,而MMP-13在肝纤维化早期表达增强,晚期减弱,存在MMP-13和TIMP-1比例失衡。③Sorafenib抑制肝纤维化大鼠TIMP-1蛋白及mRNA的表达,在BDL大鼠,sorafenib在20 mg/kg和40 mg/kg治疗组TIMP-1蛋白表达分别下调了31.69%和47.87%,mRNA表达下调44.63%和43.65%;而在DMN大鼠,sorafenib在1 mg/kg和5 mg/kg治疗量时TIMP-1蛋白表达分别下调44.14%和55.18%,mRNA表达分别下调72.03%和67.56%。④Sorafenib上调MMP-13表达。在BDL大鼠,sorafenib在20 mg/kg和40 mg/kg时MMP-13蛋白表达分别上调164.35%和175.52%,mRNA表达上调78.50%和56.07%;而在DMN大鼠,sorafenib在1 mg/kg和5 mg/kg治疗量时MMP-13蛋白表达分别上调119.74%和152.7%,mRNA表达分别上调33.87%和57.26%。MMP-13表达主要见于纤维间隔内及其周围以及汇管区的部分间质细胞胞浆中,其中部分肝细胞有强阳性表达。⑤Sorafenib上调MMP-13/TIMP-1比值。随着肝纤维化进展,MMP-13/TIMP-1比值逐渐下降,模型4wk组MMP-13/TIMP-1比值最低,在BDL大鼠为0.32±0.06,在DMN大鼠为0.51±0.07。给予sorafenib治疗后,无论是BDL大鼠还是DMN大鼠,MMP-13/TIMP-1比值提高,逆转了MMP-13/TIMP-1失衡。
结论:sorafenib治疗可以降低实验性肝纤维化大鼠α-SMA表达,减少HSC激活;另一方面,Sorafenib可抑制TIMP-1表达,上调MMP-13表达,纠正MMP-13和TIMP-1比例失衡,促进基质降解,从而发挥抗纤维化作用。
第三部分:Sorafenib对肝星状细胞胶原代谢的影响
目的:研究sorafenib对PDGF刺激的HSC胶原合成、MMP-13(或MMP-1)和TIMP-1表达的影响,探讨sorafenib调节胶原代谢的机制。
方法:原代HSC、大鼠和人肝星状细胞株HSC-T6和LX-2用于实验,应用体外HSC培养技术,采用3H-脯氨酸(3H-pro)掺入法测定HSC胶原合成。Western blot和Real-time PCR检测I型胶原、MMP-13和TIMP-1蛋白和mRNA水平。
结果:①PDGF-BB刺激可使大鼠原代肝星状细胞、HSC-T6和LX-2Ⅰ型胶原α1 mRNA表达分别上调5.45倍、2.49倍和2.20倍,使HSC-T6和LX-2Ⅰ型胶原蛋白表达分别上调1.26倍和1.10倍。2.5μM、5.0μM和10.0μM sorafenib使原代HSCⅠ型胶原mRNA下调54.90%、64.88%和64.51%;使HSC-T6 mRNA下调62.64%、67.94%和82.52%,蛋白下调21.28%、28.72%和38.30%;使LX-2 mRNA下调58.66%、67.06%和81.64%,蛋白下调17.40%、18.84%和18.84%,低于PDGF组,P<0.05。免疫细胞化学也发现sorafenib能够抑制Ⅰ型胶原蛋白的表达。②无论是否有PDGF的刺激作用,在2.5~10.0μM范围内,sorafenib浓度越高,对HSC 3H-Pro掺入的抑制作用越强,P<0.01。10.0μM sorafenib在12 h、24 h和48 h 3H-Pro掺入抑制率在HSC-T6分别为14.51%、36.67%和63.44%,在LX-2 3H-Pro掺入抑制率分别为22.69%、37.52%和71.74%。Sorafenib抑制胶原合成具有时间和剂量依赖关系。③PDGF-BB刺激可使HSC-T6和LX-2细胞TIMP-1蛋白分别升高1.29倍和2.31倍,mRNA表达上调2.57倍和2.11倍,P<0.01;Sorafenib干预24 h后可使HSC-T6和LX-2细胞TIMP-1蛋白下降,对HSC-T6在10.0μM时抑制作用最明显,而对LX-2细胞在5.0μM作用最明显。Sorafenib以剂量依赖的方式下调了HSC-T6和LX-2细胞和原代HSC TIMP-1mRNA表达。④Sorafenib仅在5.0μM时使LX-2 MMP-1蛋白表达上调约167.39%,在2.5μM和5.0μM时使原代肝星状细胞MMP-13 mRNA表达上调,而对HSC-T6 MMP-13表达基本无明显影响。
结论:sorafenib以剂量依赖方式对HSC胶原合成产生抑制作用,通过抑制HSC胶原合成,减少胶原的产生;另一方面通过从蛋白和mRNA水平抑制TIMP-1表达,减少对MMPs阻止,促进已合成的ECM降解以减少胶原的沉积。
第四部分:Sorafenib对肝纤维化大鼠及肝星状细胞胶原代谢影响的信号转导机制
目的:研究sorafenib对HSC及肝纤维化大鼠信号分子Akt及ERK表达的影响,进一步探讨sorafenib调控胶原代谢的信号转导机制。
方法:实验包括大鼠和人肝星状细胞株HSC-T6和LX-2以及BDL和DMN两种肝纤维化动物模型。应用体外细胞培养技术,采用不同浓度的sorafenib和PDGF-BB 20ng/mlg干预HSC,同时与ERK特异性阻滞剂PD98059和PI3K特异性抑制剂LY294002作对比,应用Western blot方法检测HSC和肝组织ERK、p-ERK、Akt和p-Akt的表达。采用免疫组织化学方法测定p-ERK和p-Akt在肝纤维化大鼠肝组织的表达。
结果:①Sorafenib对HSC-T6和LX-2细胞总ERK和总Akt蛋白的表达无影响:总ERK和总Akt蛋白在HSC-T6各实验组表达无差异,P值分别为0.16和0.24;在LX-2各实验组差异亦无显著性,P值分别为0.26和0.17。②Sorafenib抑制HSC-T6的信号分子ERK和Akt的磷酸化:PDGF-BB刺激可使HSC p-ERK和p-Akt表达分别提高1.27倍(P=0.071)和1.48倍(P<0.001)。sorafenib 2.5μM、5.0μM和10.0μM干预PDGF-BB激活的HSC,p-ERK蛋白表达分别下调24.24%、34.85%和50%,P<0.01;p-Akt蛋白表达分别下调25.15%、40%和50.29%,明显低于PDGF组,P<0.01。在2.5～10.0μM浓度范围内,sorafenib剂量依赖性抑制HSC-T6 p-ERK和p-Akt蛋白表达, P<0.01。③Sorafenib抑制LX-2细胞p-ERK和p-Akt蛋白的表达:PDGF-BB刺激使LX-2细胞p-ERK和p-Akt提高1.61倍和1.23倍,与对照组比较差异有显著性(P<0.01)。2.5μM、5.0μM和10.0μM的sorafenib干预PDGF-BB刺激的LX-2,p-ERK表达分别下调60.38%、69.81%和73.58%;p-Akt表达分别下调32.12%、36.50%和45.26%,低于PDGF组,P<0.05。④Sorafenib对信号分子发挥了与特异性阻断剂相似的抑制作用:不论是否有PDGF-BB刺激,ERK特异性阻断剂PD98059和Akt特异性阻断剂LY294002均分别显著抑制了HSC ERK和Akt的磷酸化,而sorafenib则同时对ERK和Akt的磷酸化具有抑制作用。sorafenib对HSC-T6细胞p-ERK和p-Akt的抑制率与PD98059和LY294002的抑制作用无差异,P值分别为0.24和0.11。在LX2细胞中与HSC-T6细胞中的结果一样。⑤Sorafenib抑制实验性肝纤维化大鼠ERK和Akt的磷酸化:Western blot显示,随着肝纤维化进展,大鼠p-ERK和p-Akt的表达逐渐增高,sorafenib治疗2周后,p-ERK和p-Akt的表达受到抑制,在20mg/kg和40mg/kg治疗组,BDL大鼠p-ERK蛋白表达分别下调39.82%和46.02%,P<0.01;p-Akt表达下调38.75%和40%,P<0.05。免疫组化显示在正常大鼠,p-ERK和p-Akt呈弱阳性表达。随着肝纤维化进展,BDL大鼠肝组织p-ERK和p-Akt表达增多,主要分布于汇管区、Disse间隙、纤维间隔的间质细胞,部分肝细胞亦呈强阳性表达。Sorafenib治疗2周后p-ERK和p-Akt表达明显减低。在DMN大鼠得到同样的结果。
结论:Sorafenib抑制了HSC和纤维化大鼠肝组织ERK和Akt的磷酸化,阻断了ERK/MAPK和PI3K/Akt信号通路;BDL和DMN大鼠肝纤维化形成过程中,p-ERK和p-Akt表达增加;Sorafenib减轻肝纤维化可能与抑制肝纤维化大鼠p-ERK和p-Akt表达有关。
Liver fibrosis, a wound-healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM), of which type I collagen predominates. It is in the early stage of cirrhosis and may be reversed under some circumstances. If the liver injury is persistent, the excess deposition of ECM disrupts the normal architecture of the liver resulting in the formation of nodules, and fibrosis will develop to liver cirrhosis. It is well established that the activated hepatic stellate cells (HSCs) play a key role in the excess synthesis and deposition of ECM following a fibrotic stimulus. HSCs are not only the major source of ECM, but also the major cells releasing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which responsible for regulating matrix degradation. The activated HSC express tyrosine kinase receptors, including platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and nerve growth factor receptor (NGFR). With these receptors binding to specific ligands, some intracellular signaling pathways were trigged. It has been shown that the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K) play important roles in proliferation, apoptosis, angiopoiesis and collagen metabolism in HSCs stimulated by different growth factors.
Multikinase inhibitor is a kind of novel multi-targeted agents studied in recent years. It has been shown to inhibit tumor cell growth and angiogenesis by blocking the receptor tyrosine kinases, such as PDGFR, VEGFR, FGFR, and NGFR. This is attributed to block signaling pathways including Ras/Raf/mitogen-activated protein kinase (MAPK) and PI3K/Akt/70-kDa ribosomal S6 kinase (p70S6K) and provide theoretical basis for antifibrotic therapy. Yoshiji H et al found imatinib, a multikinase inhibitor, markedly attenuated liver fibrosis, and decreased hepatic hydroxyproline and serum fibrosis markers. Imatinib markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner in vitro. Sunitinib, another multikinase inhibitor, resulted in significant decreases in hepatic vascular density, inflammatory infiltrate, alpha-SMA abundance, collagen expression and portal pressure. Sorafenib, a novel oral multikinase inhibitor, is more effective in inhibiting ERK1/2 and PI3K signaling pathways. Mejias M et al found sorafenib treatment resulted in a reduction in portal pressure, as well as a remarkable improvement in liver damage and intra- hepatic fibrosis, inflammation, and angiogenesis. Although sorafenib exerts in reducing portal pressure, the role of sorafenib in the reversal of fibrosis, the activation of HSCs, collagen synthesis and degradation are unclear. The effects of sorafenib on collagen metabolism in vivo and in vitro are investigated in this study, and the mechanism is also explored. The experiments are composed of four parts as below.
Part 1: The therapic effect of sorafenib on intrahepatic fibrosis of experimental rats.
Objective : To explore the therapic effect of sorafenib on liver pathological changes, hydroxyproline content and expression of type I collagen in experimental fibrotic rats.
Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson’s trichrome method. Hepatic hydroxyproline was measured using a hydroxyproline detection kit. Type I collagen protein expression in the livers were determined by Western blot, while the expression of collagenα1(I) mRNA in the livers was assessed by Real-time PCR analysis .
Results:①Hepatic fibrosis was successfully induced by BDL and by intraperitoneal injections of dimethylnitrosamine (DMN) by hematoxylin and eosin staining and Masson’s trichrome method.②Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation in BDL and DMN rats. In BDL rats after sorafenib treatment at dose of 20 mg/kg and 40 mg/kg, hepatic fibrosis score (11.92±1.35 and 8.64±2.13, respective- ly) were both lower than that in vehicle-treated group(16.97±2.82), P<0.01; In DMN rats after sorafenib treatment at dose of 1 mg/kg and 5 mg/kg, hepatic fibrosis score(12.41±3.34 and 9.65±3.02, respectively) were both lower than that in vehicle-treated group(17.17±3.65), P<0.01.③Treatment with sorafenib reduced hydroxyproline content in BDL and DMN rats. In BDL rats, hydroxyproline content (247.00±16.05μg/g and 200.00±50.81μg/g, respect- tively) at dose of 20 mg/kg and 40 mg/kg were both lower than that in vehicle- treated group(442.50±139.14μg/g), P<0.01. In DMN rats, hydroxyproline content (214.40±35.71μg/g ) at doses of 5 mg/kg was lower than that in vehicle-treated group(355.80±33.05μg/g), P<0.05.④Sorafenib down- regulated the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in BDL rats. The expression of type I collagen protein (0.54±0.02 and 0.51±0.01, respectively) at doses of 20 mg/kg and 40 mg/kg by Western blot were both lower than that in vehicle-treated group, reduced by 14.86% and 17.89% , respectively, P<0.05. The expression of collagenα1 (I) mRNA (1.77±0.33 and 1.56±0.18, respectively ) at doses of 20 mg/kg and 40mg/kg by real-time PCR were both lower than that in vehicle-treated group(3.32±0.67), reduced by 46.69% and 53.01% , respectively, P<0.01.⑤Sorafenib inhibited the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in DMN rats. The expression of type I collagen protein at doses of 1 mg/kg and 5 mg/kg reduced by 14.92% and 32.18%, respectively, P<0.05. The expression of collagenα1 (I) mRNA reduced by 43.17% and 49.78%, respectively, P<0.01. Furthermore,sorafenib 5 mg/kg was more significantly than that at dose of 1 mg/kg in inhibiting type collagen I protein and mRNA.
Conclusions: During the fibrogenesis, hydroxyproline content, type I collagen and collagenα1 (I) mRNA are upregulated in BDL and DMN rats. Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation, reduces hepatic fibrosis score and hydroxyproline content, as well as downregulats the expression of type I collagen and collagenα1 (I) mRNA. All data suggest that sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.
Part 2: The effect of sorafenib on the expression ofα-SMA,MMP-13 and TIMP-1 in experimental fibrotic rats.
Objective:To explore the effect of sorafenib on the expression ofα-SMA, MMP-13 and TIMP-1 in experimental fibrotic rats. Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks (BDL rats: 20, 40 mg/kg; DMN rats: 1, 5 mg/kg). The expression ofα-SMA were evaluated by immunohistochemistry. MMP-13 and TIMP-1 protein expression in the livers were determined by Western blot and immunohistochemistry. While the expression of MMP-13 and TIMP-1 mRNA in the livers was assessed by Real-time PCR analysis.
Results:①During fibrogenesis, interstitial cells strongly positive forα-SMA appeared along the portal area, fibrous septum, the hepatic sinusoids and the periportal area. Sorafenib reduced the number of the cells positive forα-SMA. The mean density forα-SMA were 30.72±1.87% and 18.05±4.02% in BDL rats after administration of sorafenib (20 mg/kg and 40 mg/kg), respectively, and lower than that in vehicle-treated rats (38.19±2.10%), P<0.01. In DMN rats, after sorafenib (1 mg/kg and 5 mg/kg) treatment, the mean density forα-SMA were significantly lower than that in vehicle-treated rats(23.06±2.20%, 20.63±2.50% vs 32.32±4.40%), P<0.01.②During the progress of liver fibrosis, the expression of TIMP-1 was up- regulated in BDL and DMN rats. The expression of MMP-13 was up-regulated at the early stage, wheras downregulated at end-stage of liver fibrosis. The balance between MMP-13 and TIMP-1 was disturbed.③Sorafenib inhibited the expression of TIMP-1 protein and mRNA. Sorafenib down-regulated TIMP-1 protein by 31.69% and 47.87% and mRNA by 44.63% and 43.65% in BDL rats at the dose of 20 mg/kg and 40 mg/kg, respectively. Sorafenib (1 mg/kg and 5 mg/kg) decreased the expression of TIMP-1 protein by 44.14% and 55.18% and mRNA by 72.03% and 67.56% in DMN rats, respectively.④Sorafenib upregulated the expression of MMP-13 protein and mRNA in experimental fibrotic rats. Sorafenib (20 mg/kgand 40 mg/kg) up-regulated MMP-13 protein by 164.35% and 175.52% and mRNA by 78.50% and 56.07% in BDL rats, respectively. Sorafenib increased the expression of MMP-13 protein by 119.74% and 152.7% and mRNA by 33.87% and 57.26% in DMN rats at the dose of 1 mg/kg and 5 mg/kg, respectively. Hepatocyte and interstitial cells strongly positive for MMP-13 appeared along the portal area and fibrous septum.⑤Sorafenib increased the ratio of MMP-13 to TIMP-1 and improved the disbalance between MMP-13 and TIMP-1.With the progress of liver fibrosis, the ratio of MMP-13 to TIMP-1 decreased and was the lowest in model 4wk, the value was 0.32±0.06 in BDL rats and 0.51±0.07 in DMN rats, respectively. The ratio of MMP-13 to TIMP-1 was increased after treatment with sorafenib in BDL and DMN rats and improved the disbalance between MMP-13 and TIMP-1.
Conclusions: Sorafenib has potent to reduceα-SMA expression and inhibit HSC activation. For another, sorafenib down-regulates TIMP-1 protein and mRNA and up-regulates MMP-13 protein and mRNA. It improves the disbalance between MMP-13 and TIMP-1. As a result, sorafenib promotes degradation of ECM and plays a key role in the reversal of fibrosis.
Part 3: Effects of sorafenib on the Collagen Metabolism of Hepatic Stellate Cells
Objective:To investigate the effects of sorafenib on collagen synthesis and MMP-13 (or MMP-1), TIMP-1 expressions in PDGF-stimulated HSC,so as to study the mechanism of regulating collagen metabolism of HSC with sorafenib.
Methods: Three types of cell lines, freshly isolated rat HSC, HSC-T6 and LX-2 cell lines, were used in this study. Collagen synthesis was determined by 3H-pro incorporation. Expressions of type I collagen, MMP-13 and TIMP-1 were evaluated by western blot and quantitative real-time RT-PCR. Immuocytochemistry was used to detect type I collagen andα-SMA.
Results:①Stimulation with PDGF-BB induced increase of collagen Iα1 mRNA by 5.45-, 2.49- and 2.20-fold in primary HSC , HSC-T6 and LX-2. The expression of type I collagen protein was increased by 1.26- and 1.10- fold in HSC-T6 and LX-2. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated collagen Iα1 mRNA by 54.90%, 64.88% and 64.51% in primary HSC, by 62.64%, 67.94% and 82.52% in HSC-T6 and by 58.66%, 67.06% and 81.64% in LX-2, respectively. Furthermore, sorafenib decreased type I collagen protein by 21.28%, 28.72% and 38.30% in HSC-T6 and by 17.40%, 18.84% and 18.84% in LX-2, respectively (P<0.05).The same results were obtained by immuocytochemistry in HSC-T6 and LX-2 cells.②In the range of sorafenib (2.5～10.0μM), 3H-pro incorporation could be inhibited in dose- dependent manner with or without stimulation with PDGF-BB, P<0.01. The inhibition rates with sorafenib(10.0μM) were 14.51%, 36.67% , 63.44% (HSC-T6)and 22.69%, 37.52% and 71.74% (LX-2) at 12h,24h and 48h, respectively. So sorafenib could also dramatically inhibit the collagen synthesis of HSC in dose- and time-dependent manner.③Stimulation with PDGF-BB induced increase of TIMP-1 protein by 1.29-, 2.31-fold and mRNA by 2.57-, 2.11-fold in HSC-T6 and LX-2, respectively(P<0.01). Increased TIMP-1 expression was inhibited after treatment with sorafenib for 24 h, with a significant reduction at 10.0μM in T6 cells and at 5.0μM in LX-2 cells. Sorafenib dose-dependently downregulated the expression of TIMP-1 mRNA in the three HSC.④Sorafenib (5.0μM) induced the increase of MMP-1 protein by 167.39% in LX-2. The expression of MMP-13 mRNA were upregulated at the dose of sorafenib 2.5μM and 5.0μM in primary HSC. Sorafenib had no effect on the expression of MMP-13 in HSC-T6.
Conclusions: Sorafenib inhibited collagen synthesis in primary HSCs, HSC-T6 and LX-2 cells dose-dependently. As a result, the production of ECM was decreased. For another, the expressions of TIMP-1 protein and mRNA were downregulated by treatment with sorafenib. The dissolution of ECM was increased because of abolishing the inhibition of activity of MMPs. These data demonstrate that sorafenib is a potential therapeutic agent in the treatment of liver fibrosis.
Part 4: Mechanism of sorafenib on the collagen metabolism in experimental fibrotic rats and in hepatic stellate cells
Objective:To investigate the effects of sorafenib on Akt , phosphorylated Akt,ERK and phosphorylated ERK in PDGF-stimulated HSC and in experimental fibrotic rats and further to explore the mechanism of signal transduction in regulating collagen synthesis.
Methods:Two types of HSC cell lines (HSC-T6 and LX-2) and two different fibrotic models were used in this study. Fibrosis was induced by bile duct-ligation (BDL) or intraperitoneal injections of dimethylnitrosamine. HSC were preincubated with or without sorafenib and then stimulated with PDGF-BB. The ERK inhibitor PD98059 and Akt inhibitor LY294002 were used to treat HSC. The expression of ERK, phosphorylated ERK, Akt and phosphorylated Akt in HSC and rat livers were evaluated by western blot. The phosphorylated ERK and phosphorylated Akt in liver were detected by immunohistochemistry.
Results:①Sorafenib had no effect on total ERK and total Akt in HSC-T6 and LX-2 cells. The difference among groups was no significant in HSC-T6 and LX-2 cells. P value was 0.16, 0.24, 0.26and 0.17, respectively.②Sorafenib inhibited the activation of ERK and Akt in HSC-T6 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.27-fold (P=0.071) and 1.48-fold (P<0.001) in HSC-T6 cells. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated- ERK by 24.24%, 34.85% , 50% and inhibited phosphorylated Akt by 25.15%、40% and 50.29%,respectively. The expression were lower than that in PGDF- stimulated group, P<0.01. Furthermore, sorafenib inhibited the activation of ERK and Akt in dose-dependent manner from 2.5μM to 10.0μM.③Sorafenib decreased the expression of phosphorylated ERK and phosphorylated Akt in LX-2 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.61- and 1.23-fold in LX-2, the difference was significant in statistic (P<0.01). Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated ERK by 60.38%, 69.81%, 73.58% and inhibited the activation of Akt by 32.12%、36.50% and 45.26%,respectively. The expression were less than that in PGDF-stimulated group, P<0.05.④Sorafenib played similar roles in inhibiting the activation of signal transduction molecule in HSC-T6 cells, compared with special inhibitor. Both phosphorylated ERK and phosphorylated Akt were significantly blocked by sorafenib. The inhibition of phosphorylated ERK was similar after treatment with sorafenib and PD98059, and so was the phospho-Akt inhibition by sorafenib and LY294002. P value was 0.24 and 0.11, respectively. The same results were obtained in LX-2 cells.⑤Sorafenib decreased the expression of phospho-ERK and Akt in in experimental fibrotic rats. During fibrogenesis, the expression of phospho-ERK and phospho-Akt gradually increased and decreased after administration of sorafenib (20 mg/kg and 40 mg/kg) for two weeks. The expression of phospho-ERK was downregulated by 39.82% and 46.02%, respectively, P<0.01;and the expression of phospho-Akt was decreased by 38.75% and 40%, respectively, P<0.05 ; As the evidence showed by immunochemistry, the phosphorylation of ERK and Akt were very low in normal rats. With the development of hepatic fibrosis, the positive staining cells of phospho-ERK and Akt increased obviously, and mainly resided in the interstitial cells in portal ducts, fiber septa and perisinusoidal space. Part hepatocytes were accompanied. The expressions of phospho-ERK and phospho-Akt were both decreased by administration of sorafenib. The same results were obtained in DMN rats.
Conclusions: Sorafenib blocks ERK/MAPK and PI3K/Akt ERK signal transduction pathways by inhibiting the phosphorylations of ERK and Akt in HSCs. Sorafenib ameliorates fibrosis by downregulating the expressions of phosphorylated ERK and phosphorylated Akt in liver fibrosis.
多激酶抑制剂是近年来新研发的一类多靶点药物,以PDGFR、VEGFR、干细胞因子受体(c-Kit)和Fms样酪氨酸激酶3(FLT3)等为靶点,抑制Ras/Raf/MAPK和PI-3K/Akt/p70s6k信号通路的激活而发挥抑制细胞增殖和血管生成的双重作用。多激酶抑制剂许多作用靶点在纤维化时是上调的,为分子靶向治疗提供了理论基础。Yoshiji H等发现伊马替尼显著抑制肝纤维化进展,降低肝羟脯氨酸含量,体外实验发现伊马替尼抑制PDGF-BB诱导的HSC增殖、迁移,下调I型胶原α2和α-SMA mRNA表达。Sunitinib是另一个多靶点激酶抑制剂,动物试验发现sunitinib能够减少肝内血管形成、炎症细胞的浸润及肝星状细胞的激活,降低肝脏胶原的表达和门脉压力。Sorafenib为活性更强的多靶点酪氨酸激酶抑制剂,Mejias M等发现sorafenib能够降低肝硬化大鼠的门脉压力,减轻肝组织的损伤、炎症,抑制肝内新生血管的形成。尽管sorafenib在降低门脉高压中的作用得到揭示,但sorafenib对肝纤维化的作用目前仍未明,对HSC的活化、胶原代谢的影响及其机制知之甚少。本实验从体内和体外两个方面研究sorafenib对肝纤维化大鼠和HSC胶原代谢的影响,并初步探讨其对胶原代谢的影响机制。实验内容主要包括以下4部分。
第一部分:Sorafenib对实验性大鼠肝纤维化的影响
目的:研究sorafenib对肝纤维化大鼠肝组织病理、羟脯氨酸含量以及I型胶原表达的影响。
方法:运用胆总管结扎(bile duct ligation, BDL)和二甲基亚硝胺(dimethylnitrosamine, DMN)腹腔注射的方法建立两种不同的肝纤维化动物模型。在造模的第三周和第四周给予sorafenib灌胃治疗,每天一次,共2周。肝组织切片经HE和Masson三色染色检测病理变化,比色法测定肝组织羟脯氨酸含量,Real time Q-PCR和Western blot方法检测I型胶原的表达。
结果:①HE和Masson三色染色结果证明采用胆总管结扎和二甲基亚硝胺腹腔注射两种不同的方法成功建立了肝纤维化动物模型。②Sorafenib治疗后,BDL和DMN大鼠肝内纤维组织增生减轻,炎性细胞浸润减少。肝纤维化计分在BDL大鼠于20mg/kg和40mg/kg治疗组分别为11.92±1.35和8.64±2.13,与溶剂对照组(16.97±2.82)相比显著减低,P<0.01;而在DMN大鼠肝纤维化计分在1mg/kg和5mg/kg治疗组分别为12.41±3.34和9.65±3.02,明显低于溶剂对照组(17.17±3.65),P<0.01。③Sorafenib降低了肝组织中羟脯氨酸含量,BDL大鼠在20 mg/kg和40 mg/kg治疗组肝组织羟脯氨酸含量分别为(247.00±16.05)μg/g和(200.00±50.81)μg/g,低于溶剂对照组(442.50±139.14)μg/g,P<0.05;DMN大鼠在1 mg/kg和5 mg/kg治疗组肝组织羟脯氨酸含量分别为(340.00±19.87)μg/g和(214.40±35.71)μg/g ,在5 mg/kg治疗量组降低明显,P<0.05。④Sorafenib抑制BDL大鼠肝组织Ι型胶原蛋白及mRNA表达,应用Western blot方法发现sorafenib在20 mg/kg和40 mg/kg治疗后,Ι型胶原蛋白表达分别为0.54±0.02和0.51±0.01,表达下调了14.86%和17.89%,P<0.01;Real-time PCR方法发现Ι型胶原α1 mRNA相对表达量分别为1.77±0.33和1.56±0.18,基因表达下调46.69%和53.01%,P<0.01。⑤Sorafenib剂量依赖性地抑制DMN大鼠肝组织Ι型胶原蛋白及mRNA表达,sorafenib在1 mg/kg和5 mg/kg治疗后,Ι型胶原蛋白相对表达量下调了14.92%和32.18%,P<0.05;Real-time PCR方法发现Ι型胶原α1 mRNA相对表达量分别为1.29±0.92和1.14±0.15,mRNA表达下调43.17%和49.78%,P<0.01。而且40 mg/kg治疗组比20 mg/kg治疗组对Ι型胶原抑制效果明显。
结论:胆总管结扎和腹腔注射二甲基亚硝胺诱导肝纤维化形成过程中,肝组织羟脯氨酸含量逐渐增高,I型胶原蛋白和mRNA表达提高;Sorafenib治疗后,肝内炎症和纤维组织增生减轻,纤维化计分和羟脯氨酸含量降低,I型胶原表达下调,肝纤维化程度减轻。表明sorafenib具有抗纤维化作用,提示可能是一种新型抗纤维化药物。
第二部分:Sorafenib对实验性肝纤维化大鼠α-SMA、MMP-13和TIMP-1表达的影响
目的:研究sorafenib对肝纤维化大鼠α-SMA、MMP-13和TIMP-1表达的影响。
方法:运用胆总管结扎和二甲基亚硝胺腹腔注射的方法建立两种不同肝纤维化动物模型。采用不同剂量sorafenib (BDL: 20mg/kg和40 mg/kg; DMN: 1 mg/kg和5 mg/kg)治疗2周。免疫组织化学检测α-SMA、MMP-13和TIMP-1在肝组织的表达情况,Real time Q-PCR和Western blot方法检测MMP-13和TIMP-1的表达。
结果:①随着肝纤维化的进展,α-SMA阳性着色细胞逐渐增多,阳性着色主要分布于汇管区、纤维间隔、肝窦周围及门静脉周围的间质细胞。Sorafenib明显抑制BDL和DMN大鼠肝组织α-SMA表达,sorafenib 20 mg/kg和40 mg/kg组BDL大鼠α-SMA阳性染色平均光密度分别为30.72±1.87%和18.05±4.02%,低于溶剂对照组38.19±2.10%,P<0.01;而DMN大鼠在sorafenib 1 mg/kg和5 mg/kg治疗2周后,α-SMA表达较溶剂对照组明显减弱(23.06±2.20%, 20.63±2.50% vs 32.32±4.40%),P<0.01。②肝纤维化过程中,TIMP-1表达增强,而MMP-13在肝纤维化早期表达增强,晚期减弱,存在MMP-13和TIMP-1比例失衡。③Sorafenib抑制肝纤维化大鼠TIMP-1蛋白及mRNA的表达,在BDL大鼠,sorafenib在20 mg/kg和40 mg/kg治疗组TIMP-1蛋白表达分别下调了31.69%和47.87%,mRNA表达下调44.63%和43.65%;而在DMN大鼠,sorafenib在1 mg/kg和5 mg/kg治疗量时TIMP-1蛋白表达分别下调44.14%和55.18%,mRNA表达分别下调72.03%和67.56%。④Sorafenib上调MMP-13表达。在BDL大鼠,sorafenib在20 mg/kg和40 mg/kg时MMP-13蛋白表达分别上调164.35%和175.52%,mRNA表达上调78.50%和56.07%;而在DMN大鼠,sorafenib在1 mg/kg和5 mg/kg治疗量时MMP-13蛋白表达分别上调119.74%和152.7%,mRNA表达分别上调33.87%和57.26%。MMP-13表达主要见于纤维间隔内及其周围以及汇管区的部分间质细胞胞浆中,其中部分肝细胞有强阳性表达。⑤Sorafenib上调MMP-13/TIMP-1比值。随着肝纤维化进展,MMP-13/TIMP-1比值逐渐下降,模型4wk组MMP-13/TIMP-1比值最低,在BDL大鼠为0.32±0.06,在DMN大鼠为0.51±0.07。给予sorafenib治疗后,无论是BDL大鼠还是DMN大鼠,MMP-13/TIMP-1比值提高,逆转了MMP-13/TIMP-1失衡。
结论:sorafenib治疗可以降低实验性肝纤维化大鼠α-SMA表达,减少HSC激活;另一方面,Sorafenib可抑制TIMP-1表达,上调MMP-13表达,纠正MMP-13和TIMP-1比例失衡,促进基质降解,从而发挥抗纤维化作用。
第三部分:Sorafenib对肝星状细胞胶原代谢的影响
目的:研究sorafenib对PDGF刺激的HSC胶原合成、MMP-13(或MMP-1)和TIMP-1表达的影响,探讨sorafenib调节胶原代谢的机制。
方法:原代HSC、大鼠和人肝星状细胞株HSC-T6和LX-2用于实验,应用体外HSC培养技术,采用3H-脯氨酸(3H-pro)掺入法测定HSC胶原合成。Western blot和Real-time PCR检测I型胶原、MMP-13和TIMP-1蛋白和mRNA水平。
结果:①PDGF-BB刺激可使大鼠原代肝星状细胞、HSC-T6和LX-2Ⅰ型胶原α1 mRNA表达分别上调5.45倍、2.49倍和2.20倍,使HSC-T6和LX-2Ⅰ型胶原蛋白表达分别上调1.26倍和1.10倍。2.5μM、5.0μM和10.0μM sorafenib使原代HSCⅠ型胶原mRNA下调54.90%、64.88%和64.51%;使HSC-T6 mRNA下调62.64%、67.94%和82.52%,蛋白下调21.28%、28.72%和38.30%;使LX-2 mRNA下调58.66%、67.06%和81.64%,蛋白下调17.40%、18.84%和18.84%,低于PDGF组,P<0.05。免疫细胞化学也发现sorafenib能够抑制Ⅰ型胶原蛋白的表达。②无论是否有PDGF的刺激作用,在2.5~10.0μM范围内,sorafenib浓度越高,对HSC 3H-Pro掺入的抑制作用越强,P<0.01。10.0μM sorafenib在12 h、24 h和48 h 3H-Pro掺入抑制率在HSC-T6分别为14.51%、36.67%和63.44%,在LX-2 3H-Pro掺入抑制率分别为22.69%、37.52%和71.74%。Sorafenib抑制胶原合成具有时间和剂量依赖关系。③PDGF-BB刺激可使HSC-T6和LX-2细胞TIMP-1蛋白分别升高1.29倍和2.31倍,mRNA表达上调2.57倍和2.11倍,P<0.01;Sorafenib干预24 h后可使HSC-T6和LX-2细胞TIMP-1蛋白下降,对HSC-T6在10.0μM时抑制作用最明显,而对LX-2细胞在5.0μM作用最明显。Sorafenib以剂量依赖的方式下调了HSC-T6和LX-2细胞和原代HSC TIMP-1mRNA表达。④Sorafenib仅在5.0μM时使LX-2 MMP-1蛋白表达上调约167.39%,在2.5μM和5.0μM时使原代肝星状细胞MMP-13 mRNA表达上调,而对HSC-T6 MMP-13表达基本无明显影响。
结论:sorafenib以剂量依赖方式对HSC胶原合成产生抑制作用,通过抑制HSC胶原合成,减少胶原的产生;另一方面通过从蛋白和mRNA水平抑制TIMP-1表达,减少对MMPs阻止,促进已合成的ECM降解以减少胶原的沉积。
第四部分:Sorafenib对肝纤维化大鼠及肝星状细胞胶原代谢影响的信号转导机制
目的:研究sorafenib对HSC及肝纤维化大鼠信号分子Akt及ERK表达的影响,进一步探讨sorafenib调控胶原代谢的信号转导机制。
方法:实验包括大鼠和人肝星状细胞株HSC-T6和LX-2以及BDL和DMN两种肝纤维化动物模型。应用体外细胞培养技术,采用不同浓度的sorafenib和PDGF-BB 20ng/mlg干预HSC,同时与ERK特异性阻滞剂PD98059和PI3K特异性抑制剂LY294002作对比,应用Western blot方法检测HSC和肝组织ERK、p-ERK、Akt和p-Akt的表达。采用免疫组织化学方法测定p-ERK和p-Akt在肝纤维化大鼠肝组织的表达。
结果:①Sorafenib对HSC-T6和LX-2细胞总ERK和总Akt蛋白的表达无影响:总ERK和总Akt蛋白在HSC-T6各实验组表达无差异,P值分别为0.16和0.24;在LX-2各实验组差异亦无显著性,P值分别为0.26和0.17。②Sorafenib抑制HSC-T6的信号分子ERK和Akt的磷酸化:PDGF-BB刺激可使HSC p-ERK和p-Akt表达分别提高1.27倍(P=0.071)和1.48倍(P<0.001)。sorafenib 2.5μM、5.0μM和10.0μM干预PDGF-BB激活的HSC,p-ERK蛋白表达分别下调24.24%、34.85%和50%,P<0.01;p-Akt蛋白表达分别下调25.15%、40%和50.29%,明显低于PDGF组,P<0.01。在2.5～10.0μM浓度范围内,sorafenib剂量依赖性抑制HSC-T6 p-ERK和p-Akt蛋白表达, P<0.01。③Sorafenib抑制LX-2细胞p-ERK和p-Akt蛋白的表达:PDGF-BB刺激使LX-2细胞p-ERK和p-Akt提高1.61倍和1.23倍,与对照组比较差异有显著性(P<0.01)。2.5μM、5.0μM和10.0μM的sorafenib干预PDGF-BB刺激的LX-2,p-ERK表达分别下调60.38%、69.81%和73.58%;p-Akt表达分别下调32.12%、36.50%和45.26%,低于PDGF组,P<0.05。④Sorafenib对信号分子发挥了与特异性阻断剂相似的抑制作用:不论是否有PDGF-BB刺激,ERK特异性阻断剂PD98059和Akt特异性阻断剂LY294002均分别显著抑制了HSC ERK和Akt的磷酸化,而sorafenib则同时对ERK和Akt的磷酸化具有抑制作用。sorafenib对HSC-T6细胞p-ERK和p-Akt的抑制率与PD98059和LY294002的抑制作用无差异,P值分别为0.24和0.11。在LX2细胞中与HSC-T6细胞中的结果一样。⑤Sorafenib抑制实验性肝纤维化大鼠ERK和Akt的磷酸化:Western blot显示,随着肝纤维化进展,大鼠p-ERK和p-Akt的表达逐渐增高,sorafenib治疗2周后,p-ERK和p-Akt的表达受到抑制,在20mg/kg和40mg/kg治疗组,BDL大鼠p-ERK蛋白表达分别下调39.82%和46.02%,P<0.01;p-Akt表达下调38.75%和40%,P<0.05。免疫组化显示在正常大鼠,p-ERK和p-Akt呈弱阳性表达。随着肝纤维化进展,BDL大鼠肝组织p-ERK和p-Akt表达增多,主要分布于汇管区、Disse间隙、纤维间隔的间质细胞,部分肝细胞亦呈强阳性表达。Sorafenib治疗2周后p-ERK和p-Akt表达明显减低。在DMN大鼠得到同样的结果。
结论:Sorafenib抑制了HSC和纤维化大鼠肝组织ERK和Akt的磷酸化,阻断了ERK/MAPK和PI3K/Akt信号通路;BDL和DMN大鼠肝纤维化形成过程中,p-ERK和p-Akt表达增加;Sorafenib减轻肝纤维化可能与抑制肝纤维化大鼠p-ERK和p-Akt表达有关。
Liver fibrosis, a wound-healing response to a variety of chronic stimuli, is characterized by excessive deposition of extracellular matrix (ECM), of which type I collagen predominates. It is in the early stage of cirrhosis and may be reversed under some circumstances. If the liver injury is persistent, the excess deposition of ECM disrupts the normal architecture of the liver resulting in the formation of nodules, and fibrosis will develop to liver cirrhosis. It is well established that the activated hepatic stellate cells (HSCs) play a key role in the excess synthesis and deposition of ECM following a fibrotic stimulus. HSCs are not only the major source of ECM, but also the major cells releasing matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), which responsible for regulating matrix degradation. The activated HSC express tyrosine kinase receptors, including platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR), fibroblast growth factor receptor (FGFR) and nerve growth factor receptor (NGFR). With these receptors binding to specific ligands, some intracellular signaling pathways were trigged. It has been shown that the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3-kinase (PI3K) play important roles in proliferation, apoptosis, angiopoiesis and collagen metabolism in HSCs stimulated by different growth factors.
Multikinase inhibitor is a kind of novel multi-targeted agents studied in recent years. It has been shown to inhibit tumor cell growth and angiogenesis by blocking the receptor tyrosine kinases, such as PDGFR, VEGFR, FGFR, and NGFR. This is attributed to block signaling pathways including Ras/Raf/mitogen-activated protein kinase (MAPK) and PI3K/Akt/70-kDa ribosomal S6 kinase (p70S6K) and provide theoretical basis for antifibrotic therapy. Yoshiji H et al found imatinib, a multikinase inhibitor, markedly attenuated liver fibrosis, and decreased hepatic hydroxyproline and serum fibrosis markers. Imatinib markedly attenuated PDGF-BB-induced proliferation and migration and alpha-SMA and alpha2-(I)-procollagen mRNA of activated HSC in a dose-dependent manner in vitro. Sunitinib, another multikinase inhibitor, resulted in significant decreases in hepatic vascular density, inflammatory infiltrate, alpha-SMA abundance, collagen expression and portal pressure. Sorafenib, a novel oral multikinase inhibitor, is more effective in inhibiting ERK1/2 and PI3K signaling pathways. Mejias M et al found sorafenib treatment resulted in a reduction in portal pressure, as well as a remarkable improvement in liver damage and intra- hepatic fibrosis, inflammation, and angiogenesis. Although sorafenib exerts in reducing portal pressure, the role of sorafenib in the reversal of fibrosis, the activation of HSCs, collagen synthesis and degradation are unclear. The effects of sorafenib on collagen metabolism in vivo and in vitro are investigated in this study, and the mechanism is also explored. The experiments are composed of four parts as below.
Part 1: The therapic effect of sorafenib on intrahepatic fibrosis of experimental rats.
Objective : To explore the therapic effect of sorafenib on liver pathological changes, hydroxyproline content and expression of type I collagen in experimental fibrotic rats.
Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson’s trichrome method. Hepatic hydroxyproline was measured using a hydroxyproline detection kit. Type I collagen protein expression in the livers were determined by Western blot, while the expression of collagenα1(I) mRNA in the livers was assessed by Real-time PCR analysis .
Results:①Hepatic fibrosis was successfully induced by BDL and by intraperitoneal injections of dimethylnitrosamine (DMN) by hematoxylin and eosin staining and Masson’s trichrome method.②Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation in BDL and DMN rats. In BDL rats after sorafenib treatment at dose of 20 mg/kg and 40 mg/kg, hepatic fibrosis score (11.92±1.35 and 8.64±2.13, respective- ly) were both lower than that in vehicle-treated group(16.97±2.82), P<0.01; In DMN rats after sorafenib treatment at dose of 1 mg/kg and 5 mg/kg, hepatic fibrosis score(12.41±3.34 and 9.65±3.02, respectively) were both lower than that in vehicle-treated group(17.17±3.65), P<0.01.③Treatment with sorafenib reduced hydroxyproline content in BDL and DMN rats. In BDL rats, hydroxyproline content (247.00±16.05μg/g and 200.00±50.81μg/g, respect- tively) at dose of 20 mg/kg and 40 mg/kg were both lower than that in vehicle- treated group(442.50±139.14μg/g), P<0.01. In DMN rats, hydroxyproline content (214.40±35.71μg/g ) at doses of 5 mg/kg was lower than that in vehicle-treated group(355.80±33.05μg/g), P<0.05.④Sorafenib down- regulated the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in BDL rats. The expression of type I collagen protein (0.54±0.02 and 0.51±0.01, respectively) at doses of 20 mg/kg and 40 mg/kg by Western blot were both lower than that in vehicle-treated group, reduced by 14.86% and 17.89% , respectively, P<0.05. The expression of collagenα1 (I) mRNA (1.77±0.33 and 1.56±0.18, respectively ) at doses of 20 mg/kg and 40mg/kg by real-time PCR were both lower than that in vehicle-treated group(3.32±0.67), reduced by 46.69% and 53.01% , respectively, P<0.01.⑤Sorafenib inhibited the expression of type I collagen and collagenα1 (I) mRNA in a dose-dependent manner in DMN rats. The expression of type I collagen protein at doses of 1 mg/kg and 5 mg/kg reduced by 14.92% and 32.18%, respectively, P<0.05. The expression of collagenα1 (I) mRNA reduced by 43.17% and 49.78%, respectively, P<0.01. Furthermore,sorafenib 5 mg/kg was more significantly than that at dose of 1 mg/kg in inhibiting type collagen I protein and mRNA.
Conclusions: During the fibrogenesis, hydroxyproline content, type I collagen and collagenα1 (I) mRNA are upregulated in BDL and DMN rats. Sorafenib suppresses hepatic fibrosis by inhibiting the collagen deposition and inflammation, reduces hepatic fibrosis score and hydroxyproline content, as well as downregulats the expression of type I collagen and collagenα1 (I) mRNA. All data suggest that sorafenib may be a potential therapeutic agent in the treatment of liver fibrosis.
Part 2: The effect of sorafenib on the expression ofα-SMA,MMP-13 and TIMP-1 in experimental fibrotic rats.
Objective:To explore the effect of sorafenib on the expression ofα-SMA, MMP-13 and TIMP-1 in experimental fibrotic rats. Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL) and in Wistar rats by intraperitoneal injections of dimethylnitrosamine (DMN). Sorafenib was administered orally by gavage once a day during the third and the fourth week for two weeks (BDL rats: 20, 40 mg/kg; DMN rats: 1, 5 mg/kg). The expression ofα-SMA were evaluated by immunohistochemistry. MMP-13 and TIMP-1 protein expression in the livers were determined by Western blot and immunohistochemistry. While the expression of MMP-13 and TIMP-1 mRNA in the livers was assessed by Real-time PCR analysis.
Results:①During fibrogenesis, interstitial cells strongly positive forα-SMA appeared along the portal area, fibrous septum, the hepatic sinusoids and the periportal area. Sorafenib reduced the number of the cells positive forα-SMA. The mean density forα-SMA were 30.72±1.87% and 18.05±4.02% in BDL rats after administration of sorafenib (20 mg/kg and 40 mg/kg), respectively, and lower than that in vehicle-treated rats (38.19±2.10%), P<0.01. In DMN rats, after sorafenib (1 mg/kg and 5 mg/kg) treatment, the mean density forα-SMA were significantly lower than that in vehicle-treated rats(23.06±2.20%, 20.63±2.50% vs 32.32±4.40%), P<0.01.②During the progress of liver fibrosis, the expression of TIMP-1 was up- regulated in BDL and DMN rats. The expression of MMP-13 was up-regulated at the early stage, wheras downregulated at end-stage of liver fibrosis. The balance between MMP-13 and TIMP-1 was disturbed.③Sorafenib inhibited the expression of TIMP-1 protein and mRNA. Sorafenib down-regulated TIMP-1 protein by 31.69% and 47.87% and mRNA by 44.63% and 43.65% in BDL rats at the dose of 20 mg/kg and 40 mg/kg, respectively. Sorafenib (1 mg/kg and 5 mg/kg) decreased the expression of TIMP-1 protein by 44.14% and 55.18% and mRNA by 72.03% and 67.56% in DMN rats, respectively.④Sorafenib upregulated the expression of MMP-13 protein and mRNA in experimental fibrotic rats. Sorafenib (20 mg/kgand 40 mg/kg) up-regulated MMP-13 protein by 164.35% and 175.52% and mRNA by 78.50% and 56.07% in BDL rats, respectively. Sorafenib increased the expression of MMP-13 protein by 119.74% and 152.7% and mRNA by 33.87% and 57.26% in DMN rats at the dose of 1 mg/kg and 5 mg/kg, respectively. Hepatocyte and interstitial cells strongly positive for MMP-13 appeared along the portal area and fibrous septum.⑤Sorafenib increased the ratio of MMP-13 to TIMP-1 and improved the disbalance between MMP-13 and TIMP-1.With the progress of liver fibrosis, the ratio of MMP-13 to TIMP-1 decreased and was the lowest in model 4wk, the value was 0.32±0.06 in BDL rats and 0.51±0.07 in DMN rats, respectively. The ratio of MMP-13 to TIMP-1 was increased after treatment with sorafenib in BDL and DMN rats and improved the disbalance between MMP-13 and TIMP-1.
Conclusions: Sorafenib has potent to reduceα-SMA expression and inhibit HSC activation. For another, sorafenib down-regulates TIMP-1 protein and mRNA and up-regulates MMP-13 protein and mRNA. It improves the disbalance between MMP-13 and TIMP-1. As a result, sorafenib promotes degradation of ECM and plays a key role in the reversal of fibrosis.
Part 3: Effects of sorafenib on the Collagen Metabolism of Hepatic Stellate Cells
Objective:To investigate the effects of sorafenib on collagen synthesis and MMP-13 (or MMP-1), TIMP-1 expressions in PDGF-stimulated HSC,so as to study the mechanism of regulating collagen metabolism of HSC with sorafenib.
Methods: Three types of cell lines, freshly isolated rat HSC, HSC-T6 and LX-2 cell lines, were used in this study. Collagen synthesis was determined by 3H-pro incorporation. Expressions of type I collagen, MMP-13 and TIMP-1 were evaluated by western blot and quantitative real-time RT-PCR. Immuocytochemistry was used to detect type I collagen andα-SMA.
Results:①Stimulation with PDGF-BB induced increase of collagen Iα1 mRNA by 5.45-, 2.49- and 2.20-fold in primary HSC , HSC-T6 and LX-2. The expression of type I collagen protein was increased by 1.26- and 1.10- fold in HSC-T6 and LX-2. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated collagen Iα1 mRNA by 54.90%, 64.88% and 64.51% in primary HSC, by 62.64%, 67.94% and 82.52% in HSC-T6 and by 58.66%, 67.06% and 81.64% in LX-2, respectively. Furthermore, sorafenib decreased type I collagen protein by 21.28%, 28.72% and 38.30% in HSC-T6 and by 17.40%, 18.84% and 18.84% in LX-2, respectively (P<0.05).The same results were obtained by immuocytochemistry in HSC-T6 and LX-2 cells.②In the range of sorafenib (2.5～10.0μM), 3H-pro incorporation could be inhibited in dose- dependent manner with or without stimulation with PDGF-BB, P<0.01. The inhibition rates with sorafenib(10.0μM) were 14.51%, 36.67% , 63.44% (HSC-T6)and 22.69%, 37.52% and 71.74% (LX-2) at 12h,24h and 48h, respectively. So sorafenib could also dramatically inhibit the collagen synthesis of HSC in dose- and time-dependent manner.③Stimulation with PDGF-BB induced increase of TIMP-1 protein by 1.29-, 2.31-fold and mRNA by 2.57-, 2.11-fold in HSC-T6 and LX-2, respectively(P<0.01). Increased TIMP-1 expression was inhibited after treatment with sorafenib for 24 h, with a significant reduction at 10.0μM in T6 cells and at 5.0μM in LX-2 cells. Sorafenib dose-dependently downregulated the expression of TIMP-1 mRNA in the three HSC.④Sorafenib (5.0μM) induced the increase of MMP-1 protein by 167.39% in LX-2. The expression of MMP-13 mRNA were upregulated at the dose of sorafenib 2.5μM and 5.0μM in primary HSC. Sorafenib had no effect on the expression of MMP-13 in HSC-T6.
Conclusions: Sorafenib inhibited collagen synthesis in primary HSCs, HSC-T6 and LX-2 cells dose-dependently. As a result, the production of ECM was decreased. For another, the expressions of TIMP-1 protein and mRNA were downregulated by treatment with sorafenib. The dissolution of ECM was increased because of abolishing the inhibition of activity of MMPs. These data demonstrate that sorafenib is a potential therapeutic agent in the treatment of liver fibrosis.
Part 4: Mechanism of sorafenib on the collagen metabolism in experimental fibrotic rats and in hepatic stellate cells
Objective:To investigate the effects of sorafenib on Akt , phosphorylated Akt,ERK and phosphorylated ERK in PDGF-stimulated HSC and in experimental fibrotic rats and further to explore the mechanism of signal transduction in regulating collagen synthesis.
Methods:Two types of HSC cell lines (HSC-T6 and LX-2) and two different fibrotic models were used in this study. Fibrosis was induced by bile duct-ligation (BDL) or intraperitoneal injections of dimethylnitrosamine. HSC were preincubated with or without sorafenib and then stimulated with PDGF-BB. The ERK inhibitor PD98059 and Akt inhibitor LY294002 were used to treat HSC. The expression of ERK, phosphorylated ERK, Akt and phosphorylated Akt in HSC and rat livers were evaluated by western blot. The phosphorylated ERK and phosphorylated Akt in liver were detected by immunohistochemistry.
Results:①Sorafenib had no effect on total ERK and total Akt in HSC-T6 and LX-2 cells. The difference among groups was no significant in HSC-T6 and LX-2 cells. P value was 0.16, 0.24, 0.26and 0.17, respectively.②Sorafenib inhibited the activation of ERK and Akt in HSC-T6 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.27-fold (P=0.071) and 1.48-fold (P<0.001) in HSC-T6 cells. Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated- ERK by 24.24%, 34.85% , 50% and inhibited phosphorylated Akt by 25.15%、40% and 50.29%,respectively. The expression were lower than that in PGDF- stimulated group, P<0.01. Furthermore, sorafenib inhibited the activation of ERK and Akt in dose-dependent manner from 2.5μM to 10.0μM.③Sorafenib decreased the expression of phosphorylated ERK and phosphorylated Akt in LX-2 cells. Stimulation with PDGF-BB induced increase of phosphorylated ERK and phosphorylated Akt by 1.61- and 1.23-fold in LX-2, the difference was significant in statistic (P<0.01). Sorafenib (2.5μM, 5.0μM and 10.0μM) downregulated phosphorylated ERK by 60.38%, 69.81%, 73.58% and inhibited the activation of Akt by 32.12%、36.50% and 45.26%,respectively. The expression were less than that in PGDF-stimulated group, P<0.05.④Sorafenib played similar roles in inhibiting the activation of signal transduction molecule in HSC-T6 cells, compared with special inhibitor. Both phosphorylated ERK and phosphorylated Akt were significantly blocked by sorafenib. The inhibition of phosphorylated ERK was similar after treatment with sorafenib and PD98059, and so was the phospho-Akt inhibition by sorafenib and LY294002. P value was 0.24 and 0.11, respectively. The same results were obtained in LX-2 cells.⑤Sorafenib decreased the expression of phospho-ERK and Akt in in experimental fibrotic rats. During fibrogenesis, the expression of phospho-ERK and phospho-Akt gradually increased and decreased after administration of sorafenib (20 mg/kg and 40 mg/kg) for two weeks. The expression of phospho-ERK was downregulated by 39.82% and 46.02%, respectively, P<0.01;and the expression of phospho-Akt was decreased by 38.75% and 40%, respectively, P<0.05 ; As the evidence showed by immunochemistry, the phosphorylation of ERK and Akt were very low in normal rats. With the development of hepatic fibrosis, the positive staining cells of phospho-ERK and Akt increased obviously, and mainly resided in the interstitial cells in portal ducts, fiber septa and perisinusoidal space. Part hepatocytes were accompanied. The expressions of phospho-ERK and phospho-Akt were both decreased by administration of sorafenib. The same results were obtained in DMN rats.
Conclusions: Sorafenib blocks ERK/MAPK and PI3K/Akt ERK signal transduction pathways by inhibiting the phosphorylations of ERK and Akt in HSCs. Sorafenib ameliorates fibrosis by downregulating the expressions of phosphorylated ERK and phosphorylated Akt in liver fibrosis.
引文
1 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115:209~218
2 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~8
3 Van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
4 Hartmann JT, Haap M, Kopp HG, et al. Tyrosine Kinase Inhibitors - A Review on Pharmacology, Metabolism and Side Effects. Curr Drug Metab, 2009,10(5):470~481
5 Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res, 2004,64:7099~7109
6姜慧卿,张晓岚.丹参单体IH764-3对胆总管结扎大鼠胶原降解的作用及其机制.胃肠病学和肝病学杂志, 2003,4:336~338
7王玉珍,姜慧卿,扈彩霞,等. RhoA和肌球蛋白轻链在肝纤维化大鼠肝组织中的表达.基础医学与临床, 2007,3:275~278
8 Oh SW, Kim DH, Ha JR, et al. Anti-fibrotic effects of a methylenedioxybenzene compound, CW209292 on dimethylnitrosamine-induced hepatic fibrosis in rats. Biol Pharm Bull, 2009,32:1364~1370
9 Sakaida I, Hironaka K, Terai S, et al. Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells. Life Sci, 2003,72:943~959
10 Worns MA, Weinmann A, Pfingst K, et al. Safety and efficacy of sorafenib in patients with advanced hepatocellular carcinoma in consideration of concomitant stage of liver cirrhosis. J Clin Gastroenterol, 2009,43:489~495
11 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
12 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. British journal of pharmacology, 2009,157:258~270
13邝满元,刘映霞,李映菊.肝纤维化动物模型造模方法的研究进展.现代生物医学进展, 2008,9:1768~1770
14李志钢,杨晋翔,张伟,等.肝纤维化实验动物模型的建立与评价.北京中医药大学学报(中医临床版), 2009,4:43~46
15朱跃科,申凤俊,贾继东.肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系.中华肝脏病杂志,2004,10:41~44
16 Raetsch C, Jia JD, Boigk G, et al. Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis. Gut, 2002,50:241~247
17刘成,陆雄,刘平.二甲基亚硝胺诱发大鼠肝纤维化过程中α平滑肌肌动蛋白与门脉压力的变化.中国中西医结合消化杂志, 2002,5:271~272
18 Shah VH, Bruix J. Antiangiogenic therapy: not just for cancer anymore. Hepatology, 2009,49:1066~1068
19 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
1 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
2 Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009,25:223~229
3 Hellman NE, Spector J, Robinson J, et al. Matrix metalloproteinase 13 (MMP13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1), regulated by the MAPK pathway, are both necessary for Madin-Darby canine kidney tubulogenesis. The Journal of biological chemistry, 2008,283:4272~4282
4 Vincenti MP. The matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) genes. Transcriptional and posttranscriptional regulation, signal transduction and cell-type-specific expression. Methods Mol Biol, 2001,151:121~148
5熊敏莉,吴建新.基质金属蛋白酶及其抑制因子与肝纤维化.实用医学杂志, 2008,24:4318~4321
6刘秀玲,张洪,王飞.枳椇子提取物对实验大鼠肝组织中TIMP-1与MMP-13表达的影响.中国中药杂志, 2006,13:1097~1100
7 Yoshiji H, Kuriyama S, Miyamoto Y, et al. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model. Hepatology, 2000,32:1248~1254
8 Yoshiji H, Kuriyama S, Yoshii J, et al. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology, 2002,36:850~860
9 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076
10朱跃科,申凤俊,贾继东.肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系.中华肝脏病杂志,2004,10:41~44
11刘丽,姜慧卿,张晓岚,等.丹参单体IH764-3通过下调H_2O_2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达.中国应用生理学杂志, 2007,4:482~486
12 Williams EJ, Benyon RC, Trim N, et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut, 2001,49:577~583
13 van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
14 Chang YS, Adnane J, Trail PA, et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol, 2007,59:561~574
15 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172
1 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115:209~218
2 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
3 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172
4 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
5 Friedman SL. Hepatic fibrosis --overview. Toxicology, 2008,254:120~ 129
6 Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis, 2001,21:417~426
7 Iredale JP, Benyon RC, Arthur MJ, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology, 1996,24:176~184
8 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076.
9 Van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
10 Williams EJ, Benyon RC, Trim N, et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut, 2001,49:577~583
11 Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009,25:223~229
12 Lee TF, Mak KM, Rackovsky O, et al. Downregulation of hepatic stellatecell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP). J Cell Physiol, 2010,223(3): 648~657
13 Vercelino R, Crespo I, de Souza GF, et al. S-nitroso-N-acetylcysteine attenuates liver fibrosis in cirrhotic rats. J Mol Med, 2010,88:401~411.
14 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000,275:2247~2250
15 McCrudden R, Iredale JP. Liver fibrosis, the hepatic stellate cell and tissue inhibitors of metalloproteinases. Histol Histopathol, 2000,15:1159~1168
16 Hironaka K, Sakaida I, Matsumura Y, et al. Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis. Biochem Biophys Res Commun, 2000,267:290~295
17 Yoshiji H, Kuriyama S, Miyamoto Y, et al. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model. Hepatology, 2000,32:1248~1254
18 Yoshiji H, Kuriyama S, Yoshii J, et al. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology, 2002,36:850~860
19 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076
1 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
2 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
3 Peng X, Wang B, Wang T, et al. Expression of basic fibroblast growth factor in rat liver fibrosis and hepatic stellate cells. J Huazhong Univ Sci Technolog Med Sci, 2005,25:166~169
4 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
5 Lee TF, Mak KM, Rackovsky O, et al. Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP). J Cell Physiol, 2010,223(3):648~657
6 Eichler W, Friedrichs U, Thies A, et al. Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells. Invest Ophthalmol Vis Sci, 2002,43:2767~2773
7 van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
8 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. British journal of pharmacology, 2009,157:258~270
9 Adnane L, Trail PA, Taylor I, et al. Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol, 2006,407:597~612
10 Strumberg D. Preclinical and clinical development of the oral multikinase inhibitor sorafenib in cancer treatment. Drugs Today (Barc), 2005,41:773~784
11 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004,15:255~273
12 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32:496~501
13 Svegliati-Baroni G, Ridolfi F, Di SA, et al. Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene expression in human hepatic stellate cells. Hepatology, 2001,33:1130~1140
14 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
15 Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis inhepatocellular carcinoma model PLC/PRF/5. Cancer Res, 2006,66:11851~11858
16 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
17 Benitez-Rajal J, Lorite MJ, Burt AD, et al. Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides. Am J Physiol Gastrointest Liver Physiol, 2006,291:977~986
18 Zheng SM, Jiang MD, Zeng WZ, et al. Effects of extracellular signal-regulated kinase on rat cultured hepatic stellate cells stimulated by acetaldehyde. J Dig Dis, 2007,8:148~153
19 Pinzani M, Marra F, Caligiuri A, et al. Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells. Br J Pharmacol, 1996,119:1117~1124
20 Marra F, Gentilini A, Pinzani M, et al. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology, 1997,112:1297~1306
21 Reif S, Lang A, Lindquist JN, et al. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem, 2003,278:8083~8090
22 Son G, Hines IN, Lindquist J, et al. Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis. Hepatology, 2009,50:1512~1523
23 Bridle KR, Li L, O'Neill R, et al. Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells. J Lab Clin Med, 2006,147:234~241
1 Medina J, Arroyo AG, Sanchez-Madrid F, et al. Angiogenesis in chronic inflammatory liver disease. Hepatology, 2004,39:1185~1195
2 Friedman SL. Hepatic fibrosis -- overview. Toxicology, 2008,254:120~129
3 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
4 Medina J, Arroyo AG, Sanchez-Madrid F, et al. Angiogenesis in chronic inflammatory liver disease. Hepatology, 2004,39:1185~1195
5 Friedman SL. Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol, 2004,1:98~105
6 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172.
7 Lee JS, Semela D, Iredale J, et al. Sinusoidal remodeling and angiogenesis: a new function for the liver-specific pericyte. Hepatology, 2007,45:817~825
8 Parola M, Marra F, Pinzani M. Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario. Mol Aspects Med, 2008,29:58~66
9 Kitade M, Yoshiji H, Kojima H, et al. Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats. Hepatology, 2006,44:983~991
10 Sanz-Cameno P, Martin-Vilchez S, Lara-Pezzi E, et al. Hepatitis B viruspromotes angiopoietin-2 expression in liver tissue: role of HBV x protein. Am J Pathol, 2006,169:1215~1222
11 Salcedo X, Medina J, Sanz-Cameno P, et al. The potential of angiogenesis soluble markers in chronic hepatitis C. Hepatology, 2005,42:696~701
12 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
13 Potgens AJ, Lubsen NH, van AMC, et al. Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. Am J Pathol, 1995,146:197~209
14 Potgens AJ, Westphal HR, de Waal RM, et al. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis. Biol Chem Hoppe Seyler, 1995,376:57~70
15 Millanta F, Silvestri G, Vaselli C, et al. The role of vascular endothelial growth factor and its receptor Flk-1/KDR in promoting tumour angiogenesis in feline and canine mammary carcinomas: a preliminary study of autocrine and paracrine loops. Res Vet Sci, 2006,81:350~357
16 Kwak HJ, Park MJ, Park CM, et al. Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation. Int J Cancer, 2006,118:2711~20
17 Sawano A, Iwai S, Sakurai Y, et al. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood, 2001,97:785~91
18 Kearney JB, Ambler CA, Monaco KA, et al. Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood, 2002,99:2397~407
19 Langer DA, Shah VH. Nitric oxide and portal hypertension: interface of vasoreactivity and angiogenesis. J Hepatol, 2006,44:209~216
20 Wang JF, Zhang XF, Groopman JE. Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration. J Biol Chem, 2001,276:41950~41957
21 Fukushima K, Miyamoto S, Tsukimori K, et al. Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell line. Biol Reprod, 2005,73:172~179
22 Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med, 1993,328:1828~1835
23 Marx M, Perlmutter RA, Madri JA. Modulation of platelet-derived growth factor receptor expression in microvascular endothelial cells during in vitro angiogenesis. J Clin Invest, 1994,93:131~139
24 Guo P, Hu B, Gu W, et al. Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment. Am J Pathol, 2003,162:1083~1093
25纪素英,齐颖新,姜隽,等. PDGF BB、Rho-GDIα和Akt信号通路在低切应力诱导血管内皮细胞和平滑肌细胞迁移中的作用.医用生物力学, 2009,S1:15~16
26 Sato N, Beitz JG, Kato J, et al. Platelet-derived growth factor indirectly stimulates angiogenesis in vitro. Am J Pathol, 1993,142:1119~1130
27 Brogi E, Wu T, Namiki A, et al. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation, 1994,90:649~652
28任雨笙,杜荣增,俞世强.血小板源生长因子对人血管内皮细胞DNA及胶原蛋白合成的影响.中国动脉硬化杂志, 2003,01:72~73.
29夏豪,黄从新,尹丽娅.血小板源性生长因子促进新生血管形成的机制研究.微循环学杂志, 1999,04:12~13
30 Yamashita Y, Jeschke MG, Wolf SE. Differential expression of hepatocyte growth factor in liver, kidney, lung, and spleen following burn in rats. Cytokine, 2000,12:1293~1298
31 Cramer T, Schuppan D, Bauer M, et al. Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver Int,2004,24:335~344
32 Sierra-Honigmann MR, Nath AK, Murakami C, et al. Biological action of leptin as an angiogenic factor. Science, 1998,281:1683~1686
33 Bouloumie A, Drexler HC, Lafontan M, et al. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res, 1998,83:1059~1066
34 Yamagishi S, Inagaki Y, Amano S, et al. Up-regulation of vascular endothelial growth factor and down-regulation of pigment epithelium-derived factor messenger ribonucleic acid levels in leptin-exposed cultured retinal pericytes. Int J Tissue React, 2002,24:137~142
35 Cao R, Brakenhielm E, Wahlestedt C, et al. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci U S A, 2001,98:6390~6395
36 Misztal DB, Stepien H, Komorowski J. Effect of leptin on proliferative activity and vascular endothelial growth factor (VEGF) secretion from cultured endothelial cells HECa10 in vitro. Endocr Regul, 2004,38:161~166
37 Yamagishi S, Amano S, Inagaki Y, et al. Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppressing vascular endothelial growth factor gene expression through anti-oxidative properties. Microvasc Res, 2003,65:186~190
38 Uchihara M, Izumi N, Sato C, et al. Clinical significance of elevated plasma endothelin concentration in patients with cirrhosis. Hepatology, 1992,16:95~99
39 Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002,7:91~102
40 Lee PC, Kibbe MR, Schuchert MJ, et al. Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells. Microvasc Res, 2000,60:269~280
41 Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis innormal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. J Hepatol, 2001,34:644~650
42 Varner JA. The role of vascular cell integrins alpha v beta 3 and alpha v beta 5 in angiogenesis. EXS, 1997,79:361~390
43 Jang YC, Arumugam S, Gibran NS, et al. Role of alpha(v) integrins and angiogenesis during wound repair. Wound Repair Regen, 1999,7:375-80.
44 Eliceiri BP, Cheresh DA. Role of alpha v integrins during angiogenesis. Cancer J, 2000,6 Suppl 3:S245~249
45 Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets, 2003,4:123~131
46 Hood JD, Cheresh DA. Targeted delivery of mutant Raf kinase to neovessels causes tumor regression. Cold Spring Harb Symp Quant Biol, 2002,67:285~291
47 Boudreau NJ, Varner JA. The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis. J Biol Chem, 2004,279:4862~4868
48 Christofidou-Solomidou M, Bridges M, Murphy GF, et al. Expression and function of endothelial cell alpha v integrin receptors in wound-induced human angiogenesis in human skin/SCID mice chimeras. Am J Pathol, 1997,151:975~983
49 Weis SM. Evaluating integrin function in models of angiogenesis and vascular permeability. Methods Enzymol, 2007,426:505~528
50 Tagaya M, Haring HP, Stuiver I, et al. Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury. J Cereb Blood Flow Metab, 2001,21:835~846
51 Sincock PM, Fitter S, Parton RG, et al. PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci, 1999,112 ( Pt 6):833~844
52 Calzada MJ, Zhou L, Sipes JM, et al. Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro andmodulates angiogenesis in vivo. Circ Res, 2004,94:462~470
53 Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene, 1998,17:115~121
54 Ishikawa K, Mochida S, Mashiba S, et al. Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis. Biochem Biophys Res Commun, 1999,254:587~593
55 Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000,31:141~148
56 Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells. Biochem Biophys Res Commun, 2004,317:358~362
57 Aleffi S, Petrai I, Bertolani C, et al. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology, 2005,42:1339~1348
58 Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol, 2007,170:1942~1953
59 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
60 Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology, 2003,37:674~685
61 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
62 Aleffi S, Petrai I, Bertolani C, et al. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology, 2005,42:1339~1348
63 Wang YQ, Ikeda K, Ikebe T, et al. Inhibition of hepatic stellate cell proliferation and activation by the semisynthetic analogue of fumagillin TNP-470 in rats. Hepatology, 2000,32:980~989
64 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
65 Fernandez M, Vizzutti F, Garcia-Pagan JC, et al. Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice. Gastroenterology, 2004,126:886~894
66 Fernandez M, Mejias M, Angermayr B, et al. Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats. J Hepatol, 2005,43:98~103
67 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
68 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. Br J Pharmacol, 2009,157:258~270
69 Mejias M, Garcia PE, Tiani C, et al. Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats. Hepatology, 2009,49:1245~1256
70 Shimaoka M, Springer TA. Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov, 2003,2:703~716
71 Patsenker E, Popov Y, Stickel F, et al. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis. Hepatology, 2009,50:1501~1511
72 Shah VH, Bruix J. Antiangiogenic therapy: not just for cancer anymore. Hepatology, 2009,49:1066~1068
1 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
2 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115:209~218.
3 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
4 Friedman SL. Hepatic fibrosis -- overview. Toxicology, 2008,254:120~129
5 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
6 Peng X, Wang B, Wang T, et al. Expression of basic fibroblast growth factor in rat liver fibrosis and hepatic stellate cells. J Huazhong Univ Sci Technolog Med Sci, 2005,25:166~169
7 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1241
8 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. Am J Pathol, 1996,148:785~800
9 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000,31:131~140
10 Pinzani M, Gesualdo L, Sabbah GM, et al. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest,1989,84:1786~1793
11 Yoshiji H, Noguchi R, Kuriyama S, et al. Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats. Am J Physiol Gastrointest Liver Physiol, 2005,288: 907~913
12 Li X, Eriksson U. Novel PDGF family members: PDGF-C and PDGF-D. Cytokine Growth Factor Rev, 2003,14:91~98
13 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004,15:255~273
14 Marra F, Choudhury GG, Pinzani M, et al. Regulation of platelet-derived growth factor secretion and gene expression in human liver fat-storing cells. Gastroenterology, 1994,107:1110~1117
15 Breitkopf K, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC). Cytokine, 2005,31:349~357
16袁农,王宪智.血小板衍生生长因子及其受体在肝纤维化患者肝组织中的表达及意义.中华肝脏病杂志, 2002,01:58~60
17 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32:496~501
18 Ankoma SV, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000,31:141~148
19 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
20 Corpechot C, Barbu V, Wendum D, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002,35:1010~1021
21 Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology, 2003,37:674~685
22 Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene, 1998,17:115~121
23 Paternostro C, David E, Novo E, et al. Hypoxia, angiogenesis and liver fibrogenesis in the progression of chronic liver diseases. World J Gastroenterol, 2010,16:281~288
24 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
25时磊,刘绍能,李敏,等.芪术颗粒对大鼠肝纤维化形成过程中VEGF表达的影响.世界华人消化杂志, 2009:3675~3678
26 Roskoski R Jr. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor. Biochem Biophys Res Commun, 2005,337:1~13
27 Roskoski R Jr. Structure and regulation of Kit protein-tyrosine kinase--the stem cell factor receptor. Biochem Biophys Res Commun, 2005,338:1307~1315
28 Ishiguro A. SCF (stem cell factor). Nippon Rinsho, 2005,63 Suppl 8:57~59
29 Fujio K, Evarts RP, Hu Z, et al. Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat. Lab Invest, 1994,70:511~516
30 Gaca MD, Pickering JA, Arthur MJ, et al. Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis. J Hepatol, 1999,30:850~858
31 Yamamoto T, Hartmann K, Eckes B, et al. Role of stem cell factor and monocyte chemoattractant protein-1 in the interaction between fibroblasts and mast cells in fibrosis. J Dermatol Sci, 2001,26:106~111
32 Rosenbaum J, Mavier P, Preaux AM, et al. Demonstration of a basic fibroblast growth factor-like molecule in mouse hepatic endothelial cells. Biochem Biophys Res Commun, 1989,164:1099~1104
33阎晋平,马学惠,赵元昌,等.实验性肝纤维化中碱性成纤维细胞生长因子的免疫组化研究.新消化病学杂志, 1997,10:33~35
34 Ma TY, Kikuchi M, Sarfeh IJ, et al. Basic fibroblast growth factorstimulates repair of wounded hepatocyte monolayer: modulatory role of protein kinase A and extracellular matrix. J Lab Clin Med, 1999,134:363~371
35 Fibbi G, Pucci M, Grappone C, et al. Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor. Hepatology, 1999,29:868~878
36 Oakley F, Trim N, Constandinou CM, et al. Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis. Am J Pathol, 2003,163:1849~1858
37 Ohkubo T, Sugawara Y, Sasaki K, et al. Early induction of nerve growth factor-induced genes after liver resection-reperfusion injury. J Hepatol, 2002,36:210~217
38 Oakley F, Trim N, Constandinou CM, et al. Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis. Am J Pathol, 2003,163:1849~1858
39 Trim N, Morgan S, Evans M, et al. Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation. Am J Pathol, 2000,156:1235~1243
40 Asai K, Tamakawa S, Yamamoto M, et al. Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation. Liver Int, 2006,26:595~603
41 Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998,102:538~549
42 Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest, 2007,117:539~548
43 Issa R, Williams E, Trim N, et al. Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors. Gut, 2001,48:548~557
44 Huang S, Sinicrope FA. Sorafenib Inhibits STAT3 Activation to Enhance TRAIL-Mediated Apoptosis in Human Pancreatic Cancer Cells. Mol Cancer Ther, 2010,9:742~750
45 Wolfesberger B, Tonar Z, Gerner W, et al. The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line. Res Vet Sci, 2010,88:94~100
46 Peng CL, Guo W, Ji T, et al. Sorafenib induces growth inhibition and apoptosis in human synovial sarcoma cells via inhibiting the RAF/MEK/ERK signaling pathway. Cancer Biol Ther, 2009,8:1729~1736
47 Yang F, Van Meter TE, Buettner R, et al. Sorafenib inhibits signal transducer and activator of transcription 3 signaling associated with growth arrest and apoptosis of medulloblastomas. Mol Cancer Ther, 2008,7:3519~3526
48 Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res, 2006,66:11851~11858
49 Chang YS, Adnane J, Trail PA, et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol, 2007,59:561~574
50 Panka DJ, Wang W, Atkins MB, et al. The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells. Cancer Res, 2006,66:1611~1619
51 Strumberg D. Preclinical and clinical development of the oral multikinase inhibitor sorafenib in cancer treatment. Drugs Today (Barc), 2005,41:773~784
52 Amarapurkar AD, Amarapurkar DN, Vibhav S, et al. Angiogenesis in chronic liver disease. Ann Hepatol, 2007,6:170~173
53 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
54 Mejias M, Garcia PE, Tiani C, et al. Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats. Hepatology, 2009,49:1245~1256
55 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. Br J Pharmacol, 2009,157:258~270
56 Worns MA, Weinmann A, Pfingst K, et al. Safety and efficacy of sorafenib in patients with advanced hepatocellular carcinoma in consideration of concomitant stage of liver cirrhosis. J Clin Gastroenterol, 2009,43:489~495
57 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
58 Goodman VL, Rock EP, Dagher R, et al. Approval summary: sunitinib for the treatment of imatinib refractory or intolerant gastrointestinal stromal tumors and advanced renal cell carcinoma. Clin Cancer Res, 2007,13:1367~1373
59 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
60 Daniels CE, Wilkes MC, Edens M, et al. Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis. J Clin Invest, 2004,114:1308~1316
61 Wang S, Wilkes MC, Leof EB, et al. Imatinib mesylate blocks a non-Smad TGF-beta pathway and reduces renal fibrogenesis in vivo. FASEB J, 2005,19:1~11
62 Vittal R, Zhang H, Han MK, et al. Effects of the protein kinase inhibitor, imatinib mesylate, on epithelial/mesenchymal phenotypes: implications for treatment of fibrotic diseases. J Pharmacol Exp Ther, 2007,321:35~44
63 Yoshiji H, Noguchi R, Kuriyama S, et al. Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats. Am J Physiol Gastrointest Liver Physiol, 2005,288: 907~913
64 Distler JH, Jungel A, Huber LC, et al. Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis. Arthritis Rheum, 2007,56:311~322
65 Distler JH, Distler O. Imatinib as a novel therapeutic approach for fibrotic disorders. Rheumatology (Oxford), 2009,48:2~4
2 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~8
3 Van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
4 Hartmann JT, Haap M, Kopp HG, et al. Tyrosine Kinase Inhibitors - A Review on Pharmacology, Metabolism and Side Effects. Curr Drug Metab, 2009,10(5):470~481
5 Wilhelm SM, Carter C, Tang L, et al. BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res, 2004,64:7099~7109
6姜慧卿,张晓岚.丹参单体IH764-3对胆总管结扎大鼠胶原降解的作用及其机制.胃肠病学和肝病学杂志, 2003,4:336~338
7王玉珍,姜慧卿,扈彩霞,等. RhoA和肌球蛋白轻链在肝纤维化大鼠肝组织中的表达.基础医学与临床, 2007,3:275~278
8 Oh SW, Kim DH, Ha JR, et al. Anti-fibrotic effects of a methylenedioxybenzene compound, CW209292 on dimethylnitrosamine-induced hepatic fibrosis in rats. Biol Pharm Bull, 2009,32:1364~1370
9 Sakaida I, Hironaka K, Terai S, et al. Gadolinium chloride reverses dimethylnitrosamine (DMN)-induced rat liver fibrosis with increased matrix metalloproteinases (MMPs) of Kupffer cells. Life Sci, 2003,72:943~959
10 Worns MA, Weinmann A, Pfingst K, et al. Safety and efficacy of sorafenib in patients with advanced hepatocellular carcinoma in consideration of concomitant stage of liver cirrhosis. J Clin Gastroenterol, 2009,43:489~495
11 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
12 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. British journal of pharmacology, 2009,157:258~270
13邝满元,刘映霞,李映菊.肝纤维化动物模型造模方法的研究进展.现代生物医学进展, 2008,9:1768~1770
14李志钢,杨晋翔,张伟,等.肝纤维化实验动物模型的建立与评价.北京中医药大学学报(中医临床版), 2009,4:43~46
15朱跃科,申凤俊,贾继东.肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系.中华肝脏病杂志,2004,10:41~44
16 Raetsch C, Jia JD, Boigk G, et al. Pentoxifylline downregulates profibrogenic cytokines and procollagen I expression in rat secondary biliary fibrosis. Gut, 2002,50:241~247
17刘成,陆雄,刘平.二甲基亚硝胺诱发大鼠肝纤维化过程中α平滑肌肌动蛋白与门脉压力的变化.中国中西医结合消化杂志, 2002,5:271~272
18 Shah VH, Bruix J. Antiangiogenic therapy: not just for cancer anymore. Hepatology, 2009,49:1066~1068
19 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
1 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
2 Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009,25:223~229
3 Hellman NE, Spector J, Robinson J, et al. Matrix metalloproteinase 13 (MMP13) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1), regulated by the MAPK pathway, are both necessary for Madin-Darby canine kidney tubulogenesis. The Journal of biological chemistry, 2008,283:4272~4282
4 Vincenti MP. The matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) genes. Transcriptional and posttranscriptional regulation, signal transduction and cell-type-specific expression. Methods Mol Biol, 2001,151:121~148
5熊敏莉,吴建新.基质金属蛋白酶及其抑制因子与肝纤维化.实用医学杂志, 2008,24:4318~4321
6刘秀玲,张洪,王飞.枳椇子提取物对实验大鼠肝组织中TIMP-1与MMP-13表达的影响.中国中药杂志, 2006,13:1097~1100
7 Yoshiji H, Kuriyama S, Miyamoto Y, et al. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model. Hepatology, 2000,32:1248~1254
8 Yoshiji H, Kuriyama S, Yoshii J, et al. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology, 2002,36:850~860
9 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076
10朱跃科,申凤俊,贾继东.肝纤维化过程中胶原、基质金属蛋白酶及其抑制因子表达的动态变化及相互关系.中华肝脏病杂志,2004,10:41~44
11刘丽,姜慧卿,张晓岚,等.丹参单体IH764-3通过下调H_2O_2刺激的肝星状细胞FAK水平影响MMP-13和TIMP-1的表达.中国应用生理学杂志, 2007,4:482~486
12 Williams EJ, Benyon RC, Trim N, et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut, 2001,49:577~583
13 van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
14 Chang YS, Adnane J, Trail PA, et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol, 2007,59:561~574
15 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172
1 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115:209~218
2 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
3 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172
4 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
5 Friedman SL. Hepatic fibrosis --overview. Toxicology, 2008,254:120~ 129
6 Maher JJ. Interactions between hepatic stellate cells and the immune system. Semin Liver Dis, 2001,21:417~426
7 Iredale JP, Benyon RC, Arthur MJ, et al. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology, 1996,24:176~184
8 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076.
9 Van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
10 Williams EJ, Benyon RC, Trim N, et al. Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut, 2001,49:577~583
11 Jiao J, Friedman SL, Aloman C. Hepatic fibrosis. Curr Opin Gastroenterol, 2009,25:223~229
12 Lee TF, Mak KM, Rackovsky O, et al. Downregulation of hepatic stellatecell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP). J Cell Physiol, 2010,223(3): 648~657
13 Vercelino R, Crespo I, de Souza GF, et al. S-nitroso-N-acetylcysteine attenuates liver fibrosis in cirrhotic rats. J Mol Med, 2010,88:401~411.
14 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000,275:2247~2250
15 McCrudden R, Iredale JP. Liver fibrosis, the hepatic stellate cell and tissue inhibitors of metalloproteinases. Histol Histopathol, 2000,15:1159~1168
16 Hironaka K, Sakaida I, Matsumura Y, et al. Enhanced interstitial collagenase (matrix metalloproteinase-13) production of Kupffer cell by gadolinium chloride prevents pig serum-induced rat liver fibrosis. Biochem Biophys Res Commun, 2000,267:290~295
17 Yoshiji H, Kuriyama S, Miyamoto Y, et al. Tissue inhibitor of metalloproteinases-1 promotes liver fibrosis development in a transgenic mouse model. Hepatology, 2000,32:1248~1254
18 Yoshiji H, Kuriyama S, Yoshii J, et al. Tissue inhibitor of metalloproteinases-1 attenuates spontaneous liver fibrosis resolution in the transgenic mouse. Hepatology, 2002,36:850~860
19 Murphy FR, Issa R, Zhou X, et al. Inhibition of apoptosis of activated hepatic stellate cells by tissue inhibitor of metalloproteinase-1 is mediated via effects on matrix metalloproteinase inhibition: implications for reversibility of liver fibrosis. J Biol Chem, 2002,277:11069~11076
1 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
2 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
3 Peng X, Wang B, Wang T, et al. Expression of basic fibroblast growth factor in rat liver fibrosis and hepatic stellate cells. J Huazhong Univ Sci Technolog Med Sci, 2005,25:166~169
4 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
5 Lee TF, Mak KM, Rackovsky O, et al. Downregulation of hepatic stellate cell activation by retinol and palmitate mediated by adipose differentiation-related protein (ADRP). J Cell Physiol, 2010,223(3):648~657
6 Eichler W, Friedrichs U, Thies A, et al. Modulation of matrix metalloproteinase and TIMP-1 expression by cytokines in human RPE cells. Invest Ophthalmol Vis Sci, 2002,43:2767~2773
7 van BH, Dubois EA, Osanto S, et al. New drugs; sunitinib and sorafenib. Ned Tijdschr Geneeskd, 2007,151:2888~2890
8 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. British journal of pharmacology, 2009,157:258~270
9 Adnane L, Trail PA, Taylor I, et al. Sorafenib (BAY 43-9006, Nexavar), a dual-action inhibitor that targets RAF/MEK/ERK pathway in tumor cells and tyrosine kinases VEGFR/PDGFR in tumor vasculature. Methods Enzymol, 2006,407:597~612
10 Strumberg D. Preclinical and clinical development of the oral multikinase inhibitor sorafenib in cancer treatment. Drugs Today (Barc), 2005,41:773~784
11 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004,15:255~273
12 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32:496~501
13 Svegliati-Baroni G, Ridolfi F, Di SA, et al. Intracellular signaling pathways involved in acetaldehyde-induced collagen and fibronectin gene expression in human hepatic stellate cells. Hepatology, 2001,33:1130~1140
14 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
15 Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis inhepatocellular carcinoma model PLC/PRF/5. Cancer Res, 2006,66:11851~11858
16 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
17 Benitez-Rajal J, Lorite MJ, Burt AD, et al. Phospholipase D and extracellular signal-regulated kinase in hepatic stellate cells: effects of platelet-derived growth factor and extracellular nucleotides. Am J Physiol Gastrointest Liver Physiol, 2006,291:977~986
18 Zheng SM, Jiang MD, Zeng WZ, et al. Effects of extracellular signal-regulated kinase on rat cultured hepatic stellate cells stimulated by acetaldehyde. J Dig Dis, 2007,8:148~153
19 Pinzani M, Marra F, Caligiuri A, et al. Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells. Br J Pharmacol, 1996,119:1117~1124
20 Marra F, Gentilini A, Pinzani M, et al. Phosphatidylinositol 3-kinase is required for platelet-derived growth factor's actions on hepatic stellate cells. Gastroenterology, 1997,112:1297~1306
21 Reif S, Lang A, Lindquist JN, et al. The role of focal adhesion kinase-phosphatidylinositol 3-kinase-akt signaling in hepatic stellate cell proliferation and type I collagen expression. J Biol Chem, 2003,278:8083~8090
22 Son G, Hines IN, Lindquist J, et al. Inhibition of phosphatidylinositol 3-kinase signaling in hepatic stellate cells blocks the progression of hepatic fibrosis. Hepatology, 2009,50:1512~1523
23 Bridle KR, Li L, O'Neill R, et al. Coordinate activation of intracellular signaling pathways by insulin-like growth factor-1 and platelet-derived growth factor in rat hepatic stellate cells. J Lab Clin Med, 2006,147:234~241
1 Medina J, Arroyo AG, Sanchez-Madrid F, et al. Angiogenesis in chronic inflammatory liver disease. Hepatology, 2004,39:1185~1195
2 Friedman SL. Hepatic fibrosis -- overview. Toxicology, 2008,254:120~129
3 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
4 Medina J, Arroyo AG, Sanchez-Madrid F, et al. Angiogenesis in chronic inflammatory liver disease. Hepatology, 2004,39:1185~1195
5 Friedman SL. Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications. Nat Clin Pract Gastroenterol Hepatol, 2004,1:98~105
6 Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev, 2008,88:125~172.
7 Lee JS, Semela D, Iredale J, et al. Sinusoidal remodeling and angiogenesis: a new function for the liver-specific pericyte. Hepatology, 2007,45:817~825
8 Parola M, Marra F, Pinzani M. Myofibroblast - like cells and liver fibrogenesis: Emerging concepts in a rapidly moving scenario. Mol Aspects Med, 2008,29:58~66
9 Kitade M, Yoshiji H, Kojima H, et al. Leptin-mediated neovascularization is a prerequisite for progression of nonalcoholic steatohepatitis in rats. Hepatology, 2006,44:983~991
10 Sanz-Cameno P, Martin-Vilchez S, Lara-Pezzi E, et al. Hepatitis B viruspromotes angiopoietin-2 expression in liver tissue: role of HBV x protein. Am J Pathol, 2006,169:1215~1222
11 Salcedo X, Medina J, Sanz-Cameno P, et al. The potential of angiogenesis soluble markers in chronic hepatitis C. Hepatology, 2005,42:696~701
12 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
13 Potgens AJ, Lubsen NH, van AMC, et al. Vascular permeability factor expression influences tumor angiogenesis in human melanoma lines xenografted to nude mice. Am J Pathol, 1995,146:197~209
14 Potgens AJ, Westphal HR, de Waal RM, et al. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis. Biol Chem Hoppe Seyler, 1995,376:57~70
15 Millanta F, Silvestri G, Vaselli C, et al. The role of vascular endothelial growth factor and its receptor Flk-1/KDR in promoting tumour angiogenesis in feline and canine mammary carcinomas: a preliminary study of autocrine and paracrine loops. Res Vet Sci, 2006,81:350~357
16 Kwak HJ, Park MJ, Park CM, et al. Emodin inhibits vascular endothelial growth factor-A-induced angiogenesis by blocking receptor-2 (KDR/Flk-1) phosphorylation. Int J Cancer, 2006,118:2711~20
17 Sawano A, Iwai S, Sakurai Y, et al. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood, 2001,97:785~91
18 Kearney JB, Ambler CA, Monaco KA, et al. Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood, 2002,99:2397~407
19 Langer DA, Shah VH. Nitric oxide and portal hypertension: interface of vasoreactivity and angiogenesis. J Hepatol, 2006,44:209~216
20 Wang JF, Zhang XF, Groopman JE. Stimulation of beta 1 integrin induces tyrosine phosphorylation of vascular endothelial growth factor receptor-3 and modulates cell migration. J Biol Chem, 2001,276:41950~41957
21 Fukushima K, Miyamoto S, Tsukimori K, et al. Tumor necrosis factor and vascular endothelial growth factor induce endothelial integrin repertories, regulating endovascular differentiation and apoptosis in a human extravillous trophoblast cell line. Biol Reprod, 2005,73:172~179
22 Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med, 1993,328:1828~1835
23 Marx M, Perlmutter RA, Madri JA. Modulation of platelet-derived growth factor receptor expression in microvascular endothelial cells during in vitro angiogenesis. J Clin Invest, 1994,93:131~139
24 Guo P, Hu B, Gu W, et al. Platelet-derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment. Am J Pathol, 2003,162:1083~1093
25纪素英,齐颖新,姜隽,等. PDGF BB、Rho-GDIα和Akt信号通路在低切应力诱导血管内皮细胞和平滑肌细胞迁移中的作用.医用生物力学, 2009,S1:15~16
26 Sato N, Beitz JG, Kato J, et al. Platelet-derived growth factor indirectly stimulates angiogenesis in vitro. Am J Pathol, 1993,142:1119~1130
27 Brogi E, Wu T, Namiki A, et al. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation, 1994,90:649~652
28任雨笙,杜荣增,俞世强.血小板源生长因子对人血管内皮细胞DNA及胶原蛋白合成的影响.中国动脉硬化杂志, 2003,01:72~73.
29夏豪,黄从新,尹丽娅.血小板源性生长因子促进新生血管形成的机制研究.微循环学杂志, 1999,04:12~13
30 Yamashita Y, Jeschke MG, Wolf SE. Differential expression of hepatocyte growth factor in liver, kidney, lung, and spleen following burn in rats. Cytokine, 2000,12:1293~1298
31 Cramer T, Schuppan D, Bauer M, et al. Hepatocyte growth factor and c-Met expression in rat and human liver fibrosis. Liver Int,2004,24:335~344
32 Sierra-Honigmann MR, Nath AK, Murakami C, et al. Biological action of leptin as an angiogenic factor. Science, 1998,281:1683~1686
33 Bouloumie A, Drexler HC, Lafontan M, et al. Leptin, the product of Ob gene, promotes angiogenesis. Circ Res, 1998,83:1059~1066
34 Yamagishi S, Inagaki Y, Amano S, et al. Up-regulation of vascular endothelial growth factor and down-regulation of pigment epithelium-derived factor messenger ribonucleic acid levels in leptin-exposed cultured retinal pericytes. Int J Tissue React, 2002,24:137~142
35 Cao R, Brakenhielm E, Wahlestedt C, et al. Leptin induces vascular permeability and synergistically stimulates angiogenesis with FGF-2 and VEGF. Proc Natl Acad Sci U S A, 2001,98:6390~6395
36 Misztal DB, Stepien H, Komorowski J. Effect of leptin on proliferative activity and vascular endothelial growth factor (VEGF) secretion from cultured endothelial cells HECa10 in vitro. Endocr Regul, 2004,38:161~166
37 Yamagishi S, Amano S, Inagaki Y, et al. Pigment epithelium-derived factor inhibits leptin-induced angiogenesis by suppressing vascular endothelial growth factor gene expression through anti-oxidative properties. Microvasc Res, 2003,65:186~190
38 Uchihara M, Izumi N, Sato C, et al. Clinical significance of elevated plasma endothelin concentration in patients with cirrhosis. Hepatology, 1992,16:95~99
39 Wei CL, Khoo HE, Lee KH, et al. Differential expression and localization of nitric oxide synthases in cirrhotic livers of bile duct-ligated rats. Nitric Oxide, 2002,7:91~102
40 Lee PC, Kibbe MR, Schuchert MJ, et al. Nitric oxide induces angiogenesis and upregulates alpha(v)beta(3) integrin expression on endothelial cells. Microvasc Res, 2000,60:269~280
41 Sieber CC, Sumanovski LT, Stumm M, et al. In vivo angiogenesis innormal and portal hypertensive rats: role of basic fibroblast growth factor and nitric oxide. J Hepatol, 2001,34:644~650
42 Varner JA. The role of vascular cell integrins alpha v beta 3 and alpha v beta 5 in angiogenesis. EXS, 1997,79:361~390
43 Jang YC, Arumugam S, Gibran NS, et al. Role of alpha(v) integrins and angiogenesis during wound repair. Wound Repair Regen, 1999,7:375-80.
44 Eliceiri BP, Cheresh DA. Role of alpha v integrins during angiogenesis. Cancer J, 2000,6 Suppl 3:S245~249
45 Kumar CC. Integrin alpha v beta 3 as a therapeutic target for blocking tumor-induced angiogenesis. Curr Drug Targets, 2003,4:123~131
46 Hood JD, Cheresh DA. Targeted delivery of mutant Raf kinase to neovessels causes tumor regression. Cold Spring Harb Symp Quant Biol, 2002,67:285~291
47 Boudreau NJ, Varner JA. The homeobox transcription factor Hox D3 promotes integrin alpha5beta1 expression and function during angiogenesis. J Biol Chem, 2004,279:4862~4868
48 Christofidou-Solomidou M, Bridges M, Murphy GF, et al. Expression and function of endothelial cell alpha v integrin receptors in wound-induced human angiogenesis in human skin/SCID mice chimeras. Am J Pathol, 1997,151:975~983
49 Weis SM. Evaluating integrin function in models of angiogenesis and vascular permeability. Methods Enzymol, 2007,426:505~528
50 Tagaya M, Haring HP, Stuiver I, et al. Rapid loss of microvascular integrin expression during focal brain ischemia reflects neuron injury. J Cereb Blood Flow Metab, 2001,21:835~846
51 Sincock PM, Fitter S, Parton RG, et al. PETA-3/CD151, a member of the transmembrane 4 superfamily, is localised to the plasma membrane and endocytic system of endothelial cells, associates with multiple integrins and modulates cell function. J Cell Sci, 1999,112 ( Pt 6):833~844
52 Calzada MJ, Zhou L, Sipes JM, et al. Alpha4beta1 integrin mediates selective endothelial cell responses to thrombospondins 1 and 2 in vitro andmodulates angiogenesis in vivo. Circ Res, 2004,94:462~470
53 Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene, 1998,17:115~121
54 Ishikawa K, Mochida S, Mashiba S, et al. Expressions of vascular endothelial growth factor in nonparenchymal as well as parenchymal cells in rat liver after necrosis. Biochem Biophys Res Commun, 1999,254:587~593
55 Ankoma-Sey V, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000,31:141~148
56 Wang YQ, Luk JM, Ikeda K, et al. Regulatory role of vHL/HIF-1alpha in hypoxia-induced VEGF production in hepatic stellate cells. Biochem Biophys Res Commun, 2004,317:358~362
57 Aleffi S, Petrai I, Bertolani C, et al. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells. Hepatology, 2005,42:1339~1348
58 Novo E, Cannito S, Zamara E, et al. Proangiogenic cytokines as hypoxia-dependent factors stimulating migration of human hepatic stellate cells. Am J Pathol, 2007,170:1942~1953
59 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
60 Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology, 2003,37:674~685
61 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
62 Aleffi S, Petrai I, Bertolani C, et al. Upregulation of proinflammatory and proangiogenic cytokines by leptin in human hepatic stellate cells.Hepatology, 2005,42:1339~1348
63 Wang YQ, Ikeda K, Ikebe T, et al. Inhibition of hepatic stellate cell proliferation and activation by the semisynthetic analogue of fumagillin TNP-470 in rats. Hepatology, 2000,32:980~989
64 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
65 Fernandez M, Vizzutti F, Garcia-Pagan JC, et al. Anti-VEGF receptor-2 monoclonal antibody prevents portal-systemic collateral vessel formation in portal hypertensive mice. Gastroenterology, 2004,126:886~894
66 Fernandez M, Mejias M, Angermayr B, et al. Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats. J Hepatol, 2005,43:98~103
67 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
68 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. Br J Pharmacol, 2009,157:258~270
69 Mejias M, Garcia PE, Tiani C, et al. Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats. Hepatology, 2009,49:1245~1256
70 Shimaoka M, Springer TA. Therapeutic antagonists and conformational regulation of integrin function. Nat Rev Drug Discov, 2003,2:703~716
71 Patsenker E, Popov Y, Stickel F, et al. Pharmacological inhibition of integrin alphavbeta3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis. Hepatology, 2009,50:1501~1511
72 Shah VH, Bruix J. Antiangiogenic therapy: not just for cancer anymore. Hepatology, 2009,49:1066~1068
1 Parsons CJ, Takashima M, Rippe RA. Molecular mechanisms of hepatic fibrogenesis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S79~84
2 Bataller R, Brenner DA. Liver fibrosis. J Clin Invest, 2005,115:209~218.
3 Kisseleva T, Brenner DA. Role of hepatic stellate cells in fibrogenesis and the reversal of fibrosis. J Gastroenterol Hepatol, 2007,22 Suppl 1:S73~78
4 Friedman SL. Hepatic fibrosis -- overview. Toxicology, 2008,254:120~129
5 Marra F, Arrighi MC, Fazi M, et al. Extracellular signal-regulated kinase activation differentially regulates platelet-derived growth factor's actions in hepatic stellate cells, and is induced by in vivo liver injury in the rat. Hepatology, 1999,30:951~958
6 Peng X, Wang B, Wang T, et al. Expression of basic fibroblast growth factor in rat liver fibrosis and hepatic stellate cells. J Huazhong Univ Sci Technolog Med Sci, 2005,25:166~169
7 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1241
8 Pinzani M, Milani S, Herbst H, et al. Expression of platelet-derived growth factor and its receptors in normal human liver and during active hepatic fibrogenesis. Am J Pathol, 1996,148:785~800
9 Carloni V, Pinzani M, Giusti S, et al. Tyrosine phosphorylation of focal adhesion kinase by PDGF is dependent on ras in human hepatic stellate cells. Hepatology, 2000,31:131~140
10 Pinzani M, Gesualdo L, Sabbah GM, et al. Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells. J Clin Invest,1989,84:1786~1793
11 Yoshiji H, Noguchi R, Kuriyama S, et al. Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats. Am J Physiol Gastrointest Liver Physiol, 2005,288: 907~913
12 Li X, Eriksson U. Novel PDGF family members: PDGF-C and PDGF-D. Cytokine Growth Factor Rev, 2003,14:91~98
13 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004,15:255~273
14 Marra F, Choudhury GG, Pinzani M, et al. Regulation of platelet-derived growth factor secretion and gene expression in human liver fat-storing cells. Gastroenterology, 1994,107:1110~1117
15 Breitkopf K, Roeyen C, Sawitza I, et al. Expression patterns of PDGF-A, -B, -C and -D and the PDGF-receptors alpha and beta in activated rat hepatic stellate cells (HSC). Cytokine, 2005,31:349~357
16袁农,王宪智.血小板衍生生长因子及其受体在肝纤维化患者肝组织中的表达及意义.中华肝脏病杂志, 2002,01:58~60
17 Ikura Y, Morimoto H, Ogami M, et al. Expression of platelet-derived growth factor and its receptor in livers of patients with chronic liver disease. J Gastroenterol, 1997,32:496~501
18 Ankoma SV, Wang Y, Dai Z. Hypoxic stimulation of vascular endothelial growth factor expression in activated rat hepatic stellate cells. Hepatology, 2000,31:141~148
19 Yoshiji H, Kuriyama S, Yoshii J, et al. Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis. Gut, 2003,52:1347~1354
20 Corpechot C, Barbu V, Wendum D, et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology, 2002,35:1010~1021
21 Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology, 2003,37:674~685
22 Ankoma-Sey V, Matli M, Chang KB, et al. Coordinated induction of VEGF receptors in mesenchymal cell types during rat hepatic wound healing. Oncogene, 1998,17:115~121
23 Paternostro C, David E, Novo E, et al. Hypoxia, angiogenesis and liver fibrogenesis in the progression of chronic liver diseases. World J Gastroenterol, 2010,16:281~288
24 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
25时磊,刘绍能,李敏,等.芪术颗粒对大鼠肝纤维化形成过程中VEGF表达的影响.世界华人消化杂志, 2009:3675~3678
26 Roskoski R Jr. Signaling by Kit protein-tyrosine kinase--the stem cell factor receptor. Biochem Biophys Res Commun, 2005,337:1~13
27 Roskoski R Jr. Structure and regulation of Kit protein-tyrosine kinase--the stem cell factor receptor. Biochem Biophys Res Commun, 2005,338:1307~1315
28 Ishiguro A. SCF (stem cell factor). Nippon Rinsho, 2005,63 Suppl 8:57~59
29 Fujio K, Evarts RP, Hu Z, et al. Expression of stem cell factor and its receptor, c-kit, during liver regeneration from putative stem cells in adult rat. Lab Invest, 1994,70:511~516
30 Gaca MD, Pickering JA, Arthur MJ, et al. Human and rat hepatic stellate cells produce stem cell factor: a possible mechanism for mast cell recruitment in liver fibrosis. J Hepatol, 1999,30:850~858
31 Yamamoto T, Hartmann K, Eckes B, et al. Role of stem cell factor and monocyte chemoattractant protein-1 in the interaction between fibroblasts and mast cells in fibrosis. J Dermatol Sci, 2001,26:106~111
32 Rosenbaum J, Mavier P, Preaux AM, et al. Demonstration of a basic fibroblast growth factor-like molecule in mouse hepatic endothelial cells. Biochem Biophys Res Commun, 1989,164:1099~1104
33阎晋平,马学惠,赵元昌,等.实验性肝纤维化中碱性成纤维细胞生长因子的免疫组化研究.新消化病学杂志, 1997,10:33~35
34 Ma TY, Kikuchi M, Sarfeh IJ, et al. Basic fibroblast growth factorstimulates repair of wounded hepatocyte monolayer: modulatory role of protein kinase A and extracellular matrix. J Lab Clin Med, 1999,134:363~371
35 Fibbi G, Pucci M, Grappone C, et al. Functions of the fibrinolytic system in human Ito cells and its control by basic fibroblast and platelet-derived growth factor. Hepatology, 1999,29:868~878
36 Oakley F, Trim N, Constandinou CM, et al. Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis. Am J Pathol, 2003,163:1849~1858
37 Ohkubo T, Sugawara Y, Sasaki K, et al. Early induction of nerve growth factor-induced genes after liver resection-reperfusion injury. J Hepatol, 2002,36:210~217
38 Oakley F, Trim N, Constandinou CM, et al. Hepatocytes express nerve growth factor during liver injury: evidence for paracrine regulation of hepatic stellate cell apoptosis. Am J Pathol, 2003,163:1849~1858
39 Trim N, Morgan S, Evans M, et al. Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation. Am J Pathol, 2000,156:1235~1243
40 Asai K, Tamakawa S, Yamamoto M, et al. Activated hepatic stellate cells overexpress p75NTR after partial hepatectomy and undergo apoptosis on nerve growth factor stimulation. Liver Int, 2006,26:595~603
41 Iredale JP, Benyon RC, Pickering J, et al. Mechanisms of spontaneous resolution of rat liver fibrosis. Hepatic stellate cell apoptosis and reduced hepatic expression of metalloproteinase inhibitors. J Clin Invest, 1998,102:538~549
42 Iredale JP. Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ. J Clin Invest, 2007,117:539~548
43 Issa R, Williams E, Trim N, et al. Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors. Gut, 2001,48:548~557
44 Huang S, Sinicrope FA. Sorafenib Inhibits STAT3 Activation to Enhance TRAIL-Mediated Apoptosis in Human Pancreatic Cancer Cells. Mol Cancer Ther, 2010,9:742~750
45 Wolfesberger B, Tonar Z, Gerner W, et al. The tyrosine kinase inhibitor sorafenib decreases cell number and induces apoptosis in a canine osteosarcoma cell line. Res Vet Sci, 2010,88:94~100
46 Peng CL, Guo W, Ji T, et al. Sorafenib induces growth inhibition and apoptosis in human synovial sarcoma cells via inhibiting the RAF/MEK/ERK signaling pathway. Cancer Biol Ther, 2009,8:1729~1736
47 Yang F, Van Meter TE, Buettner R, et al. Sorafenib inhibits signal transducer and activator of transcription 3 signaling associated with growth arrest and apoptosis of medulloblastomas. Mol Cancer Ther, 2008,7:3519~3526
48 Liu L, Cao Y, Chen C, et al. Sorafenib blocks the RAF/MEK/ERK pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hepatocellular carcinoma model PLC/PRF/5. Cancer Res, 2006,66:11851~11858
49 Chang YS, Adnane J, Trail PA, et al. Sorafenib (BAY 43-9006) inhibits tumor growth and vascularization and induces tumor apoptosis and hypoxia in RCC xenograft models. Cancer Chemother Pharmacol, 2007,59:561~574
50 Panka DJ, Wang W, Atkins MB, et al. The Raf inhibitor BAY 43-9006 (Sorafenib) induces caspase-independent apoptosis in melanoma cells. Cancer Res, 2006,66:1611~1619
51 Strumberg D. Preclinical and clinical development of the oral multikinase inhibitor sorafenib in cancer treatment. Drugs Today (Barc), 2005,41:773~784
52 Amarapurkar AD, Amarapurkar DN, Vibhav S, et al. Angiogenesis in chronic liver disease. Ann Hepatol, 2007,6:170~173
53 Valfre dBL, Novo E, Cannito S, et al. Angiogenesis and liver fibrogenesis. Histol Histopathol, 2009,24:1323~1341
54 Mejias M, Garcia PE, Tiani C, et al. Beneficial effects of sorafenib on splanchnic, intrahepatic, and portocollateral circulations in portal hypertensive and cirrhotic rats. Hepatology, 2009,49:1245~1256
55 Hennenberg M, Trebicka J, Stark C, et al. Sorafenib targets dysregulated Rho kinase expression and portal hypertension in rats with secondary biliary cirrhosis. Br J Pharmacol, 2009,157:258~270
56 Worns MA, Weinmann A, Pfingst K, et al. Safety and efficacy of sorafenib in patients with advanced hepatocellular carcinoma in consideration of concomitant stage of liver cirrhosis. J Clin Gastroenterol, 2009,43:489~495
57 Di MM, Daniele B, Perrone F. Targeted therapies: Role of sorafenib in HCC patients with compromised liver function. Nat Rev Clin Oncol, 2009,6:505~506
58 Goodman VL, Rock EP, Dagher R, et al. Approval summary: sunitinib for the treatment of imatinib refractory or intolerant gastrointestinal stromal tumors and advanced renal cell carcinoma. Clin Cancer Res, 2007,13:1367~1373
59 Tugues S, Fernandez VG, Munoz LJ, et al. Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats. Hepatology, 2007,46:1919~1926
60 Daniels CE, Wilkes MC, Edens M, et al. Imatinib mesylate inhibits the profibrogenic activity of TGF-beta and prevents bleomycin-mediated lung fibrosis. J Clin Invest, 2004,114:1308~1316
61 Wang S, Wilkes MC, Leof EB, et al. Imatinib mesylate blocks a non-Smad TGF-beta pathway and reduces renal fibrogenesis in vivo. FASEB J, 2005,19:1~11
62 Vittal R, Zhang H, Han MK, et al. Effects of the protein kinase inhibitor, imatinib mesylate, on epithelial/mesenchymal phenotypes: implications for treatment of fibrotic diseases. J Pharmacol Exp Ther, 2007,321:35~44
63 Yoshiji H, Noguchi R, Kuriyama S, et al. Imatinib mesylate (STI-571) attenuates liver fibrosis development in rats. Am J Physiol Gastrointest Liver Physiol, 2005,288: 907~913
64 Distler JH, Jungel A, Huber LC, et al. Imatinib mesylate reduces production of extracellular matrix and prevents development of experimental dermal fibrosis. Arthritis Rheum, 2007,56:311~322
65 Distler JH, Distler O. Imatinib as a novel therapeutic approach for fibrotic disorders. Rheumatology (Oxford), 2009,48:2~4
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