PKIP在大鼠肝纤维化发生机制中的作用研究
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摘要
肝纤维化(hepatic fibrosis)是肝硬化乃至肝功能衰竭的共同病理基础和必经阶段,是影响慢性肝病的重要环节,因此肝纤维化的预防、治疗乃至逆转是阻断肝硬化的关键环节。肝纤维化也成为国内外学者研究慢性肝病的焦点。我国是肝病高发国家,以慢性乙型、丙型病毒性肝炎最为常见,在目前尚无良好的抗病毒药物的情况下,阻止或逆转肝纤维化进程成为治疗慢性肝病的重要对策。
生理状态下,HSCs在维生素A代谢和维持肝脏构架(产生细胞外间质和通过收缩作用调节窦状隙血流)中起重要作用。在慢性肝损伤过程中HSCs经历了表型转化(激活),变成了高增殖活性的类维生素A缺乏细胞(表达α-SMA),激活的HSCs在肝纤维化,肝硬化,门脉高压和肝癌形成过程中具有重要的作用。激活的HSCs获得不断增殖,合成大量的ECM分子,分泌细胞因子和生长因子,迁移和收缩的能力。肝星状细胞在塑料培养皿中进行体外培养时使HSCs的活化过程得到很好地重现。
在肝纤维化和肝硬化过程中,Raf-1/MEK/ERK1,2信号通路处于激活状态。Raf-1/MEK/ERK1,2信号通路的激活可以促进细胞生长,分化和迁移。许多不同的生长因子受体,包括PDGF和EGF受体通过小G蛋白Ras结合和聚集Raf-1激酶进而激活ERK/MAPK信号通路。Raf-1磷酸化MEK接着磷酸化和激活ERK。磷酸化的ERK进入细胞核通过结合多种转录因子来调节基因表达,进而促进了致纤维化蛋白分子的表达。
Yeung和他的同事们首次证明RKIP通过直接与Raf-1上的激酶位点相互作用抑制ERK/MAPK信号通路。RKIP是广泛表达的高度保守的胞浆蛋白,与其它的激酶抑制剂没有同源性,是人们目前发现的唯一的Raf-1蛋白天然抑制剂。在非磷酸状态下,RKIP通过与Raf-1结合阻止Raf-1磷酸化,干扰Raf/MEK结合,抑制MEK和下游分子的活化来达到负向调节Raf-1/MEK/ERK1,2信号通路的作用。磷酸化的RKIP从Raf-1上解离出来与GRK-2结合并阻断其活性,GRK-2是G蛋白偶联受体(GPCRs)的负反馈抑制蛋白。GRK-2使GPCRs磷酸化,从而将G蛋白与GPCRs解离导致G蛋白信号通路的活化。有数据表明RKIP被PKC磷酸化后能同时激活Raf-1/MEK/ERK1,2和GPCR信号通路。近些年来,RKIP被证明是一个抑制肿瘤细胞侵袭扩散的蛋白分子,包括前列腺癌,恶性黑色素瘤,乳腺癌,胰岛细胞癌,直肠癌和肝癌。RKIP在MDCK表皮细胞中的作用则相反,RKIP过表达能使MDCK由表皮细胞转化为成纤维样细胞并且促进细胞迁移。Locostatin(是一种不具有抗菌作用的黄恶唑酮衍生物)能够特异地消除RKIP对Raf-1的抑制作用,同时也是MDCK表皮细胞迁移抑制剂。进一步的研究证明RKIP在正向调节细胞与基层黏附和负向调节细胞与细胞的黏附过程中具有重要作用。
HSCs细胞形态改变和运动迁移构成了一系列肝纤维化生物过程的重要部分。尽管RKIP在MDCK表皮细胞和高侵袭力肿瘤细胞中的作用已经得到揭示,但是RKIP在肝纤维化形成时HSCs中的作用却知之甚少。在本实验中,我们将揭示RKIP在肝纤维化组织的表达及其在HSCs细胞增殖和迁移中的作用。实验内容主要包括以下3部分:
第一部分:肝纤维化大鼠肝组织中RKIP、p-RKIP、ERK和p-ERK的动态表达情况
目的:研究RKIP在胆总管结扎肝纤维化大鼠肝脏组织的表达情况。
方法:运用胆总管结扎方法制作大鼠肝纤维化模型,模型组分别于结扎后1周、2周、3周、4周麻醉动物,假手术组与4周模型组同批麻醉,留取肝脏标本。组织切片经HE和Masson三色染色检测病理变化,Western blot和免疫组织化学方法检测RKIP在肝组织的表达,用Western blot方法检测RKIP和ERK的磷酸化水平。
结果:①HE和Masson三色染色结果证明胆总管结扎大鼠肝纤维化模型制作成功。②RKIP在大鼠肝组织的免疫组织化学定位:RKIP在正常大鼠肝组织的肝细胞、胆管周围细胞、血管内皮细胞和肝窦周围细胞中均有表达,分布在细胞胞浆和胞膜;随着肝纤维化的发展,大鼠肝脏中RKIP阳性细胞明显减少,与肝细胞相比RKIP在汇管区和纤维间隔的成维细胞中表达较少,但在肝细胞胞膜中的表达增强。造模1~4周大鼠肝组织RKIP的阳性面积占总面积的百分比分别为(87.1±1.4) %,(77.2±2.2) %,(60.9±2.3) %和(48.2±2.2) %;2~4周大鼠肝组织RKIP的表达显著性低于正常对照组(89.2±1.3) %,P<0.05。③Western blot分析结果显示,RKIP在模型组3~4周肝纤维化组织的表达较假手术组下降,假手术组(105.7±4.9) %,模型组1~4周其表达分别(104.0±4.2) %,(103.1±3.5) %,(54.5±2.8) %和(41.0±1.8) %。ERK的相对表达量没有明显变化,假手术组表达为(66.8±2.3) %,模型组1~4周其表达分别(70.4±2.3) %,(69.3±2.0) %,(68.1±1.4) %和(67.4±2.4 ) %。随着肝纤维化的发展,RKIP和ERK的磷酸化水平逐渐增多。假手术组p-RKIP表达为(12.4±1.9) %,模型组1~4周其表达分别(43.6±2.2) %,(45.0±2.6) %,(83.9±2.9) %和(89.7±3.5) %。p-ERK假手术组表达为(25.8±3.2) %,模型组1~4周其表达分别(95.5±3.8) %,(132.1±2.7) %,(277.7±4.8) %和(332.9±2.4) %。
结论:胆总管结扎大鼠肝纤维化形成过程中,肝脏组织磷酸化ERK表达明显增加,存在Raf-1/MEK/ERK1,2信号转导通路的活化。RKIP表达水平下降,同时磷酸化RKIP水平升高,肝纤维化过程中肝脏组织Raf-1/MEK/ERK1,2信号转导途径的激活与RKIP的蛋白表达下降和磷酸化增加有关。
第二部分:RKIP在大鼠原代肝星状细胞活化中的作用
目的:体外分离大鼠原代HSCs,研究RKIP在大鼠原代肝星状细胞激活前后的表达情况。
方法:应用台盼兰染色,观察细胞存活率。应用荧光显微镜观察刚分离大鼠原代HSCs的自发荧光,以做特异性细胞鉴定。应用单克隆抗体α-SMA做免疫细胞和Western blot以鉴定原代HSCs体外培养后的活化状态。RT-real time PCR和Western blot方法检测RKIP在原代HSCs的表达情况,用Western blot方法检测RKIP、Raf-1和ERK1/2磷酸化水平。
结果:①采用原位灌注和密度梯度离心方法成功分离出大鼠原代HSCs。②台盼兰染色观察,细胞存活率大于92%,经过自发荧光特性鉴定纯度大于95%,每只大鼠肝星状细胞的得率大约为1.5×107至2.0×107。③在荧光显微镜(328nm)下观察,刚分离的原代HSCs呈自发蓝色荧光。在倒置显微镜下观察刚分离的HSCs呈圆形,胞浆中含有较多脂滴,细胞核位于细胞的一侧。培养至8天,大鼠HSCs呈活化状态,脂滴消失,变成梭形样细胞。④免疫细胞化学染色显示,刚分离的肝星状细胞α-SMA染色阴性;原代培养8天的肝星状细胞α-SMA阳性染色达100%。Western blot分析在42 KD的位置上出现一条杂交带,从杂交信号强度可知,大鼠肝星状细胞活化之后α-SMA表达明显增加。⑤RT-real time PCR分析结果显示,原代培养8天的肝星状细胞和刚分离的肝星状细胞之间RKIP基因表达无明显差异。Western blot分析在23 KD的位置上出现一条杂交带,从杂交信号强度可知,与刚分离的细胞(0.926±0.008)相比,大鼠肝星状细胞活化之后RKIP表达明显下降(0.377±0.009)。⑥Western blot分析结果显示,大鼠肝星状细胞活化之后RKIP、Raf-1和ERK的磷酸化水平却明显增多。刚分离的肝星状细胞p-RKIP、p-Raf-1和p-ERK表达分别为(1.087±0.021),(0.053±0.009)和(0.011±0.002)。而活化后的肝星状细胞p-RKIP、p-Raf-1和p-ERK表达分别表达为(1.721±0.081),(0.216±0.019)和(0.922±0.014)。
结论:采用原位灌注和密度梯度离心方法成功分离大鼠原代HSCs。原代HSCs体外培养活化之后信号通路Raf-1/MEK/ERK1,2处于激活状态,而RKIP表达则明显下降,p-RKIP表达则明显增加。
第三部分:RKIP对大鼠肝星状细胞增殖和迁移的影响
目的:观察RKIP对大鼠肝星状细胞增殖、凋亡和迁移能力的影响。
方法:大鼠肝星状细胞株HSC-T6的培养采用含10%FCS的DMEM培养液,重组质粒pCMV5-HA-RKIP或者空载体用Lipofectamine 2000包裹,按照说明书操作进行转染48 h后,弃去培养基;质粒pCMV5-HA-RKIP转染36 h后用50μM locostatin或者0.1% DMSO处理12 h,弃去培养基最后收集细胞提取细胞总蛋白以用于Western blot分析。一抗封闭时采用抗RKIP、pRKIP、Raf-1、pRaf-1、ERK1/2、pERK1/2和β-actin蛋白抗体测定。RKIP过表达质粒转染后的HSC-T6采用MTT法测定HSCs增殖;采用TUNEL试剂盒测定HSCs凋亡率。G418筛选RKIP稳定转染株,稳定转染的细胞用Transwell小室对细胞进行迁移评估;细胞爬片在含10% FCS的DMEM培养液中生长融合,然后用无菌的枪头刮开一个小伤口。在刮伤后0, 12和24小时的时间点,用伤口愈合实验测定HSCs迁移率。
结果:①为了检查RKIP在活化的HSCs中的作用,HSC-T6细胞株用RKIP质粒转染后RKIP表达比对照组明显增加(RKIP/β-actin, 0.673±0.016 vs 0.227±0.025, P<0.01)。而相对应,Raf-1和ERK磷酸化水平明显降低(pRaf/Raf, 0.027±0.006 vs 0.853±0.022, P<0.01; pERK/ERK, 0.293±0.012 vs 1.027±0.060, P<0.01)。Locostatin处理后,Western blot结果显示RKIP过表达降低(RKIP/β-actin, 0.338±0.016, vs 0.673±0.016, P<0.01)。而相对应RKIP、Raf-1和ERK磷酸化水平增加(pRKIP/RKIP, 1.090±0.128 vs 0.332±0.024, P<0.01; pRaf/Raf, 0.216±0.015 vs 0.027±0.006, P<0.01; pERK/ERK, 0.790±0.028 vs 0.293±0.012, P<0.01)。②相对于空白对照组RKIP过表达抑制HSCs增殖(0.981±0.020 vs 0.860±0.022, P<0.01)。但是相对于空白对照组,RKIP过表达对HSCs凋亡没有明显的影响。③Transwell assay结果显示,相对于空白对照组(124.00±6.00),RKIP过表达组、RKIP +Locostatin组和Locostatin组细胞迁移数分别为(161.00±9.17)、(43.00±7.94)和(10.00±3.61)。RKIP过表达可以显著促进HSC-T6细胞迁移,而locostatin具有相反作用。为了进一步确认RKIP在细胞迁移中的作用,我们又采用伤口愈合实验(wound closure assay)进行测定。生长融合后的HSC-T6细胞刮开一个小口,24小时之后过表达RKIP的HSC-T6已经重新融合,而空白对照组融合率只有73.2%。Locostatin明显抑制HSC-T6细胞的伤口愈合率,只有3.3%。Wound closure assay显示相对于对照组,RKIP易位过表达可以促进细胞迁移,locostatin则抑制伤口愈合。
结论:在HSCs中RKIP可以调节Raf-1/MEK/ERK1,2信号通路的活化。体外细胞培养研究表明,RKIP过表达可以抑制HSCs的细胞增殖,但是促进HSCs的细胞迁移。
Chronic damage results in a progressive accumulation of scarring proteins (hepatic fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Hepatic fibrosis is the important link that affects the chronic liver disease. Therefore, prevention and treatment of hepatic fibrosis is the focus of chronic liver disease. HBV and HCV type hepatitis are the most mainly chronic liver diseases in our country. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.
Physiologically, hepatic stellate cells (HSCs) play a pivotal role in vitamin A metabolism and in maintaining the liver’s architecture by producing components of extracellular matrix and regulating sinusoidal blood flow by contractility. During chronic liver injury, HSCs undergo phenotypical transformation (i.e. activation) to actively proliferate retinoid-deficient cells that express theα-smooth muscle actin (α-SMA) known as activated HSCs. Activated HSCs play an essential role in the pathogenesis of liver fibrosis and cirrhosis, the development of portal hypertension, and in the progression of liver cancer. Activated HSCs have the ability to proliferate, to synthesize a large variety of ECM molecules, to secrete cytokines and growth factors, and to migrate and contract. This activation process of HSCs is reproducible in vitro.
The extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway has been shown to be activated in liver fibrosis and cirrhosis and is involved in cell growth, differentiation, and migration. Many different growth factor receptors, including PDGF receptor and EGF receptor, activate the ERK/MAPK signaling pathway through small G-protein Ras, which consequently binds Raf-1 kinase and thereby recruits Raf-1 to the inner surface of the cell membrane. After this event, Raf-1 phosphorylates MEK, which in turn phosphorylates and activates ERK. Phosphorylated ERK translocates into the nucleus and regulates gene expression via interactions with various transcription factors and subsequently, results in fibrosis related protein expression.
Yeung et al, who identified the Raf kinase inhibitor protein (RKIP) as a protein that directly interacts with the kinase domain of Raf-1, first demonstrated that RKIP acts as an inhibitor of the ERK/MAPK signaling pathway. RKIP is a widely expressed and highly conserved cytosolic protein that does not share any significant homology with other kinase inhibitors. In its non-phosphorylated form, RKIP negatively regulates the Raf-1/MEK/ERK1,2 pathway by interfering with the activity of Raf-1, disrupting the Raf/MEK interaction, and preventing the activation of MEK and downstream components. In its phosphorylated state, RKIP dissociates from Raf-1 and combines with GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP by PKC stimulates both the Raf-1/MEK/ERK1,2 and GPCR pathways. In recent years, RKIP has been identified as a member of a novel class of molecules that suppress metastasis, with evidences from prostate cancer, malignant melanomas, breast cancer, insulinomas, colorectal cancer and hepatocellular carcinoma. Such activity appears to be opposite to that in MDCK epithelial cells, in which RKIP overexpression converts the cells to fibroblast-like morphology and promotes migration. Locostatin, a non-antibacterial oxazolidinone derivative, was discovered to abrogate RKIP’s ability to bind and inhibit Raf-1 kinase by disrupting a protein-protein interaction. Locostatin also inhibits epithelial cell sheet migration. Another study suggests that RKIP plays important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion and negatively controlling cell-cell adhesion.
Although the importance of RKIP in MDCK epithelial cells and metastatic cells of tumors has been well documented, there is no report so far on the role of RKIP in HSC behavior and hepatic fibrosis. The present study investigates the expression of RKIP in liver fibrogenesis and the involvement of RKIP in HSC proliferation and migration. The experiments contained three parts as below:
Part 1: The dynamic expressions of RKIP, p-RKIP, ERK and p-ERK in liver of the bile duct ligated rats
Objective:To explore the dynamic expressions of RKIP, p-RKIP, p-ERK and ERK in liver of the bile duct ligated rats.
Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL). Livers in model group were harvested at fixed timepoints: 1wk, 2wk, 3wk and 4wk after operation. Livers in sham operation group were harvested at 4wk after operation. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson’s trichrome method. RKIP protein expression and phosphorylatin of RKIP and ERK in the livers were determined by Western blot, while the distribution of RKIP in the livers was assessed immunohistochemistically.
Results:①Hematoxylin and eosin staining of liver established the bile duct ligated rats.②The location of RKIP in rat liver by immunohistochemistry assay: RKIP was located in the cytoplasm and plasma membrane, and expressed in hepatocytes, bile duct epitheliums, vascular endothelial cells and sinusoidal endothelial cells of the normal rat liver. With the development of hepatic fibrosis, the positive cells of RKIP decreased. RKIP expression was decreased in the myofibroblast of portal ducts and fiber septa, but was increased in the plasma membrane of hepatocytes. The positive areas of RKIP in the rat livers in model groups at week 1 to 4 were (87.1±1.4) %, (77.2±2.2) %, (60.9±2.3) % and (48.2±2.2) %. RKIP expression in model groups at week 2 to 4 was all lower than that in control group ((89.2±1.3) %, P<0.05).③Western blot analysis: RKIP expression in model groups at week 3 to 4 was decreased compared with the control group ((105.7±4.9) %, P<0.05). RKIP expression in model groups at week 1 to 4 was (104.0±4.2) %, (103.1±3.5) %, (54.5±2.8) % and (41.0±1.8) %. ERK expression was not changed, with (66.8±2.3) % in control group, and (70.4±2.3) %, (69.3±2.0) %, (68.1±1.4) %, (67.4±2.4) % in model groups at week 1 to 4. With the development of hepatic fibrosis, phosphorylatin of RKIP and ERK in the liver was increased. The expression of p-RKIP in control group was (12.4±1.9) %, with (43.6±2.2) %, (45.0±2.6) %, (83.9±2.9) % and (89.7±3.5) % in model groups at week 1 to 4. The expression of p-RKIP in control group was (25.8±3.2) %, with (95.5±3.8) %, (132.1±2.7) %, (277.7±4.8) % and (332.9±2.4) % in model groups at week 1 to 4.
Conclusions: With the development of liver fibrosis, phosphorylatin of ERK in the livers was increased and Raf-1/MEK/ERK1,2 signaling pathway was activated in liver of the bile duct ligated rats. The activation of Raf-1/MEK/ERK1,2 signaling pathway was correlated with decreased RKIP expression and increased phosphorylated RKIP in the livers of bile duct ligated rats.
Part 2: The RKIP expressions during rat HSC activation in vitro Objective: To isolate rat HSCs, and investigate the RKIP expressions during HSC activation.
Methods: The viability of all cells was verified by phase contrast microscopy as well as the ability to exclude Trypan Blue. To evaluate the purity of the cultures, HSCs were tested by immunofluorescence. With the use ofα-SMA immunoreactivity and Western Blot as an activation parameter, HSCs were tested during culture in vitro. RKIP protein expression and phosphorylatin of RKIP and ERK in the HSCs were determined by Western blot, while the RKIP gene expression was assessed by real time RT-PCR.
Results:①Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz.②Cell viability was greater than 92% as determined by Trypan Blue exclusion. HSC purity, as assessed by phase-contrast microscopy and vitamin A autofluorescence immediately after plating, was greater than 95%, with a yield ranging from 1.5×107 to 2.0×107 HSC/rat.③Primary HSCs show blue under ultraviolet light (328nm). The primary HSC is in rich of retinoid which squeezes the nuclear to one side. After 8 days culture, rat HSC is activated. Activated HSC loses retinoid and becomes myofibroblast-like cell.④Rat HSCs were checked byα-SMA staining with monoclonal antibody at day 1 (quiescent,α-SMA negative cells) and day 8 (activated,α-SMA positive cells). Western blots also showed that the activated HSCs wereα-SMA positive.⑤By real time RT-PCR analysis, RKIP mRNA expression was not changed during rat HSC culture in vitro. RKIP protein expression was significantly decreased in activated HSCs compared with those in quiescent status (RKIP/β-actin, 0.377±0.009, vs 0.926±0.008, P<0.01).⑥RKIP, Raf, and ERK phosphorylations were significantly increased (pRKIP/RKIP, 1.721±0.081 vs 1.087±0.021, P<0.01; pRaf/Raf, 0.216±0.019 vs 0.053±0.009, P<0.01; pERK/ERK, 0.922±0.014 vs 0.011±0.002, P<0.01). These results indicate that RKIP downregulation and RKIP phosphorylation upregulation activated the ERK/MAPK pathway.
Conclusions: Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz. RKIP protein expression is downregulated and high phosphorylation of RKIP is followed by activated Raf/MEK/ERK pathway after activation of rat HSCs, suggesting that this protein is a possible factor in regulation of HSC cell proliferation and migration.
Part 3: The role of RKIP in HSC proliferation and migration
Objective: To investigate the role of RKIP in HSC proliferation, apoptosis, and migration.
Methods: Rat HSC cell line (HSC-T6) was used in this study, and cultured in HEPES-buffered DMEM. For transfection, pCMV5-HA-RKIP or empty vector plasmid (control) was added to HSC-T6 when 60%-70% confluence using Lipofectamine 2000 reagent. 48 hours later, the medium was renewed. After 36 hours of transient transfection, HSC-T6 cells in 6-well dishes were treated with 0.1% DMSO carrier solvent or with 50μM locostatin for 12 hours. The medium was renewed, and cells were rinsed with PBS thrice and extracted for SDS-PAGE in lysis buffer. Western blot analysis was undertaken by using the following primary antibodies: RKIP, pRKIP, Raf-1, pRaf-1, ERK1/2, pERK1/2 andβ-actin. HSC proliferation and apoptosis were evaluated with 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. To cell migration assay, G418 was added for the selection of stable clones. The method for Transwell cell migration assay was used. For wound closure assay, HSC-T6 cells were grown to confluence in DMEM supplemented with 10% FCS and then scratch wounded with a sterile plastic micropipette tip. At 0, 12, and 24 hours, photographs were taken at the same position of the wound.
Results:①HSC-T6 cells transfected with RKIP plasmids overexpressed RKIP (RKIP/β-actin, 0.673±0.016, vs 0.227±0.025, P<0.01), and the phosphorylation of Raf-1 and ERK were significantly decreased in these cells (pRaf/Raf, 0.027±0.006 vs 0.853±0.022, P<0.01; pERK/ERK, 0.293±0.012 vs 1.027±0.060, P<0.01). Locostatin significantly decreased RKIP expression in transfected cells (RKIP/β-actin, 0.338±0.016, vs 0.673±0.016, P<0.01). Locostatin also increased the phosphorylation of RKIP, Raf-1, and ERK (pRKIP/RKIP, 1.090±0.128 vs 0.332±0.024, P<0.01; pRaf/Raf, 0.216±0.015 vs 0.027±0.006, P<0.01; pERK/ERK, 0.790±0.028 vs 0.293±0.012, P<0.01).②The proliferation of HSCs was significantly inhibited in cells overexpressing RKIP compared with those in the empty vector group (0.981±0.020 vs 0.860±0.022, P<0.01), while RKIP overexpression did not significantly affect HSC apoptosis.③RKIP overexpression significantly increased HSC-T6 cell migration rates compared to the control (161.00±9.17 vs 124.00±6.00, P<0.01), and locostatin abrogated this effect (43.00±7.94 vs 161.00±9.17, P<0.01), and HSC-T6 cell treated with locostatin (10.00±3.61 vs 161.00±9.17, P<0.01). To further confirm the effect of RKIP on cell migration, RKIP-overexpressing HSC-T6 cells were grown to confluence and then scrape wounded. Within 24 hours, RKIP-overexpressing cells grew to confluence again while cells transfected with the empty vector grew only to 73.2%. Locostatin significantly delayed wound closure of HSC-T6 cells as they grew only to 3.3%. Wound closure assay showed that overexpression of RKIP promoted wound closure while locostatin inhibited wound closure.
Conclusions: RKIP inhibits Raf-1/MEK/ERK1,2 signaling and this inhibition impedes HSCs proliferation. RKIP promotes HSCs migration and wound closure.
生理状态下,HSCs在维生素A代谢和维持肝脏构架(产生细胞外间质和通过收缩作用调节窦状隙血流)中起重要作用。在慢性肝损伤过程中HSCs经历了表型转化(激活),变成了高增殖活性的类维生素A缺乏细胞(表达α-SMA),激活的HSCs在肝纤维化,肝硬化,门脉高压和肝癌形成过程中具有重要的作用。激活的HSCs获得不断增殖,合成大量的ECM分子,分泌细胞因子和生长因子,迁移和收缩的能力。肝星状细胞在塑料培养皿中进行体外培养时使HSCs的活化过程得到很好地重现。
在肝纤维化和肝硬化过程中,Raf-1/MEK/ERK1,2信号通路处于激活状态。Raf-1/MEK/ERK1,2信号通路的激活可以促进细胞生长,分化和迁移。许多不同的生长因子受体,包括PDGF和EGF受体通过小G蛋白Ras结合和聚集Raf-1激酶进而激活ERK/MAPK信号通路。Raf-1磷酸化MEK接着磷酸化和激活ERK。磷酸化的ERK进入细胞核通过结合多种转录因子来调节基因表达,进而促进了致纤维化蛋白分子的表达。
Yeung和他的同事们首次证明RKIP通过直接与Raf-1上的激酶位点相互作用抑制ERK/MAPK信号通路。RKIP是广泛表达的高度保守的胞浆蛋白,与其它的激酶抑制剂没有同源性,是人们目前发现的唯一的Raf-1蛋白天然抑制剂。在非磷酸状态下,RKIP通过与Raf-1结合阻止Raf-1磷酸化,干扰Raf/MEK结合,抑制MEK和下游分子的活化来达到负向调节Raf-1/MEK/ERK1,2信号通路的作用。磷酸化的RKIP从Raf-1上解离出来与GRK-2结合并阻断其活性,GRK-2是G蛋白偶联受体(GPCRs)的负反馈抑制蛋白。GRK-2使GPCRs磷酸化,从而将G蛋白与GPCRs解离导致G蛋白信号通路的活化。有数据表明RKIP被PKC磷酸化后能同时激活Raf-1/MEK/ERK1,2和GPCR信号通路。近些年来,RKIP被证明是一个抑制肿瘤细胞侵袭扩散的蛋白分子,包括前列腺癌,恶性黑色素瘤,乳腺癌,胰岛细胞癌,直肠癌和肝癌。RKIP在MDCK表皮细胞中的作用则相反,RKIP过表达能使MDCK由表皮细胞转化为成纤维样细胞并且促进细胞迁移。Locostatin(是一种不具有抗菌作用的黄恶唑酮衍生物)能够特异地消除RKIP对Raf-1的抑制作用,同时也是MDCK表皮细胞迁移抑制剂。进一步的研究证明RKIP在正向调节细胞与基层黏附和负向调节细胞与细胞的黏附过程中具有重要作用。
HSCs细胞形态改变和运动迁移构成了一系列肝纤维化生物过程的重要部分。尽管RKIP在MDCK表皮细胞和高侵袭力肿瘤细胞中的作用已经得到揭示,但是RKIP在肝纤维化形成时HSCs中的作用却知之甚少。在本实验中,我们将揭示RKIP在肝纤维化组织的表达及其在HSCs细胞增殖和迁移中的作用。实验内容主要包括以下3部分:
第一部分:肝纤维化大鼠肝组织中RKIP、p-RKIP、ERK和p-ERK的动态表达情况
目的:研究RKIP在胆总管结扎肝纤维化大鼠肝脏组织的表达情况。
方法:运用胆总管结扎方法制作大鼠肝纤维化模型,模型组分别于结扎后1周、2周、3周、4周麻醉动物,假手术组与4周模型组同批麻醉,留取肝脏标本。组织切片经HE和Masson三色染色检测病理变化,Western blot和免疫组织化学方法检测RKIP在肝组织的表达,用Western blot方法检测RKIP和ERK的磷酸化水平。
结果:①HE和Masson三色染色结果证明胆总管结扎大鼠肝纤维化模型制作成功。②RKIP在大鼠肝组织的免疫组织化学定位:RKIP在正常大鼠肝组织的肝细胞、胆管周围细胞、血管内皮细胞和肝窦周围细胞中均有表达,分布在细胞胞浆和胞膜;随着肝纤维化的发展,大鼠肝脏中RKIP阳性细胞明显减少,与肝细胞相比RKIP在汇管区和纤维间隔的成维细胞中表达较少,但在肝细胞胞膜中的表达增强。造模1~4周大鼠肝组织RKIP的阳性面积占总面积的百分比分别为(87.1±1.4) %,(77.2±2.2) %,(60.9±2.3) %和(48.2±2.2) %;2~4周大鼠肝组织RKIP的表达显著性低于正常对照组(89.2±1.3) %,P<0.05。③Western blot分析结果显示,RKIP在模型组3~4周肝纤维化组织的表达较假手术组下降,假手术组(105.7±4.9) %,模型组1~4周其表达分别(104.0±4.2) %,(103.1±3.5) %,(54.5±2.8) %和(41.0±1.8) %。ERK的相对表达量没有明显变化,假手术组表达为(66.8±2.3) %,模型组1~4周其表达分别(70.4±2.3) %,(69.3±2.0) %,(68.1±1.4) %和(67.4±2.4 ) %。随着肝纤维化的发展,RKIP和ERK的磷酸化水平逐渐增多。假手术组p-RKIP表达为(12.4±1.9) %,模型组1~4周其表达分别(43.6±2.2) %,(45.0±2.6) %,(83.9±2.9) %和(89.7±3.5) %。p-ERK假手术组表达为(25.8±3.2) %,模型组1~4周其表达分别(95.5±3.8) %,(132.1±2.7) %,(277.7±4.8) %和(332.9±2.4) %。
结论:胆总管结扎大鼠肝纤维化形成过程中,肝脏组织磷酸化ERK表达明显增加,存在Raf-1/MEK/ERK1,2信号转导通路的活化。RKIP表达水平下降,同时磷酸化RKIP水平升高,肝纤维化过程中肝脏组织Raf-1/MEK/ERK1,2信号转导途径的激活与RKIP的蛋白表达下降和磷酸化增加有关。
第二部分:RKIP在大鼠原代肝星状细胞活化中的作用
目的:体外分离大鼠原代HSCs,研究RKIP在大鼠原代肝星状细胞激活前后的表达情况。
方法:应用台盼兰染色,观察细胞存活率。应用荧光显微镜观察刚分离大鼠原代HSCs的自发荧光,以做特异性细胞鉴定。应用单克隆抗体α-SMA做免疫细胞和Western blot以鉴定原代HSCs体外培养后的活化状态。RT-real time PCR和Western blot方法检测RKIP在原代HSCs的表达情况,用Western blot方法检测RKIP、Raf-1和ERK1/2磷酸化水平。
结果:①采用原位灌注和密度梯度离心方法成功分离出大鼠原代HSCs。②台盼兰染色观察,细胞存活率大于92%,经过自发荧光特性鉴定纯度大于95%,每只大鼠肝星状细胞的得率大约为1.5×107至2.0×107。③在荧光显微镜(328nm)下观察,刚分离的原代HSCs呈自发蓝色荧光。在倒置显微镜下观察刚分离的HSCs呈圆形,胞浆中含有较多脂滴,细胞核位于细胞的一侧。培养至8天,大鼠HSCs呈活化状态,脂滴消失,变成梭形样细胞。④免疫细胞化学染色显示,刚分离的肝星状细胞α-SMA染色阴性;原代培养8天的肝星状细胞α-SMA阳性染色达100%。Western blot分析在42 KD的位置上出现一条杂交带,从杂交信号强度可知,大鼠肝星状细胞活化之后α-SMA表达明显增加。⑤RT-real time PCR分析结果显示,原代培养8天的肝星状细胞和刚分离的肝星状细胞之间RKIP基因表达无明显差异。Western blot分析在23 KD的位置上出现一条杂交带,从杂交信号强度可知,与刚分离的细胞(0.926±0.008)相比,大鼠肝星状细胞活化之后RKIP表达明显下降(0.377±0.009)。⑥Western blot分析结果显示,大鼠肝星状细胞活化之后RKIP、Raf-1和ERK的磷酸化水平却明显增多。刚分离的肝星状细胞p-RKIP、p-Raf-1和p-ERK表达分别为(1.087±0.021),(0.053±0.009)和(0.011±0.002)。而活化后的肝星状细胞p-RKIP、p-Raf-1和p-ERK表达分别表达为(1.721±0.081),(0.216±0.019)和(0.922±0.014)。
结论:采用原位灌注和密度梯度离心方法成功分离大鼠原代HSCs。原代HSCs体外培养活化之后信号通路Raf-1/MEK/ERK1,2处于激活状态,而RKIP表达则明显下降,p-RKIP表达则明显增加。
第三部分:RKIP对大鼠肝星状细胞增殖和迁移的影响
目的:观察RKIP对大鼠肝星状细胞增殖、凋亡和迁移能力的影响。
方法:大鼠肝星状细胞株HSC-T6的培养采用含10%FCS的DMEM培养液,重组质粒pCMV5-HA-RKIP或者空载体用Lipofectamine 2000包裹,按照说明书操作进行转染48 h后,弃去培养基;质粒pCMV5-HA-RKIP转染36 h后用50μM locostatin或者0.1% DMSO处理12 h,弃去培养基最后收集细胞提取细胞总蛋白以用于Western blot分析。一抗封闭时采用抗RKIP、pRKIP、Raf-1、pRaf-1、ERK1/2、pERK1/2和β-actin蛋白抗体测定。RKIP过表达质粒转染后的HSC-T6采用MTT法测定HSCs增殖;采用TUNEL试剂盒测定HSCs凋亡率。G418筛选RKIP稳定转染株,稳定转染的细胞用Transwell小室对细胞进行迁移评估;细胞爬片在含10% FCS的DMEM培养液中生长融合,然后用无菌的枪头刮开一个小伤口。在刮伤后0, 12和24小时的时间点,用伤口愈合实验测定HSCs迁移率。
结果:①为了检查RKIP在活化的HSCs中的作用,HSC-T6细胞株用RKIP质粒转染后RKIP表达比对照组明显增加(RKIP/β-actin, 0.673±0.016 vs 0.227±0.025, P<0.01)。而相对应,Raf-1和ERK磷酸化水平明显降低(pRaf/Raf, 0.027±0.006 vs 0.853±0.022, P<0.01; pERK/ERK, 0.293±0.012 vs 1.027±0.060, P<0.01)。Locostatin处理后,Western blot结果显示RKIP过表达降低(RKIP/β-actin, 0.338±0.016, vs 0.673±0.016, P<0.01)。而相对应RKIP、Raf-1和ERK磷酸化水平增加(pRKIP/RKIP, 1.090±0.128 vs 0.332±0.024, P<0.01; pRaf/Raf, 0.216±0.015 vs 0.027±0.006, P<0.01; pERK/ERK, 0.790±0.028 vs 0.293±0.012, P<0.01)。②相对于空白对照组RKIP过表达抑制HSCs增殖(0.981±0.020 vs 0.860±0.022, P<0.01)。但是相对于空白对照组,RKIP过表达对HSCs凋亡没有明显的影响。③Transwell assay结果显示,相对于空白对照组(124.00±6.00),RKIP过表达组、RKIP +Locostatin组和Locostatin组细胞迁移数分别为(161.00±9.17)、(43.00±7.94)和(10.00±3.61)。RKIP过表达可以显著促进HSC-T6细胞迁移,而locostatin具有相反作用。为了进一步确认RKIP在细胞迁移中的作用,我们又采用伤口愈合实验(wound closure assay)进行测定。生长融合后的HSC-T6细胞刮开一个小口,24小时之后过表达RKIP的HSC-T6已经重新融合,而空白对照组融合率只有73.2%。Locostatin明显抑制HSC-T6细胞的伤口愈合率,只有3.3%。Wound closure assay显示相对于对照组,RKIP易位过表达可以促进细胞迁移,locostatin则抑制伤口愈合。
结论:在HSCs中RKIP可以调节Raf-1/MEK/ERK1,2信号通路的活化。体外细胞培养研究表明,RKIP过表达可以抑制HSCs的细胞增殖,但是促进HSCs的细胞迁移。
Chronic damage results in a progressive accumulation of scarring proteins (hepatic fibrosis) that, with increasing severity, alters tissue structure and function, leading to cirrhosis and liver failure. Hepatic fibrosis is the important link that affects the chronic liver disease. Therefore, prevention and treatment of hepatic fibrosis is the focus of chronic liver disease. HBV and HCV type hepatitis are the most mainly chronic liver diseases in our country. Understanding the complex intercellular interactions regulating liver fibrogenesis is of increasing importance in view of predicted increases in chronic liver disease and the current paucity of effective therapies.
Physiologically, hepatic stellate cells (HSCs) play a pivotal role in vitamin A metabolism and in maintaining the liver’s architecture by producing components of extracellular matrix and regulating sinusoidal blood flow by contractility. During chronic liver injury, HSCs undergo phenotypical transformation (i.e. activation) to actively proliferate retinoid-deficient cells that express theα-smooth muscle actin (α-SMA) known as activated HSCs. Activated HSCs play an essential role in the pathogenesis of liver fibrosis and cirrhosis, the development of portal hypertension, and in the progression of liver cancer. Activated HSCs have the ability to proliferate, to synthesize a large variety of ECM molecules, to secrete cytokines and growth factors, and to migrate and contract. This activation process of HSCs is reproducible in vitro.
The extracellular signal-regulated kinases (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway has been shown to be activated in liver fibrosis and cirrhosis and is involved in cell growth, differentiation, and migration. Many different growth factor receptors, including PDGF receptor and EGF receptor, activate the ERK/MAPK signaling pathway through small G-protein Ras, which consequently binds Raf-1 kinase and thereby recruits Raf-1 to the inner surface of the cell membrane. After this event, Raf-1 phosphorylates MEK, which in turn phosphorylates and activates ERK. Phosphorylated ERK translocates into the nucleus and regulates gene expression via interactions with various transcription factors and subsequently, results in fibrosis related protein expression.
Yeung et al, who identified the Raf kinase inhibitor protein (RKIP) as a protein that directly interacts with the kinase domain of Raf-1, first demonstrated that RKIP acts as an inhibitor of the ERK/MAPK signaling pathway. RKIP is a widely expressed and highly conserved cytosolic protein that does not share any significant homology with other kinase inhibitors. In its non-phosphorylated form, RKIP negatively regulates the Raf-1/MEK/ERK1,2 pathway by interfering with the activity of Raf-1, disrupting the Raf/MEK interaction, and preventing the activation of MEK and downstream components. In its phosphorylated state, RKIP dissociates from Raf-1 and combines with GRK-2, a negative regulator of G-protein coupled receptors (GPCRs). Available data indicate that the phosphorylation of RKIP by PKC stimulates both the Raf-1/MEK/ERK1,2 and GPCR pathways. In recent years, RKIP has been identified as a member of a novel class of molecules that suppress metastasis, with evidences from prostate cancer, malignant melanomas, breast cancer, insulinomas, colorectal cancer and hepatocellular carcinoma. Such activity appears to be opposite to that in MDCK epithelial cells, in which RKIP overexpression converts the cells to fibroblast-like morphology and promotes migration. Locostatin, a non-antibacterial oxazolidinone derivative, was discovered to abrogate RKIP’s ability to bind and inhibit Raf-1 kinase by disrupting a protein-protein interaction. Locostatin also inhibits epithelial cell sheet migration. Another study suggests that RKIP plays important roles in the regulation of cell adhesion, positively controlling cell-substratum adhesion and negatively controlling cell-cell adhesion.
Although the importance of RKIP in MDCK epithelial cells and metastatic cells of tumors has been well documented, there is no report so far on the role of RKIP in HSC behavior and hepatic fibrosis. The present study investigates the expression of RKIP in liver fibrogenesis and the involvement of RKIP in HSC proliferation and migration. The experiments contained three parts as below:
Part 1: The dynamic expressions of RKIP, p-RKIP, ERK and p-ERK in liver of the bile duct ligated rats
Objective:To explore the dynamic expressions of RKIP, p-RKIP, p-ERK and ERK in liver of the bile duct ligated rats.
Methods:Hepatic fibrosis was induced in Sprague-Dawley rats by bile duct ligation (BDL). Livers in model group were harvested at fixed timepoints: 1wk, 2wk, 3wk and 4wk after operation. Livers in sham operation group were harvested at 4wk after operation. Histopathological changes were evaluated by hematoxylin and eosin staining and by Masson’s trichrome method. RKIP protein expression and phosphorylatin of RKIP and ERK in the livers were determined by Western blot, while the distribution of RKIP in the livers was assessed immunohistochemistically.
Results:①Hematoxylin and eosin staining of liver established the bile duct ligated rats.②The location of RKIP in rat liver by immunohistochemistry assay: RKIP was located in the cytoplasm and plasma membrane, and expressed in hepatocytes, bile duct epitheliums, vascular endothelial cells and sinusoidal endothelial cells of the normal rat liver. With the development of hepatic fibrosis, the positive cells of RKIP decreased. RKIP expression was decreased in the myofibroblast of portal ducts and fiber septa, but was increased in the plasma membrane of hepatocytes. The positive areas of RKIP in the rat livers in model groups at week 1 to 4 were (87.1±1.4) %, (77.2±2.2) %, (60.9±2.3) % and (48.2±2.2) %. RKIP expression in model groups at week 2 to 4 was all lower than that in control group ((89.2±1.3) %, P<0.05).③Western blot analysis: RKIP expression in model groups at week 3 to 4 was decreased compared with the control group ((105.7±4.9) %, P<0.05). RKIP expression in model groups at week 1 to 4 was (104.0±4.2) %, (103.1±3.5) %, (54.5±2.8) % and (41.0±1.8) %. ERK expression was not changed, with (66.8±2.3) % in control group, and (70.4±2.3) %, (69.3±2.0) %, (68.1±1.4) %, (67.4±2.4) % in model groups at week 1 to 4. With the development of hepatic fibrosis, phosphorylatin of RKIP and ERK in the liver was increased. The expression of p-RKIP in control group was (12.4±1.9) %, with (43.6±2.2) %, (45.0±2.6) %, (83.9±2.9) % and (89.7±3.5) % in model groups at week 1 to 4. The expression of p-RKIP in control group was (25.8±3.2) %, with (95.5±3.8) %, (132.1±2.7) %, (277.7±4.8) % and (332.9±2.4) % in model groups at week 1 to 4.
Conclusions: With the development of liver fibrosis, phosphorylatin of ERK in the livers was increased and Raf-1/MEK/ERK1,2 signaling pathway was activated in liver of the bile duct ligated rats. The activation of Raf-1/MEK/ERK1,2 signaling pathway was correlated with decreased RKIP expression and increased phosphorylated RKIP in the livers of bile duct ligated rats.
Part 2: The RKIP expressions during rat HSC activation in vitro Objective: To isolate rat HSCs, and investigate the RKIP expressions during HSC activation.
Methods: The viability of all cells was verified by phase contrast microscopy as well as the ability to exclude Trypan Blue. To evaluate the purity of the cultures, HSCs were tested by immunofluorescence. With the use ofα-SMA immunoreactivity and Western Blot as an activation parameter, HSCs were tested during culture in vitro. RKIP protein expression and phosphorylatin of RKIP and ERK in the HSCs were determined by Western blot, while the RKIP gene expression was assessed by real time RT-PCR.
Results:①Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz.②Cell viability was greater than 92% as determined by Trypan Blue exclusion. HSC purity, as assessed by phase-contrast microscopy and vitamin A autofluorescence immediately after plating, was greater than 95%, with a yield ranging from 1.5×107 to 2.0×107 HSC/rat.③Primary HSCs show blue under ultraviolet light (328nm). The primary HSC is in rich of retinoid which squeezes the nuclear to one side. After 8 days culture, rat HSC is activated. Activated HSC loses retinoid and becomes myofibroblast-like cell.④Rat HSCs were checked byα-SMA staining with monoclonal antibody at day 1 (quiescent,α-SMA negative cells) and day 8 (activated,α-SMA positive cells). Western blots also showed that the activated HSCs wereα-SMA positive.⑤By real time RT-PCR analysis, RKIP mRNA expression was not changed during rat HSC culture in vitro. RKIP protein expression was significantly decreased in activated HSCs compared with those in quiescent status (RKIP/β-actin, 0.377±0.009, vs 0.926±0.008, P<0.01).⑥RKIP, Raf, and ERK phosphorylations were significantly increased (pRKIP/RKIP, 1.721±0.081 vs 1.087±0.021, P<0.01; pRaf/Raf, 0.216±0.019 vs 0.053±0.009, P<0.01; pERK/ERK, 0.922±0.014 vs 0.011±0.002, P<0.01). These results indicate that RKIP downregulation and RKIP phosphorylation upregulation activated the ERK/MAPK pathway.
Conclusions: Primary rat HSCs were isolated successfully by sequential digestion of the liver with Pronase and collagenase, followed by single step density gradient centrifugation with Nycodenz. RKIP protein expression is downregulated and high phosphorylation of RKIP is followed by activated Raf/MEK/ERK pathway after activation of rat HSCs, suggesting that this protein is a possible factor in regulation of HSC cell proliferation and migration.
Part 3: The role of RKIP in HSC proliferation and migration
Objective: To investigate the role of RKIP in HSC proliferation, apoptosis, and migration.
Methods: Rat HSC cell line (HSC-T6) was used in this study, and cultured in HEPES-buffered DMEM. For transfection, pCMV5-HA-RKIP or empty vector plasmid (control) was added to HSC-T6 when 60%-70% confluence using Lipofectamine 2000 reagent. 48 hours later, the medium was renewed. After 36 hours of transient transfection, HSC-T6 cells in 6-well dishes were treated with 0.1% DMSO carrier solvent or with 50μM locostatin for 12 hours. The medium was renewed, and cells were rinsed with PBS thrice and extracted for SDS-PAGE in lysis buffer. Western blot analysis was undertaken by using the following primary antibodies: RKIP, pRKIP, Raf-1, pRaf-1, ERK1/2, pERK1/2 andβ-actin. HSC proliferation and apoptosis were evaluated with 3-(4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. To cell migration assay, G418 was added for the selection of stable clones. The method for Transwell cell migration assay was used. For wound closure assay, HSC-T6 cells were grown to confluence in DMEM supplemented with 10% FCS and then scratch wounded with a sterile plastic micropipette tip. At 0, 12, and 24 hours, photographs were taken at the same position of the wound.
Results:①HSC-T6 cells transfected with RKIP plasmids overexpressed RKIP (RKIP/β-actin, 0.673±0.016, vs 0.227±0.025, P<0.01), and the phosphorylation of Raf-1 and ERK were significantly decreased in these cells (pRaf/Raf, 0.027±0.006 vs 0.853±0.022, P<0.01; pERK/ERK, 0.293±0.012 vs 1.027±0.060, P<0.01). Locostatin significantly decreased RKIP expression in transfected cells (RKIP/β-actin, 0.338±0.016, vs 0.673±0.016, P<0.01). Locostatin also increased the phosphorylation of RKIP, Raf-1, and ERK (pRKIP/RKIP, 1.090±0.128 vs 0.332±0.024, P<0.01; pRaf/Raf, 0.216±0.015 vs 0.027±0.006, P<0.01; pERK/ERK, 0.790±0.028 vs 0.293±0.012, P<0.01).②The proliferation of HSCs was significantly inhibited in cells overexpressing RKIP compared with those in the empty vector group (0.981±0.020 vs 0.860±0.022, P<0.01), while RKIP overexpression did not significantly affect HSC apoptosis.③RKIP overexpression significantly increased HSC-T6 cell migration rates compared to the control (161.00±9.17 vs 124.00±6.00, P<0.01), and locostatin abrogated this effect (43.00±7.94 vs 161.00±9.17, P<0.01), and HSC-T6 cell treated with locostatin (10.00±3.61 vs 161.00±9.17, P<0.01). To further confirm the effect of RKIP on cell migration, RKIP-overexpressing HSC-T6 cells were grown to confluence and then scrape wounded. Within 24 hours, RKIP-overexpressing cells grew to confluence again while cells transfected with the empty vector grew only to 73.2%. Locostatin significantly delayed wound closure of HSC-T6 cells as they grew only to 3.3%. Wound closure assay showed that overexpression of RKIP promoted wound closure while locostatin inhibited wound closure.
Conclusions: RKIP inhibits Raf-1/MEK/ERK1,2 signaling and this inhibition impedes HSCs proliferation. RKIP promotes HSCs migration and wound closure.
引文
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