索拉菲尼诱导肝星状细胞程序性细胞死亡的作用及其机制研究
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摘要
肝纤维化(liver fibrosis)是一种损伤-愈合反应，其主要特点是慢性肝损伤后的细胞外基质(extracellular matrix, ECM)沉积。ECM主要来源于活化的肝星状细胞(hepatic stellate cell, HSC)，它在纤维化致病过程中起关键性作用，因此，对其深入研究为抗纤维化治疗提供了潜在、重要的理论基础，诱导HSC死亡可能成为有效的抗肝纤维化策略。理想的诱导细胞死亡策略是在诱导HSC死亡的同时不引起肝脏炎症反应。程序性细胞死亡(programmed cell death, PCD)就是一种有组织的、受控的细胞清除过程；PCD与“坏死”不同，并不引起炎症反应。
程序性细胞死亡是指细胞受到某些因素刺激或接受某种信号后，细胞为了维持其内环境稳定而发生的一种主动性消亡过程，是生长发育和维持组织内环境稳态的关键。根据垂死细胞（dying cells）的不同形态学特征可分为：凋亡和自噬性细胞死亡(autophagic cell death，ACD)，又称作“Ⅰ和Ⅱ型程序性细胞死亡”。近年来，自噬性细胞死亡被受广大学者所关注。自噬(autophagy)是一种溶酶体降解过程，它可将损伤的或过剩的细胞元件分解成基本的活性成分使之可再利用。自噬过程包括合成包绕靶目标的膜型结构、自噬体形成、与溶酶体融合形成自噬溶酶体、酸性溶酶体水解酶降解靶目标。凋亡(apoptosis)是机体在一定的生理或病理条件下,经由启动内部机制,最终导致内源性内切酶激活,自己结束生命的过程;它是主动死亡过程,在整个过程中涉及到一系列凋亡相关基因的表达变化,同时细胞发生特征性形态学改变。
此外，最新研究发现自噬与凋亡间有复杂的相互联系，因为参与自噬和凋亡过程的某些分子存在交叉。诸如：两种关键的自噬蛋白Beclin1和Atg5，在凋亡途径中也发挥了重要的作用；同时参与凋亡的caspases和Bcl-2家族成员也可通过裂解或绑定Beclin1的方式调控自噬。最新研究报道表明，自噬通过降解长寿蛋白和细胞器促使活化HSC存活，通过降解HSC内脂滴为其活化提供能量，促使肝纤维化发生。亦有研究表明：生育三烯酚类通过诱导活化的胰星状细胞凋亡和自噬发挥其抗胰纤维化的作用。因此，自噬对细胞的作用具有两面性，可以说是一把双刃剑，既可作为细胞生长的“朋友”，又可能成为杀死细胞“敌人”，这取决于疾病进展的不同阶段、细胞周围环境的变化和治疗干预措施的不同。已有大量的研究证实：多种抗肿瘤药物bufalin、cannabidiol、cucurbitacin等均可通过诱导肿瘤细胞发生自噬性细胞死亡而发挥其抗肿瘤作用。
索拉菲尼是一种靶向性Raf/ERK信号通路多靶点受体酪氨酸激酶抑制剂，它可抑制多种肿瘤细胞的增生并诱导其凋亡，是FDA批准的最早应用于治疗肝癌的口服药。索拉菲尼靶向抑制Raf丝氨酸/苏氨酸激酶以及下游的MEK/ERK信号通路，抑制多种肿瘤细胞的增殖并促进细胞凋亡，并与其对PI3K/Akt/p70S6K信号通路的磷酸化的抑制作用有关。有报道：索拉菲尼干预多种肿瘤细胞后可诱导其自噬水平增高，这与索拉菲尼调控多条自噬相关信号通路有关，诸如： MAPK/ERK、Akt/mTOR/p70S6K和JNK/c-Jun信号通路等。早期的研究表明索拉菲尼可显著降低大鼠肝纤维化模型的门脉压力和血管形成，新的研究发现索拉菲尼具有潜在的抗肝纤维化功效。本课题组的研究已证实索拉菲尼可抑制活化的HSC增殖并诱导其凋亡。然而，索拉菲尼的抗肝纤维化作用及其与程序性细胞死亡间的关系尚不明确。
由此我们推测索拉菲尼的抗肝纤维化作用，除通过其抑制HSC增殖并诱导HSC凋亡外，还可能通过诱导HSC自噬性细胞死亡实现。本课题旨在研究索拉菲尼是否可诱导人HSC-LX2细胞株以及大鼠原代HSC发生自噬性细胞死亡，并探讨自噬和凋亡间的关系及可能的分子机制。实验由以下三部分组成：
第一部分：索拉菲尼诱导肝星状细胞凋亡和自噬性细胞死亡
目的：研究索拉菲尼对HSC凋亡和自噬性细胞死亡的影响。
方法：采用IV型胶原酶和链酶原位灌注肝组织和Nycodenz梯度密度离心技术分离提取大鼠原代HSC，应用α-SMA免疫细胞化学染色和荧光染色方法鉴定原代HSC。索拉菲尼(2.5μmol/L，5.0μmol/L，10.0μmol/L)干预HSC-LX2细胞株6h、12h或24h，四甲基偶氮唑盐(methylthiazolyl tetrazolium, MTT)比色法检测细胞存活率、碘化丙啶(Propidium iodide, PI)/膜联蛋白(Annexin V)联合标记流式细胞术检测HSC凋亡率。HSC-LX2细胞株预孵育凋亡抑制剂Z-VAD-FEK(20μmol/L)或坏死抑制剂IM-54(10μmol/L)1h后，与5μmol/L索拉菲尼共孵育11h，MTT法检测细胞存活率、流式细胞术检测Annexin Ⅴ+细胞数和PI+细胞数百分率变化。索拉菲尼(5μmol/L)干预原代HSC或HSC-LX2细胞株12h后，透射电镜和吖啶橙染色法观察HSC中自噬的激活，激光共聚焦显微镜观察自噬标记蛋白LC3的变化，RT-PCR法检测索拉菲尼对HSC中自噬相关基因Beclin1、Atg7和Atg5mRNA的影响，WesternBlot法检测索拉菲尼对HSC自噬相关蛋白表达LC3、Beclin1、Atg7、Atg5和p62/SQSTM1表达的影响，以及自噬抑制剂3-MA(5mmol/L)抑制自噬对这些蛋白表达的影响。
结果：①HSC获得率为1.4～2.3×107/只，细胞纯度和存活率均大于90%。刚提取的HSC呈圆形，胞浆内富含脂滴，在波长328nm的荧光显微镜下观察，能激发出蓝色荧光。HSC培养14天后，静止的细胞被激活变为梭形或星形，富含维生素A的脂滴消失。②大鼠原代HSC性质鉴定。未活化HSC内不表达α-SMA；当细胞培养14天后，完全活化的HSC胞浆内有α-SMA表达。③索拉菲尼抑制HSC-LX2细胞存活率并促凋亡。MTT和流式细胞术检测显示索拉菲尼呈时间与浓度依赖性抑制HSC存活率并促凋亡，其中5μmol/L索拉菲尼干预12h后细胞存活率即由对照组的100.00%降低至50.19±0.19(P<0.001)，而凋亡率(41.22±2.425%)较对照组(7.73±1.34%)显著增高(P<0.001)。④索拉菲尼诱导HSC细胞非凋亡性细胞死亡。预孵育Z-VAD-FEK后细胞存活率和单用索拉菲尼组间无差异且均较对照组降低(P<0.001)，但PI+细胞率(54.19±4.03%)显著高于Annexin V+细胞率(10.22±2.03%)(P<0.001)。预孵育IM-54后细胞存活率(50.36±4.73%)仍较对照组降低(P<0.001)，PI+细胞率和Annexin V+细胞率较索拉菲尼组均无明显差异。⑤透射电镜和吖啶橙染色观察HSC形态变化。应用5μmol/L索拉菲尼干预HSC12h后，电镜细胞呈现自噬特征，诸如大量双层膜囊泡，囊泡内包含部分胞浆物质，可见包裹的细胞器如线粒体，溶酶体激活，双层膜囊泡与溶酶体融合；荧光显微镜下可见大量点状橙红色荧光颗粒散布于胞浆内细胞核周围。⑥索拉菲尼诱导HSC细胞形成“自噬潮”(autophagic flux)。HSC预孵育溶酶体抑制剂E64d和pepstatinA1h后，与5μmol/L索拉菲尼共孵育11h，Western blot显示预孵育E64d和pepstatin A组LC3Ⅱ/Ⅰ较单用索拉菲尼组进一步增加；激光共聚焦显微镜下预孵育E64d和pepstatin A组原代HSC中LC3绿色斑点进一步增多。⑦RT-PCR检测自噬相关基因Beclin1, Atg7和Atg5的mRNA表达增加。⑧Western blot法检测索拉菲尼干预后自噬相关蛋白Beclin1、Atg5和LC3Ⅱ/Ⅰ表达显著增加，p62表达显著下降；而3-MA可逆转上述改变。
结论：索拉菲尼可通过诱导凋亡和自噬性细胞死亡两种途径，促使HSC发生细胞死亡。HSC启动自噬性死亡途径，对细胞自身的胞浆物质及细胞器进行吞噬、消化和清除。
第二部分：索拉菲尼诱导肝星状细胞自噬性细胞死亡及其与凋亡的关系
目的：探讨索拉菲尼诱导HSC凋亡和自噬性细胞死亡间的相互关系。
方法：应用5μmol/L索拉菲尼干预不同时间点(0h,3h,6h,12h,24h)或不同浓度索拉菲尼(2.5μmol/L,5μmol/L,10μmol/L)干预HSC-LX212h，Western blot法检测自噬和凋亡标志蛋白LC3II/I和Cleaved-PARP变化。不同浓度索拉菲尼(2.5μmol/L,5μmol/L,10μmol/L)干预12h，吖啶橙染色或PI/Annexin V联合标记后流式细胞术检测AVOs或凋亡细胞数变化；Western blot法检测自噬相关蛋白Beclin1和凋亡相关蛋白cleaved-caspase-3,-8变化。预孵育Z-VAD-FEK(20μmol/L)1h后，与10μmol/L索拉菲尼共孵育11h。Western blot法检测自噬相关蛋白LC3II/I和Beclin1变化。自噬抑制剂3-MA(5mmol/L)预孵育1h后，与5μmol/L索拉菲尼共孵育11h。流式细胞术检测凋亡率，Western blot法检测自噬相关蛋白Beclin1和凋亡相关蛋白cleaved-caspase-3,-8变化。应用Atg5-siRNA技术干扰LX2后，5μmol/L索拉菲尼干预12h，Western blot法检测自噬和凋亡相关蛋白变化。
结果：①索拉菲尼诱导自噬先于凋亡。Western blot显示：索拉菲尼干预24h后Cleaved-PARP显著增加，而LC3II/I水平在干预6h后即出现显著性增加，12h达高峰，24h恢复到基线水平。②不同浓度索拉菲尼对自噬和凋亡的影响不同。Western blot显示索拉菲尼(5μmol/L)组LC3II/I显著增加(P<0.01)；而索拉菲尼(10μmol/L)组LC3II/I明显下降，同时Cleaved-PARP显著增加(P<0.01)。流式细胞学结果显示：索拉菲尼(5μmol/L)组AVOs细胞百分率(62.19±6.11%)显著多于凋亡细胞百分比(38.28±4.91%)，(P<0.05)，索拉菲尼(10μmol/L)组凋亡水平(95.90±3.33%)较索拉菲尼(5μmol/L)组显著增加(P<0.01)，而自噬水平(73.18±5.04%)无明显改变。③活化的caspases裂解Beclin1。Western Blot显示：索拉菲尼(10μmol/L)组cleaved-caspase-3,-8表达增强的同时Beclin1明显下降。Z-VAD-FMK可逆转索拉菲尼(10μmol/L)诱导的Beclin1水平降低，并且使LC3Ⅱ/Ⅰ增加。④抑制自噬可促进索拉菲尼诱导的凋亡。预孵育3-MA后，流式细胞术结果显示：凋亡细胞率(81.94±5.34%)较单一索拉菲尼干预组(42.34±3.60%)显著增多(P<0.05)。Western blot显示预孵育3-MA后可显著增加凋亡相关蛋白水平，如cleaved-caspase-3,-8和cleaved-PARP。Atg5-siRNA可抑制索拉菲尼(5μmol/L)诱导的LC3Ⅱ/Ⅰ增加，同时使cleaved-caspase-3,-8和cleaved-PARP表达增加。
结论：索拉菲尼干预HSC后自噬性细胞死亡先于凋亡而发生，当凋亡启动后自噬性细胞死亡被抑制，这种作用可能与活化的caspases断裂Beclin1有关。阻断索拉菲尼诱导的自噬后可代偿性增加凋亡水平，而这种凋亡的增加是作为一种程序性细胞死亡的替代方式。
第三部分：索拉菲尼对肝星状细胞程序性细胞死亡影响的信号转导机制
目的：探讨索拉菲尼对HSC细胞内Akt/mTOR/p70S6K和JNK/c-Jun信号转导通路的调节作用。
方法：5μmol/L索拉菲尼干预HSC-LX2细胞株(0h,0.5h,1.5h,3h,6h,12h,24h)，Western blot检测Akt、p-Akt、mTOR、p-mTOR、p70S6K、p-p70S6K、JNK和p-JNK的蛋白表达变化，激光共聚焦显微镜观察p-c-Jun核转位。以5μmol/L索拉菲尼、25μmol/L LY294002干预HSC-LX2细胞株12h，吖啶橙染色或PI/Annexin V联合标记后流式细胞术检测AVOs和凋亡细胞数变化。以5μmol/L索拉菲尼、10μmol/LSP600125干预HSC-LX2细胞株12h，Western blot检测JNK、p-JNK、c-Jun、p-c-Jun、Beclin1和LC3Ⅱ/Ⅰ变化。
结果：①索拉菲尼抑制HSC-LX2细胞内Akt/mTOR/p70S6K信号通路的磷酸化。索拉菲尼干预后p-Akt/Akt、 p-mTOR/mTOR和p-p70S6K/p70S6K分别较对照组降低；②索拉菲尼可通过Akt/mTOR/p70S6K信号通路诱导凋亡。流式细胞术显示LY294002+索拉菲尼组细胞凋亡率(69.44±6.71%)较索拉菲尼组(38.22±4.66%)增加了53%；而LY294002对AVOs细胞百分率无影响。③索拉菲尼通过JNK/c-Jun信号通路诱导自噬，并激活HSC-LX2细胞内JNK/c-Jun信号通路。索拉菲尼干预后p-JNK/JNK较对照组显著增加，激光共聚焦显微镜观察p-c-Jun增加并转位入核。SP600125抑制了JNK/c-Jun信号分子的磷酸化水平，同时也抑制了索拉菲尼诱导的LC3II/I和Beclin1增加。
结论：索拉菲尼可抑制Akt/mTOR/p70S6k信号通路并活化JNK/c-Jun信号通路，其中Akt/mTOR/p70S6k途径有助于凋亡发生，而JNK/c-Jun信号通路直接参与索拉菲尼诱导的自噬事件。
Hepatic fibrosis is a wound-healing response characterized by theaccumulation of extracellular matrix (ECM) proteins as results of chronicliver injury. Activation of hepatic stellate cells (HSCs), the main source ofECM proteins, represents a crucial event in the pathogenic sequence offibrosis and thus, provides an important framework to define potentialstrategies for anti-fibrotic therapy. Beside the inhibition of fibrogeniccytokines, the induction of HSCs death is now considered an effectivestrategy for the resolution of hepatic fibrosis. The ideal cell death strategyshould selectively target activated HSCs while maintaining quiescent HSCsbut not alert the hepatic inflammatory response. In contrast to theunorganized way of death “necrosis” that triggers the inflammatory response,apoptosis and autophagy proceed with the participation of organized andcontrolled cellular processes, which achieve a cleaner cellular execution.
Death by apoptosis commonly signals through the activation of initiatorand executioner caspases, resulting in the formation of apoptotic bodies thatare removed by phagocytes. The autophagic pathway involves the formationof a membrane around a targeted region of the cell, resulting in theautophagosome formation, which fuses with a lysosome to form anautophagolysosome where the contents are degraded via acidic lysosomalhydrolases. Although apoptosis is the primary mechanism ofchemotherapy-induced cell death, an alternative mode of cell death, termedautophagic cell death, has emerged recently as an important mechanism ofcell death. However, at present, manipulation of autophagy in favor of celldeath is still confusing and controversial due to the paradoxical roles ofautophagy in cell survival and cell death. In addition, the latest research found that there is complex interaction between autophagy and apoptosis,because some moleculars which play an important role in autophagy alsoparticipate in apoptosis, such as beclin1and Atg5; meanwhile the caspaseand Bcl-2family which are involved in apoptosis regulate autophagy bycleaving or binding Beclin1. Most recently, autophagy has been implicatedin the release of lipid droplets from HSCs that further promoted fibrogenesisof activated HSCs. Paradoxically, Rickmann et al. have reported thattocotrienols counteracted pancreatic fibrogenesis through the induction ofapoptosis and autophagy in activated pancreatic stellate cells. Therefore,autophagy is a double-edged sword which is a friend as well as an emeny forcell death depending on the different stages of the disease, cellular nichesand different treatment. There are a large number of studies showing thatvarious anti-cancer drugs play a role in anti-tumor effect by inducing tumorcells to undergo autophagic cell death, such as bufalin, cannabidiol,cucurbitacin and so on.
Sorafenib is a multiple receptor tyrosine kinase inhibitor targeting theRaf/ERK signaling pathways. It is reported that: sorafenib could inducekinds of tumor cell to undergo autophagy due to its effect on regulatingmultiple autophagy-related signaling pathways, such as: MAPK/ERK, Akt/mTOR/p70S6K and JNK/c-Jun signaling pathway.. There has been a recentinterest regarding the potential efficacy of sorafenib in the treatment of themost common and progressive forms of fibrosis. Previous studies indicatedthat sorafenib induced a significant decrease in portal pressure andangiogenesis in rats with liver fibrosis. Our previous study showed thatsorafenib inhibited proliferation and induced apoptosis in activated HSCs.However, the molecular mechanisms involved in sorafenib anti-fibroticeffects have not been completely characterized.
Thus, we hypothesize that sorafenib has a therapeutic effect on liverfibrosis by inhibiting proliferation meanwhile inducing apoptosis, or byinducing HSC autophagic cell death in HSC.The aim of this project is toexplore sorafenib’s effect on inducing autophagic cell death in human HSC-LX2cell lines and primary rat HSCs as well as the relationshipbetween autophagy and apoptosis and potential molecular mechanism. Theexperiment consists of the following three parts:
Part1: Sorafenib induces the apoptosis and ACD of HSCs
Objective: To study the effect of sorafenib on apoptosis and ACD
Methods:To obtained primary HSCs, rat liver tissues sequentialdigestion with pronase and collagenase, followed by single step densitygradient centrifugation with Nycodenz. The identification of HSC was madeby using fluorescent microscope and alpha-SMA immunocytochemicalstaining. Different concentration of Sorafenib (2.5μmol/L,5.0μmol/L,10.0μmol/L) treated HSC-LX2cell lines at the time point of6h,12h or24h,then use MTT assay to detect cell viability, Annexin-V/Propidium iodide (PI)double-labeled the apoptosis of HSCs rate by flow cytometry. HSC-LX2celllines were preincubated by apoptosis inhibitor Z-VAD-FEK (20μmol/L) orthe necrosis inhibitor IM-54(10μmol/L) for1hour, then co-incubated with5μmol/L sorafenib for another11h. The cell viability was also determinedby MTT assay, while the changes of the percentage of Annexin V+cellnumber and PI+cells was dectected by flow cytometry. After primary HSCor HSC-LX2cell lines were treated by sorafenib (5μmol/L) for12h,transmission electron microscopy and acridine orange staining were used toabserve the autophagy activation of HSCs, laser confocal microscope wasused to track the changes of autophagy marker protein LC3; RT-PCR methodwas explored to detect autophagy-related gene Beclin1, Atg7and Atg5aftersorafenib treatment. Western Blot was used to study the effect of sorafeniband autophagy inhibitor’s on the expression of autophagy-related proteinLC3, Beclin1, Atg7, Atg5, and p62/SQSTM1.
Results:①The average havest rate of HSC cells was1.2to2.0×107perrat and the cell purity and survival rate were more than90%. At the verybeginning the extracted HSCs were round and cytoplasm contained a greatdeal of lipid droplets, emitting blue ray under the320nm fluorescence microscope. After the HSCs were cultured for10days, the rested cells wereactivated and present a shape of fusiform or star, and the lipid droplets rich invitamin A disappeard.②The identification of the characteristic of primaryrat HSC. Inactivated HSCs had no expression of alpha-SMA; when the cellswere cultured for14days, fully activated HSCs did have alpha-SMAexpression in cytoplasmic.③sorafenib inhibited HSC-LX2cell survivaland promoted apoptosis. The MTT assay and flow cytometry results showedthat sorafenib inhibited HSCs survival and promoted cell apoptosis in atime-and concentration-dependent manner,5μmol/L sorafenib treatment for12h made the cell viability reduced to50.19±0.19%(P <0.001) comparedwith100%of the control group, and the apoptosis rate (41.22±2.425%)was significantly higher than the control group (7.73±1.34%)(P <0.01).④sorafenib induced the cell non-apoptotic cell death in HSC. The Z-VAD-FEKPre-incubation group had no difference in cell survival rate with sorafenibgroup, but less than the control group (P <0.01).However, the PI+rate(54.19±4.03%) was significantly higher than Annexin V+rate (10.22±2.03%)(P <0.01). The cell viability (50.36±4.73%) of IM-54Pre-incubation group was still less than the control group (P <0.01),both PI+rate and Annexin V+rate had no significantly differences with sorafenibtreated group.⑤The observation of HSCs morphological changes byacridine orange staining and transmission electron microscopy. After5μmol/L sorafenib treated HSCs for12h, the electron microscopy showedthe characteristics of autophagy: a large number of vesicles withdouble-membran which contained cytoplasmic substances, sometimes itshowed wrapped organelles such as mitochondria. Besides, lysosomal wasactivated which was fused with bilayer membrane vesicle; Under thefluorescence microscopy, large numbers of particles with orange flurescencewere dispersed in the cytoplasm around the nucleus.⑥"Autophagy tide"was induced by sorafenib in HSCs. The HSCs were pre-incubated withlysosomal inhibitors E64d and pepstatin A for1h, after then co-incubatedwith5μmol/L sorafenib for another11h. Western blot displayed the LC3II/I exrepssion is further increased in the E64d and pepstatin A pre-incubationgroup compared with only sorafenib treated group; Under the confocalmicroscope the primary HSC of both E64d and pepstatin A pre-incubationgroup showed increase LC3green spots.⑦RT-PCR detected theexpression of autophagy-related gene Beclin1, Atg7and Atg5geneincreased.⑧Western blot results showed that autophagy-related proteinBeclin1, Atg5and LC3II/I were significantly increased, while p62decreased significantly after sorafenib treatment which could be reversed byadding3-MA.
Conclusion: Sorafenib could promote death of HSCs by inducingapoptosis and autophagic cell death. It is speculated that the HSCs couldphagocyte, digestion and eliminate cytoplasm substances and organelles byactivating autophagic death pathway.
Part II: Sorafenib induces programmed cell death in hepatic stellate cellby coordinating the cross-talk between apoptosis and autophagy
Objective: To probe the cross-talk between apoptosis and autophagyinduced by sorafenib.
Methods:5μmol/L sorafenib treated HSCs at different time points (0h,3h,6h,12h,24h) or different concentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) treated HSCs for12h. To study the HSCs’ autophagyand apoptosis, the expression of blot LC3II/I and Cleaved-PARP wasdetected by Western blot. After HSCs were treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,acridine orange staining or PI/Annexin V double staining were used to detectthe AVOs and changes of the apoptotic cell number; Western blot methodwas explored to detect the expression of autophagy-related protein Beclin1and apoptosis-related proteins cleaved-caspase-3,-8. HSCs werepre-incubated with Z-VAD-FEK (20μmol/L) for1h then incubated with10μmol/L sorafenib for another11h. Western blot was used to detect theautophagy-related protein LC3II/I and Beclin1change. And after autophagy inhibitor3-MA (5mmol/L) pre-incubated for1h and5μmol/L sorafenibincubated for another11h, apoptosis rate of HSCs was detected by flowcytometry, and the expression of autophagy-related protein Beclin1andapoptosis-related proteins cleaved-caspase-3,-8were detected by Westernblot. In addition, Atg5-siRNA technology is applied to interfere LX2, after5μmol/L sorafenib treated for12h, the autophagy and apoptosis-relatedprotein changes were checked by Western blot.
Results:①Autophagy induced by sorafenib is prior to apoptosis. Thewestern blot results showed that: after the HSCs were treated with sorafenibfor24h, Cleaved-PARP expression was significantly increased, while LC3II/I expression was significantly increased at the time point of6h, andreached the peak at12h. when treatment time extended to24h, the LC3II/Iexpression returned to baseline levels.②Different concentrations ofsorafenib had different effect on autophagy and apoptosis in HSCs. Westernblot showed that in sorafenib (5μmol/L) group LC3II/I expression wassignificantly increased (P <0.01); In sorafenib group (10μmol/L), LC3II/Iexpression was decreased, but Cleaved-PARP expression was significantincreased (P <0.01). Flow cytometry results showed that in sorafenib group(5μmol/L), the AVOS cell percentage (62.19±6.11%) was significantlyhigher than the percentage of apoptotic cells (38.28±4.91%)(P <0.01),while in sorafenib (10μmol/L) group, the apoptosis rate (95.90±3.33%)was significantly higher than in sorafenib (5μmol/L) group (P <0.01),butthere was no significant change of the autophagy rate(73.18±5.04%).③The activated caspases cleaved Beclin1. The Western Blot resultsdemonstrated that in sorafenib (10μmol/L) group,the expression of thecleaved-caspase-3,-8enhanced, but Beclin1decreased. Z-VAD-FMK canreverse this situation, and increase the LC3II/I expression.④Inhibition ofautophagy can promote sorafenib-induced apoptosis. After pre-incubationwith3-MA, HSCs were detected by flow cytometry. The results showed thatAnnexin V-positive cells (81.94±5.34%) were much higher the sorafenibtreated group (42.34±3.60%)(P<0.01). Western blot showed that the expression of the apoptosis-related proteins, such as cleaved-caspase-3,-8and cleaved-PARP could significantly increase after pre-incubated with3-MA. At the mean time, Atg5-siRNA could inhibit sorafenib-inducedincrease of LC3II/I, meanwhile increase expression of the cleaved-caspase-3,-8and cleaved-PARP.
Conclusion: The HSCs treated with sorafenib undergo autophagic celldeath pior to apoptosis, autophagic cell death was inhibited when theapoptosis was started, which may due to the activated caspases whichinvolves in cleavaging Beclin1. After blocking the autophagy induced bysorafenib, the apoptosis increased in a compensatory manner, which is analternative way as a kind of programmed cell death.
Part III: The signal transduction mechanism of sorafenib inducingprogrammed cell death in hepatic stellate cells
Objective: To investigate the regulatory function of sorafenib onAkt/mTOR/p70S6K and JNK/c-Jun signal transduction pathway in HSCcells.
Methods: HSC-LX2cell line was treated with5μmol/L sorafenib atdifferent time porints (0h,0.5h,1.5h,3h,6h,12h,24h), expression ofAkt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, JNK and p-JNK,weredetected by Western blot. Laser scanning confocal microscopy was exploredto track pc-Jun nuclear translocation. After HSCs was treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,Acridine orange staining or Annexin-V/Propidium iodide (PI) double-labeledstaining by flow cytometry and changes of the apoptotic cellss number; afterHSCs were treated with5μmol/L sorafenib and10μmol/L SP600125for12h, Western blot was used to detect JNK, p-JNK, c-Jun, p-c-Jun, Beclin1andLC3II/I expression.
Results:①Sorafenib inhibited phosphorylation of Akt/mTOR/p70S6Ksignaling pathway in HSC-LX2cells. The ratio of p-Akt/Akt,p-mTOR/mTOR and p-p70S6K/p70S6K were reduced (P<0.01) respectively compared to control group.②Sorafenib induced cell apoptosis byAkt/mTOR/p70S6K signal pathway. Flow cytometry revealed that the rate ofapoptosis in LY294002+sorafenib group (69.44±6.71%) increased by about53%compared to the sorafenib group (38.22±4.66%) while LY294002hadno effect on the AVOs cell percentage.③Sorafenib initiated autophagythrough JNK/c-Jun signaling pathway. Sorafenib activated phosphorylationof JNK/c-Jun signaling pathway in HSC-LX2cells. The ratio of p-JNK/JNKsignificantly increased compared to the control group by sorafenibintervention, a significant increase of p-c-Jun and its translocation to thenucleus were proved by confocal macrophage. SP600125inhibited thephosphorylation level of JNK/c-Jun and also inhibited the sorafenib-inducedLC3II/I and Beclin1increasing.
Conclusion: Sorafenib inhibited Akt/mTOR/p70S6k signaling pathwayand activated JNK/c-Jun signaling pathway, in which Akt/mTOR/p70S6kpathway leaded to apoptosis, and JNK/c-Jun signal pathway participated insorafenib-induced autophagy directly.
程序性细胞死亡是指细胞受到某些因素刺激或接受某种信号后，细胞为了维持其内环境稳定而发生的一种主动性消亡过程，是生长发育和维持组织内环境稳态的关键。根据垂死细胞（dying cells）的不同形态学特征可分为：凋亡和自噬性细胞死亡(autophagic cell death，ACD)，又称作“Ⅰ和Ⅱ型程序性细胞死亡”。近年来，自噬性细胞死亡被受广大学者所关注。自噬(autophagy)是一种溶酶体降解过程，它可将损伤的或过剩的细胞元件分解成基本的活性成分使之可再利用。自噬过程包括合成包绕靶目标的膜型结构、自噬体形成、与溶酶体融合形成自噬溶酶体、酸性溶酶体水解酶降解靶目标。凋亡(apoptosis)是机体在一定的生理或病理条件下,经由启动内部机制,最终导致内源性内切酶激活,自己结束生命的过程;它是主动死亡过程,在整个过程中涉及到一系列凋亡相关基因的表达变化,同时细胞发生特征性形态学改变。
此外，最新研究发现自噬与凋亡间有复杂的相互联系，因为参与自噬和凋亡过程的某些分子存在交叉。诸如：两种关键的自噬蛋白Beclin1和Atg5，在凋亡途径中也发挥了重要的作用；同时参与凋亡的caspases和Bcl-2家族成员也可通过裂解或绑定Beclin1的方式调控自噬。最新研究报道表明，自噬通过降解长寿蛋白和细胞器促使活化HSC存活，通过降解HSC内脂滴为其活化提供能量，促使肝纤维化发生。亦有研究表明：生育三烯酚类通过诱导活化的胰星状细胞凋亡和自噬发挥其抗胰纤维化的作用。因此，自噬对细胞的作用具有两面性，可以说是一把双刃剑，既可作为细胞生长的“朋友”，又可能成为杀死细胞“敌人”，这取决于疾病进展的不同阶段、细胞周围环境的变化和治疗干预措施的不同。已有大量的研究证实：多种抗肿瘤药物bufalin、cannabidiol、cucurbitacin等均可通过诱导肿瘤细胞发生自噬性细胞死亡而发挥其抗肿瘤作用。
索拉菲尼是一种靶向性Raf/ERK信号通路多靶点受体酪氨酸激酶抑制剂，它可抑制多种肿瘤细胞的增生并诱导其凋亡，是FDA批准的最早应用于治疗肝癌的口服药。索拉菲尼靶向抑制Raf丝氨酸/苏氨酸激酶以及下游的MEK/ERK信号通路，抑制多种肿瘤细胞的增殖并促进细胞凋亡，并与其对PI3K/Akt/p70S6K信号通路的磷酸化的抑制作用有关。有报道：索拉菲尼干预多种肿瘤细胞后可诱导其自噬水平增高，这与索拉菲尼调控多条自噬相关信号通路有关，诸如： MAPK/ERK、Akt/mTOR/p70S6K和JNK/c-Jun信号通路等。早期的研究表明索拉菲尼可显著降低大鼠肝纤维化模型的门脉压力和血管形成，新的研究发现索拉菲尼具有潜在的抗肝纤维化功效。本课题组的研究已证实索拉菲尼可抑制活化的HSC增殖并诱导其凋亡。然而，索拉菲尼的抗肝纤维化作用及其与程序性细胞死亡间的关系尚不明确。
由此我们推测索拉菲尼的抗肝纤维化作用，除通过其抑制HSC增殖并诱导HSC凋亡外，还可能通过诱导HSC自噬性细胞死亡实现。本课题旨在研究索拉菲尼是否可诱导人HSC-LX2细胞株以及大鼠原代HSC发生自噬性细胞死亡，并探讨自噬和凋亡间的关系及可能的分子机制。实验由以下三部分组成：
第一部分：索拉菲尼诱导肝星状细胞凋亡和自噬性细胞死亡
目的：研究索拉菲尼对HSC凋亡和自噬性细胞死亡的影响。
方法：采用IV型胶原酶和链酶原位灌注肝组织和Nycodenz梯度密度离心技术分离提取大鼠原代HSC，应用α-SMA免疫细胞化学染色和荧光染色方法鉴定原代HSC。索拉菲尼(2.5μmol/L，5.0μmol/L，10.0μmol/L)干预HSC-LX2细胞株6h、12h或24h，四甲基偶氮唑盐(methylthiazolyl tetrazolium, MTT)比色法检测细胞存活率、碘化丙啶(Propidium iodide, PI)/膜联蛋白(Annexin V)联合标记流式细胞术检测HSC凋亡率。HSC-LX2细胞株预孵育凋亡抑制剂Z-VAD-FEK(20μmol/L)或坏死抑制剂IM-54(10μmol/L)1h后，与5μmol/L索拉菲尼共孵育11h，MTT法检测细胞存活率、流式细胞术检测Annexin Ⅴ+细胞数和PI+细胞数百分率变化。索拉菲尼(5μmol/L)干预原代HSC或HSC-LX2细胞株12h后，透射电镜和吖啶橙染色法观察HSC中自噬的激活，激光共聚焦显微镜观察自噬标记蛋白LC3的变化，RT-PCR法检测索拉菲尼对HSC中自噬相关基因Beclin1、Atg7和Atg5mRNA的影响，WesternBlot法检测索拉菲尼对HSC自噬相关蛋白表达LC3、Beclin1、Atg7、Atg5和p62/SQSTM1表达的影响，以及自噬抑制剂3-MA(5mmol/L)抑制自噬对这些蛋白表达的影响。
结果：①HSC获得率为1.4～2.3×107/只，细胞纯度和存活率均大于90%。刚提取的HSC呈圆形，胞浆内富含脂滴，在波长328nm的荧光显微镜下观察，能激发出蓝色荧光。HSC培养14天后，静止的细胞被激活变为梭形或星形，富含维生素A的脂滴消失。②大鼠原代HSC性质鉴定。未活化HSC内不表达α-SMA；当细胞培养14天后，完全活化的HSC胞浆内有α-SMA表达。③索拉菲尼抑制HSC-LX2细胞存活率并促凋亡。MTT和流式细胞术检测显示索拉菲尼呈时间与浓度依赖性抑制HSC存活率并促凋亡，其中5μmol/L索拉菲尼干预12h后细胞存活率即由对照组的100.00%降低至50.19±0.19(P<0.001)，而凋亡率(41.22±2.425%)较对照组(7.73±1.34%)显著增高(P<0.001)。④索拉菲尼诱导HSC细胞非凋亡性细胞死亡。预孵育Z-VAD-FEK后细胞存活率和单用索拉菲尼组间无差异且均较对照组降低(P<0.001)，但PI+细胞率(54.19±4.03%)显著高于Annexin V+细胞率(10.22±2.03%)(P<0.001)。预孵育IM-54后细胞存活率(50.36±4.73%)仍较对照组降低(P<0.001)，PI+细胞率和Annexin V+细胞率较索拉菲尼组均无明显差异。⑤透射电镜和吖啶橙染色观察HSC形态变化。应用5μmol/L索拉菲尼干预HSC12h后，电镜细胞呈现自噬特征，诸如大量双层膜囊泡，囊泡内包含部分胞浆物质，可见包裹的细胞器如线粒体，溶酶体激活，双层膜囊泡与溶酶体融合；荧光显微镜下可见大量点状橙红色荧光颗粒散布于胞浆内细胞核周围。⑥索拉菲尼诱导HSC细胞形成“自噬潮”(autophagic flux)。HSC预孵育溶酶体抑制剂E64d和pepstatinA1h后，与5μmol/L索拉菲尼共孵育11h，Western blot显示预孵育E64d和pepstatin A组LC3Ⅱ/Ⅰ较单用索拉菲尼组进一步增加；激光共聚焦显微镜下预孵育E64d和pepstatin A组原代HSC中LC3绿色斑点进一步增多。⑦RT-PCR检测自噬相关基因Beclin1, Atg7和Atg5的mRNA表达增加。⑧Western blot法检测索拉菲尼干预后自噬相关蛋白Beclin1、Atg5和LC3Ⅱ/Ⅰ表达显著增加，p62表达显著下降；而3-MA可逆转上述改变。
结论：索拉菲尼可通过诱导凋亡和自噬性细胞死亡两种途径，促使HSC发生细胞死亡。HSC启动自噬性死亡途径，对细胞自身的胞浆物质及细胞器进行吞噬、消化和清除。
第二部分：索拉菲尼诱导肝星状细胞自噬性细胞死亡及其与凋亡的关系
目的：探讨索拉菲尼诱导HSC凋亡和自噬性细胞死亡间的相互关系。
方法：应用5μmol/L索拉菲尼干预不同时间点(0h,3h,6h,12h,24h)或不同浓度索拉菲尼(2.5μmol/L,5μmol/L,10μmol/L)干预HSC-LX212h，Western blot法检测自噬和凋亡标志蛋白LC3II/I和Cleaved-PARP变化。不同浓度索拉菲尼(2.5μmol/L,5μmol/L,10μmol/L)干预12h，吖啶橙染色或PI/Annexin V联合标记后流式细胞术检测AVOs或凋亡细胞数变化；Western blot法检测自噬相关蛋白Beclin1和凋亡相关蛋白cleaved-caspase-3,-8变化。预孵育Z-VAD-FEK(20μmol/L)1h后，与10μmol/L索拉菲尼共孵育11h。Western blot法检测自噬相关蛋白LC3II/I和Beclin1变化。自噬抑制剂3-MA(5mmol/L)预孵育1h后，与5μmol/L索拉菲尼共孵育11h。流式细胞术检测凋亡率，Western blot法检测自噬相关蛋白Beclin1和凋亡相关蛋白cleaved-caspase-3,-8变化。应用Atg5-siRNA技术干扰LX2后，5μmol/L索拉菲尼干预12h，Western blot法检测自噬和凋亡相关蛋白变化。
结果：①索拉菲尼诱导自噬先于凋亡。Western blot显示：索拉菲尼干预24h后Cleaved-PARP显著增加，而LC3II/I水平在干预6h后即出现显著性增加，12h达高峰，24h恢复到基线水平。②不同浓度索拉菲尼对自噬和凋亡的影响不同。Western blot显示索拉菲尼(5μmol/L)组LC3II/I显著增加(P<0.01)；而索拉菲尼(10μmol/L)组LC3II/I明显下降，同时Cleaved-PARP显著增加(P<0.01)。流式细胞学结果显示：索拉菲尼(5μmol/L)组AVOs细胞百分率(62.19±6.11%)显著多于凋亡细胞百分比(38.28±4.91%)，(P<0.05)，索拉菲尼(10μmol/L)组凋亡水平(95.90±3.33%)较索拉菲尼(5μmol/L)组显著增加(P<0.01)，而自噬水平(73.18±5.04%)无明显改变。③活化的caspases裂解Beclin1。Western Blot显示：索拉菲尼(10μmol/L)组cleaved-caspase-3,-8表达增强的同时Beclin1明显下降。Z-VAD-FMK可逆转索拉菲尼(10μmol/L)诱导的Beclin1水平降低，并且使LC3Ⅱ/Ⅰ增加。④抑制自噬可促进索拉菲尼诱导的凋亡。预孵育3-MA后，流式细胞术结果显示：凋亡细胞率(81.94±5.34%)较单一索拉菲尼干预组(42.34±3.60%)显著增多(P<0.05)。Western blot显示预孵育3-MA后可显著增加凋亡相关蛋白水平，如cleaved-caspase-3,-8和cleaved-PARP。Atg5-siRNA可抑制索拉菲尼(5μmol/L)诱导的LC3Ⅱ/Ⅰ增加，同时使cleaved-caspase-3,-8和cleaved-PARP表达增加。
结论：索拉菲尼干预HSC后自噬性细胞死亡先于凋亡而发生，当凋亡启动后自噬性细胞死亡被抑制，这种作用可能与活化的caspases断裂Beclin1有关。阻断索拉菲尼诱导的自噬后可代偿性增加凋亡水平，而这种凋亡的增加是作为一种程序性细胞死亡的替代方式。
第三部分：索拉菲尼对肝星状细胞程序性细胞死亡影响的信号转导机制
目的：探讨索拉菲尼对HSC细胞内Akt/mTOR/p70S6K和JNK/c-Jun信号转导通路的调节作用。
方法：5μmol/L索拉菲尼干预HSC-LX2细胞株(0h,0.5h,1.5h,3h,6h,12h,24h)，Western blot检测Akt、p-Akt、mTOR、p-mTOR、p70S6K、p-p70S6K、JNK和p-JNK的蛋白表达变化，激光共聚焦显微镜观察p-c-Jun核转位。以5μmol/L索拉菲尼、25μmol/L LY294002干预HSC-LX2细胞株12h，吖啶橙染色或PI/Annexin V联合标记后流式细胞术检测AVOs和凋亡细胞数变化。以5μmol/L索拉菲尼、10μmol/LSP600125干预HSC-LX2细胞株12h，Western blot检测JNK、p-JNK、c-Jun、p-c-Jun、Beclin1和LC3Ⅱ/Ⅰ变化。
结果：①索拉菲尼抑制HSC-LX2细胞内Akt/mTOR/p70S6K信号通路的磷酸化。索拉菲尼干预后p-Akt/Akt、 p-mTOR/mTOR和p-p70S6K/p70S6K分别较对照组降低；②索拉菲尼可通过Akt/mTOR/p70S6K信号通路诱导凋亡。流式细胞术显示LY294002+索拉菲尼组细胞凋亡率(69.44±6.71%)较索拉菲尼组(38.22±4.66%)增加了53%；而LY294002对AVOs细胞百分率无影响。③索拉菲尼通过JNK/c-Jun信号通路诱导自噬，并激活HSC-LX2细胞内JNK/c-Jun信号通路。索拉菲尼干预后p-JNK/JNK较对照组显著增加，激光共聚焦显微镜观察p-c-Jun增加并转位入核。SP600125抑制了JNK/c-Jun信号分子的磷酸化水平，同时也抑制了索拉菲尼诱导的LC3II/I和Beclin1增加。
结论：索拉菲尼可抑制Akt/mTOR/p70S6k信号通路并活化JNK/c-Jun信号通路，其中Akt/mTOR/p70S6k途径有助于凋亡发生，而JNK/c-Jun信号通路直接参与索拉菲尼诱导的自噬事件。
Hepatic fibrosis is a wound-healing response characterized by theaccumulation of extracellular matrix (ECM) proteins as results of chronicliver injury. Activation of hepatic stellate cells (HSCs), the main source ofECM proteins, represents a crucial event in the pathogenic sequence offibrosis and thus, provides an important framework to define potentialstrategies for anti-fibrotic therapy. Beside the inhibition of fibrogeniccytokines, the induction of HSCs death is now considered an effectivestrategy for the resolution of hepatic fibrosis. The ideal cell death strategyshould selectively target activated HSCs while maintaining quiescent HSCsbut not alert the hepatic inflammatory response. In contrast to theunorganized way of death “necrosis” that triggers the inflammatory response,apoptosis and autophagy proceed with the participation of organized andcontrolled cellular processes, which achieve a cleaner cellular execution.
Death by apoptosis commonly signals through the activation of initiatorand executioner caspases, resulting in the formation of apoptotic bodies thatare removed by phagocytes. The autophagic pathway involves the formationof a membrane around a targeted region of the cell, resulting in theautophagosome formation, which fuses with a lysosome to form anautophagolysosome where the contents are degraded via acidic lysosomalhydrolases. Although apoptosis is the primary mechanism ofchemotherapy-induced cell death, an alternative mode of cell death, termedautophagic cell death, has emerged recently as an important mechanism ofcell death. However, at present, manipulation of autophagy in favor of celldeath is still confusing and controversial due to the paradoxical roles ofautophagy in cell survival and cell death. In addition, the latest research found that there is complex interaction between autophagy and apoptosis,because some moleculars which play an important role in autophagy alsoparticipate in apoptosis, such as beclin1and Atg5; meanwhile the caspaseand Bcl-2family which are involved in apoptosis regulate autophagy bycleaving or binding Beclin1. Most recently, autophagy has been implicatedin the release of lipid droplets from HSCs that further promoted fibrogenesisof activated HSCs. Paradoxically, Rickmann et al. have reported thattocotrienols counteracted pancreatic fibrogenesis through the induction ofapoptosis and autophagy in activated pancreatic stellate cells. Therefore,autophagy is a double-edged sword which is a friend as well as an emeny forcell death depending on the different stages of the disease, cellular nichesand different treatment. There are a large number of studies showing thatvarious anti-cancer drugs play a role in anti-tumor effect by inducing tumorcells to undergo autophagic cell death, such as bufalin, cannabidiol,cucurbitacin and so on.
Sorafenib is a multiple receptor tyrosine kinase inhibitor targeting theRaf/ERK signaling pathways. It is reported that: sorafenib could inducekinds of tumor cell to undergo autophagy due to its effect on regulatingmultiple autophagy-related signaling pathways, such as: MAPK/ERK, Akt/mTOR/p70S6K and JNK/c-Jun signaling pathway.. There has been a recentinterest regarding the potential efficacy of sorafenib in the treatment of themost common and progressive forms of fibrosis. Previous studies indicatedthat sorafenib induced a significant decrease in portal pressure andangiogenesis in rats with liver fibrosis. Our previous study showed thatsorafenib inhibited proliferation and induced apoptosis in activated HSCs.However, the molecular mechanisms involved in sorafenib anti-fibroticeffects have not been completely characterized.
Thus, we hypothesize that sorafenib has a therapeutic effect on liverfibrosis by inhibiting proliferation meanwhile inducing apoptosis, or byinducing HSC autophagic cell death in HSC.The aim of this project is toexplore sorafenib’s effect on inducing autophagic cell death in human HSC-LX2cell lines and primary rat HSCs as well as the relationshipbetween autophagy and apoptosis and potential molecular mechanism. Theexperiment consists of the following three parts:
Part1: Sorafenib induces the apoptosis and ACD of HSCs
Objective: To study the effect of sorafenib on apoptosis and ACD
Methods:To obtained primary HSCs, rat liver tissues sequentialdigestion with pronase and collagenase, followed by single step densitygradient centrifugation with Nycodenz. The identification of HSC was madeby using fluorescent microscope and alpha-SMA immunocytochemicalstaining. Different concentration of Sorafenib (2.5μmol/L,5.0μmol/L,10.0μmol/L) treated HSC-LX2cell lines at the time point of6h,12h or24h,then use MTT assay to detect cell viability, Annexin-V/Propidium iodide (PI)double-labeled the apoptosis of HSCs rate by flow cytometry. HSC-LX2celllines were preincubated by apoptosis inhibitor Z-VAD-FEK (20μmol/L) orthe necrosis inhibitor IM-54(10μmol/L) for1hour, then co-incubated with5μmol/L sorafenib for another11h. The cell viability was also determinedby MTT assay, while the changes of the percentage of Annexin V+cellnumber and PI+cells was dectected by flow cytometry. After primary HSCor HSC-LX2cell lines were treated by sorafenib (5μmol/L) for12h,transmission electron microscopy and acridine orange staining were used toabserve the autophagy activation of HSCs, laser confocal microscope wasused to track the changes of autophagy marker protein LC3; RT-PCR methodwas explored to detect autophagy-related gene Beclin1, Atg7and Atg5aftersorafenib treatment. Western Blot was used to study the effect of sorafeniband autophagy inhibitor’s on the expression of autophagy-related proteinLC3, Beclin1, Atg7, Atg5, and p62/SQSTM1.
Results:①The average havest rate of HSC cells was1.2to2.0×107perrat and the cell purity and survival rate were more than90%. At the verybeginning the extracted HSCs were round and cytoplasm contained a greatdeal of lipid droplets, emitting blue ray under the320nm fluorescence microscope. After the HSCs were cultured for10days, the rested cells wereactivated and present a shape of fusiform or star, and the lipid droplets rich invitamin A disappeard.②The identification of the characteristic of primaryrat HSC. Inactivated HSCs had no expression of alpha-SMA; when the cellswere cultured for14days, fully activated HSCs did have alpha-SMAexpression in cytoplasmic.③sorafenib inhibited HSC-LX2cell survivaland promoted apoptosis. The MTT assay and flow cytometry results showedthat sorafenib inhibited HSCs survival and promoted cell apoptosis in atime-and concentration-dependent manner,5μmol/L sorafenib treatment for12h made the cell viability reduced to50.19±0.19%(P <0.001) comparedwith100%of the control group, and the apoptosis rate (41.22±2.425%)was significantly higher than the control group (7.73±1.34%)(P <0.01).④sorafenib induced the cell non-apoptotic cell death in HSC. The Z-VAD-FEKPre-incubation group had no difference in cell survival rate with sorafenibgroup, but less than the control group (P <0.01).However, the PI+rate(54.19±4.03%) was significantly higher than Annexin V+rate (10.22±2.03%)(P <0.01). The cell viability (50.36±4.73%) of IM-54Pre-incubation group was still less than the control group (P <0.01),both PI+rate and Annexin V+rate had no significantly differences with sorafenibtreated group.⑤The observation of HSCs morphological changes byacridine orange staining and transmission electron microscopy. After5μmol/L sorafenib treated HSCs for12h, the electron microscopy showedthe characteristics of autophagy: a large number of vesicles withdouble-membran which contained cytoplasmic substances, sometimes itshowed wrapped organelles such as mitochondria. Besides, lysosomal wasactivated which was fused with bilayer membrane vesicle; Under thefluorescence microscopy, large numbers of particles with orange flurescencewere dispersed in the cytoplasm around the nucleus.⑥"Autophagy tide"was induced by sorafenib in HSCs. The HSCs were pre-incubated withlysosomal inhibitors E64d and pepstatin A for1h, after then co-incubatedwith5μmol/L sorafenib for another11h. Western blot displayed the LC3II/I exrepssion is further increased in the E64d and pepstatin A pre-incubationgroup compared with only sorafenib treated group; Under the confocalmicroscope the primary HSC of both E64d and pepstatin A pre-incubationgroup showed increase LC3green spots.⑦RT-PCR detected theexpression of autophagy-related gene Beclin1, Atg7and Atg5geneincreased.⑧Western blot results showed that autophagy-related proteinBeclin1, Atg5and LC3II/I were significantly increased, while p62decreased significantly after sorafenib treatment which could be reversed byadding3-MA.
Conclusion: Sorafenib could promote death of HSCs by inducingapoptosis and autophagic cell death. It is speculated that the HSCs couldphagocyte, digestion and eliminate cytoplasm substances and organelles byactivating autophagic death pathway.
Part II: Sorafenib induces programmed cell death in hepatic stellate cellby coordinating the cross-talk between apoptosis and autophagy
Objective: To probe the cross-talk between apoptosis and autophagyinduced by sorafenib.
Methods:5μmol/L sorafenib treated HSCs at different time points (0h,3h,6h,12h,24h) or different concentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) treated HSCs for12h. To study the HSCs’ autophagyand apoptosis, the expression of blot LC3II/I and Cleaved-PARP wasdetected by Western blot. After HSCs were treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,acridine orange staining or PI/Annexin V double staining were used to detectthe AVOs and changes of the apoptotic cell number; Western blot methodwas explored to detect the expression of autophagy-related protein Beclin1and apoptosis-related proteins cleaved-caspase-3,-8. HSCs werepre-incubated with Z-VAD-FEK (20μmol/L) for1h then incubated with10μmol/L sorafenib for another11h. Western blot was used to detect theautophagy-related protein LC3II/I and Beclin1change. And after autophagy inhibitor3-MA (5mmol/L) pre-incubated for1h and5μmol/L sorafenibincubated for another11h, apoptosis rate of HSCs was detected by flowcytometry, and the expression of autophagy-related protein Beclin1andapoptosis-related proteins cleaved-caspase-3,-8were detected by Westernblot. In addition, Atg5-siRNA technology is applied to interfere LX2, after5μmol/L sorafenib treated for12h, the autophagy and apoptosis-relatedprotein changes were checked by Western blot.
Results:①Autophagy induced by sorafenib is prior to apoptosis. Thewestern blot results showed that: after the HSCs were treated with sorafenibfor24h, Cleaved-PARP expression was significantly increased, while LC3II/I expression was significantly increased at the time point of6h, andreached the peak at12h. when treatment time extended to24h, the LC3II/Iexpression returned to baseline levels.②Different concentrations ofsorafenib had different effect on autophagy and apoptosis in HSCs. Westernblot showed that in sorafenib (5μmol/L) group LC3II/I expression wassignificantly increased (P <0.01); In sorafenib group (10μmol/L), LC3II/Iexpression was decreased, but Cleaved-PARP expression was significantincreased (P <0.01). Flow cytometry results showed that in sorafenib group(5μmol/L), the AVOS cell percentage (62.19±6.11%) was significantlyhigher than the percentage of apoptotic cells (38.28±4.91%)(P <0.01),while in sorafenib (10μmol/L) group, the apoptosis rate (95.90±3.33%)was significantly higher than in sorafenib (5μmol/L) group (P <0.01),butthere was no significant change of the autophagy rate(73.18±5.04%).③The activated caspases cleaved Beclin1. The Western Blot resultsdemonstrated that in sorafenib (10μmol/L) group,the expression of thecleaved-caspase-3,-8enhanced, but Beclin1decreased. Z-VAD-FMK canreverse this situation, and increase the LC3II/I expression.④Inhibition ofautophagy can promote sorafenib-induced apoptosis. After pre-incubationwith3-MA, HSCs were detected by flow cytometry. The results showed thatAnnexin V-positive cells (81.94±5.34%) were much higher the sorafenibtreated group (42.34±3.60%)(P<0.01). Western blot showed that the expression of the apoptosis-related proteins, such as cleaved-caspase-3,-8and cleaved-PARP could significantly increase after pre-incubated with3-MA. At the mean time, Atg5-siRNA could inhibit sorafenib-inducedincrease of LC3II/I, meanwhile increase expression of the cleaved-caspase-3,-8and cleaved-PARP.
Conclusion: The HSCs treated with sorafenib undergo autophagic celldeath pior to apoptosis, autophagic cell death was inhibited when theapoptosis was started, which may due to the activated caspases whichinvolves in cleavaging Beclin1. After blocking the autophagy induced bysorafenib, the apoptosis increased in a compensatory manner, which is analternative way as a kind of programmed cell death.
Part III: The signal transduction mechanism of sorafenib inducingprogrammed cell death in hepatic stellate cells
Objective: To investigate the regulatory function of sorafenib onAkt/mTOR/p70S6K and JNK/c-Jun signal transduction pathway in HSCcells.
Methods: HSC-LX2cell line was treated with5μmol/L sorafenib atdifferent time porints (0h,0.5h,1.5h,3h,6h,12h,24h), expression ofAkt, p-Akt, mTOR, p-mTOR, p70S6K, p-p70S6K, JNK and p-JNK,weredetected by Western blot. Laser scanning confocal microscopy was exploredto track pc-Jun nuclear translocation. After HSCs was treated by differentconcentrations of sorafenib (2.5μmol/L,5μmol/L,10μmol/L) for12h,Acridine orange staining or Annexin-V/Propidium iodide (PI) double-labeledstaining by flow cytometry and changes of the apoptotic cellss number; afterHSCs were treated with5μmol/L sorafenib and10μmol/L SP600125for12h, Western blot was used to detect JNK, p-JNK, c-Jun, p-c-Jun, Beclin1andLC3II/I expression.
Results:①Sorafenib inhibited phosphorylation of Akt/mTOR/p70S6Ksignaling pathway in HSC-LX2cells. The ratio of p-Akt/Akt,p-mTOR/mTOR and p-p70S6K/p70S6K were reduced (P<0.01) respectively compared to control group.②Sorafenib induced cell apoptosis byAkt/mTOR/p70S6K signal pathway. Flow cytometry revealed that the rate ofapoptosis in LY294002+sorafenib group (69.44±6.71%) increased by about53%compared to the sorafenib group (38.22±4.66%) while LY294002hadno effect on the AVOs cell percentage.③Sorafenib initiated autophagythrough JNK/c-Jun signaling pathway. Sorafenib activated phosphorylationof JNK/c-Jun signaling pathway in HSC-LX2cells. The ratio of p-JNK/JNKsignificantly increased compared to the control group by sorafenibintervention, a significant increase of p-c-Jun and its translocation to thenucleus were proved by confocal macrophage. SP600125inhibited thephosphorylation level of JNK/c-Jun and also inhibited the sorafenib-inducedLC3II/I and Beclin1increasing.
Conclusion: Sorafenib inhibited Akt/mTOR/p70S6k signaling pathwayand activated JNK/c-Jun signaling pathway, in which Akt/mTOR/p70S6kpathway leaded to apoptosis, and JNK/c-Jun signal pathway participated insorafenib-induced autophagy directly.
引文
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