RhoA/ROCK信号转导通路抑制剂-C3转移酶和法舒地尔抑制大鼠肝星状细胞迁移
|
摘要
肝纤维化(hepatic fibrosis)是机体对慢性肝损伤的创伤-愈合反应,是各种慢性肝病发展至肝硬化的中间环节和前期病变。因此,探讨肝纤维化的发病机制为其治疗提供可靠依据、早期逆转肝纤维化具有重要的临床价值。肝星状细胞(hepatic stellate cell, HSC)在肝纤维化的形成中起关键作用。在肝损伤及各种慢性肝病时,HSC被激活,转换为肌成纤维细胞(myofibroblastic like cells) ,表达α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA),合成各种细胞外基质成分,并且具有舒缩、黏附和定向迁移的能力。各种细胞因子和细胞信号转导途径在HSC活化过程中起着非常重要的作用。
RhoA家族蛋白是细胞骨架肌动蛋白的重要调节分子,并作为信号分子参与众多与细胞骨架有关的细胞信号传导。RhoA主要增加细胞的收缩力,促进粘着斑连接和应力纤维的装配。Rho激酶(Rho-kinase, ROCK)是目前研究最清楚的RhoA下游效应分子,作用于肌球蛋白轻链磷酸酶(myosin light chain phosphatase, MLCP)使其失活,使胞浆内肌球蛋白轻链(myosin light chain, MLC)磷酸化水平升高,肌动-肌球蛋白交联增加,从而促进肌动蛋白微丝骨架的聚合,引起细胞收缩、黏附和迁移。
Rho蛋白与GTP结合后呈激活状态,与GDP结合呈失活状态。溶血磷脂酸(lysophosphatidic acid, LPA)是第一个被发现能激活Rho的物质;C3转移酶(C3 transferase)引起的ADP核糖基化可使Rho蛋白失活;法舒地尔(fasudil)是ROCK抑制剂。近年来有研究证实Rho家族蛋白在细胞迁移中发挥重要作用,Rho信号通路相关的细胞迁移与心血管、肿瘤和纤维化疾病有关。我们以前的研究工作发现整合素(integrin)-黏着斑激酶(focal adhesion kinase, FAK)信号途径参与HSC的迁移过程,黏着斑相关非激酶(focal adhesion related non-kinase, FRNK)对HSC迁移具有抑制作用。对整合素信号通路研究发现,基质不断提供信号给迁移细胞Rho蛋白影响细胞迁移。而RhoA/ROCK信号转导通路对HSC的行为影响尚不清楚。
本课题从活化的HSC细胞生物学基础研究出发,结合整体和离体试验,探讨RhoA/ROCK信号通路在肝纤维化形成中的作用以及对HSC形态、迁移和黏附的影响,为临床肝纤维化的治疗提供理论依据。实验共分三部分。
第一部分:大鼠肝纤维化组织RhoA、p-MLC和α-SMA的动态表达
目的:观察RhoA/ROCK信号转导通路关键信号分子RhoA、磷酸化的肌球蛋白轻链(phosphorylated myosin light chain, p-MLC(Thr18/Ser19))在实验性肝纤维化大鼠不同阶段肝组织中的表达规律,同时观察细胞骨架成分α-SMA在肝纤维化大鼠肝组织中的表达变化,探讨RhoA/ROCK信号转导通路在肝纤维化发生中的作用。
方法:采用胆总管结扎(bile duct ligation, BDL)方法建立大鼠肝纤维化模型;分别与手术后1wk、2wk、3wk、4wk麻醉动物,假手术组4wk麻醉动物,留取肝组织;HE及Masson三色染色观察肝纤维化大鼠的病理组织学变化;用免疫组织化学和Western blot检测RhoA、p-MLC和α-SMA的表达,用逆转录聚合酶链式反应(RT-PCR)检测RhoA的表达。
结果:①肝纤维化大鼠动物模型的建立:HE及Masson三色染色显示:假手术组肝小叶结构完整,肝板排列整齐。模型组随着肝纤维化逐渐发展,出现肝脏广泛结缔组织增生,中央静脉周围出现胶原纤维沉积,肝小叶结构紊乱甚至形成假小叶。②免疫组织化学显示:假手术组大鼠肝脏RhoA呈阴性表达,造模1周汇管区间质细胞呈弱阳性表达,2～3周后,着色细胞逐渐增多,在汇管区、纤维间隔、肝窦周围及增生的胆管周围细胞出现棕黄色着色,第4周着色面积进一步扩大,少数细胞可见细胞质阳性染色;造模1wk、2 wk、3 wk、4 wk大鼠肝组织内RhoA阳性分布面积为5.32%±0.41%,13.43%±2.13%,22.14%±3.22%,31.48%±2.31%,均显著高于对照组(0.23%±0.14%),P<0.05。免疫组织化学显示假手术组大鼠肝脏α-SMA在血管壁平滑肌细胞有弱阳性表达,随着纤维化进展,阳性着色细胞逐渐增多,汇管区、Disse间隙、纤维间隔及增生的胆管周围细胞出现棕黄色着色。正常大鼠肝组织中p-MLC仅在血管壁平滑肌细胞有弱阳性表达;随着肝纤维化的进展,血管壁表达增强,尤其以静脉血管壁明显,并在肝窦周围出现阳性表达细胞;造模1wk、2 wk、3 wk、4 wk大鼠肝组织内p-MLC阳性分布面积为13.47%±1.02%,20.05%±2.22%,28.31%±2.05%,35.41%±3.08%,与假手术组(2.01%±0.51%)比较,均有显著性差异,p<0.05。③假手术组、BDL组在503bp处出现了RhoA基因的特异性条带,375 bp处出现了内参照β-actin的条带。通过对电泳条带光密度扫描分析,假手术组大鼠肝脏有弱RhoA基因表达(43.23%±5.03%);与假手术组比较,BDL组随着造模时间延长,RhoA基因表达逐渐上调;造模1wk、2 wk、3 wk、4 wk基因表达分别为71.22%±1.06%,78.49%±2.24%,81.31%±3.21%,96.38%±2.12%,与假手术组比较均有统计学意义,p<0.05;造模4 wk RhoA mRNA表达是假手术组的2.23倍。④Western blot在24kD的位置上出现了阳性杂交带,为RhoA的蛋白表达。从杂交信号的强度可知,假手术组大鼠肝组织中有微弱RhoA蛋白表达,随着造模时间延长,蛋白表达逐渐增加,1～4 wk分别增加了4.34倍、9.95倍、19.85倍、22.12倍。Western blot分析显示在18 kD的位置上出现阳性杂交带为p-MLC的蛋白表达,从杂交信号的强度可知,假手术组大鼠肝组织中有少量p-MLC表达,随着肝纤维化加重表达逐渐增加,4 wk达高峰,是1 wk的5.00倍,假手术组的43.16倍。在43kD的位置上出现的阳性杂交带为α-SMA蛋白表达,造模1～4 wk表达呈上升趋势, 1～4 wk蛋白表达分别是假手术组的2.37倍,7.08倍,13.14倍和17.62倍,与对照组比较均有显著性差异,P<0.05。RhoA、p-MLC分别与α-SMA呈显著性正相关(r=0.98,P<0.01,r=0.976,P<0.01)。
结论:肝纤维化形成过程中RhoA、p-MLC和α-SMA表达逐渐增加,提示RhoA/ROCK信号转导通路和细胞骨架的变化在肝纤维化形成与发展中起一定作用。
第二部分:C3转移酶和法舒地尔对大鼠肝星状细胞黏附和迁移的影响
目的:观察RhoA信号通路激动剂LPA、RhoA抑制剂-C3转移酶和ROCK抑制剂-fasudil对HSC迁移和黏附的影响。
方法:本研究应用体外细胞培养技术,采用甲苯胺蓝法了解LPA诱导HSC黏附的情况和C3转移酶及fasudil对HSC黏附的抑制作用;采用改良的Boyden双腔系统了解LPA对HSC迁移的诱导作用和C3转移酶及fasudil对HSC迁移的抑制作用;应用激光共聚焦显微镜观察C3转移酶及fasudil对HSC形态的影响。
结果:①共聚焦显微镜观察,正常活化的HSC呈星状形态,伸展良好。当C3转移酶与HSC共同培养24 h后,细胞变园,呈收缩形态。不同浓度fasudil作用于HSC后,细胞伴有多个树突样隆起,呈现无力状态。②LPA、C3转移酶和fasudil分别与HSC培养24 h后,与对照组比较,LPA使细胞增殖活性增加了12.24%;而C3使细胞增殖活性下降了73.26%;C3抑制了LPA诱导的HSC增殖,与对照组比较下降了46.51%。fasudil与HSC共同培养后,未见明显不良反应,fasudil浓度增加后,细胞增殖活性降低,与对照组比较,分别下降了11.6%,26.7%,37.2%,51.2%。细胞增殖活性与fasudil浓度呈负相关(r= -0.9489, P <0.01)。③与对照组相比,LPA使HSC黏附率增加25.58% ;C3转移酶使HSC黏附率下降50.00%;而C3转移酶对LPA诱导的HSC黏附有抑制作用,黏附率下降了40.63 %;含有C3转移酶的两组相比无统计学差异,P>0.05,二者与对照组比较,差异均有显著性,P<0.05。fasudil抑制了LPA诱导的HSC黏附,随着干预浓度增加,HSC黏附率下降;与对照组相比,fasudil 12.5、25、50、100μmol/L四种浓度的黏附抑制率分别为13.31%,36.72%,41.71%,62.43%,与对照组比较均有统计学意义,P<0.05。④对照组细胞迁移数为4.3×103,LPA使HSC迁移细胞数明显增多(25.8×103),为对照组的6倍;C3转移酶与HSC共同培养24 h后,迁移细胞数量明显减少(2.1×103),与对照组比较下降了51.16%;而C3转移酶对LPA诱导的HSC迁移有抑制作用(2.8×103),与单纯C3转移酶与HSC共同培养24 h比较无统计学意义,P>0.05;与对照组比较差异有显著性,P<0.05。fasudil抑制了LPA诱导的HSC迁移,随着fasudil干预浓度增加,细胞迁移数量下降,100μmol/L的fasudil组比单纯LPA组HSC迁移数量下降了80%。
结论:RhoA/ROCK信号转导通路抑制剂C3转移酶和fasudil对LPA诱导的HSC增殖、黏附和迁移具有抑制作用。
第三部分:RhoA/ROCK信号转导通路对大鼠HSC迁移影响的机制研究
目的:探讨RhoA/ROCK信号转导通路抑制剂对LPA诱导的HSC黏附、迁移抑制作用的分子机制。
方法:本研究应用体外细胞培养技术,采用Western blot和RT-PCR共扩增技术检测RhoA、p-MLC和α-SMA的表达情况。用激光扫描共聚焦显微镜(LSCM)检验细胞[Ca2+]i
结果:①Western blot结果显示在24kD的位置上出现了阳性杂交带,为RhoA的蛋白表达,经光密度测定分析:经LPA诱导后,HSC RhoA蛋白表达量(4326.48±213.32)明显高于对照组(2335.54±211.81),增加了46.02 %。经统计学处理有显著性差异,P<0.01。②C3转移酶与HSC共同培养24 h后,经光密度测定分析显示:HSC RhoA蛋白表达(621.25±33.54)明显下降,与对照组(2335.54±211.81)比较,下降了73.40%;而C3抑制了LPA诱导的HSC RhoA蛋白表达(789.35±56.71),与对照组比较下降了66.21%。③RT-PCR共扩增检测RhoA和内参照β-actin基因表达,在503bp处出现了特异性的RhoA基因条带,375bp处出现了内参照β-actin的特异性片段,扫描结果分析显示:与对照组(0.25±0.01)比较,LPA使HSC RhoA mRNA表达(0.48±0.02)增加47.92%,经统计学分析有显著性差异,P<0.01。④通过测定RhoA mRNA的变化,能够从基因水平反映出C3转移酶对HSC RhoA蛋白合成的影响。C3转移酶与HSC共同培养24 h后,通过对电泳条带的光密度扫描显示:C3转移酶使HSC RhoA mRNA表达(0.08±0.01)明显下降,较对照组(0.25±0.01)比较下降了68.00%。而C3转移酶抑制了LPA诱导的HSC RhoA mRNA表达(0.09±0.01),较对照组(0.25±0.01)比较下降了64.00%;含有C3转移酶的两组相比无统计学差异,P>0.05,二者与对照组比较,差异均有显著性,P<0.05。⑤LPA增加了HSC p-MLC、α-SMA蛋白表达。Western blot结果分析显示:对照组p-MLC和α-SMA的蛋白表达分别为(1675.24±28.22),(1986.32±78.62),LPA诱导后,p-MLC和α-SMA的蛋白表达分别为(2532.29±283.24),(3143.35±278.61),与对照组比较, p-MLC和α-SMA蛋白表达分别增加了33.85%,36.81%。⑥Western blot结果分析显示:C3转移酶与HSC共同培养24 h后,与对照组比较,HSC p-MLC和α-SMA蛋白表达分别下降70.45%,68.34%;C3转移酶同样抑制LPA诱导的HSC p-MLC和α-SMA蛋白表达,与对照组比较, HSC p-MLC和α-SMA蛋白表达分别下降63.24%,61.83%。⑦不同浓度fasudil与HSC共同培养后,经光密度测定分析,对照组α-SMA蛋白表达量为(2336.53±329.51),fasudil抑制了LPA诱导HSCα-SMA蛋白表达, 12.5、25、50和100μmol/L四种浓度α-SMA蛋白表达量分别为(2015.78±231.56),(1865.37±203.12),(1549.64±187.13),(1364.72±140.88),与对照组比较分别下降了13.74%,20.16%,33.69%,41.61%;fasudil对LPA诱导HSC p-MLC(Thr18/Ser19)的蛋白表达有抑制作用,与对照组(2687.32±320.14)比较明显下降,fasudil 12.5、25、50和100μmol/L四种浓度p-MLC的蛋白表达量分别为(2105.49±305.73),(1783.57±216.83),(1544.31±158.67)和(1042.83±146.09),与对照组比较下降了21.66%,33.64%,42.54%和61.22%。fasudil干预浓度越高,p-MLC和α-SMA蛋白表达下降趋势越明显。⑧与对照组比较,C3转移酶与HSC共同培育24 h后,[Ca2+]i荧光强度轻微下降了2.6%,两者相比无显著性差异,P>0.05;而C3对LPA诱导HSC [Ca2+]i也无明显影响,与对照组比较,差异无显著性,P>0.05。⑨fasudil与HSC共同培育24 h后,对HSC [Ca2+]i进行检测,fasudil 12.5、25、50、100μmol/L四种浓度的HSC的荧光强度基本一致,各组之间以及与对照组比较,无统计学差异,P>0.05。
结论:活化的HSC表达RhoA/ROCK信号通路关键信号分子RhoA和p-MLC。RhoA抑制剂- C3转移酶对HSC RhoA蛋白表达有明显抑制作用,并且从基因水平抑制其合成,同时抑制了RhoA/ROCK下游信号分子p-MLC的表达,对细胞骨架成分α-SMA的表达有抑制作用; ROCK抑制剂-fasudil对p-MLC和α-SMA的表达均有抑制作用,并呈现浓度依赖关系。应用RhoA/ROCK信号通路抑制剂干预HSC后,胞浆[Ca2+]i没有发生变化,说明它是非Ca2+依赖的信号通路。
Actually, hepatic fibrosis(HF) is a repairing process of the body towards the injury. Now it is widely accepted that HF is the inevitable and preexisting stage for lots of chronic hepatic disease developing into liver cirrhosis(LC)eventually. Investigation of mechanism of liver fibrosis and reversing the disease are very important for clinical treatment. Hepatic stellate cell(HSC)plays a key role in the pathogenesis of HF. Following chronic liver injury, HSC undergos transdifferentiation to an activated myofibroblastic phenotype with expression ofα-smooth muscle actin (α-SMA) and synthesizes various extracellular matrix protein components, which has the ability of contraction, adhesion and migration. All sorts of cytokines and signal transduction pathway exert very important roles in the activation of HSC.
RhoA family proteins are important regulatory molecules of actin cytoskeleton, which take part in all kinds of cell signal transduction associated with cytoskeleton. RhoA mainly increases cell contractility by promoting the assembly of focal adhesion and actin stress fibers. Rho-kinase(ROCK)is now the most distinct downstream signal molecule of RhoA, which can increase the level of myosin light chain phosphorylation though inactivation of MLC phosphatase, cause cross linking of actin and myosin, which promotes actin microfilament polymerization and results in cell contraction, migration and adhesion.
RhoA proteins generally cycle between an active GTP-bound, conformation and an inactive GDP-bound conformation. Lysophosphatidic acid (LPA) is first identifyed to activate Rho; C3 transferase can inactivate RhoA by making ribosylation of ADP; and fasudil is an inhibitor of ROCK. Recent years some studies have confirmed that Rho family proteins play potent roles in cell migration. Cell migration related with RhoA/ROCK signaling pathways has correlation with cardiovascular diseases, tumours and fibrotic diseases. In our past study, integrin-FAK signal pathway was found to take part in the HSC migration process, Focal adhesion related non-kinase (FRNK) inhibited the HSC migration. Studies on integrin signalling suggested that the substratum continuously feed signals to Rho proteins in migrating cells to influence migration rate. But RhoA/ROCK signaling pathways how to affect HSC migration process has not been proved.
This research will focus on the basis of activation of HSC, through experiments in vivo and in vitro, discuss the roles of RhoA/ROCK signaling pathways in the hepatic fibrogenesis and the effects on the contraction, adhesion and migration of HSC. We hope that it would provide theoretical basis for therapy of liver fibrosis. The experiments contain three parts as below:
Part 1: The Dynamic Expressions of RhoA、p-MLC andα-SMA during Hepatic Fibrogenesis in Rats
Objective: To observe the expression changes of the key signaling molecules RhoA and phosphorylated myosin light chain belonged to RhoA/ROCK signaling pathways in rat liver tissue during hepatic fibrogenesis, meanwhile, to observe the expression ofα-SMA which represents cytoskeleton element and to discuss the role of Rho/ROCK signaling pathways in the hepatic fibrogenesis.
Methods:A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the formation of liver fibrosis. Liver tissues were obtained at 1st、2nd、3rd and 4th week, respectively, in the operation group, and at 4th week in the sham operation group. The liver histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson’s trichrome method. Western blotting and Immunohistochemistry were used to determine the expressions of RhoA、p-MLC andα-SMA. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of RhoA mRNA.
Results:①Building of liver fibrosis model: The results of Hematoxylin and eosin staining and Masson's trichrome methods showed that in sham-operated group, the structure of liver lobule was integrin and the array of the liver flank was in order. But in model groups, the connective tissue hyperplasiaed broadly and the collagen fiber deposited around the central vein. The structure of the lobule was disturbed even the pseudo-lobules were formed.②The outcome of immunohistochemistry showed that the expression of RhoA protein was negative. After 1st week, there becomed some spot-like staining mainly in the interstitial cells in portal tract, the positive cells of RhoA increased a lot after BDL 2nd and 3rd week, they were mainly situated in portal ducts, fiber septa, perisinusoidal cells and around the bile ducts, the staining area enlarged at 4th week. The positive areas of RhoA in the rat livers in model groups at week 1 to 4 (5.32%±0.41%,13.43%±2.13%,22.14%±3.22%,31.48%±2.31%) were all larger than that in control group(0.23%±0.14%), P<0.05; The expression ofα-SMA was weak located in the smooth muscle cells of vessel wall, with the development of liver fibrosis, the expression ofα-SMA increased, there was brown staining in portal ducts, disse space, fiber septa, and around the bile ducts. There was weak staining for p-MLC in vessel walls in the sham operation group, with the development of hepatic fibrosis, the expression of p-MLC was increased especially in the walls of venules, and there was positive staining in the perisinusoidal cells. The positive areas of p-MLC in the rat livers in model groups at week 1 to 4 (13.47%±1.02%,20.05%±2.22%,28.31%±2.05%,35.41%±3.08%) were all larger than that in control group(2.01%±0.51%),and the difference was significant, P<0.05.③By RT-PCR analysis, RhoA mRNA appeared in the liver of sham-operated group (43%±5%), and was upregulated after bile duct ligation, during the liver fibrosis, the expression of RhoA mRNA was 71.22%±1.06%,78.49%±2.24%,81.31%±3.21%,96.38%±2.12%, respectively, after BDL1-4 week, compared with the sham-operated group, and the difference was significant, P<0.05. The expression of RhoA mRNA reached the peak value at the 4th week, which was 2.23 times of that of the sham-operated group.④RhoA, the molecular weight is 24 kD, Western blot analysis showed that in the sham-operated group, the RhoA was expressed a little, but with the development of liver fibrosis, the expression of it increased in model group, the expression increased 4.34, 9.95, 19.85, 22.12 times than that of the sham-operated group. p-MLC , the molecular weight is 18 kD, Western blot analysis showed that the p-MLC was expressed weak in the sham-operated group, The expression level of it was highest in the fourth week and 5 times of that of 1 wk and 43.16 times of that of the sham-operated group. With the development of liver fibrosis, the expression ofα-SMA in model group at week 1 to 4 increased 2.37, 7.08, 13.14, 17.62 times than that of the sham-operated group, the difference was significant, P<0.05. RhoA and p-MLC correlated withα-SMA positively, respectively (r=0.981, P<0.01, r=0.976, P<0.01).
Conclusions: In liver fibrogenesis, the expression of RhoA、p-MLC andα-SMA increased obviously. It suggested that RhoA/ROCK signaling pathways and cytoskeleton changes play a pivotal role in the formation and development of liver fibrosis.
Part 2: Effects of RhoA/ROCK signaling pathway inhibitors-C3 transferase and fasudil on the rat HSC adhesion and migration Objective: To observe the effects of RhoA/ROCK signaling pathway agonist-LPA, RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil on the HSC adhesion and migration.
Methods: The adhesive effects of LPA, C3 transferase and fasudil on HSC were examined by toluidine blue colorimetric assay,and the effects of migration were evaluated by improved Boyden chamber. Morphological alteration of HSC treated with C3 transferase and fasudil was observed with laser scanning confocal microscopy (LSCM).
Results:①Normal activited HSC was well spread. By contrast, HSC treated with C3 transferase changed to round and retractile shape, HSC treated with fasudil (50μM) changed to an elongated morphology with prominent dendritic processes, and appeared powerless shape.②Compared with the control group, LPA increased the HSC proliferation, the increation rate was 12.24%, while C3 transferase inhibited the HSC proliferation, the inhibition rate was 73.26%. C3 transferase inhibited LPA-induced HSC proliferation, compared with the control group, the inhibition rate was 46.51%. Fasudil inhibited HSC proliferation, the inhibition rates were 11.6%, 26.7%, 37.2%, 51.2%, at 12.5, 25, 50 and 100μmol/L, respectively. HSC proliferation correlated with fasudil concention negatively (r=-0.949, P<0.01).③compared with the control group, the adhesion rate increased 25.58% in LPA group, and decreased 50% in C3 group. While C3 transferase inhibited LPA-induced HSC adhesion, the adhesion rate decreased 40.63%. Fasudil inhibited LPA-induced HSC adhesion, and it was in concention-dependent manner; compared with the control group. The adhesive inhibition rates of fasudil 12.5, 25, 50, 100μmol/L were 13.31%, 36.72%, 41.71%, 62.43%, respectively. There was significant difference among the different groups, P<0.05.④LPA induced HSC migration, the cell number of migrating to the bottom chamber increased 6 times in LPA group compared with that of the control group. C3 transferase inhibited HSC migration, simultaneously, inhibited LPA-induced HSC migration, compared with the control group, and there was statistic difference, P<0.01. Fasudil inhibited LPA induced HSC migration, and it was in concention-dependent manner; the cell number of migrating of fasudil 100μmol/L decreased 80% compared with that of the LPA group.
Conclusions: RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil inhibited the LPA-induced HSC proliferation, adhesion and migration. Part 3: The molecular mechanism of the inhibiting migration of RhoA/ROCK signaling pathway inhibitors on rat HSC
Objective: To explore the molecular mechanism of RhoA/ROCK signaling pathway inhibitors inhibiting HSC migration induced by LPA.
Methods: HSC was cultured in vitro, and the expressions of RhoA, p-MLC andα-SMA were assessed using Western blot and RT-PCR assay. The intracellular free calcium was observed with laser scanning confocal microscopy (LSCM).
Results:①Western blot showed: RhoA expression (4326.48±213.32) increased obviously after cultivated with LPA, compared with the control group (2335.54±211.81), up to 46.02%,P<0.01.②When HSC was cultured with C3 transferase for 24 h, The protein expression of RhoA decreased obviously, compared with the control group, down to 73.40 %, meanwhile C3 transferase inhibited LPA-induced HSC RhoA expression, compared with the control group, down to 66.21%.③HSC was induced by LPA for 24 h, the expression of RhoA mRNA was examined by RT-PCR. Compared with the control group, the expression of RhoA mRNA increased 47.92%.④When HSC was cultured with C3 for 24 h, the expression of RhoA mRNA decreased obviously, Compared with the control group, it reduced 68 %. C3 transferase inhibited LPA-induced expression of HSC RhoA mRNA, compared with the control group, it reduced 64 %.⑤LPA increased the protein expressions of p-MLC andα-SMA, compared with the sham-operated group, it increased 33.85%, 36.81%, respectively.⑥Western blot analysis showed: when HSC was cultured with C3 transferase for 24h, the protein expressions of p-MLC andα-SMA decreased 70.45%, 68.34%, respectively, than that in the control group. While C3 transferase inhibited LPA-induced protein expressions of p-MLC andα-SMA, compared with the control group, they decreased 63.24%, 61.83%, respectively.⑦Western blot showed: the expression number ofα-SMA was 2336.53±329.51 in the control group. The protein expressions ofα-SMA in the fasudil 12.5、25、50、100μmol/L group were 2015.78±231.56, 1865.37±203.12, 1549.64±187.13 and 1364.72±140.88, respectively, compared with the control group, it reduced 13.74%, 20.16%, 33.69%, 41.61%, respectively. Fasudil inhibited the expression of p-MLC, the protein expression number of p-MLC in the fasudil 12.5、25、50、100μmol/L group were 2105.49±305.73, 1783.57±216.83, 1544.31±158.67, 1042.83±146.09, respectively, so it reduced 21.66%, 33.64%, 42.54%, 61.22% than that in the control group. The expressions ofα-SMA and p-MLC were in fasudil concention-dependent manners.⑧Compared with HSC in the control group, calcium fluorescence intensity of HSC treated by C3 for 24 h mildly decreased 2.6%, there was no statistic difference between them, P>0.05; C3 transferase had no effect on LPA-induced HSC calcium fluorescence intensity, compared with the control group, there was no significant difference, P>0.05.⑨HSC was treated with fasudil for 24 h, calcium fluorescence intensities of fasudil 12.5、25、50、100μmol/L group were the same on the whole. The results showed that there was no difference among 4 different groups, P>0.05, and compared with the control group, there was no statistic difference, P>0.05.
Conclusions: Activated HSC expressed the key signaling molecules RhoA and p-MLC belonged to RhoA/ROCK signaling pathways. RhoA inhibitor-C3 transferase inhibited the expression and transcription of RhoA, and the expression of downstream factor p-MLC was inhibited in the meantime, the expression ofα-SMA was also inhibited. ROCK inhibitor-fasudil inhibited the expressions ofα-SMA and p-MLC, and they were in concention manners. RhoA/ROCK signaling pathway inhibitors had no effect on intracellular free calcium. It belonged to Ca2+-independent signaling pathway.
RhoA家族蛋白是细胞骨架肌动蛋白的重要调节分子,并作为信号分子参与众多与细胞骨架有关的细胞信号传导。RhoA主要增加细胞的收缩力,促进粘着斑连接和应力纤维的装配。Rho激酶(Rho-kinase, ROCK)是目前研究最清楚的RhoA下游效应分子,作用于肌球蛋白轻链磷酸酶(myosin light chain phosphatase, MLCP)使其失活,使胞浆内肌球蛋白轻链(myosin light chain, MLC)磷酸化水平升高,肌动-肌球蛋白交联增加,从而促进肌动蛋白微丝骨架的聚合,引起细胞收缩、黏附和迁移。
Rho蛋白与GTP结合后呈激活状态,与GDP结合呈失活状态。溶血磷脂酸(lysophosphatidic acid, LPA)是第一个被发现能激活Rho的物质;C3转移酶(C3 transferase)引起的ADP核糖基化可使Rho蛋白失活;法舒地尔(fasudil)是ROCK抑制剂。近年来有研究证实Rho家族蛋白在细胞迁移中发挥重要作用,Rho信号通路相关的细胞迁移与心血管、肿瘤和纤维化疾病有关。我们以前的研究工作发现整合素(integrin)-黏着斑激酶(focal adhesion kinase, FAK)信号途径参与HSC的迁移过程,黏着斑相关非激酶(focal adhesion related non-kinase, FRNK)对HSC迁移具有抑制作用。对整合素信号通路研究发现,基质不断提供信号给迁移细胞Rho蛋白影响细胞迁移。而RhoA/ROCK信号转导通路对HSC的行为影响尚不清楚。
本课题从活化的HSC细胞生物学基础研究出发,结合整体和离体试验,探讨RhoA/ROCK信号通路在肝纤维化形成中的作用以及对HSC形态、迁移和黏附的影响,为临床肝纤维化的治疗提供理论依据。实验共分三部分。
第一部分:大鼠肝纤维化组织RhoA、p-MLC和α-SMA的动态表达
目的:观察RhoA/ROCK信号转导通路关键信号分子RhoA、磷酸化的肌球蛋白轻链(phosphorylated myosin light chain, p-MLC(Thr18/Ser19))在实验性肝纤维化大鼠不同阶段肝组织中的表达规律,同时观察细胞骨架成分α-SMA在肝纤维化大鼠肝组织中的表达变化,探讨RhoA/ROCK信号转导通路在肝纤维化发生中的作用。
方法:采用胆总管结扎(bile duct ligation, BDL)方法建立大鼠肝纤维化模型;分别与手术后1wk、2wk、3wk、4wk麻醉动物,假手术组4wk麻醉动物,留取肝组织;HE及Masson三色染色观察肝纤维化大鼠的病理组织学变化;用免疫组织化学和Western blot检测RhoA、p-MLC和α-SMA的表达,用逆转录聚合酶链式反应(RT-PCR)检测RhoA的表达。
结果:①肝纤维化大鼠动物模型的建立:HE及Masson三色染色显示:假手术组肝小叶结构完整,肝板排列整齐。模型组随着肝纤维化逐渐发展,出现肝脏广泛结缔组织增生,中央静脉周围出现胶原纤维沉积,肝小叶结构紊乱甚至形成假小叶。②免疫组织化学显示:假手术组大鼠肝脏RhoA呈阴性表达,造模1周汇管区间质细胞呈弱阳性表达,2～3周后,着色细胞逐渐增多,在汇管区、纤维间隔、肝窦周围及增生的胆管周围细胞出现棕黄色着色,第4周着色面积进一步扩大,少数细胞可见细胞质阳性染色;造模1wk、2 wk、3 wk、4 wk大鼠肝组织内RhoA阳性分布面积为5.32%±0.41%,13.43%±2.13%,22.14%±3.22%,31.48%±2.31%,均显著高于对照组(0.23%±0.14%),P<0.05。免疫组织化学显示假手术组大鼠肝脏α-SMA在血管壁平滑肌细胞有弱阳性表达,随着纤维化进展,阳性着色细胞逐渐增多,汇管区、Disse间隙、纤维间隔及增生的胆管周围细胞出现棕黄色着色。正常大鼠肝组织中p-MLC仅在血管壁平滑肌细胞有弱阳性表达;随着肝纤维化的进展,血管壁表达增强,尤其以静脉血管壁明显,并在肝窦周围出现阳性表达细胞;造模1wk、2 wk、3 wk、4 wk大鼠肝组织内p-MLC阳性分布面积为13.47%±1.02%,20.05%±2.22%,28.31%±2.05%,35.41%±3.08%,与假手术组(2.01%±0.51%)比较,均有显著性差异,p<0.05。③假手术组、BDL组在503bp处出现了RhoA基因的特异性条带,375 bp处出现了内参照β-actin的条带。通过对电泳条带光密度扫描分析,假手术组大鼠肝脏有弱RhoA基因表达(43.23%±5.03%);与假手术组比较,BDL组随着造模时间延长,RhoA基因表达逐渐上调;造模1wk、2 wk、3 wk、4 wk基因表达分别为71.22%±1.06%,78.49%±2.24%,81.31%±3.21%,96.38%±2.12%,与假手术组比较均有统计学意义,p<0.05;造模4 wk RhoA mRNA表达是假手术组的2.23倍。④Western blot在24kD的位置上出现了阳性杂交带,为RhoA的蛋白表达。从杂交信号的强度可知,假手术组大鼠肝组织中有微弱RhoA蛋白表达,随着造模时间延长,蛋白表达逐渐增加,1～4 wk分别增加了4.34倍、9.95倍、19.85倍、22.12倍。Western blot分析显示在18 kD的位置上出现阳性杂交带为p-MLC的蛋白表达,从杂交信号的强度可知,假手术组大鼠肝组织中有少量p-MLC表达,随着肝纤维化加重表达逐渐增加,4 wk达高峰,是1 wk的5.00倍,假手术组的43.16倍。在43kD的位置上出现的阳性杂交带为α-SMA蛋白表达,造模1～4 wk表达呈上升趋势, 1～4 wk蛋白表达分别是假手术组的2.37倍,7.08倍,13.14倍和17.62倍,与对照组比较均有显著性差异,P<0.05。RhoA、p-MLC分别与α-SMA呈显著性正相关(r=0.98,P<0.01,r=0.976,P<0.01)。
结论:肝纤维化形成过程中RhoA、p-MLC和α-SMA表达逐渐增加,提示RhoA/ROCK信号转导通路和细胞骨架的变化在肝纤维化形成与发展中起一定作用。
第二部分:C3转移酶和法舒地尔对大鼠肝星状细胞黏附和迁移的影响
目的:观察RhoA信号通路激动剂LPA、RhoA抑制剂-C3转移酶和ROCK抑制剂-fasudil对HSC迁移和黏附的影响。
方法:本研究应用体外细胞培养技术,采用甲苯胺蓝法了解LPA诱导HSC黏附的情况和C3转移酶及fasudil对HSC黏附的抑制作用;采用改良的Boyden双腔系统了解LPA对HSC迁移的诱导作用和C3转移酶及fasudil对HSC迁移的抑制作用;应用激光共聚焦显微镜观察C3转移酶及fasudil对HSC形态的影响。
结果:①共聚焦显微镜观察,正常活化的HSC呈星状形态,伸展良好。当C3转移酶与HSC共同培养24 h后,细胞变园,呈收缩形态。不同浓度fasudil作用于HSC后,细胞伴有多个树突样隆起,呈现无力状态。②LPA、C3转移酶和fasudil分别与HSC培养24 h后,与对照组比较,LPA使细胞增殖活性增加了12.24%;而C3使细胞增殖活性下降了73.26%;C3抑制了LPA诱导的HSC增殖,与对照组比较下降了46.51%。fasudil与HSC共同培养后,未见明显不良反应,fasudil浓度增加后,细胞增殖活性降低,与对照组比较,分别下降了11.6%,26.7%,37.2%,51.2%。细胞增殖活性与fasudil浓度呈负相关(r= -0.9489, P <0.01)。③与对照组相比,LPA使HSC黏附率增加25.58% ;C3转移酶使HSC黏附率下降50.00%;而C3转移酶对LPA诱导的HSC黏附有抑制作用,黏附率下降了40.63 %;含有C3转移酶的两组相比无统计学差异,P>0.05,二者与对照组比较,差异均有显著性,P<0.05。fasudil抑制了LPA诱导的HSC黏附,随着干预浓度增加,HSC黏附率下降;与对照组相比,fasudil 12.5、25、50、100μmol/L四种浓度的黏附抑制率分别为13.31%,36.72%,41.71%,62.43%,与对照组比较均有统计学意义,P<0.05。④对照组细胞迁移数为4.3×103,LPA使HSC迁移细胞数明显增多(25.8×103),为对照组的6倍;C3转移酶与HSC共同培养24 h后,迁移细胞数量明显减少(2.1×103),与对照组比较下降了51.16%;而C3转移酶对LPA诱导的HSC迁移有抑制作用(2.8×103),与单纯C3转移酶与HSC共同培养24 h比较无统计学意义,P>0.05;与对照组比较差异有显著性,P<0.05。fasudil抑制了LPA诱导的HSC迁移,随着fasudil干预浓度增加,细胞迁移数量下降,100μmol/L的fasudil组比单纯LPA组HSC迁移数量下降了80%。
结论:RhoA/ROCK信号转导通路抑制剂C3转移酶和fasudil对LPA诱导的HSC增殖、黏附和迁移具有抑制作用。
第三部分:RhoA/ROCK信号转导通路对大鼠HSC迁移影响的机制研究
目的:探讨RhoA/ROCK信号转导通路抑制剂对LPA诱导的HSC黏附、迁移抑制作用的分子机制。
方法:本研究应用体外细胞培养技术,采用Western blot和RT-PCR共扩增技术检测RhoA、p-MLC和α-SMA的表达情况。用激光扫描共聚焦显微镜(LSCM)检验细胞[Ca2+]i
结果:①Western blot结果显示在24kD的位置上出现了阳性杂交带,为RhoA的蛋白表达,经光密度测定分析:经LPA诱导后,HSC RhoA蛋白表达量(4326.48±213.32)明显高于对照组(2335.54±211.81),增加了46.02 %。经统计学处理有显著性差异,P<0.01。②C3转移酶与HSC共同培养24 h后,经光密度测定分析显示:HSC RhoA蛋白表达(621.25±33.54)明显下降,与对照组(2335.54±211.81)比较,下降了73.40%;而C3抑制了LPA诱导的HSC RhoA蛋白表达(789.35±56.71),与对照组比较下降了66.21%。③RT-PCR共扩增检测RhoA和内参照β-actin基因表达,在503bp处出现了特异性的RhoA基因条带,375bp处出现了内参照β-actin的特异性片段,扫描结果分析显示:与对照组(0.25±0.01)比较,LPA使HSC RhoA mRNA表达(0.48±0.02)增加47.92%,经统计学分析有显著性差异,P<0.01。④通过测定RhoA mRNA的变化,能够从基因水平反映出C3转移酶对HSC RhoA蛋白合成的影响。C3转移酶与HSC共同培养24 h后,通过对电泳条带的光密度扫描显示:C3转移酶使HSC RhoA mRNA表达(0.08±0.01)明显下降,较对照组(0.25±0.01)比较下降了68.00%。而C3转移酶抑制了LPA诱导的HSC RhoA mRNA表达(0.09±0.01),较对照组(0.25±0.01)比较下降了64.00%;含有C3转移酶的两组相比无统计学差异,P>0.05,二者与对照组比较,差异均有显著性,P<0.05。⑤LPA增加了HSC p-MLC、α-SMA蛋白表达。Western blot结果分析显示:对照组p-MLC和α-SMA的蛋白表达分别为(1675.24±28.22),(1986.32±78.62),LPA诱导后,p-MLC和α-SMA的蛋白表达分别为(2532.29±283.24),(3143.35±278.61),与对照组比较, p-MLC和α-SMA蛋白表达分别增加了33.85%,36.81%。⑥Western blot结果分析显示:C3转移酶与HSC共同培养24 h后,与对照组比较,HSC p-MLC和α-SMA蛋白表达分别下降70.45%,68.34%;C3转移酶同样抑制LPA诱导的HSC p-MLC和α-SMA蛋白表达,与对照组比较, HSC p-MLC和α-SMA蛋白表达分别下降63.24%,61.83%。⑦不同浓度fasudil与HSC共同培养后,经光密度测定分析,对照组α-SMA蛋白表达量为(2336.53±329.51),fasudil抑制了LPA诱导HSCα-SMA蛋白表达, 12.5、25、50和100μmol/L四种浓度α-SMA蛋白表达量分别为(2015.78±231.56),(1865.37±203.12),(1549.64±187.13),(1364.72±140.88),与对照组比较分别下降了13.74%,20.16%,33.69%,41.61%;fasudil对LPA诱导HSC p-MLC(Thr18/Ser19)的蛋白表达有抑制作用,与对照组(2687.32±320.14)比较明显下降,fasudil 12.5、25、50和100μmol/L四种浓度p-MLC的蛋白表达量分别为(2105.49±305.73),(1783.57±216.83),(1544.31±158.67)和(1042.83±146.09),与对照组比较下降了21.66%,33.64%,42.54%和61.22%。fasudil干预浓度越高,p-MLC和α-SMA蛋白表达下降趋势越明显。⑧与对照组比较,C3转移酶与HSC共同培育24 h后,[Ca2+]i荧光强度轻微下降了2.6%,两者相比无显著性差异,P>0.05;而C3对LPA诱导HSC [Ca2+]i也无明显影响,与对照组比较,差异无显著性,P>0.05。⑨fasudil与HSC共同培育24 h后,对HSC [Ca2+]i进行检测,fasudil 12.5、25、50、100μmol/L四种浓度的HSC的荧光强度基本一致,各组之间以及与对照组比较,无统计学差异,P>0.05。
结论:活化的HSC表达RhoA/ROCK信号通路关键信号分子RhoA和p-MLC。RhoA抑制剂- C3转移酶对HSC RhoA蛋白表达有明显抑制作用,并且从基因水平抑制其合成,同时抑制了RhoA/ROCK下游信号分子p-MLC的表达,对细胞骨架成分α-SMA的表达有抑制作用; ROCK抑制剂-fasudil对p-MLC和α-SMA的表达均有抑制作用,并呈现浓度依赖关系。应用RhoA/ROCK信号通路抑制剂干预HSC后,胞浆[Ca2+]i没有发生变化,说明它是非Ca2+依赖的信号通路。
Actually, hepatic fibrosis(HF) is a repairing process of the body towards the injury. Now it is widely accepted that HF is the inevitable and preexisting stage for lots of chronic hepatic disease developing into liver cirrhosis(LC)eventually. Investigation of mechanism of liver fibrosis and reversing the disease are very important for clinical treatment. Hepatic stellate cell(HSC)plays a key role in the pathogenesis of HF. Following chronic liver injury, HSC undergos transdifferentiation to an activated myofibroblastic phenotype with expression ofα-smooth muscle actin (α-SMA) and synthesizes various extracellular matrix protein components, which has the ability of contraction, adhesion and migration. All sorts of cytokines and signal transduction pathway exert very important roles in the activation of HSC.
RhoA family proteins are important regulatory molecules of actin cytoskeleton, which take part in all kinds of cell signal transduction associated with cytoskeleton. RhoA mainly increases cell contractility by promoting the assembly of focal adhesion and actin stress fibers. Rho-kinase(ROCK)is now the most distinct downstream signal molecule of RhoA, which can increase the level of myosin light chain phosphorylation though inactivation of MLC phosphatase, cause cross linking of actin and myosin, which promotes actin microfilament polymerization and results in cell contraction, migration and adhesion.
RhoA proteins generally cycle between an active GTP-bound, conformation and an inactive GDP-bound conformation. Lysophosphatidic acid (LPA) is first identifyed to activate Rho; C3 transferase can inactivate RhoA by making ribosylation of ADP; and fasudil is an inhibitor of ROCK. Recent years some studies have confirmed that Rho family proteins play potent roles in cell migration. Cell migration related with RhoA/ROCK signaling pathways has correlation with cardiovascular diseases, tumours and fibrotic diseases. In our past study, integrin-FAK signal pathway was found to take part in the HSC migration process, Focal adhesion related non-kinase (FRNK) inhibited the HSC migration. Studies on integrin signalling suggested that the substratum continuously feed signals to Rho proteins in migrating cells to influence migration rate. But RhoA/ROCK signaling pathways how to affect HSC migration process has not been proved.
This research will focus on the basis of activation of HSC, through experiments in vivo and in vitro, discuss the roles of RhoA/ROCK signaling pathways in the hepatic fibrogenesis and the effects on the contraction, adhesion and migration of HSC. We hope that it would provide theoretical basis for therapy of liver fibrosis. The experiments contain three parts as below:
Part 1: The Dynamic Expressions of RhoA、p-MLC andα-SMA during Hepatic Fibrogenesis in Rats
Objective: To observe the expression changes of the key signaling molecules RhoA and phosphorylated myosin light chain belonged to RhoA/ROCK signaling pathways in rat liver tissue during hepatic fibrogenesis, meanwhile, to observe the expression ofα-SMA which represents cytoskeleton element and to discuss the role of Rho/ROCK signaling pathways in the hepatic fibrogenesis.
Methods:A rat model of common bile duct ligation (BDL)-induced hepatic fibrosis was used to assess the formation of liver fibrosis. Liver tissues were obtained at 1st、2nd、3rd and 4th week, respectively, in the operation group, and at 4th week in the sham operation group. The liver histopathological changes were evaluated by hematoxylin and eosin staining, and by Masson’s trichrome method. Western blotting and Immunohistochemistry were used to determine the expressions of RhoA、p-MLC andα-SMA. Reverse transcription-polymerase chain reaction (RT-PCR) was used to determine the expression of RhoA mRNA.
Results:①Building of liver fibrosis model: The results of Hematoxylin and eosin staining and Masson's trichrome methods showed that in sham-operated group, the structure of liver lobule was integrin and the array of the liver flank was in order. But in model groups, the connective tissue hyperplasiaed broadly and the collagen fiber deposited around the central vein. The structure of the lobule was disturbed even the pseudo-lobules were formed.②The outcome of immunohistochemistry showed that the expression of RhoA protein was negative. After 1st week, there becomed some spot-like staining mainly in the interstitial cells in portal tract, the positive cells of RhoA increased a lot after BDL 2nd and 3rd week, they were mainly situated in portal ducts, fiber septa, perisinusoidal cells and around the bile ducts, the staining area enlarged at 4th week. The positive areas of RhoA in the rat livers in model groups at week 1 to 4 (5.32%±0.41%,13.43%±2.13%,22.14%±3.22%,31.48%±2.31%) were all larger than that in control group(0.23%±0.14%), P<0.05; The expression ofα-SMA was weak located in the smooth muscle cells of vessel wall, with the development of liver fibrosis, the expression ofα-SMA increased, there was brown staining in portal ducts, disse space, fiber septa, and around the bile ducts. There was weak staining for p-MLC in vessel walls in the sham operation group, with the development of hepatic fibrosis, the expression of p-MLC was increased especially in the walls of venules, and there was positive staining in the perisinusoidal cells. The positive areas of p-MLC in the rat livers in model groups at week 1 to 4 (13.47%±1.02%,20.05%±2.22%,28.31%±2.05%,35.41%±3.08%) were all larger than that in control group(2.01%±0.51%),and the difference was significant, P<0.05.③By RT-PCR analysis, RhoA mRNA appeared in the liver of sham-operated group (43%±5%), and was upregulated after bile duct ligation, during the liver fibrosis, the expression of RhoA mRNA was 71.22%±1.06%,78.49%±2.24%,81.31%±3.21%,96.38%±2.12%, respectively, after BDL1-4 week, compared with the sham-operated group, and the difference was significant, P<0.05. The expression of RhoA mRNA reached the peak value at the 4th week, which was 2.23 times of that of the sham-operated group.④RhoA, the molecular weight is 24 kD, Western blot analysis showed that in the sham-operated group, the RhoA was expressed a little, but with the development of liver fibrosis, the expression of it increased in model group, the expression increased 4.34, 9.95, 19.85, 22.12 times than that of the sham-operated group. p-MLC , the molecular weight is 18 kD, Western blot analysis showed that the p-MLC was expressed weak in the sham-operated group, The expression level of it was highest in the fourth week and 5 times of that of 1 wk and 43.16 times of that of the sham-operated group. With the development of liver fibrosis, the expression ofα-SMA in model group at week 1 to 4 increased 2.37, 7.08, 13.14, 17.62 times than that of the sham-operated group, the difference was significant, P<0.05. RhoA and p-MLC correlated withα-SMA positively, respectively (r=0.981, P<0.01, r=0.976, P<0.01).
Conclusions: In liver fibrogenesis, the expression of RhoA、p-MLC andα-SMA increased obviously. It suggested that RhoA/ROCK signaling pathways and cytoskeleton changes play a pivotal role in the formation and development of liver fibrosis.
Part 2: Effects of RhoA/ROCK signaling pathway inhibitors-C3 transferase and fasudil on the rat HSC adhesion and migration Objective: To observe the effects of RhoA/ROCK signaling pathway agonist-LPA, RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil on the HSC adhesion and migration.
Methods: The adhesive effects of LPA, C3 transferase and fasudil on HSC were examined by toluidine blue colorimetric assay,and the effects of migration were evaluated by improved Boyden chamber. Morphological alteration of HSC treated with C3 transferase and fasudil was observed with laser scanning confocal microscopy (LSCM).
Results:①Normal activited HSC was well spread. By contrast, HSC treated with C3 transferase changed to round and retractile shape, HSC treated with fasudil (50μM) changed to an elongated morphology with prominent dendritic processes, and appeared powerless shape.②Compared with the control group, LPA increased the HSC proliferation, the increation rate was 12.24%, while C3 transferase inhibited the HSC proliferation, the inhibition rate was 73.26%. C3 transferase inhibited LPA-induced HSC proliferation, compared with the control group, the inhibition rate was 46.51%. Fasudil inhibited HSC proliferation, the inhibition rates were 11.6%, 26.7%, 37.2%, 51.2%, at 12.5, 25, 50 and 100μmol/L, respectively. HSC proliferation correlated with fasudil concention negatively (r=-0.949, P<0.01).③compared with the control group, the adhesion rate increased 25.58% in LPA group, and decreased 50% in C3 group. While C3 transferase inhibited LPA-induced HSC adhesion, the adhesion rate decreased 40.63%. Fasudil inhibited LPA-induced HSC adhesion, and it was in concention-dependent manner; compared with the control group. The adhesive inhibition rates of fasudil 12.5, 25, 50, 100μmol/L were 13.31%, 36.72%, 41.71%, 62.43%, respectively. There was significant difference among the different groups, P<0.05.④LPA induced HSC migration, the cell number of migrating to the bottom chamber increased 6 times in LPA group compared with that of the control group. C3 transferase inhibited HSC migration, simultaneously, inhibited LPA-induced HSC migration, compared with the control group, and there was statistic difference, P<0.01. Fasudil inhibited LPA induced HSC migration, and it was in concention-dependent manner; the cell number of migrating of fasudil 100μmol/L decreased 80% compared with that of the LPA group.
Conclusions: RhoA inhibitor-C3 transferase and ROCK inhibitor-fasudil inhibited the LPA-induced HSC proliferation, adhesion and migration. Part 3: The molecular mechanism of the inhibiting migration of RhoA/ROCK signaling pathway inhibitors on rat HSC
Objective: To explore the molecular mechanism of RhoA/ROCK signaling pathway inhibitors inhibiting HSC migration induced by LPA.
Methods: HSC was cultured in vitro, and the expressions of RhoA, p-MLC andα-SMA were assessed using Western blot and RT-PCR assay. The intracellular free calcium was observed with laser scanning confocal microscopy (LSCM).
Results:①Western blot showed: RhoA expression (4326.48±213.32) increased obviously after cultivated with LPA, compared with the control group (2335.54±211.81), up to 46.02%,P<0.01.②When HSC was cultured with C3 transferase for 24 h, The protein expression of RhoA decreased obviously, compared with the control group, down to 73.40 %, meanwhile C3 transferase inhibited LPA-induced HSC RhoA expression, compared with the control group, down to 66.21%.③HSC was induced by LPA for 24 h, the expression of RhoA mRNA was examined by RT-PCR. Compared with the control group, the expression of RhoA mRNA increased 47.92%.④When HSC was cultured with C3 for 24 h, the expression of RhoA mRNA decreased obviously, Compared with the control group, it reduced 68 %. C3 transferase inhibited LPA-induced expression of HSC RhoA mRNA, compared with the control group, it reduced 64 %.⑤LPA increased the protein expressions of p-MLC andα-SMA, compared with the sham-operated group, it increased 33.85%, 36.81%, respectively.⑥Western blot analysis showed: when HSC was cultured with C3 transferase for 24h, the protein expressions of p-MLC andα-SMA decreased 70.45%, 68.34%, respectively, than that in the control group. While C3 transferase inhibited LPA-induced protein expressions of p-MLC andα-SMA, compared with the control group, they decreased 63.24%, 61.83%, respectively.⑦Western blot showed: the expression number ofα-SMA was 2336.53±329.51 in the control group. The protein expressions ofα-SMA in the fasudil 12.5、25、50、100μmol/L group were 2015.78±231.56, 1865.37±203.12, 1549.64±187.13 and 1364.72±140.88, respectively, compared with the control group, it reduced 13.74%, 20.16%, 33.69%, 41.61%, respectively. Fasudil inhibited the expression of p-MLC, the protein expression number of p-MLC in the fasudil 12.5、25、50、100μmol/L group were 2105.49±305.73, 1783.57±216.83, 1544.31±158.67, 1042.83±146.09, respectively, so it reduced 21.66%, 33.64%, 42.54%, 61.22% than that in the control group. The expressions ofα-SMA and p-MLC were in fasudil concention-dependent manners.⑧Compared with HSC in the control group, calcium fluorescence intensity of HSC treated by C3 for 24 h mildly decreased 2.6%, there was no statistic difference between them, P>0.05; C3 transferase had no effect on LPA-induced HSC calcium fluorescence intensity, compared with the control group, there was no significant difference, P>0.05.⑨HSC was treated with fasudil for 24 h, calcium fluorescence intensities of fasudil 12.5、25、50、100μmol/L group were the same on the whole. The results showed that there was no difference among 4 different groups, P>0.05, and compared with the control group, there was no statistic difference, P>0.05.
Conclusions: Activated HSC expressed the key signaling molecules RhoA and p-MLC belonged to RhoA/ROCK signaling pathways. RhoA inhibitor-C3 transferase inhibited the expression and transcription of RhoA, and the expression of downstream factor p-MLC was inhibited in the meantime, the expression ofα-SMA was also inhibited. ROCK inhibitor-fasudil inhibited the expressions ofα-SMA and p-MLC, and they were in concention manners. RhoA/ROCK signaling pathway inhibitors had no effect on intracellular free calcium. It belonged to Ca2+-independent signaling pathway.
引文
1 Nan JX, Park EJ, Kang HC, et al. Anti-fibrotic effects of a hot-water extract from Salvia miltiorrhiza roots on liver fibrosis induced by biliary obstruction in rats. J Pharm Pharmacol, 2001, 53:197-204
2 姜慧卿, 张晓岚, 王占魁, 等. 大鼠肝纤维化过程中肝组织黏着斑激酶表达增加. 基础医学与临床, 2005, 25:49-53
3 Wang XM, Yu DM, Mc Caughan, et al. Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology, 2005, 42:935-945
4 Tufvesson E, Westergren-Thorsson G. Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration. J Cell Sci, 2003, 116(Pt 23):4857-4864
5 Tanaka J , Watanabe T, Nakamura N , et al. Morphological and biochemical analyses of contractile proteins (actin ,myosin ,caldesmon and tropomyosin) in normal and transformed cells. J Cell Sci, 1993, 104(Pt 2) :595-601
6 Foo ITH , Naylor IL ,Timmons MJ , et al. Intracellular actin as a marker for myofibroblasts in vitro. Lab Invest, 1992, 67 :727-733
7 Hellerbrand C, Stefanovic B, Giordano F. The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo. J Hepatol, 1999, 30:77-87
8 Kinnman N, Francoz C, Barbu V, et al. The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis. Lab Invest, 2003, 83:163-173
9 Yu E, Choe G, Gong G, et al. Expression of alpha-smooth muscle actin in liver diseases. J Korean Med Sci, 1993, 8:367-373
10 武希润,吕敏和,王琦,等.α-平滑肌肌动蛋白表达及血浆转化生长因子β-1 变化在肝纤维化发生发展中的作用. 中华肝脏病杂志,2004,12:400-402
11 Ridley AJ. Rho GTPases and cell migration. J Cell Sci, 2001, 114(Pt 15):2713-2722
12 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
13 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
14 Arthur WT, Noren NK, Burridge K. Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion. Biol Res, 2002, 35:239-246
15 Van Aelst L, Symons M. Role of Rho family GTPases in epithelial morphogenesis. Genes Dev, 2002, 16:1032-1054
16 李志利, 姜世忠. RhoA-ROK 通路在平滑肌收缩中的作用. 生理科学进展, 2005, 36:341-344
17 Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science, 1996, 273:245-248
18 Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci, 2001, 22:32-39
19 Vale RD, Milligan RA. The way things move: looking under the hood of molecular motor proteins. Science, 2000 ,288:88-95
1 Gressner AM. Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: a key event in hepatic fibrogenesis.Kidney. Int Suppl, 1996, 54:S39-45
2 Anne J, Ridley. Rho GTPases and cell migration. Journal of Cell Science, 2001, 114:2713-2722
3 王玉珍, 姜慧卿, 扈彩霞. Rho 信号转导通路于细胞迁移. 医学综述, 2006,12:651-653
4 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
5 辛华, 郑雅娟, 韩振国, 等. 内皮细胞 Rho 及 Rho 激酶在肉瘤细胞向血管外游走过程中的作用. 中国病理生理杂志, 2004, 20:950-953
6 Nakayama M, Amano M, Katsumi A, et al. Rho-kinase and myosin II activities are required for cell type and environment specific migration. Genes Cells, 2005, 10:107-117
7 Shimizu Y, Dobashi K, Iizuka K, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med, 2001, 163:210-217
8 Ridley AJ. Rho GTPases and cell migration. J Cell Sci, 2001, 114:2713-2722
9 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
10 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
11 Parsons J K, Martin K H, Slack J K. Focal adhension kinase :a regulatorof focal adhension dynamics and cell movement. Oncogene, 2000, 19:5606-5613
12 Hocking DC, Sottile J, Langenbach KJ. Stimulation of integrin-mediated cell contractility by fibronectin polymerization. J Biol Chem, 2000, 275:10673-10682.
13 Gohla A, Harhammer R, Schultz G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem, 1998, 273:4653-4659
14 Ridley AJ, Hall A. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J, 1994, 13:2600-2610
15 Borbiev T, Nurmukhambetova S, Liu F, et al. Introduction of C3 exoenzyme into cultured endothelium by lipofectamine. Anal Biochem, 2000, 285:260-264
16 Nobes C D, Hall A. Rho,rac,and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers,lamellipodia,and filopodia. Cell, 1995, 81:53-62
17 Ishizaki T, Naito M, Fujisawa K, et al. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett, 1997, 404:118-124
18 Nishikimi T, Matsuoka H. Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effect of rho-kinase inhibitor in hypertensive glomerulosclerosis. J Pharmacol Sci, 2006, 100:22-28
1 Clemens MG, Zhang JX. Regulation of sinusoidal perfusion: in vivo methodology and control by endothelins. Semin Liver Dis, 1999, 19:383-396
2 Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through Rho-kinase-mediated alterations in the acto-myosin cytoskeleton. Hepatology, 2001, 33:74-80
3 Hall A. Rho GTPases and the actin cytoskeletion. Science, 1998, 279:509-514
4 Rockey DC, Housset CN, Friedman SL. Activation-dependent contractili -ty of rat hepatic lipocytes in culture and in vivo. J Clin Invest, 1993, 92 :1795-1804
5 Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell, 1996, 84:359-369
6 Amano M, Chihara K, Kimura K, et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science, 1997 , 275:1308-1311
7 Kato M, Iwamoto H, Higashi N, et al. Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells. J Hepatol, 1999, 31:91-99
8 Yee HF Jr. Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton. Hepatology, 1998, 28:843-850
9 Barber SC, Mellor H, Gampel A, et al. S1P and LPA trigger Schwann cell actin changes and migration. Eur J Neurosci, 2004, 19:3142-3150
10 Fukushima M, Nakamuta M, Kohjima M, et al. Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver Int, 2005, 25:829-838
11 Matsui T, Amano M, Yamamoto T, et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J,1996, 15:2208-2216
12 Borbiev T,Nurmukhambetova S,Liu F,et al. Introduction of C3 exoenzyme into cultured endothelium by lipofectamine. Anal Biochem, 2000, 285:260-264
13 Kawada N, Seki S, Kuroki T, et al. ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1. Biochem Biophys Res Commun, 1999, 266:296-300
14 郑惠珍, 赵克森, 黄巧冰. Rho 激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用. 中华烧伤杂志, 2005, 21:181-184
15 Nohria A, Grunert ME, Rikitake Y, et al. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circ Res, 2006, 99:1426-1432
16 王立安, 赵俊霞. 钙对细胞骨架的调控及其在生命活动中的重要作用. 生物学杂志, 1999,16:6-7
17 Rockey DC, Housset CN, Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest, 1993, 92 :1795-1804
18 扬丽, 刘小菁, 王琼, 等. 4,5,6-三羟基异黄酮对大鼠肝星状细胞骨架及胞浆内游离钙的作用. 中华肝脏病杂志, 2003, 11:634-635
19 Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev, 2003, 83:1325-1358
20 李志力, 姜世忠. RhoA/ROK 通路在平滑肌收缩中的作用. 生理科学进展, 2005, 36:341-344
21 Himpens B, Kitazawa T, Somlyo AP. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Arch, 1990, 417:21-28
22 Yamagata S, Ichinose M, Sugiura H, et al. Effect of a calcium sensitization modulator, Y-27632, on isolated human bronchus andpulmonary artery. Pulm Pharmacol Ther, 2000, 13:25-29
23 Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem, 1999, 274:37385-37390
24 Kawano Y, Fukata Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol, 1999,147:1023-1038
25 Amano M, Ito M, Kimura K, et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem, 1996, 271:20246-20249
1 Ahmed F, Wyckoff J, Lin E Y , et al. GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res, 2002, 62:7166-7169
2 Anne J, Ridley. Rho GTPases and cell migration. Journal of Cell Science, 2001, 114:2713-2722
3 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005,280:10624-10635
4 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
5 解翼虎, 覃文新, 万大方, 等. Rho 蛋白. 生命的化学, 2002, 22:21-24
6 Etienne-Manneville S, Hall A. RhoGTPases in cell biology. Nature, 2002, 420:629-635
7 Nobes C D, Hall A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol, 1999, 144:1235 -1244
8 Nobes C D, Hall A. Rho,rac,and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers,lamellipodia,and filopodia. Cell, 1995, 81:53-62
9 Stephens L, Ellson C, Hawkins P. Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr Opin Cell Biol, 2002, 14:203-213
10 Price L S, Leng J, Schwartz M A, et al. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell, 1998, 9:1863 -1871
11 Schmitz A A, Govek E E, Bottner B, et al. Rho GTPases: signaling, migration, and invasion. Exp Cell Res, 2000, 261:1 -12
12 Kawano Y, Fukata Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit of myosin phosphatase by Rho-kinase in vivo. J Cell Biol, 1999, 147:831-844
13 Sander E E, Ten Klooster J P, Van Delft S, et al. Rac downregulates Rho activity:reciprocal balance between both GPTases determines cellular morphology and migratory behavior. J Cell Biol, 1999, 147:1009-1022
14 Kheradmand F, Werner E., Tremble P, et al. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science, 1998, 280:898 -902
15 Waterman-Storer C M, Salmon E. Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr Opin Cell Biol, 1999, 11:61-67
16 Ishizaki T, Morishima Y, Okamoto M, et al. Coordination of microtubulesand the actin cytoskeleton by the Rho effector mDial. Nat Cell Biol, 2001, 3:8 -14
17 Daub H, Gevaert K, Vandekerckhove J , et al. Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16. J Biol Chem, 2001, 276:1677 -1680
18 Etienne-Manneville S, Hall A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell, 2001, 106:489-498
19 Nakayama M, Amano M, Katsumi A, et al. Rho-kinase and myosin II activities are required for cell type and environment specific migration. Genes Cells, 2005, 10:107-117
20 辛华, 郑雅娟, 韩振国, 等. 内皮细胞 Rho 及 Rho 激酶在肉瘤细胞向血管外游走过程中的作用. 中国病理生理杂志, 2004, 20:950-953
21 Adam L, Vadlamudi R, Mandal M, et al. Regulation of microfilament reorganization and invasiveness of breast cancer cells by kinade dead p21-activated kinase-1. J Biol Chem, 2000, 275:12041-12050
22 Satoh S, Yamaguchi T, Hitomi A, et al. Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction. Eur J Pharmacol, 2002, 455:169-174
23 Shimizu Y, Dobashi K, Iizuka K, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med, 2001, 163:210-217
24 Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through Rho-kinase-mediated alterations in the acto-myosin cytoskeleton. Hepatology, 2001, 33:74-80
25 Masamune A, Kikuta K, Satoh M, et al. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol, 2003 , 140:1292-1302
1 Jansen PLM. Non-alcoholic steatohepatitis. Eur J Gastroenterol Hepatol, 2004, 16:1079-1085
2 Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest, 1990, 86:1641-1648
3 Schmitt-Graff A, Chzkroun G, Gabbiani G. Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis.Virchows Arch A Pathol Anat Histopathol, 1993, 422:99-107
4 Saile B, Matthes N, Neubauer K, et al. Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol, 2002, 283:G435-444.
5 Ramadori G, Saile B. Portal tract fibrogenesis in the liver. Lab Invest, 2004, 84:153-159
6 Goddard CJR, Smith A, Hoyland JA, et al. Localization and semiquantitative assessment of hepatic procollagen mRNA in primarybiliary cirrhosis. Gut, 1998, 43:433-440
7 Kinnman N, Housset C, Peribiliary myofibroblasts in biliary type liver fibrosis. Front Biosci, 2002, 7:d496-503
8 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
9 Racine-Samson L, Rockey DC, Bissell DM. The role of alpha1beta1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem, 1997, 272:30911-30917
10 Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature, 1994, 372:425-432
11 McCullough AJ, Bugianesi E, Marchesini G, et al. Gender-dependent alterations in serum leptin in alcoholic cirrhosis. Gastroenterology, 1998, 115:947-953
12 Ikejima K, Honda H,Yoshikawa M, et al. Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals.Hepatology,2001, 34:288-297
13 Leclercq IA, Farrell GC, Schriemer R, et al. Leptin is essential for the hepatic fibrogenic response to chronic liver injury. J Hepatol, 2002,
37:206-213
14 Hegyi K, Fulop K, Kovacs K, et al. Leptin-induced signal transduction pathways. Cell Biol Int, 2004,28:159-169
15 Saxena NK, Ikeda K, Rockey DC, et al. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology, 2002, 35:762-771
16 Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci, 2002, 7:d793-807
17 Inagaki Y, Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem, 1994,269:14828-14834
18 Ritzenthaler JD, Goldstein RH, Fine A, et al. Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene. Biochem J, 1991, 280:157-162
19 Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology.Biochem Cell Biol, 2003, 81:355-363
20 Bradham DM, Igarashi A, Potter RL, et al. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol, 1991, 114:1285-1294
21 Williams EJ, Gaca MD, Brigstock DR, et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000, 32:754-761
22 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004, 15:255-273
23 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
24 Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol, 1997, 9:180-186
25 Gentilini A, Marra F, Gentilini P, et al. Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells. J Hepatol, 2000, 32:227-234
26 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
27 Schnabl B, Bradham CA, Bennett BL, et al. TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. Hepatology, 2001, 34:953-963
28 Reimann T, Hempel U, Krauwald S, et al. Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells. FEBS Lett, 1997, 403:57-560
29 Hanafusa H, Ninomiya-Tsuji J, Masuyama N, et al. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J Biol Chem, 1999, 274:27161-27167
30 Tsukada S, Westwick JK, Ikejima K, et al. SMAD and p38 MAPK signaling pathways independently regulate alpha1(I) collagen gene expression in unstimulated and transforming growth factor-beta-stimulated hepatic stellate cells. J Biol Chem, 2005, 280:10055-10064
31 Cao Q, Mak KM, Lieber CS. DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2002, 283:G1051-1061
32 Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol, 2000,18:621-663
33 Elsharkawy AM, Wright MC, Hay RT, et al. Persistent activation of nuclear factor-kappaB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkappaB family proteins. Hepatology, 1999, 30:761-769
34 Lang A, Schoonhoven R, Tuvia S, et al. Nuclear factor kappaB in proliferation, activation, and apoptosis in rat hepatic stellate cells. J Hepatol, 2000, 33:49-58
35 Lee KS, Buck M, Houglum K, et al. Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression. J Clin Invest, 1995, 96:2461-2468
36 Lang A, Schrum LW, Schoonhoven R, et al. Expression of small heatshock protein alphaB-crystallin is induced after hepatic stellate cell activation. Am J Physiol Gastrointest Liver Physiol, 2000, 279:G1333-1342
37 Liu XJ, Yang L, Luo FM, et al. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA microarray. World J Gastroenterol, 2004, 10:1600-1607
38 Knittel T, Mehde M, Grundmann A, et al. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat.Histochem Cell Biol, 2000, 113:443-453
39 Olaso E, Ikeda K, Eng FJ, et al. DDR2 receptor promotes MMP-2- mediated proliferation and invasion by hepatic stellate cells. J Clin Invest, 2001, 108:1369-1378
40 Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis, 2001, 21:373-384
41 Iredale JP, Goddard S, Murphy G, et al. Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes. Clin Sci (Lond), 1995, 89:75-81
42 Wang DR, Sato M, Li LN,et al. Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells.Cell Struct Funct, 2003, 28:505-513
43 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
44 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
45 McGuire RF, Bissell DM, Boyles J, et al. Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver. Hepatology, 1992, 15:989-997
46 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
47 Knittel T, Mehde M,Grundmann A, et al. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat. Histochem Cell Biol, 2000, 113:443-453
48 Issa R, Zhou X,Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking G Gastroenterology, 2004, 126:1795-1808
49 Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Semin Liver Dis, 2001, 21:427-436.
50 Rocca P, Cocuzza E, Rasetti R, et al. Predictors of psychiatric disorders in liver transplantation candidates: logistic regression models. Liver Transpl, 2003, 9:721-726
2 姜慧卿, 张晓岚, 王占魁, 等. 大鼠肝纤维化过程中肝组织黏着斑激酶表达增加. 基础医学与临床, 2005, 25:49-53
3 Wang XM, Yu DM, Mc Caughan, et al. Fibroblast activation protein increases apoptosis, cell adhesion, and migration by the LX-2 human stellate cell line. Hepatology, 2005, 42:935-945
4 Tufvesson E, Westergren-Thorsson G. Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration. J Cell Sci, 2003, 116(Pt 23):4857-4864
5 Tanaka J , Watanabe T, Nakamura N , et al. Morphological and biochemical analyses of contractile proteins (actin ,myosin ,caldesmon and tropomyosin) in normal and transformed cells. J Cell Sci, 1993, 104(Pt 2) :595-601
6 Foo ITH , Naylor IL ,Timmons MJ , et al. Intracellular actin as a marker for myofibroblasts in vitro. Lab Invest, 1992, 67 :727-733
7 Hellerbrand C, Stefanovic B, Giordano F. The role of TGFbeta1 in initiating hepatic stellate cell activation in vivo. J Hepatol, 1999, 30:77-87
8 Kinnman N, Francoz C, Barbu V, et al. The myofibroblastic conversion of peribiliary fibrogenic cells distinct from hepatic stellate cells is stimulated by platelet-derived growth factor during liver fibrogenesis. Lab Invest, 2003, 83:163-173
9 Yu E, Choe G, Gong G, et al. Expression of alpha-smooth muscle actin in liver diseases. J Korean Med Sci, 1993, 8:367-373
10 武希润,吕敏和,王琦,等.α-平滑肌肌动蛋白表达及血浆转化生长因子β-1 变化在肝纤维化发生发展中的作用. 中华肝脏病杂志,2004,12:400-402
11 Ridley AJ. Rho GTPases and cell migration. J Cell Sci, 2001, 114(Pt 15):2713-2722
12 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
13 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
14 Arthur WT, Noren NK, Burridge K. Regulation of Rho family GTPases by cell-cell and cell-matrix adhesion. Biol Res, 2002, 35:239-246
15 Van Aelst L, Symons M. Role of Rho family GTPases in epithelial morphogenesis. Genes Dev, 2002, 16:1032-1054
16 李志利, 姜世忠. RhoA-ROK 通路在平滑肌收缩中的作用. 生理科学进展, 2005, 36:341-344
17 Kimura K, Ito M, Amano M, et al. Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Science, 1996, 273:245-248
18 Fukata Y, Amano M, Kaibuchi K. Rho-Rho-kinase pathway in smooth muscle contraction and cytoskeletal reorganization of non-muscle cells. Trends Pharmacol Sci, 2001, 22:32-39
19 Vale RD, Milligan RA. The way things move: looking under the hood of molecular motor proteins. Science, 2000 ,288:88-95
1 Gressner AM. Transdifferentiation of hepatic stellate cells (Ito cells) to myofibroblasts: a key event in hepatic fibrogenesis.Kidney. Int Suppl, 1996, 54:S39-45
2 Anne J, Ridley. Rho GTPases and cell migration. Journal of Cell Science, 2001, 114:2713-2722
3 王玉珍, 姜慧卿, 扈彩霞. Rho 信号转导通路于细胞迁移. 医学综述, 2006,12:651-653
4 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
5 辛华, 郑雅娟, 韩振国, 等. 内皮细胞 Rho 及 Rho 激酶在肉瘤细胞向血管外游走过程中的作用. 中国病理生理杂志, 2004, 20:950-953
6 Nakayama M, Amano M, Katsumi A, et al. Rho-kinase and myosin II activities are required for cell type and environment specific migration. Genes Cells, 2005, 10:107-117
7 Shimizu Y, Dobashi K, Iizuka K, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med, 2001, 163:210-217
8 Ridley AJ. Rho GTPases and cell migration. J Cell Sci, 2001, 114:2713-2722
9 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
10 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005, 280:10624-10635
11 Parsons J K, Martin K H, Slack J K. Focal adhension kinase :a regulatorof focal adhension dynamics and cell movement. Oncogene, 2000, 19:5606-5613
12 Hocking DC, Sottile J, Langenbach KJ. Stimulation of integrin-mediated cell contractility by fibronectin polymerization. J Biol Chem, 2000, 275:10673-10682.
13 Gohla A, Harhammer R, Schultz G. The G-protein G13 but not G12 mediates signaling from lysophosphatidic acid receptor via epidermal growth factor receptor to Rho. J Biol Chem, 1998, 273:4653-4659
14 Ridley AJ, Hall A. Signal transduction pathways regulating Rho-mediated stress fibre formation: requirement for a tyrosine kinase. EMBO J, 1994, 13:2600-2610
15 Borbiev T, Nurmukhambetova S, Liu F, et al. Introduction of C3 exoenzyme into cultured endothelium by lipofectamine. Anal Biochem, 2000, 285:260-264
16 Nobes C D, Hall A. Rho,rac,and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers,lamellipodia,and filopodia. Cell, 1995, 81:53-62
17 Ishizaki T, Naito M, Fujisawa K, et al. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions. FEBS Lett, 1997, 404:118-124
18 Nishikimi T, Matsuoka H. Molecular mechanisms and therapeutic strategies of chronic renal injury: renoprotective effect of rho-kinase inhibitor in hypertensive glomerulosclerosis. J Pharmacol Sci, 2006, 100:22-28
1 Clemens MG, Zhang JX. Regulation of sinusoidal perfusion: in vivo methodology and control by endothelins. Semin Liver Dis, 1999, 19:383-396
2 Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through Rho-kinase-mediated alterations in the acto-myosin cytoskeleton. Hepatology, 2001, 33:74-80
3 Hall A. Rho GTPases and the actin cytoskeletion. Science, 1998, 279:509-514
4 Rockey DC, Housset CN, Friedman SL. Activation-dependent contractili -ty of rat hepatic lipocytes in culture and in vivo. J Clin Invest, 1993, 92 :1795-1804
5 Lauffenburger DA, Horwitz AF. Cell migration: a physically integrated molecular process. Cell, 1996, 84:359-369
6 Amano M, Chihara K, Kimura K, et al. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science, 1997 , 275:1308-1311
7 Kato M, Iwamoto H, Higashi N, et al. Role of Rho small GTP binding protein in the regulation of actin cytoskeleton in hepatic stellate cells. J Hepatol, 1999, 31:91-99
8 Yee HF Jr. Rho directs activation-associated changes in rat hepatic stellate cell morphology via regulation of the actin cytoskeleton. Hepatology, 1998, 28:843-850
9 Barber SC, Mellor H, Gampel A, et al. S1P and LPA trigger Schwann cell actin changes and migration. Eur J Neurosci, 2004, 19:3142-3150
10 Fukushima M, Nakamuta M, Kohjima M, et al. Fasudil hydrochloride hydrate, a Rho-kinase (ROCK) inhibitor, suppresses collagen production and enhances collagenase activity in hepatic stellate cells. Liver Int, 2005, 25:829-838
11 Matsui T, Amano M, Yamamoto T, et al. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. EMBO J,1996, 15:2208-2216
12 Borbiev T,Nurmukhambetova S,Liu F,et al. Introduction of C3 exoenzyme into cultured endothelium by lipofectamine. Anal Biochem, 2000, 285:260-264
13 Kawada N, Seki S, Kuroki T, et al. ROCK inhibitor Y-27632 attenuates stellate cell contraction and portal pressure increase induced by endothelin-1. Biochem Biophys Res Commun, 1999, 266:296-300
14 郑惠珍, 赵克森, 黄巧冰. Rho 激酶在烧伤大鼠血清诱导的血管内皮细胞骨架变化中的作用. 中华烧伤杂志, 2005, 21:181-184
15 Nohria A, Grunert ME, Rikitake Y, et al. Rho kinase inhibition improves endothelial function in human subjects with coronary artery disease. Circ Res, 2006, 99:1426-1432
16 王立安, 赵俊霞. 钙对细胞骨架的调控及其在生命活动中的重要作用. 生物学杂志, 1999,16:6-7
17 Rockey DC, Housset CN, Friedman SL. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest, 1993, 92 :1795-1804
18 扬丽, 刘小菁, 王琼, 等. 4,5,6-三羟基异黄酮对大鼠肝星状细胞骨架及胞浆内游离钙的作用. 中华肝脏病杂志, 2003, 11:634-635
19 Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev, 2003, 83:1325-1358
20 李志力, 姜世忠. RhoA/ROK 通路在平滑肌收缩中的作用. 生理科学进展, 2005, 36:341-344
21 Himpens B, Kitazawa T, Somlyo AP. Agonist-dependent modulation of Ca2+ sensitivity in rabbit pulmonary artery smooth muscle. Pflugers Arch, 1990, 417:21-28
22 Yamagata S, Ichinose M, Sugiura H, et al. Effect of a calcium sensitization modulator, Y-27632, on isolated human bronchus andpulmonary artery. Pulm Pharmacol Ther, 2000, 13:25-29
23 Feng J, Ito M, Ichikawa K, et al. Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase. J Biol Chem, 1999, 274:37385-37390
24 Kawano Y, Fukata Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit (MBS) of myosin phosphatase by Rho-kinase in vivo. J Cell Biol, 1999,147:1023-1038
25 Amano M, Ito M, Kimura K, et al. Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J Biol Chem, 1996, 271:20246-20249
1 Ahmed F, Wyckoff J, Lin E Y , et al. GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res, 2002, 62:7166-7169
2 Anne J, Ridley. Rho GTPases and cell migration. Journal of Cell Science, 2001, 114:2713-2722
3 Zhou H, Kramer RH. Integrin engagement differentially modulates epithelial cell motility by Rho/ROCK and PAK1. J Biol Chem, 2005,280:10624-10635
4 Wang R, Clark RA, Mosher DF, et al. Fibronectin's central cell-binding domain supports focal adhesion formation and Rho signal transduction. J Biol Chem, 2005, 280:28803-28810
5 解翼虎, 覃文新, 万大方, 等. Rho 蛋白. 生命的化学, 2002, 22:21-24
6 Etienne-Manneville S, Hall A. RhoGTPases in cell biology. Nature, 2002, 420:629-635
7 Nobes C D, Hall A. Rho GTPases control polarity, protrusion, and adhesion during cell movement. J Cell Biol, 1999, 144:1235 -1244
8 Nobes C D, Hall A. Rho,rac,and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers,lamellipodia,and filopodia. Cell, 1995, 81:53-62
9 Stephens L, Ellson C, Hawkins P. Roles of PI3Ks in leukocyte chemotaxis and phagocytosis. Curr Opin Cell Biol, 2002, 14:203-213
10 Price L S, Leng J, Schwartz M A, et al. Activation of Rac and Cdc42 by integrins mediates cell spreading. Mol Biol Cell, 1998, 9:1863 -1871
11 Schmitz A A, Govek E E, Bottner B, et al. Rho GTPases: signaling, migration, and invasion. Exp Cell Res, 2000, 261:1 -12
12 Kawano Y, Fukata Y, Oshiro N, et al. Phosphorylation of myosin-binding subunit of myosin phosphatase by Rho-kinase in vivo. J Cell Biol, 1999, 147:831-844
13 Sander E E, Ten Klooster J P, Van Delft S, et al. Rac downregulates Rho activity:reciprocal balance between both GPTases determines cellular morphology and migratory behavior. J Cell Biol, 1999, 147:1009-1022
14 Kheradmand F, Werner E., Tremble P, et al. Role of Rac1 and oxygen radicals in collagenase-1 expression induced by cell shape change. Science, 1998, 280:898 -902
15 Waterman-Storer C M, Salmon E. Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr Opin Cell Biol, 1999, 11:61-67
16 Ishizaki T, Morishima Y, Okamoto M, et al. Coordination of microtubulesand the actin cytoskeleton by the Rho effector mDial. Nat Cell Biol, 2001, 3:8 -14
17 Daub H, Gevaert K, Vandekerckhove J , et al. Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16. J Biol Chem, 2001, 276:1677 -1680
18 Etienne-Manneville S, Hall A. Integrin-mediated activation of Cdc42 controls cell polarity in migrating astrocytes through PKCzeta. Cell, 2001, 106:489-498
19 Nakayama M, Amano M, Katsumi A, et al. Rho-kinase and myosin II activities are required for cell type and environment specific migration. Genes Cells, 2005, 10:107-117
20 辛华, 郑雅娟, 韩振国, 等. 内皮细胞 Rho 及 Rho 激酶在肉瘤细胞向血管外游走过程中的作用. 中国病理生理杂志, 2004, 20:950-953
21 Adam L, Vadlamudi R, Mandal M, et al. Regulation of microfilament reorganization and invasiveness of breast cancer cells by kinade dead p21-activated kinase-1. J Biol Chem, 2000, 275:12041-12050
22 Satoh S, Yamaguchi T, Hitomi A, et al. Fasudil attenuates interstitial fibrosis in rat kidneys with unilateral ureteral obstruction. Eur J Pharmacol, 2002, 455:169-174
23 Shimizu Y, Dobashi K, Iizuka K, et al. Contribution of small GTPase Rho and its target protein rock in a murine model of lung fibrosis. Am J Respir Crit Care Med, 2001, 163:210-217
24 Tangkijvanich P, Tam SP, Yee HF Jr. Wound-induced migration of rat hepatic stellate cells is modulated by endothelin-1 through Rho-kinase-mediated alterations in the acto-myosin cytoskeleton. Hepatology, 2001, 33:74-80
25 Masamune A, Kikuta K, Satoh M, et al. Rho kinase inhibitors block activation of pancreatic stellate cells. Br J Pharmacol, 2003 , 140:1292-1302
1 Jansen PLM. Non-alcoholic steatohepatitis. Eur J Gastroenterol Hepatol, 2004, 16:1079-1085
2 Maher JJ, McGuire RF. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest, 1990, 86:1641-1648
3 Schmitt-Graff A, Chzkroun G, Gabbiani G. Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis.Virchows Arch A Pathol Anat Histopathol, 1993, 422:99-107
4 Saile B, Matthes N, Neubauer K, et al. Rat liver myofibroblasts and hepatic stellate cells differ in CD95-mediated apoptosis and response to TNF-alpha. Am J Physiol Gastrointest Liver Physiol, 2002, 283:G435-444.
5 Ramadori G, Saile B. Portal tract fibrogenesis in the liver. Lab Invest, 2004, 84:153-159
6 Goddard CJR, Smith A, Hoyland JA, et al. Localization and semiquantitative assessment of hepatic procollagen mRNA in primarybiliary cirrhosis. Gut, 1998, 43:433-440
7 Kinnman N, Housset C, Peribiliary myofibroblasts in biliary type liver fibrosis. Front Biosci, 2002, 7:d496-503
8 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
9 Racine-Samson L, Rockey DC, Bissell DM. The role of alpha1beta1 integrin in wound contraction. A quantitative analysis of liver myofibroblasts in vivo and in primary culture. J Biol Chem, 1997, 272:30911-30917
10 Zhang Y, Proenca R, Maffei M, et al. Positional cloning of the mouse obese gene and its human homologue. Nature, 1994, 372:425-432
11 McCullough AJ, Bugianesi E, Marchesini G, et al. Gender-dependent alterations in serum leptin in alcoholic cirrhosis. Gastroenterology, 1998, 115:947-953
12 Ikejima K, Honda H,Yoshikawa M, et al. Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals.Hepatology,2001, 34:288-297
13 Leclercq IA, Farrell GC, Schriemer R, et al. Leptin is essential for the hepatic fibrogenic response to chronic liver injury. J Hepatol, 2002,
37:206-213
14 Hegyi K, Fulop K, Kovacs K, et al. Leptin-induced signal transduction pathways. Cell Biol Int, 2004,28:159-169
15 Saxena NK, Ikeda K, Rockey DC, et al. Leptin in hepatic fibrosis: evidence for increased collagen production in stellate cells and lean littermates of ob/ob mice. Hepatology, 2002, 35:762-771
16 Gressner AM, Weiskirchen R, Breitkopf K, et al. Roles of TGF-beta in hepatic fibrosis. Front Biosci, 2002, 7:d793-807
17 Inagaki Y, Truter S, Ramirez F. Transforming growth factor-beta stimulates alpha 2(I) collagen gene expression through a cis-acting element that contains an Sp1-binding site. J Biol Chem, 1994,269:14828-14834
18 Ritzenthaler JD, Goldstein RH, Fine A, et al. Transforming-growth-factor-beta activation elements in the distal promoter regions of the rat alpha 1 type I collagen gene. Biochem J, 1991, 280:157-162
19 Leask A, Abraham DJ. The role of connective tissue growth factor, a multifunctional matricellular protein, in fibroblast biology.Biochem Cell Biol, 2003, 81:355-363
20 Bradham DM, Igarashi A, Potter RL, et al. Connective tissue growth factor: a cysteine-rich mitogen secreted by human vascular endothelial cells is related to the SRC-induced immediate early gene product CEF-10. J Cell Biol, 1991, 114:1285-1294
21 Williams EJ, Gaca MD, Brigstock DR, et al. Increased expression of connective tissue growth factor in fibrotic human liver and in activated hepatic stellate cells. J Hepatol, 2000, 32:754-761
22 Bonner JC. Regulation of PDGF and its receptors in fibrotic diseases. Cytokine Growth Factor Rev, 2004, 15:255-273
23 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
24 Robinson MJ, Cobb MH. Mitogen-activated protein kinase pathways. Curr Opin Cell Biol, 1997, 9:180-186
25 Gentilini A, Marra F, Gentilini P, et al. Phosphatidylinositol-3 kinase and extracellular signal-regulated kinase mediate the chemotactic and mitogenic effects of insulin-like growth factor-I in human hepatic stellate cells. J Hepatol, 2000, 32:227-234
26 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
27 Schnabl B, Bradham CA, Bennett BL, et al. TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells. Hepatology, 2001, 34:953-963
28 Reimann T, Hempel U, Krauwald S, et al. Transforming growth factor-beta1 induces activation of Ras, Raf-1, MEK and MAPK in rat hepatic stellate cells. FEBS Lett, 1997, 403:57-560
29 Hanafusa H, Ninomiya-Tsuji J, Masuyama N, et al. Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression. J Biol Chem, 1999, 274:27161-27167
30 Tsukada S, Westwick JK, Ikejima K, et al. SMAD and p38 MAPK signaling pathways independently regulate alpha1(I) collagen gene expression in unstimulated and transforming growth factor-beta-stimulated hepatic stellate cells. J Biol Chem, 2005, 280:10055-10064
31 Cao Q, Mak KM, Lieber CS. DLPC decreases TGF-beta1-induced collagen mRNA by inhibiting p38 MAPK in hepatic stellate cells. Am J Physiol Gastrointest Liver Physiol, 2002, 283:G1051-1061
32 Karin M, Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu Rev Immunol, 2000,18:621-663
33 Elsharkawy AM, Wright MC, Hay RT, et al. Persistent activation of nuclear factor-kappaB in cultured rat hepatic stellate cells involves the induction of potentially novel Rel-like factors and prolonged changes in the expression of IkappaB family proteins. Hepatology, 1999, 30:761-769
34 Lang A, Schoonhoven R, Tuvia S, et al. Nuclear factor kappaB in proliferation, activation, and apoptosis in rat hepatic stellate cells. J Hepatol, 2000, 33:49-58
35 Lee KS, Buck M, Houglum K, et al. Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression. J Clin Invest, 1995, 96:2461-2468
36 Lang A, Schrum LW, Schoonhoven R, et al. Expression of small heatshock protein alphaB-crystallin is induced after hepatic stellate cell activation. Am J Physiol Gastrointest Liver Physiol, 2000, 279:G1333-1342
37 Liu XJ, Yang L, Luo FM, et al. Association of differentially expressed genes with activation of mouse hepatic stellate cells by high-density cDNA microarray. World J Gastroenterol, 2004, 10:1600-1607
38 Knittel T, Mehde M, Grundmann A, et al. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat.Histochem Cell Biol, 2000, 113:443-453
39 Olaso E, Ikeda K, Eng FJ, et al. DDR2 receptor promotes MMP-2- mediated proliferation and invasion by hepatic stellate cells. J Clin Invest, 2001, 108:1369-1378
40 Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells.Semin Liver Dis, 2001, 21:373-384
41 Iredale JP, Goddard S, Murphy G, et al. Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes. Clin Sci (Lond), 1995, 89:75-81
42 Wang DR, Sato M, Li LN,et al. Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells.Cell Struct Funct, 2003, 28:505-513
43 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
44 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
45 McGuire RF, Bissell DM, Boyles J, et al. Role of extracellular matrix in regulating fenestrations of sinusoidal endothelial cells isolated from normal rat liver. Hepatology, 1992, 15:989-997
46 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
47 Knittel T, Mehde M,Grundmann A, et al. Expression of matrix metalloproteinases and their inhibitors during hepatic tissue repair in the rat. Histochem Cell Biol, 2000, 113:443-453
48 Issa R, Zhou X,Constandinou CM, et al. Spontaneous recovery from micronodular cirrhosis: evidence for incomplete resolution associated with matrix cross-linking G Gastroenterology, 2004, 126:1795-1808
49 Iredale JP. Hepatic stellate cell behavior during resolution of liver injury. Semin Liver Dis, 2001, 21:427-436.
50 Rocca P, Cocuzza E, Rasetti R, et al. Predictors of psychiatric disorders in liver transplantation candidates: logistic regression models. Liver Transpl, 2003, 9:721-726