己酮可可碱通过Raf途径抑制大鼠肝星状细胞增殖、促进凋亡
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摘要
肝纤维化(hepatic fibrosis)是在肝脏慢性损伤与修复愈合的过程中形成的一种病理改变,细胞外间质(extracellular matrix,ECM)的过度合成与沉积是其主要特征。肝星状细胞(hepatic stellate cell,HSC),也称贮脂细胞,并不是肝脏的主要细胞,仅占肝脏细胞的5%。在正常肝脏组织中,HSC处于静息状态;然而,在肝损伤时HSC可被激活发生明显的形态、结构和功能变化:分泌多种细胞因子、增殖和合成各种ECM的能力明显增强。因此,肝纤维化主要是由于HSC的激活导致ECM的大量增加,引起肝功能的损伤,并最终导致肝硬化。在HSC中各种信号转导通路共同参与肝纤维化的形成,因此,研究不同的信号转导途径,同时寻找新的信号转导通路对肝纤维化的形成机制进行系统研究,有助于对其发病机制更全面更深刻的理解。
Raf-1蛋白激酶由648个氨基酸残基组成,分子质量为70～74kD,活化后具有丝/苏氨酸蛋白激酶活性。Raf激酶的3个同工酶包括A-Raf、B-Raf与C-Raf(Raf-1)。Raf激酶主要功能是通过Ras/Raf/MEK/ERK通路,调节多种细胞功能,引起细胞的增殖和分化。此外,有研究表明Raf-1激活通过MEK/MERK通路介导抑制细胞凋亡。Raf-1激活有赖于酪氨酸蛋白激酶的活化。在Ras/Raf-1增殖信号途径中,Raf-1位于Ras的下游,其激活的初始事件是生长因子激活特异的受体酪氨酸激酶,活化的受体首先作用于Grb2-Sos蛋白复合物,Sos可使Ras活化,后者进一步激活Raf-1。Raf-1被激活后即继续激活其下游的MEK和MAPK,最终通过对转录调节因子表达的影响而将细胞增殖信号传递到细胞核内。
表皮生长因子(epidermal growth factor,EGF)系一种常见的细胞因子,对上皮细胞、成纤维细胞及平滑肌细胞都有促进增殖的作用。
己酮可可碱(pentoxifylline,PTX)为甲基黄嘌呤衍生物,是磷酸二酯酶抑制剂,目前主要用于外周血管病的治疗。体外研究显示PTX具有增加细胞内cAMP及改善血流动力学、免疫抑制和抗纤维化的作用。
迄今,Ras/Raf/MEK/Erk信号传递通路为近年来信号传导方面研究最活跃的热点之一,很多研究表明该通路具有调控细胞增殖、分化等功能,但在肝纤维化发生中的作用研究较少。Raf是该信号通路中的重要一环。Raf-1能否被激活、是否能准确地下传信号对于细胞是否出现增殖效应至关重要。为此,我们应用体外细胞培养技术,探讨Ras/Raf信号通路阻断剂PTX对大鼠HSC中Raf磷酸化水平的抑制作用,从而为肝纤维化的发病机制和有效治疗提供理论依据。
目的:探讨Ras/Raf信号通路阻断剂己酮可可碱对EGF刺激的大鼠HSC中Raf磷酸化水平的抑制作用,以及该信号通路对HSC增殖、凋亡的影响。
方法:应用含8%胎牛血清、100 IU·mL-1青霉素、100μg·mL-1链霉素、4 mmol·L-1谷氨酰胺及1 mol·L-1 HEPES的DMEM培养液,37℃、5%CO2条件下体外培养HSC。利用EGF诱导激活HSC后,以一定浓度的PTX阻断Ras/Raf通路。采用Western blotting方法检测HSC中p-Raf的蛋白表达;采用流式细胞学的方法检测PTX对HSC凋亡的影响;采用MTT法等测定PTX对HSC的增殖抑制作用。
结果:①PTX抑制HSC中Raf磷酸化水平。Western blotting方法分析示:EGF+PTX(40μg·mL-1)组p-Raf表达较EGF+PTX(20μg·mL-1)组间无明显差异(P>0.05);EGF组p-Raf表达明显较对照组增高(P<0.01);EGF+PTX(10μg·mL-1)组p-Raf表达较对照组减少了31.87%(P<0.01)。PTX干预浓度越高,蛋白表达下降趋势越明显,并呈剂量依赖性。用PTX干预EGF刺激的HSC,EGF+PTX(2h)组p-Raf表达较与对照组相比无明显差异(P>0.05);PTX干预时间越长,p-Raf表达下降趋势越明显,呈时间依赖性。②PTX对HSC凋亡的影响:流式细胞学方法测定显示: PTX干预HSC 24h后,与空白对照组、EGF组、PTX(4h)组与PTX(8h)组相比,可明显促进细胞的凋亡。表明在EGF激活p-Raf后,抑制HSC凋亡,而PTX抑制p-Raf表达后,可有效的促进细胞凋亡。③PTX抑制HSC增殖。倒置显微镜下形态学观察,新传代的HSC呈圆形,富含折光脂滴。接种1 h部分细胞开始黏附,4.5 h伸展,24 h细胞突起明显伸长、增多。随培养时间延长,细胞渐呈典型星状形态,突起多而长,并呈簇状生长。应用PTX干预HSC 4h后,HSC的星状或梭状突起开始回缩,逐渐变为缩水状的圆形细胞,细胞间隙增大,由致密状变为网格状,12h开始有细胞脱落,24 h脱落细胞明显增多,悬浮于上清中,但细胞膜一直保持完整。MTT法:EGF刺激HSC后,与对照组相比,细胞生长明显,有统计学差异,P<0.01;EGF+PTX(4h)组、EGF+PTX(8h)组、EGF+PTX(24h)组与对照组相比细胞增殖明显受到抑制,均有显著的统计学差异,P<0.01。
结论:Ras/Raf信号通路阻断剂PTX能够抑制EGF刺激的大鼠HSC中Raf-1的磷酸化水平,并呈时间和剂量依赖性。特异性阻断Ras/Raf信号通路能够抑制EGF刺激的HSC增殖、促进其凋亡。
Hepatic fibrosis is a wound-healing process in livers with chronic injury and is characterized by the excess production and deposition of extracellular matrix (ECM) components. Hepatic stellate cell (HSC), also known as fat-storing cell, are nonparenchymal cells that represent 5% of the resident cells in the liver. In the normal liver, most HSC are in quiescent; however, in response to liver injury, these cells undergo an activation process that induces changes in their structure and function. Functional changes include the expression of cell surface receptors, increased cell proliferation, and the augmentation in synthesis of ECM proteins. In fact, activated HSC are the primary source of the ECM proteins responsible for liver fibrosis, which can impair normal liver function and ultimately lead to cirrhosis and organ failure. Many signal transduction pathways are involved in the liver fibrogenesis in HSC. So we should look for a new signal transduction pathway and investigate the mechanism of liver fibrogenesis.
Raf is a family of 3 serine/threonine–specific kinases (A-Raf, B-Raf, and Raf-1) ubiquitously expressed throughout embryonic development. The principal function of the Raf protein kinases appears to be participation in the highly conserved Ras/Raf/ MEK/ extracellular signal-regulated kinase (ERK) intracellular signaling pathway, which has been implicated in the transduction of signals directing cell proliferation and differentiation. In addition, Raf-1 activation of the MEK/ERK pathway has been associated with inhibition of apoptosis, and leading to cell survival.
Raf-1 activation depends on activate to tyrosine protein kinase (TPK). In the proliferation process mediated by Ras/Raf pathway, Raf is downstream to Ras. In the beginning, growth factor activates to specific receptor tyrosine kinase, activated receptor acts to Ras and induces Ras activation. The latter further activates to Raf, MEK and ERK. In the end the proliferation signal passes to cell intranuclear by effect to transcription regulatory factor’s expression.
Epidermal growth factor (EGF) is one of the cytokines. EGF can act to epthelium、fibroblast and smooth muscle cells, which induces them proliferative.
Pentoxifylline (PTX), a trisubstituted xanthine-derived phosphodiesterase inhibitor, is currently used in the treatment of peripheral vascular disorders because of its effects on erythrocyte deformability and tissue oxygen delivery. In vitro studies, it also increase intracellular cAMP, improves haemodynamics, immunosuppression and antifibrogenic.
Up to now, Ras/Raf/MEK/Erk pathway is the most activated field of research in recent years. Many studies show that this pathway plays a role to regulate cells’proliferation and differentiation. But there are a few researches in hepatic fibrosis. Raf is one of the important roles to the pathway. For this reason, we make HSC culture in vitro, to investigate PTX how to effect on Raf phosphorylation in this pathway, in order to find a new way to cure hepatic fibrosis.
Objective: To investigate the inhibition role of PTX on Raf-1 phosphorylation in rat HSC and effects of Ras/Raf signal pathway on HSC proliferation and apoptosis.
Methods: HSC were cultured in DMEM with 8% fetal bovine serum. After being stimulated by EGF, HSC were treated with PTX to block Ras/Raf signal pathway. The protein levels of p-Raf were analyzed by Western blotting. The apoptosis of PTX on HSC was evaluated by flow cytomertry(FCM). The proliferative inhibition of PTX on HSC was evaluated by MTT assay.
Results:①The inhibition role of PTX on the phosphorylation of myosin phosphatase in rat HSC activated by EGF: The level of p-Raf protein expression by Western blotting in group of EGF (2.89±0.37) was significantly higher than that of control (2.40±0.05), P<0.01. In the group of PTX (24 h) with EGF, the level of p-Raf protein expression (1.63±0.28) was significantly lower than that of control, P<0.01. No significant difference was found between the protein expression of the control group (2.40±0.05) and the group of EGF with PTX (2 h) (2.42±0.42). The levels of p-Raf protein expression had significant difference after HSC exposured to 5、10、20μmol·L-1 PTX with EGF for 24 h compared with that of control group, P<0.01, and the levels were 1.84±0.35、1.63±0.28 and 1.45±0.29, respectively. But there was no difference between 20μmol·L-1 PTX group (1.45±0.29) and 40μmol·L-1 PTX group (1.43±0.30), (P>0.05).②The inhibition effect of PTX on HSC proliferation. Observed with inverted microscope, in the group of PTX with EGF, the cell shrunk and the cell accreted. The cells obviously increased after 24h. MTT results displayed that compared with control group, only EGF stimulated the proliferation of HSC, the proliferation rate was 34.12%, P<0.01. In the group of PTX with EGF at 4h, 8h and 24h, the HSC proliferation was inhibited. The proliferation inhibition rates respectively were 19.09%, 34.18% and 54.45%, P<0.01. There was significant difference between the control and PTX treated groups in the HSC proliferation.③The inhibition effect of PTX on the HSC apoptosis. FCM showed that EGF stimulated the proliferation and inhibited the apoptosis of HSC. The apoptosis rate of HSC, stimulated by the EGF, was 1.72±0.26, P<0.01; In the group of PTX with EGF at 4h, 8h and 24h, the apoptosis of HSC was promoted compared with that of control group (3.42±0.23). The proliferation inhibition rates respectively were 4.17±0.59,5.53±0.24 and 7.10±0.46, P<0.01.
Conclusions: PTX can inhibit the Raf phosphorylation in rat HSC, and inhibit proliferation and promote apoptosis of HSC via Ras/Raf signaling pathway.
Raf-1蛋白激酶由648个氨基酸残基组成,分子质量为70～74kD,活化后具有丝/苏氨酸蛋白激酶活性。Raf激酶的3个同工酶包括A-Raf、B-Raf与C-Raf(Raf-1)。Raf激酶主要功能是通过Ras/Raf/MEK/ERK通路,调节多种细胞功能,引起细胞的增殖和分化。此外,有研究表明Raf-1激活通过MEK/MERK通路介导抑制细胞凋亡。Raf-1激活有赖于酪氨酸蛋白激酶的活化。在Ras/Raf-1增殖信号途径中,Raf-1位于Ras的下游,其激活的初始事件是生长因子激活特异的受体酪氨酸激酶,活化的受体首先作用于Grb2-Sos蛋白复合物,Sos可使Ras活化,后者进一步激活Raf-1。Raf-1被激活后即继续激活其下游的MEK和MAPK,最终通过对转录调节因子表达的影响而将细胞增殖信号传递到细胞核内。
表皮生长因子(epidermal growth factor,EGF)系一种常见的细胞因子,对上皮细胞、成纤维细胞及平滑肌细胞都有促进增殖的作用。
己酮可可碱(pentoxifylline,PTX)为甲基黄嘌呤衍生物,是磷酸二酯酶抑制剂,目前主要用于外周血管病的治疗。体外研究显示PTX具有增加细胞内cAMP及改善血流动力学、免疫抑制和抗纤维化的作用。
迄今,Ras/Raf/MEK/Erk信号传递通路为近年来信号传导方面研究最活跃的热点之一,很多研究表明该通路具有调控细胞增殖、分化等功能,但在肝纤维化发生中的作用研究较少。Raf是该信号通路中的重要一环。Raf-1能否被激活、是否能准确地下传信号对于细胞是否出现增殖效应至关重要。为此,我们应用体外细胞培养技术,探讨Ras/Raf信号通路阻断剂PTX对大鼠HSC中Raf磷酸化水平的抑制作用,从而为肝纤维化的发病机制和有效治疗提供理论依据。
目的:探讨Ras/Raf信号通路阻断剂己酮可可碱对EGF刺激的大鼠HSC中Raf磷酸化水平的抑制作用,以及该信号通路对HSC增殖、凋亡的影响。
方法:应用含8%胎牛血清、100 IU·mL-1青霉素、100μg·mL-1链霉素、4 mmol·L-1谷氨酰胺及1 mol·L-1 HEPES的DMEM培养液,37℃、5%CO2条件下体外培养HSC。利用EGF诱导激活HSC后,以一定浓度的PTX阻断Ras/Raf通路。采用Western blotting方法检测HSC中p-Raf的蛋白表达;采用流式细胞学的方法检测PTX对HSC凋亡的影响;采用MTT法等测定PTX对HSC的增殖抑制作用。
结果:①PTX抑制HSC中Raf磷酸化水平。Western blotting方法分析示:EGF+PTX(40μg·mL-1)组p-Raf表达较EGF+PTX(20μg·mL-1)组间无明显差异(P>0.05);EGF组p-Raf表达明显较对照组增高(P<0.01);EGF+PTX(10μg·mL-1)组p-Raf表达较对照组减少了31.87%(P<0.01)。PTX干预浓度越高,蛋白表达下降趋势越明显,并呈剂量依赖性。用PTX干预EGF刺激的HSC,EGF+PTX(2h)组p-Raf表达较与对照组相比无明显差异(P>0.05);PTX干预时间越长,p-Raf表达下降趋势越明显,呈时间依赖性。②PTX对HSC凋亡的影响:流式细胞学方法测定显示: PTX干预HSC 24h后,与空白对照组、EGF组、PTX(4h)组与PTX(8h)组相比,可明显促进细胞的凋亡。表明在EGF激活p-Raf后,抑制HSC凋亡,而PTX抑制p-Raf表达后,可有效的促进细胞凋亡。③PTX抑制HSC增殖。倒置显微镜下形态学观察,新传代的HSC呈圆形,富含折光脂滴。接种1 h部分细胞开始黏附,4.5 h伸展,24 h细胞突起明显伸长、增多。随培养时间延长,细胞渐呈典型星状形态,突起多而长,并呈簇状生长。应用PTX干预HSC 4h后,HSC的星状或梭状突起开始回缩,逐渐变为缩水状的圆形细胞,细胞间隙增大,由致密状变为网格状,12h开始有细胞脱落,24 h脱落细胞明显增多,悬浮于上清中,但细胞膜一直保持完整。MTT法:EGF刺激HSC后,与对照组相比,细胞生长明显,有统计学差异,P<0.01;EGF+PTX(4h)组、EGF+PTX(8h)组、EGF+PTX(24h)组与对照组相比细胞增殖明显受到抑制,均有显著的统计学差异,P<0.01。
结论:Ras/Raf信号通路阻断剂PTX能够抑制EGF刺激的大鼠HSC中Raf-1的磷酸化水平,并呈时间和剂量依赖性。特异性阻断Ras/Raf信号通路能够抑制EGF刺激的HSC增殖、促进其凋亡。
Hepatic fibrosis is a wound-healing process in livers with chronic injury and is characterized by the excess production and deposition of extracellular matrix (ECM) components. Hepatic stellate cell (HSC), also known as fat-storing cell, are nonparenchymal cells that represent 5% of the resident cells in the liver. In the normal liver, most HSC are in quiescent; however, in response to liver injury, these cells undergo an activation process that induces changes in their structure and function. Functional changes include the expression of cell surface receptors, increased cell proliferation, and the augmentation in synthesis of ECM proteins. In fact, activated HSC are the primary source of the ECM proteins responsible for liver fibrosis, which can impair normal liver function and ultimately lead to cirrhosis and organ failure. Many signal transduction pathways are involved in the liver fibrogenesis in HSC. So we should look for a new signal transduction pathway and investigate the mechanism of liver fibrogenesis.
Raf is a family of 3 serine/threonine–specific kinases (A-Raf, B-Raf, and Raf-1) ubiquitously expressed throughout embryonic development. The principal function of the Raf protein kinases appears to be participation in the highly conserved Ras/Raf/ MEK/ extracellular signal-regulated kinase (ERK) intracellular signaling pathway, which has been implicated in the transduction of signals directing cell proliferation and differentiation. In addition, Raf-1 activation of the MEK/ERK pathway has been associated with inhibition of apoptosis, and leading to cell survival.
Raf-1 activation depends on activate to tyrosine protein kinase (TPK). In the proliferation process mediated by Ras/Raf pathway, Raf is downstream to Ras. In the beginning, growth factor activates to specific receptor tyrosine kinase, activated receptor acts to Ras and induces Ras activation. The latter further activates to Raf, MEK and ERK. In the end the proliferation signal passes to cell intranuclear by effect to transcription regulatory factor’s expression.
Epidermal growth factor (EGF) is one of the cytokines. EGF can act to epthelium、fibroblast and smooth muscle cells, which induces them proliferative.
Pentoxifylline (PTX), a trisubstituted xanthine-derived phosphodiesterase inhibitor, is currently used in the treatment of peripheral vascular disorders because of its effects on erythrocyte deformability and tissue oxygen delivery. In vitro studies, it also increase intracellular cAMP, improves haemodynamics, immunosuppression and antifibrogenic.
Up to now, Ras/Raf/MEK/Erk pathway is the most activated field of research in recent years. Many studies show that this pathway plays a role to regulate cells’proliferation and differentiation. But there are a few researches in hepatic fibrosis. Raf is one of the important roles to the pathway. For this reason, we make HSC culture in vitro, to investigate PTX how to effect on Raf phosphorylation in this pathway, in order to find a new way to cure hepatic fibrosis.
Objective: To investigate the inhibition role of PTX on Raf-1 phosphorylation in rat HSC and effects of Ras/Raf signal pathway on HSC proliferation and apoptosis.
Methods: HSC were cultured in DMEM with 8% fetal bovine serum. After being stimulated by EGF, HSC were treated with PTX to block Ras/Raf signal pathway. The protein levels of p-Raf were analyzed by Western blotting. The apoptosis of PTX on HSC was evaluated by flow cytomertry(FCM). The proliferative inhibition of PTX on HSC was evaluated by MTT assay.
Results:①The inhibition role of PTX on the phosphorylation of myosin phosphatase in rat HSC activated by EGF: The level of p-Raf protein expression by Western blotting in group of EGF (2.89±0.37) was significantly higher than that of control (2.40±0.05), P<0.01. In the group of PTX (24 h) with EGF, the level of p-Raf protein expression (1.63±0.28) was significantly lower than that of control, P<0.01. No significant difference was found between the protein expression of the control group (2.40±0.05) and the group of EGF with PTX (2 h) (2.42±0.42). The levels of p-Raf protein expression had significant difference after HSC exposured to 5、10、20μmol·L-1 PTX with EGF for 24 h compared with that of control group, P<0.01, and the levels were 1.84±0.35、1.63±0.28 and 1.45±0.29, respectively. But there was no difference between 20μmol·L-1 PTX group (1.45±0.29) and 40μmol·L-1 PTX group (1.43±0.30), (P>0.05).②The inhibition effect of PTX on HSC proliferation. Observed with inverted microscope, in the group of PTX with EGF, the cell shrunk and the cell accreted. The cells obviously increased after 24h. MTT results displayed that compared with control group, only EGF stimulated the proliferation of HSC, the proliferation rate was 34.12%, P<0.01. In the group of PTX with EGF at 4h, 8h and 24h, the HSC proliferation was inhibited. The proliferation inhibition rates respectively were 19.09%, 34.18% and 54.45%, P<0.01. There was significant difference between the control and PTX treated groups in the HSC proliferation.③The inhibition effect of PTX on the HSC apoptosis. FCM showed that EGF stimulated the proliferation and inhibited the apoptosis of HSC. The apoptosis rate of HSC, stimulated by the EGF, was 1.72±0.26, P<0.01; In the group of PTX with EGF at 4h, 8h and 24h, the apoptosis of HSC was promoted compared with that of control group (3.42±0.23). The proliferation inhibition rates respectively were 4.17±0.59,5.53±0.24 and 7.10±0.46, P<0.01.
Conclusions: PTX can inhibit the Raf phosphorylation in rat HSC, and inhibit proliferation and promote apoptosis of HSC via Ras/Raf signaling pathway.
引文
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8 Friedman, SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000, 275(4):2247~2250
9 Kolch W, Meaningful relationships: The regulation of the Ras/Raf / MEK/ERK pathway by p rotein interactions[J]. Biochem J, 2000, 351:289-305
10 Wilhelm SM, Carter C, Tang L, et al. BAY 4329006 exhibits broad spectrum oral anti2tumor activity and targets the Raf /MEK/ERK pathway and recep tor tyrosine kinases involvedin tumor progression and angiogenesis [J]. Cancer Res, 2004, 64:7099-7109
11 Kolch W, Heidecker G, Kochs G, et al. Pretein kinase C activates Raf-1 by direct phosphorylation. Nature, 1993, 364:249-252
12 Schaap D, Van der Wal J, Howe LR, et al. Adominant-negative mutant of raf block mitogen-activated protein kinase activation by growth factors an d oncogenic p21ras . Biol Clmm, 1993, 268:20232-20236
13 Piazuelo E, Jinenez P, Lanas A. Platelet-derived growth factor and epidermal growth factor play a major role in human colonic fibroblast repair activities[J]. Eur Surg Res, 2000, 32(3):191
14 Roberts PJ, Der CJ. Targeting the Raf-MEK-ERKmitogen -activated protein in kinase cascade for the treatment of cancer. Oncogene, 2007, 26(22): 3291-3310
15 Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/CIP1 expression in human epidermoid carcinoma A431 cells. Cell Single, 2006, 18(2):244-255.
16 Massimo Pinzani, Fabio Marra, Alessandra Caligiuri, et al. Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells. British Journal of Pharmacology, 1996, 119:1117-1124
17 Trakul N, Menard RE, Schade GR, et al. Raf kinaseinhibitory protein regulates Raf-1 but not B-Raf kinase activation. J Biol Chem, 2005, 280(26):24931-24940
18 Park KS, Kim JA, Chai KJ. Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line. Exp Mol Med, 2000, 32(3):120-126
19 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
20 Preaux AM, Mallat A, Rosonbaum J, et al. Pentoxifylline inhibitis growth and collagen synthesis of cultured human hepatic myofibroblast-like cells[J]. J Hepatology, 1997, 26(2): 315
21 Windmeoer C, Gressner AM. Effect of pentoxifylline on the fibrogenic functions of cultured rat liver fat storing cells and myofibroblasts[J]. Biochem Pharmacol, 1996, 51(5): 577
22 Kim KM, Yoon JM, Gwak GY, et al. Bile acid-mediated inducion of cyclooxygenase-2 and Mcl-1 in hepatic stellate cells. Biochem Bilphys Res Commun, 2006, 342(4):1108-13
23 Verma-Gandhu M, Peterson MR. Effect of fetuin, a TGFbeta antagonist and pentoxifylline, a cytokine antagonist on hepatic stellate cellfuction and fibrotic parameters in fibrosis. Eru J Pharmacol, 2007, 572(2-3):220-227
24 Kanno N, Lesage G, Phinizy JL, et al. Stimulation of alpha2-adrenergic receptor inhibits cholangiocarcinomagrowth through modulation of Raf-1 and B-Raf activities. Hepatology, 2002, 35(6):1329-1340
1 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000, 275:2247–2250
2 Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-β in hepatic fibrosis. Front Biosci, 2002, 7:793-807
3 Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci, 2002, 7:1720-1726
4 Schmidt EK, Fichelson S, Feller SM. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. BMC Biol, 2004, 2:7
5 Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Singnal, 2003, 15(5):463-469
6 McCubrey JA, Steelman LS, Chappell WH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta, 2007, 1773(8):1263-84
7 Okabe H, Lee SH, Phuchareon J, Albertson DG, et al. A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation. PloS ONE, 2006, 1:128
8 Stevenson JP, Yao KS, Gallagher M, et al. Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132(CGP 69846A). J Clin Oncol, 1999, 17(7):2227-36
9 Hickey FB, Enqland K, Cotter TG. Bcr-Abl regulates osteopontin transcription via Ras, PI-3K, Apkc, Raf-1, and MEK. J Leukoc Biol, 2005, 78(1):289-300
10 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induces syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
11 Lue H, Kapurniotu A, Fingerle-Rowson G, et al. Rapid and transient activation of the ERK/MAPK signaling pathway by macrophage migration inhibitory factor(MIF) and dependence on JAB1/CSN5 and Src kinase activity. Cell Signal, 2006, 18(5):688-703
12 Beazely MA, Alan JK, Watts VJ. Protein kinase C andepidermal growth factor stimulation of Raf-1 potentiates adenylyl cyclase type 6 activation in intact cells. Mol Pharmacol, 2005, 67(1):250-259
13 Kim SE, Choi KY. EGF reportor is involved in WNT3a-mediated proliferation and motility of NIH3T3 cells via ERK pathway activation. Cell Signal, 2007,19(7): 1554-64
14 Krotum RL, Johnson HJ, Costanzo DL, et al. The molecular scaffold kinase suppressor of Ras is a modifier of RasV12-induced and replicative senescence. Mol Cell Biol, 2006, 26(6):2202-2214
15 Zhou B, Wu LJ, Tashiro S, et al. Activation of exracellular signal-regulated kinase during silibinin-protected, isoprotereno-induce apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent. Acta Pharmacol Sin, 2007, 28(6):803-810
16 Ebisch A, Staber PB, Delavar A, et al. Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. Cancer Res, 2006, 66(7):3401-3408
17 Park KS, Kim JA, Chai KJ. Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line. Exp Mol Med, 2000, 32(3):120-126
18 Ellis C, Liu XQ, Anderson D, et al. Tyrosine phosphorylation of GAP and GAP-associated protein in lymphoid and fibroblast cells expressing lck. Oncogene,1991, 6(6):895-901
19 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
20 Janssen RA, Veenstra KG, Jonasch P, et al. Ras- and Raf-induced down-modulation of non-muscle tropomyosin are MEK-independent. J Biol Chem, 1998, 27:32182-32186
21 Zheng X, Ravatn R, Lin Y, et al. Gene expression of TPA induced differentiation in HL-60 cells by DNA microarray analysis. Nucleic Acids Res, 2002, 30(20):4489-4499
22 Kuo WL, Chung KC, Rosner MR. Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathway: roles for Ras and Src. Mol Cell Biol, 1997, 17(8):4633-4643
23 Davis JM, Navolanic PM, Weinstein-Oppenheimer CR, et al. Raf-1 and Bcl-2 induce distinct and common pathway that contribute to breast cancer drug resistance. Clin Cancer Res, 2003, 9(3):1161-1170
24 Blaqosklonny MV, Schulte T, Nquyen P, et al. Taxol- induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res, 1996, 56(8):1851-1854
25 Yu C, Rahmani M, Almenara J, et al. Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitoradaphostin proceeds through a Raf-1/MEK/ERK-and AKT-dependent process. Oncogene, 2004, 23(7):1364-1376
26 Ravi RK, Weber E, McMahon M, et al. Activated and Raf-1 causes growth arrest in human small cell lung cancer cells. J Clin Invest, 1998, 101(1):153-159
27 Dodqe JE, Okano M, Dick F, et al. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem, 2005, 280(18):17986-91
28 Liu AM, Wong YH. Activation of nuclear factor {kappa}B by somatostatin type 2 receptor in pancreatic acinar AR42J cells invoves G{alpha}14 and multiple signaling components: a mechanism requiring protein kinase C, calmodulin -dependent kinaseII, ERK and c-Src. J Biol Chem, 2005, 280(41):34617-25
29 Solit DB, Ivy SP, Kopil C, et al. Phase I trail of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer. Clin Cancer Res, 2007, 13(6):1775-82
30 Stancato LF, Silverstein AM, Owens-Grillo JK, et al. The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal groeth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase. J Biol Chem, 1997, 272(7):4013-4020
31 Yu R, Hebbar V, Kim DW, et al. Resveratrol inhibits phorbol ester and UV-induced activator protein 1 activationby interfering with mitogen-activated protein kinase pathway. Mol Pharmcol, 2001, 60(1):217-224
32 Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci, 2002, 7:1720-1726
33 Shi MN, Zheng WD, Zhang LJ, et al. Effect of IL-10 on the expression of HSC growth factor in hepatic fibrosis rat. World J Gastroenterol, 2005, 11(31):4788-4793
34 Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/ CIP1 expression in human epidermoid carcinoma A431 cells. Cell Single, 2006, 18(2):244-255
35 Reimann T, Hempel U, Krautwald S. Transforming growth factor- beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.FEBS Letters, 1997, 403(1):57-60
36 Axmann A, Seidel D, Reimann T. Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism. Biochemical and Biophysical Research Communications, 1998, 249(2):456-460
2 Schulze A, Lehmann K, Jefferies HB, et al: Analysis of the transcrip tional p rogram induced by Raf in ep ithelial cells[J].GenesDev, 2001, 15: 981-994
3 Belsches AP, Haskell MD, Parsons SJ. Role of c-Src tyrosine kinase in EGF-induced mitogenesis. Front Biosci, 1997, 2:501-518
4 Zani M, Gesualdo L, Sabbah GM, et al. Effects of platlet-derived growth factor and other polypeptide mitogens on DNA synthesis and trowty of cultures rat liver fat storing cell. J Clin invest, 1989, 84:1786
5 刘绍能,姚乃礼,殷海波.芪术颗粒对大鼠肝纤维化模型TGF- β 1 ,EGF 表达的影响.中国药理与临床,2001, 17(5):24-25
6 Raetsch C, Jia JD, Boigk G, et al. Pentoxifylline downregulates profibrogenic cytokines and procollagen expression in rat secondary biliary fibrosis, Gut, 2002, 50:241-247
7 Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Sem Liv Dis, 2001, 21(3):427~436
8 Friedman, SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000, 275(4):2247~2250
9 Kolch W, Meaningful relationships: The regulation of the Ras/Raf / MEK/ERK pathway by p rotein interactions[J]. Biochem J, 2000, 351:289-305
10 Wilhelm SM, Carter C, Tang L, et al. BAY 4329006 exhibits broad spectrum oral anti2tumor activity and targets the Raf /MEK/ERK pathway and recep tor tyrosine kinases involvedin tumor progression and angiogenesis [J]. Cancer Res, 2004, 64:7099-7109
11 Kolch W, Heidecker G, Kochs G, et al. Pretein kinase C activates Raf-1 by direct phosphorylation. Nature, 1993, 364:249-252
12 Schaap D, Van der Wal J, Howe LR, et al. Adominant-negative mutant of raf block mitogen-activated protein kinase activation by growth factors an d oncogenic p21ras . Biol Clmm, 1993, 268:20232-20236
13 Piazuelo E, Jinenez P, Lanas A. Platelet-derived growth factor and epidermal growth factor play a major role in human colonic fibroblast repair activities[J]. Eur Surg Res, 2000, 32(3):191
14 Roberts PJ, Der CJ. Targeting the Raf-MEK-ERKmitogen -activated protein in kinase cascade for the treatment of cancer. Oncogene, 2007, 26(22): 3291-3310
15 Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/CIP1 expression in human epidermoid carcinoma A431 cells. Cell Single, 2006, 18(2):244-255.
16 Massimo Pinzani, Fabio Marra, Alessandra Caligiuri, et al. Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells. British Journal of Pharmacology, 1996, 119:1117-1124
17 Trakul N, Menard RE, Schade GR, et al. Raf kinaseinhibitory protein regulates Raf-1 but not B-Raf kinase activation. J Biol Chem, 2005, 280(26):24931-24940
18 Park KS, Kim JA, Chai KJ. Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line. Exp Mol Med, 2000, 32(3):120-126
19 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
20 Preaux AM, Mallat A, Rosonbaum J, et al. Pentoxifylline inhibitis growth and collagen synthesis of cultured human hepatic myofibroblast-like cells[J]. J Hepatology, 1997, 26(2): 315
21 Windmeoer C, Gressner AM. Effect of pentoxifylline on the fibrogenic functions of cultured rat liver fat storing cells and myofibroblasts[J]. Biochem Pharmacol, 1996, 51(5): 577
22 Kim KM, Yoon JM, Gwak GY, et al. Bile acid-mediated inducion of cyclooxygenase-2 and Mcl-1 in hepatic stellate cells. Biochem Bilphys Res Commun, 2006, 342(4):1108-13
23 Verma-Gandhu M, Peterson MR. Effect of fetuin, a TGFbeta antagonist and pentoxifylline, a cytokine antagonist on hepatic stellate cellfuction and fibrotic parameters in fibrosis. Eru J Pharmacol, 2007, 572(2-3):220-227
24 Kanno N, Lesage G, Phinizy JL, et al. Stimulation of alpha2-adrenergic receptor inhibits cholangiocarcinomagrowth through modulation of Raf-1 and B-Raf activities. Hepatology, 2002, 35(6):1329-1340
1 Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem, 2000, 275:2247–2250
2 Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-β in hepatic fibrosis. Front Biosci, 2002, 7:793-807
3 Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci, 2002, 7:1720-1726
4 Schmidt EK, Fichelson S, Feller SM. PI3 kinase is important for Ras, MEK and Erk activation of Epo-stimulated human erythroid progenitors. BMC Biol, 2004, 2:7
5 Chong H, Vikis HG, Guan KL. Mechanisms of regulating the Raf kinase family. Cell Singnal, 2003, 15(5):463-469
6 McCubrey JA, Steelman LS, Chappell WH, et al. Roles of the Raf/MEK/ERK pathway in cell growth, malignant transformation and drug resistance. Biochim Biophys Acta, 2007, 1773(8):1263-84
7 Okabe H, Lee SH, Phuchareon J, Albertson DG, et al. A critical role for FBXW8 and MAPK in cyclin D1 degradation and cancer cell proliferation. PloS ONE, 2006, 1:128
8 Stevenson JP, Yao KS, Gallagher M, et al. Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132(CGP 69846A). J Clin Oncol, 1999, 17(7):2227-36
9 Hickey FB, Enqland K, Cotter TG. Bcr-Abl regulates osteopontin transcription via Ras, PI-3K, Apkc, Raf-1, and MEK. J Leukoc Biol, 2005, 78(1):289-300
10 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induces syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
11 Lue H, Kapurniotu A, Fingerle-Rowson G, et al. Rapid and transient activation of the ERK/MAPK signaling pathway by macrophage migration inhibitory factor(MIF) and dependence on JAB1/CSN5 and Src kinase activity. Cell Signal, 2006, 18(5):688-703
12 Beazely MA, Alan JK, Watts VJ. Protein kinase C andepidermal growth factor stimulation of Raf-1 potentiates adenylyl cyclase type 6 activation in intact cells. Mol Pharmacol, 2005, 67(1):250-259
13 Kim SE, Choi KY. EGF reportor is involved in WNT3a-mediated proliferation and motility of NIH3T3 cells via ERK pathway activation. Cell Signal, 2007,19(7): 1554-64
14 Krotum RL, Johnson HJ, Costanzo DL, et al. The molecular scaffold kinase suppressor of Ras is a modifier of RasV12-induced and replicative senescence. Mol Cell Biol, 2006, 26(6):2202-2214
15 Zhou B, Wu LJ, Tashiro S, et al. Activation of exracellular signal-regulated kinase during silibinin-protected, isoprotereno-induce apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent. Acta Pharmacol Sin, 2007, 28(6):803-810
16 Ebisch A, Staber PB, Delavar A, et al. Two transforming C-RAF germ-line mutations identified in patients with therapy-related acute myeloid leukemia. Cancer Res, 2006, 66(7):3401-3408
17 Park KS, Kim JA, Chai KJ. Molecular assembly of mitogen-activated protein kinase module in ras-transformed NIH3T3 cell line. Exp Mol Med, 2000, 32(3):120-126
18 Ellis C, Liu XQ, Anderson D, et al. Tyrosine phosphorylation of GAP and GAP-associated protein in lymphoid and fibroblast cells expressing lck. Oncogene,1991, 6(6):895-901
19 Wang W, Jobbagy Z, Bird TH, et al. Cell signaling through the protein kinases cAMP-dependent protein kinase, protein kinase Cepsilon, and RAF-1 regulates amphotropic murine leukemia virus envelope protein-induced syncytium formation. J Biol Chem, 2005, 280(17):16772-16783
20 Janssen RA, Veenstra KG, Jonasch P, et al. Ras- and Raf-induced down-modulation of non-muscle tropomyosin are MEK-independent. J Biol Chem, 1998, 27:32182-32186
21 Zheng X, Ravatn R, Lin Y, et al. Gene expression of TPA induced differentiation in HL-60 cells by DNA microarray analysis. Nucleic Acids Res, 2002, 30(20):4489-4499
22 Kuo WL, Chung KC, Rosner MR. Differentiation of central nervous system neuronal cells by fibroblast-derived growth factor requires at least two signaling pathway: roles for Ras and Src. Mol Cell Biol, 1997, 17(8):4633-4643
23 Davis JM, Navolanic PM, Weinstein-Oppenheimer CR, et al. Raf-1 and Bcl-2 induce distinct and common pathway that contribute to breast cancer drug resistance. Clin Cancer Res, 2003, 9(3):1161-1170
24 Blaqosklonny MV, Schulte T, Nquyen P, et al. Taxol- induced apoptosis and phosphorylation of Bcl-2 protein involves c-Raf-1 and represents a novel c-Raf-1 signal transduction pathway. Cancer Res, 1996, 56(8):1851-1854
25 Yu C, Rahmani M, Almenara J, et al. Induction of apoptosis in human leukemia cells by the tyrosine kinase inhibitoradaphostin proceeds through a Raf-1/MEK/ERK-and AKT-dependent process. Oncogene, 2004, 23(7):1364-1376
26 Ravi RK, Weber E, McMahon M, et al. Activated and Raf-1 causes growth arrest in human small cell lung cancer cells. J Clin Invest, 1998, 101(1):153-159
27 Dodqe JE, Okano M, Dick F, et al. Inactivation of Dnmt3b in mouse embryonic fibroblasts results in DNA hypomethylation, chromosomal instability, and spontaneous immortalization. J Biol Chem, 2005, 280(18):17986-91
28 Liu AM, Wong YH. Activation of nuclear factor {kappa}B by somatostatin type 2 receptor in pancreatic acinar AR42J cells invoves G{alpha}14 and multiple signaling components: a mechanism requiring protein kinase C, calmodulin -dependent kinaseII, ERK and c-Src. J Biol Chem, 2005, 280(41):34617-25
29 Solit DB, Ivy SP, Kopil C, et al. Phase I trail of 17-allylamino-17-demethoxygeldanamycin in patients with advanced cancer. Clin Cancer Res, 2007, 13(6):1775-82
30 Stancato LF, Silverstein AM, Owens-Grillo JK, et al. The hsp90-binding antibiotic geldanamycin decreases Raf levels and epidermal groeth factor signaling without disrupting formation of signaling complexes or reducing the specific enzymatic activity of Raf kinase. J Biol Chem, 1997, 272(7):4013-4020
31 Yu R, Hebbar V, Kim DW, et al. Resveratrol inhibits phorbol ester and UV-induced activator protein 1 activationby interfering with mitogen-activated protein kinase pathway. Mol Pharmcol, 2001, 60(1):217-224
32 Pinzani M. PDGF and signal transduction in hepatic stellate cells. Front Biosci, 2002, 7:1720-1726
33 Shi MN, Zheng WD, Zhang LJ, et al. Effect of IL-10 on the expression of HSC growth factor in hepatic fibrosis rat. World J Gastroenterol, 2005, 11(31):4788-4793
34 Liu ZM, Huang HS. As2O3-induced c-Src/EGFR/ERK signaling is via Sp1 binding sites to stimulate p21WAF1/ CIP1 expression in human epidermoid carcinoma A431 cells. Cell Single, 2006, 18(2):244-255
35 Reimann T, Hempel U, Krautwald S. Transforming growth factor- beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells.FEBS Letters, 1997, 403(1):57-60
36 Axmann A, Seidel D, Reimann T. Transforming growth factor-beta1-induced activation of the Raf-MEK-MAPK signaling pathway in rat lung fibroblasts via a PKC-dependent mechanism. Biochemical and Biophysical Research Communications, 1998, 249(2):456-460