【作者】 王妍；

【导师】 姜慧卿；

【作者基本信息】 河北医科大学 ， 内科学， 2010， 博士

【摘要】 肝纤维化(liver fibrosis)是对包括病毒、自身免疫、药物、酒精、胆汁淤积以及代谢疾病在内的各种病因所致的慢性肝损伤的修复反应,是一种以细胞外基质(extracellularmatrix, ECM)的过度沉积为特征的病理生理过程。肝星状细胞(hepatic stellate cell, HSC)的激活是肝纤维化发生的中心环节,活化的HSC是以I型胶原为主的细胞外基质的主要来源,在肝纤维化发展过程中起重要作用。各种致病因素作用于肝脏使静止的HSC激活转化为具有成纤维和收缩作用的肌纤维母细胞,在形态学上由静止的贮存维生素A的储脂细胞转变为表达细胞骨架蛋白α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)的活化的HSC,并不断增殖,持续活化而促进肝纤维化的发展。已有多项研究证实在急慢性肝损伤动物模型中,肝纤维化的逆转伴随着HSC增殖的减少和HSC凋亡的增多,因而抑制HSC增殖、诱导HSC凋亡成为治疗肝纤维化的关键。血小板衍生生长因子(platelet-derived growth factor, PDGF)是目前发现的HSC最强的有丝分裂原。PDGF与HSC表面的PDGF受体(PDGF receptor, PDGFR)结合以自分泌的形式活化下游的细胞信号转导途径,使HSC由静止状态活化成为分泌基质的肌成纤维细胞、促进HSC增殖以及分泌胶原。PDGF可以活化HSC内多条与HSC生物学行为有关的信号传导通路。细胞外信号调节激酶(Extracellular regulated kinase, ERK)是丝裂原活化的蛋白激酶(mitogen-activated protein kinase, MAPK)家族成员之一,此信号通路顺序通过MAPK激激酶(MAPK kinase kinase, MAPKKK, Raf),MAPK/ERK激酶(MAPK/ERK kinase, MAPKK, MEK)作为MAPK激酶(MAPK kinase),ERK即MAPK,依次传导细胞增殖信号。PDGF通过激活Ras继而激活下游Raf/MEK/ERK级联信号活化MAPK/ERK信号通路,促进HSC增殖,在肝纤维化形成过程中起重要作用。磷脂酰肌醇-3-激酶(phosphatidylinositol 3-kinase, PI3K)/Akt/核糖体40S小亚基S6蛋白激酶(70-kDa ribosomal S6 kinase, p70S6K)信号通路可被包括PDGF在内的多种生长因子和促有丝分裂原活化,对调控细胞周期、促进细胞分化和细胞生长起重要作用。MAPK/ERK和PI3K/Akt/p70S6K信号通路对HSC的DNA合成、细胞生存和胶原合成发挥关键作用,阻断任一途径皆可抑制HSC增殖和I型胶原分泌,诱导HSC凋亡。Sorafenib是较早应用于临床的一种口服多靶点酪氨酸激酶抑制剂,目前已在全球广泛用于包括肾癌、肝癌在内的多种恶性肿瘤的治疗。Sorafenib靶向受体酪氨酸激酶PDGFR-β和血管内皮生长因子受体-2 (vascular endothelial growth factor receptor-2, VEGFR-2),同时靶向抑制Raf丝氨酸/苏氨酸激酶以及下游的MEK/ERK信号通路,抑制肿瘤细胞的增殖、促进细胞凋亡,并与其对Raf/MEK/ERK和PI3K/Akt/p70S6K信号通路的磷酸化的抑制有关。有研究表明sorafenib对肿瘤细胞的增殖抑制伴随着细胞周期蛋白(Cyclin)和细胞周期依赖性蛋白激酶(Cyclin-dependent kinase, Cdk)表达下降;其对肿瘤细胞诱导凋亡的作用伴有Mcl-1、Bcl-2的下调和Fas/Fas配体(Fas ligand, Fas-L)的升高。此外,已有研究证实sorafenib可以缓解胆总管结扎大鼠肝纤维化程度,而另一种与sorafenib作用靶点相类似的酪氨酸激酶抑制剂sunitinib可以减少肝纤维化大鼠肝脏α-SMA的表达和胶原的沉积,在体外能够抑制HSC的活力和胶原表达。由此我们推测sorafenib可能对肝纤维化具有治疗作用,这种作用可能通过其抑制HSC增殖并诱导HSC凋亡实现。本课题旨在研究口服sorafenib对两种肝纤维化模型-胆总管结扎(bile duct ligation, BDL)和二甲基亚硝胺(dimethylnitrosamine, DMN)诱导的肝纤维化的影响及信号转导机制;sorafenib对鼠HSC株T6和人HSC株LX2以及大鼠原代HSC增殖和凋亡的作用及可能的分子机制。实验由以下四部分组成:第一部分:口服sorafenib对大鼠肝纤维化的影响及信号转导机制目的:研究口服不同剂量sorafenib对BDL和DMM诱导的大鼠肝纤维化的治疗作用。方法:运用BDL方法和DMN腹腔注射两种方法建立大鼠肝纤维化模型,治疗组于模型建立第3周、第4周分两个剂量(BDL:20mg/kg,40mg/kg;DMN:1mg/kg,5mg/kg)干预动物,假手术组与治疗组均于第4周取材,模型组分别于模型建立1 wk、2 wk、3 wk、4 wk取材。组织切片经HE和Masson三色染色检测模型建立过程中的动态病理变化及治疗后病理改变,利用病理图像分析软件测定肝组织Masson染色胶原面密度;测定血清ALT、AST、胆红素及白蛋白含量;Western Blot检测ERK、phospho-ERK、Akt、phospho-Akt、p70S6K、phospho-p70S6K在肝组织的表达;免疫组织化学方法检测phospho-ERK、phospho-Akt、phospho-p70S6K及α-SMA的表达。结果:①HE和Masson三色染色结果证明BDL和DMN诱导的大鼠肝纤维化模型建立成功。BDL模型肝脏可见胆管增生明显,大量胶原沉积在小胆管周围,肝脏广泛纤维结缔组织增生包绕分割肝小叶形成假小叶;DMN模型肝脏可见出血灶及炎细胞浸润、肝细胞坏死并被纤维化间隔取代。Sorafenib治疗后上述病理改变缓解,两个模型的高剂量组尤为明显。②口服sorafenib对BDL及DMN肝纤维化大鼠肝功能的影响。两个模型大鼠肝功能于造模4 wk血清ALT、AST和BIL均明显高于对照组,ALB低于对照组;sorafenib治疗对BDL模型肝功能没有明显影响;sorafenib 1 mg/kg治疗DMN模型改善了大鼠肝功能,ALT和BIL均有不同程度的降低,ALB较溶剂对照组升高;而sorafenib 5 mg/kg治疗组表现为加重肝功能损伤,使AST和ALT分别较溶剂组显著增高了101.10% (P<0.001)和20.08% (P<0.001)。③口服sorafenib抑制BDL和DMN大鼠肝脏ECM沉积。在BDL和DMN肝纤维化模型中,口服sorafenib后半定量Masson染色胶原面积密度均明显下降(P<0.001),并呈剂量依赖性。④免疫组织化学检测α-SMA在纤维化大鼠肝脏的表达变化。在BDL和DMN模型肝组织中,造模1 wk~4 wk肝组织α-SMA的阳性面积逐渐增高,均显著高于正常对照组,sorafenib治疗组较溶剂组明显下降(P<0.001),呈剂量依赖关系。⑤免疫组织化学方法以及Western blot检测肝纤维化不同时间点以及sorafenib治疗前后ERK、Akt、p70S6K、p-ERK、p-Akt和p-p70S6K在纤维化大鼠肝脏的表达变化。p-ERK、p-Akt和p-p70S6K在肝细胞、血管内皮细胞以及HSC中均有表达,随着造模时间延长,阳性细胞数目明显增加,4 wk达到最高,sorafenib治疗后表达下降;Western blot测定p-ERK/ERK、p-Akt/Akt和p-p70S6K/p70S6K的比值在两种模型的造模过程中表达逐渐增高,到4 wk时达到最高,sorafenib治疗后,各信号分子磷酸化水平下降,高剂组(BDL:40 mg/kg;DMN:5 mg/kg)变化最显著(P<0.001)。结论:sorafenib能够缓解BDL和DMN诱导的肝纤维化程度,这种治疗作用是通过抑制ERK和Akt/p70S6K信号通路的活化继而减少HSC的激活和ECM的沉积实现的。第二部分:Sorafenib抑制肝星状细胞增殖及其对细胞周期的调控目的:研究sorafenib对HSC增殖和细胞周期的影响。方法:采用原位循环灌流和密度梯度离心技术分离大鼠原代HSC,荧光显微镜观察刚分离静止的原代HSCs,应用单克隆抗体α-SMA行免疫细胞化学染色鉴定活化的原代HSC。Sorafenib不同浓度(2.5μmol/L,5.0μmol/L,10.0μmol/L)和时间点(12 h,24 h,48 h,72 h)干预鼠PDGF活化的HSC株T6和人HSC株LX2以及原代HSC,四甲基偶氮唑盐(methyl thiazolyl tetrazolium, MTT)比色法检测细胞活力变化、[3H]标记的胸腺嘧啶脱氧核苷([3H]-Thymidine, [3H]-TdR)掺入法测定细胞DNA合成;流式细胞学分析细胞周期变化;Western Blot检测Cyclin-D1和Cdk-4的表达。结果:①采用肝脏原位灌注链酶和胶原酶并以Nycodenz为介质一步法密度梯度离心成功分离出大鼠原代HSCs。细胞的得率为1.2×10~7至2.0×10~7/只,细胞存活率和纯度均大于90%。在倒置显微镜下观察刚分离的HSC呈圆形,胞浆中含有较多脂滴,在波长328 nm的荧光显微镜下观察,刚分离的原代HSC呈自发蓝色荧光。培养至10天,大鼠HSCs呈活化状态,脂滴消失,变成梭形肌成纤维样细胞。②原代HSC性质鉴定。刚分离的HSC免疫细胞化学染色α-SMA表达阴性;原代培养10天后α-SMA阳性染色率达100%。③Sorafenib抑制原代HSC、HSC-T6和HSC-LX2细胞活力。MTT检测显示不论PDGF-BB刺激与否,sorafenib均呈时间与浓度依赖性抑制以上三种HSC的活力,其中sorafenib高剂量组(10μmol/L)使T6、原代HSC以及LX2细胞活力分别由对照组的100.00%降低至16.37±3.85% (P<0.001)、22.49±1.86% (P<0.001)和15.49±2.16% (P<0.001)。④Sorafenib呈浓度依赖性地抑制PDGF-BB刺激的HSC DNA合成。[3H]-TdR掺入法测定结果显示sorafenib高剂量组(10μmol/L)干预T6和LX2细胞24 h后,细胞cpm值分别由PDGF刺激组的10023.00±2442.25和4416.00±667.63降低至891.50±160.33 (P<0.001)和1411.17±908.39 (P<0.001)。⑤Sorafenib使T6和LX2细胞S期细胞比例上升;G0/G1期和G2/M期细胞比例下降。⑥Sorafenib抑制T6和LX2 Cyclin-D1和Cdk-4的蛋白表达。Western blot结果显示sorafenib对PDGF-BB诱导而增高的Cyclin-D1和Cdk-4具有抑制作用,其中sorafenib对于Cyclin-D1的抑制作用呈现剂量依赖性。结论:Sorafenib能够抑制HSC增殖,这种作用抑制细胞周期相关蛋白Cyclin-D1和Cdk-4的表达,调控细胞周期停滞于S期有关。第三部分:Sorafenib促进肝星状细胞的凋亡目的:研究sorafenib对HSC凋亡以及对凋亡相关蛋白的影响。方法:Sorafenib (2.5μmol/L,5.0μmol/L,10.0μmol/L)干预HSC-T6和LX2细胞株12 h或24 h。透射电镜观察细胞形态学改变;膜联蛋白(Annexin-V)/碘化丙啶(Propidium iodide, PI)联合标记流式细胞术检测HSC凋亡率;末段脱氧核苷酸转移酶介导的脱氧三磷酸尿苷缺口末段标记(terminal deoxynucleotidy transferrase UTP-nick end labeling, TUNEL)检测HSC凋亡。Sorafenib (2.5μmol/L,5.0μmol/L,10.0μmol/L)预处理HSC 2 h后,干预PDGF刺激的T6和LX2细胞共24 h。测定Caspase-3活性;RT-real time PCR测定Bcl-2、Bax、Caspase-3 mRNA的表达;Western blot检测Bcl-2、Bax、Fas、Fas-L以及Caspase-3蛋白表达。结果:①透射电镜下观察HSC形态变化。浓度为10μmol/L的Sorafenib干预HSC 12 h后,细胞呈现凋亡特征:细胞体积变小,染色质凝集,细胞核质比减少,新月体形成,核膜破损,粗面内质网扩张。②Sorafenib提高HSC凋亡率。2.5μmol/L,5μmol/L和10μmol/L sorafenib作用于T6和LX2细胞,凋亡率逐渐升高,均呈浓度和时间依赖关系,其中T6细胞10μmol/L sorafenib组12 h凋亡率(72.62±4.05%)较对照组增高了2.74倍(P<0.001);LX2细胞(92.87±2.81%)较对照组增高了2.15倍(P<0.001)。③10μmol/L sorafenib分别提高T6和LX2细胞TUNEL染色阳性细胞率5.28倍(P<0.001)和3.24倍(P<0.001)。④Western blot结果显示sorafenib抑制了两个细胞株Bcl-2的表达,升高了Bax、Fas、Fas-L和Caspase-3的表达,Bax/Bcl-2比值也相应增加,呈浓度依赖关系;Bcl-2、Bax和Caspase-3的mRNA表达趋势与其蛋白变化趋一致。⑤PDGF抑制Caspase-3活性;10μmol/L sorafenib分别将T6细胞和LX2细胞Caspase-3的活性提高了2.83倍(P<0.001)和1.58倍(P=0.021)。结论:Sorafenib能够诱导HSC凋亡,这种作用与抑制Bcl-2、提高Bax、Fas、Fas-L表达和Caspase-3的活性有关。第四部分:Sorafenib对肝星状细胞增殖与凋亡影响的信号转导机制目的:研究sorafenib对HSC细胞内MAPK/ERK以及Akt/p70S6K信号转导通路的调节作用。方法:不同浓度sorafenib(2.5μmol/L,5.0μmol/L,10μmol/L)干预PDGF-BB (20 ng/ml)刺激的HSC-T6和LX2细胞株,或不经PDGF刺激直接以sorafenib干预共24 h,Western blot检测ERK、Akt、p70S6K、p-ERK、p-Akt和p-p70S6K的蛋白表达变化。以10μmol/L sorafenib、25μmol/L LY294002和50μmol/L PD98059同时干预经或不经PDGF-BB (20 ng/ml)刺激的HSC-T6和LX2细胞株,横向比较sorafenib与两种抑制剂对以上信号分子的作用。结果:①Sorafenib抑制PDGF-BB激活的T6和LX2细胞内ERK和Akt/p70S6K信号通路的磷酸化。PDGF-BB刺激对两种细胞株中3种信号分子磷酸化水平均有升高作用;sorafenib使三种信号分子磷酸化水平与PDGF-BB刺激组相比均受到明显抑制,并且呈剂量依赖性。Sorafenib (10μmol/L)组使T6细胞p-ERK/ERK、p-Akt/Akt和p-p70S6K/p70S6K分别较PDGF刺激组降低了50.00% (P=0.001) , 46.71% (P<0.001)和24.73% (P<0.001);使LX2细胞p-ERK/ERK、p-Akt/Akt和p-p70S6K/p70S6K分别较PDGF刺激组降低了61.11% (P<0.001) , 37.50% (P=0.001)和19.70% (P<0.001)。②Sorafenib发挥了与ERK和Akt/p70S6K信号通路相应阻断剂类似的抑制作用。不论PDGF-BB刺激与否,PD98059和LY294002均分别显著抑制了ERK和Akt/p70S6K信号分子的磷酸化水平,而sorafenib则同时抑制了这两条信号通路的磷酸化与LY294002和PD98059的抑制作用相比无明显差异。结论:Sorafenib可能通过对ERK和Akt/p70S6K两条细胞内信号通路磷酸化的抑制实现抑制HSC增殖、促进HSC凋亡的作用。更多还原

【Abstract】 Liver fibrosis characterized by the excessive accumulation of extracellular matrix represents the pathologic response of a sustained wound healing response to chronic liver disease induced by a variety of causes, including viral, autoimmune, drug-related, ethanol, cholestatic and metabolic damage. A key role in hepatic fibrogenesis is attributed to activated hepatic stellate cells (HSCs), which have been identified as major collagen-producing cells in an injured liver and are responsible for excessive deposition of extracellular matrix (ECM), of which type I collagen predominates.Following liver injury of any etiology, HSCs undergo a response known as“activation”, which is the transition of quiescent cells into proliferative, fibrogenic and contractile myofibroblasts. Morphological changes associated with HSC activation include a loss of vitamin A stores and appearance of the cytoskeleton proteinα-smooth muscle actin (α-SMA). Numerous studies, performed in animal models of acute or chronic liver injury, have shown a potential reversibility of liver fibrosis associated with inhibition of HSC proliferation and induction of HSC apoptosis. Consequently,inhibition of HSC proliferation and induction of HSC apoptosis become major antifibrotic therapeutic strategies.The most potent mitogenic factor for HSCs is platelet-derived growth factor (PDGF), which combines with PDGF receptor (PDGFR) on the surface of HSCs and activates intracellular signal transduction as autocrine factors, stimulate cell proliferation, synthesis of ECM including collagens and activate quiescent HSCs to matrix-secreting myofibroblasts. Multiple signaling pathways are implicated in HSC proliferation activated by PDGF.Extracellular signal-regulated kinase (ERK) is an important member of the mitogen-activated protein kinase (MAPK) family, which is composed of three core units: mitogen-activated protein kinase kinase kinase (MAPKKK, Raf), mitogen-activated protein kinase kinase (MAPKK, MEK) and MAPK (ERK). It has been found that MAPK/ERK signal pathway is involved in hepatic fibrosis and activation of Ras due to PDGF is followed by sequential activation of Raf, MEK, and ERK. The phosphatidylinositol 3-kinase (PI3K)/Akt/70-kDa ribosomal S6 kinase (p70S6K) signaling pathway is activated by mitogens and growth factors including PDGF, and is required for cell cycle progression, cell differentiation, and cell growth. Both MAPK/ERK and PI3K/Akt/p70S6K signal pathways play crucial roles in the DNA synthesis, collagen synthesis and cell survival in HSCs. Inhibiting either of the pathways blocks HSC proliferation and type I collagen synthesis and induces HSC apoptosis.Sorafenib is farthest along in clinical development and has been approved in several countries worldwide for treatment of a wide variety of tumors including renal cell carcinoma, hepatocellular carcinoma, et al. As a multikinase inhibitor, sorafenib potently blocks the tyrosine kinases of vascular endothelial growth factor receptor-2 (VEGFR-2) and PDGFR-β, as well as the Raf serine/threonine kinases along the Raf/MEK/ERK pathway, which is known to be important in tumor cell signaling and tumor cell proliferation. Simultaneouly, sorafenib decreased the phosphorylations of Raf/MEK/ERK and PI3K/Akt/p70S6K signal pathways. It has also been reported that sorafenib inhibits proliferation accompanied by the inhibition of Cyclins and Cyclin-dependent kinases (Cdks) and induces apoptosis accompanied by the inhibition of the down-regulation of myeloid cell leukemia-1 (Mcl-1), Bcl-2 and up-regulation of Fas/Fas ligand (Fas-L) in multiple human tumor cell lines. Furthermore, Sorafenib could substantially decrease the extensive deposition of fibrillar collagen in common bile duct ligation (BDL) rats. Another multitargeted receptor tyrosine kinase inhibitor sunitinib induce significant decreases ofα-SMA expression and ECM accumulation of rat liver fibrosis and down-regulates HSC viability and collagen expression. We speculate the anti-fibrotic effects of sorafenib on liver fibrosis in vivo and inhibition of proliferation as well as promotion of apoptosis of HSCs in vitro.The purpose of the present study is to investigate the impact of sorafenib on liver fibrosis in two animal models: BDL and dimethylnitrosamine (DMN) induced liver fibrosis models and the effects of sorafenib on the proliferation and apoptosis of HSC cell lines T6, LX2 and primary rat HSCs, as well as the possibly associated molecular mechanisms. The experiments contain four parts as blow:Part 1: The effects of oral sorafenib treatment on liver fibrosis and the potential mechanismObjective: To explore the impacts of sorafenib on liver fibrosis induced by common bile duct ligated (BDL) and dimethylnitrosamine (DMN). Methods: Hepatic fibrosis was induced by BDL and intraperitoneal injections of DMN. Sorafenib 20 mg/kg and 40 mg/kg in BDL rats, 1 mg/kg and 5 mg/kg in DMN rats or vehicle were administered orally by gavage once a day during the third and the fourth week. Livers in model group were harvested at fixed time points: 1 wk, 2 wk, 3 wk and 4 wk after operation. Livers in sham operation group were harvested at 4 wk after operation. Histopathological changes were evaluated by hematoxylin and eosin (HE) staining and by Masson’s trichrome method. The latter was quantified for collagen by analyzing Masson-stained area as a percentage of total area. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin (BIL) and albumin (ALB) were evaluated in samples of serum. The protein expressions of ERK, phospho-ERK, Akt, phospho-Akt, p70S6K and phospho-p70S6K were determined by Western blot, while the distribution of phospho-ERK phospho-Akt, phospho-p70S6K andα-SMA in the livers was assessed by immunohistochemistry.Results: (1) HE and Masson’s trichrome staining of liver confirmed the establishment of hepatic fibrosis models via BDL and DMN. The liver tissues of BDL rats appeared to exhibit a marked increase in the number of bile ductules and an extensive deposition of collagen, which contributed to the formation of pseudolobuli. DMN-induced liver injury and fibrosis exhibited extensive hemorrhagic necrosis and lobular architecture with thin bands of reticulin joining central areas. Sorafenib, especially the higher concentration group, attenuated the histopathologic changes of both fibrotic models. (2) Both fibrotic models developed hepatic injury as evidenced by significantly higher plasma concentrations of AST, ALT, BIL and lower concentration of ALB. Sorafenib treatment did not change the injury of liver function in BDL rats. In DMN rats, 1 mg/kg sorafenib ameliorated the increase of ALT and BIL, and the decrease of ALB. 5 mg/kg sorafenib increased AST and ALT levels significantly with a 101.10% (P<0.001) and a 20.08% (P<0.001) increase as compared to vehicle-treated group respectively. (3) Sorafenib attenuated the collagen deposition in a dose-dependent fashion in both fibrotic models by analyzing Masson-stained area (P<0.001). (4) The expression ofα-SMA of both models at week 1 to 4 in liver during the process of liver fibrogenesis increased by immunohistochemistry analysis. Sorafenib treatment decreased the positive cells ofα-SMA in a dose-dependent manner (P<0.001). (5) The relative protein expressions of phospho-ERK/ERK, phospho-Akt/Akt, phospho-p70S6K/p70S6K by Western blot and the positive areas of phospho-ERK, phospho-Akt and phospho-p70S6K by immunohistochemistry were increased during the process of liver fibrogenesis in both models. Sorafenib treatment diminished those increased the phosphorylations of ERK, Akt and p70S6K in a dose-dependent manner. Furthermore, the phosphorylations of higher concentration groups (BDL: 40 mg/kg; DMN: 5 mg/kg) decreased to the most degree (P<0.001).Conclusions: Sorafenib inhibited the phosphorylation of ERK and Akt/p70S6K signaling pathways, consequently suppressed the activation of HSCs, by which sorafenib attenuated liver fibrosis.Part 2: Sorafenib inhibits HSC proliferation and regulates HSC cell cycleObjective: To explore the effects of sorafenib on HSC proliferation and cell cycle.Methods: Primary rat HSCs were isolated by circulating perfusion of the liver and density gradient centrifugation. The quiescent HSCs immediately after plating were tested by fluorescence microscope to evaluate the purity of the cultures andα-SMA monoclonal antibody was used to identify the activated primary HSCs by immunocytochemistry. T6, LX2 and primary HSCs were preincubated with or without sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h, and then stimulated with or without PDGF-BB for 12 h, 24 h, 48 h and 72 h. The viability of HSCs was detected by 3-(4, 5-dimethylthiazol-2-yl)-3, 5- diphenyltetrazolium bromide (MTT) assay. DNA synthesis was explored by [3H]-Thymidine ([3H]-TdR) incorporation assay. Cell cycles of HSCs were analyzed by flow cytometry. The protein expressions of Cyclin-D1 and Cdk-4 were determined by Western blot analysis.Results: (1) 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. Cell viability and cell purity were greater than 90%, with a yield ranging from 1.2×107 to 2.0×107 HSCs/rat. The primary HSC immediately after plating is round and in rich of lipid droplet under the inverted microscope, while HSCs show blue under the ultraviolet light (λ=328 nm). After 10 days culture, activated HSCs lose retinoid and become fusiform myofibroblast-like cell. (2) To indentify the primary HSCs identificationα-SMA staining was performed at day 1 (quiescent,α-SMA negative cells) and day 10 (activated,α-SMA positive cells) by immunocytochemistry. (3) Sorafenib inhibited the viabilities of T6, LX2 and primary HSCs with or without the stimulation of PDGF-BB time- and dose-dependently. Sorafenib (10μmol/L) decreased the viabilities of T6 (16.37±3.85%, P<0.001), LX2 (22.49±1.86%, P<0.001) and primary HSCs (15.49±2.16%, P<0.001) compared with those of the control group (100%). (4) Sorafenib inhibited the DNA synthesis of T6 and LX2 activated by PDGF-BB dose-dependently. Sorafenib (10μmol/L) significantly decreased the cpm of T6 (891.50±160.33) compared with that of PDGF group (10023.00±2442.25) (P<0.001), and decreased the cpm of LX2 (1411.17±908.39) compared with that of PDGF group (4416.00±667.63) (P<0.001). (5) Cell cycle analysis of T6 and LX2 cells showed an increase in S phase cells and a decrease in G1 and M phase. (6) Western blot analysis indicated that sorafenib reduced the increased Cyclin-D1 and Cdk-4 protein levels by PDGF-BB in both T6 and LX2 cells. The inhibition of Cyclin-D1 of sorafenib is in a dose-dependent manner.Conclusions: Sorafenib diminished the expressions of Cyclin-D1 and Cdk-4, which contributed to the S-phase arrest of cell cycle and the inhibition of HSC proliferation.Part 3: Sorafenib induces HSC apoptosisObjective: To investigate the effects of sorafenib on HSC apoptosis and apoptosis regulatory proteins.Methods: T6 and LX2 cells were incubated with sorafenib (2.5, 5.0, and 10.0μmol/L) for 12 h or 24 h. Morphological examination via transmission electron microscopy (TEM) evaluation and apoptosis rate assay by the Annexin-V/Propidium iodide (PI) double-labeled flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique were performed. HSCs were preincubated with sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h and stimulated by PDGF-BB for another 22 h. Caspase-3 activity was detected, and the protein expressions of Bcl-2, Bax, Fas, Fas-L, and Caspase-3 were analyzed by Western blot. The mRNA levels of Bcl-2, Bax, and Caspase-3 were measured by RT real-time PCR.Results: (1) Morphological changes of T6 and LX2 cells after treatment with sorafenib (10μmol/L) for 12 h under the TEM showed that cells became smaller, the chromatins condensed along inside the nuclear membrane, the crescent cell nuclear formed, the nuclear-cytoplasmic ratio decreased, caryotheca was damaged, and the endoplasmic reticulum dilated. (2) Sorafenib induced the apoptosis of HSCs. Sorafenib (2.5, 5.0, and 10.0μmol/L) increased HSC apoptotic rates of T6 and LX2 cells in dose- and time-dependent manner. The apoptotic rate of T6 cells treated with 10.0μmol/L sorafenib for 12 h (72.62±4.05%) was 2.74 times higher than the control group (P<0.001), and that of LX2 cells (92.87±2.81%) was 2.15 times higher than the control group (P<0.001). (3) Sorafenib (2.5, 5.0, and 10.0μmol/L) up-regulated the positive-T6 and LX2 cells of TUNEL staining by 5.28 and 3.24 times respectively compared with the control groups (P<0.001). (4) Sorafenib reduced Bcl-2 protein and mRNA levels and increased the mRNA expressions of Bax and Caspase-3 as well as the protein levels of Bax, Fas, Fas-L and Caspase-3 in both T6 and LX2 cells. The ratio of Bcl-2 to Bax decreased. (5) PDGF inhibited the activity of Caspase-3, whereas 10μmol/L sorafenib increased the activity of Caspase-3 in both T6 and LX2 cells by 2.83 times (P<0.001) and 1.58 times (P=0.021) respectively compared with the control groups.Conclusions: Sorafenib induced HSC apoptosis, which was related to the down-regulation of Bcl-2 and up-regulations of Bax, Fas, Fas-L and Caspase-3.Part 4: The mechanism of intracellular signal transduction that contribute to the impacts of sorafenib on HSC proliferation and apoptosisObjective: To explore the regulation of sorafenib on MAPK/ERK and Akt/p70S6K signalling pathways in HSCs.Methods: T6 and LX2 cells were preincubated with or without sorafenib (2.5, 5.0, and 10.0μmol/L) for 2 h, and then stimulated with or without PDGF-BB (20 ng/ml) for 22 h. Simultaneously, T6 and LX2 cells were treated with sorafenib, LY294002 (25μmol/L), or PD98059 (50μmol/L), stimulated with or without PDGF-BB. The protein expressions of ERK, Akt, p70S6K, p-ERK, p-Akt and p-p70S6K were determined by Western blot analysis.Results: (1) Sorafenib inhibited the phosphorylations of ERK and Akt/p70S6K signals increased by PDGF-BB. PDGF-BB up regulated the ratios of p-ERK/ERK, p-Akt/Akt and p-p70S6K/p70S6K, which were diminished by sorafenib dose-dependently. Sorafenib (10μmol/L) in T6 cells decreased the ratios of p-ERK/ERK, p-Akt/Akt and p-p70S6K/p70S6K by 50.00% (P=0.001), 46.71% (P<0.001), and 24.73% (P<0.001) compared with PDGF-BB group. Those of LX2 cells were decreased by 61.11% (P<0.001), 37.50% (P=0.001), and 19.70% (P<0.001) compared with PDGF-BB group. (2) Sorafenib showed the inhibiting effects on ERK and Akt/p70S6K signals similar to the specific blocking agent of the two pathways. PD98059 and LY294002 were effective in inhibition of phosphorylation of ERK and Akt/p70S6K, respectively. Sorafenib inhibited both the phophorylated ERK and Akt/p70S6K, and no significant different impact was found between sorafenib vs PD98059 and sorafenib vs LY294002 on p-ERK and p-Akt respectively.Conclusions: Sorafenib inhibited HSC proliferation and induced apoptosis probably via down-regulation of phosphorylations of ERK and Akt/p70S6K signaling pathways.更多还原