【作者】 王玉珍；

【导师】 姜慧卿；

【作者基本信息】 河北医科大学 ， 内科学， 2007， 博士

【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。更多还原

【Abstract】 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 RatsObjective: 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 HSCObjective: 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.更多还原