【作者】 曹洁；

【导师】 沈薇；

【作者基本信息】 重庆医科大学 ， 内科学， 2013， 博士

【摘要】 背景与目的：非酒精性脂肪性肝病（nonalcoholic fatty liver disease，NAFLD）是一种由遗传-环境-代谢应激相关因素所致的以肝细胞内脂肪堆积为主要表现的临床病理综合征。在我国，NAFLD的发病率日趋升高，并已成为严重威胁人民健康的主要疾病之一。NAFLD包括单纯性脂肪肝、非酒精性脂肪性肝炎（nonalcoholic steatohepatitis，NASH）、脂肪性肝纤维化和肝硬化。单纯性脂肪肝进展缓慢，NASH时肝内巨噬细胞、星状细胞活化，导致肝内炎症、纤维化的发生发展，最终可进展为肝硬化、原发性肝癌等终末期肝病。最近研究发现，NASH患者并发心脑血管疾病的风险也明显上升，如动脉粥样硬化、脑梗死、心肌梗死等。因此，是否存在NASH是影响NAFLD患者预后的重要因素。至今，NASH的发病机制仍未完全阐明。血中游离脂肪酸（free fatty acids,FFAs）浓度升高、肝细胞脂性病变及凋亡增加是NASH的重要特征。当循环中FFAs水平升高超过肝脏的氧化和代谢能力时，脂质在肝脏异位沉积、炎性因子释放和凋亡信号启动，同时肝细胞对炎性反应和各种损伤因素的敏感性增高，由此导致的肝细胞功能障碍或死亡称为脂毒性；由此引起的肝细胞凋亡，称为脂毒性凋亡。肝细胞脂毒性凋亡在NASH的发生进展中是重要的始动环节并且参与整个发病过程。目前NASH与肝细胞脂毒性凋亡的机制尚不完全明了。内质网（endoplasmic reticulum, ER）功能复杂，主要与蛋白的加工成熟、钙离子储存、脂质代谢、类固醇激素的合成及细胞的解毒相关。肝细胞富含ER, ER也是维持肝细胞正常功能的重要细胞器，众多肝脏致病因素均能破坏ER稳态，从而导致内质网应激（endoplasmic reticulum stress,ER stress）。ER stress包括未折叠蛋白反应（unfolded protein response，UPR）和非UPR两种途径。蛋白质的不正确折叠和/或错误折叠引发的ER stress反应称UPR，在哺乳动物细胞中由3种ER感应蛋白介导,即需肌醇酶1（inositol-requiring protein1, IRE1）、双链RNA依赖的蛋白激酶样ER激酶（PKR-like ER kinase, PERK）和活化转录因6（activatingtranscription factor6, ATF6）3种跨膜蛋白。非UPR包括磷脂酰肌醇-3激酶/蛋白激酶B（phosphatidylinositide-3-OH kinase/protein kinase B,PI3K/Akt）、丝裂原活化蛋白激酶/细胞外信号调节激酶（mitogen-activated protein kinase/extracellular signal-regulated proteinkinases，MEK/ERK）、糖原合成酶激酶-3（glycogen synthase kinase-3，GSK-3）、细胞核转录因子κB（nuclear transcription factor，NF-κB）、Toll样受体（Toll-like receptor，TLR）等。这些通路分别对ER stress介导的细胞凋亡、炎性反应以及免疫反应有重要影响。早期的ER stress在维持细胞内环境的稳态方面起着重要的作用，但是过度的ER stress则会引起细胞凋亡，造成细胞和组织的损害。虽然已经有报道指出，ER stress与NASH肝细胞脂毒性凋亡密切相关，但是有关ER stress在脂毒性凋亡中的作用机制仍有待进一步阐明。因此，本课题运用饱和脂肪酸软脂酸钠诱导人正常肝细胞株L02和肝癌细胞株HepG2建立肝细胞脂变模型，从ER stress UPR和非UPR入手，研究软脂酸钠对UPR（PERK、IRE1、ATF6）和非UPR（glycogen synthase kinase-3β，GSK-3β）信号通路的影响以及UPR和非UPR信号途径对肝细胞脂毒性凋亡的调控作用，进而揭示软脂酸钠对肝细胞脂毒性的作用机制，这不仅有助于阐明NASH的发病机制，也为NASH的治疗奠定新的理论基础。方法：一软脂酸钠诱导L02和HepG2细胞发生脂毒性凋亡1.软脂酸钠诱导L02和HepG2细胞建立肝细胞脂变模型用不同浓度的软脂酸钠及不同时间点（12、24、48h）诱导L02和HepG2细胞，通过MTT方法检测细胞活力，筛选出软脂酸钠最佳诱导浓度建模；通过甘油三酯测定、油红O染色检测细胞内甘油三酯含量和脂质沉积情况。2.软脂酸钠诱导脂变L02和HepG2细胞凋亡通过Annexin V-PE/7-AAD流式细胞术、Hoechst33258细胞凋亡荧光染色以及透射电子显微镜检测脂变模型肝细胞的凋亡率、观察凋亡细胞的形态变化。二UPR调节软脂酸钠诱导肝细胞脂毒性凋亡的作用和机制1.软脂酸钠诱导L02和HepG2细胞后，ER stress标志分子表达及UPR信号通路的激活情况软脂酸钠诱导细胞0、12、24、48h，收集细胞，提取胞浆蛋白、核蛋白和总RNA，通过Western blot和RT-PCR方法检测GRP78、磷酸化PERK（p-PERK）、ATF4和CHOP蛋白以及未剪接型XBP-1（uXBP-1）和剪接型XBP-1（XBP-1s）mRNA的表达，比较各时间点上述蛋白和mRNA表达水平较0h的变化情况。2.沉默PERK基因对软脂酸钠诱导的PERK/ATF4/CHOP通路阻断及脂变肝细胞凋亡的影响将沉默效果最佳的PERK shRNA质粒和Control shRNA质粒转染L02和HepG2细胞24h，再予软脂酸钠诱导细胞48h，收集细胞，提取胞浆蛋白和核蛋白，通过Western blot、Annexin V-PE/7-AAD流式细胞术方法，对比PERK shRNA+软脂酸钠组与Control shRNA+软脂酸钠组总PERK（t-PERK）、ATF4和CHOP蛋白表达水平及细胞凋亡率的变化。三GSK-3β（非UPR）调节软脂酸钠诱导肝细胞脂毒性凋亡的作用和可能机制1.软脂酸钠诱导L02和HepG2细胞后，GSK-3β蛋白的表达水平以及GSK-3β在脂变肝细胞凋亡中的作用软脂酸钠诱导细胞0、12、24、48h，收集细胞，提取胞浆蛋白，Western blot方法检测磷酸化GSK-3β（p-GSK-3β，Ser9磷酸化）、总GSK-3β（t-GSK-3β）蛋白的表达，比较各时间点p-GSK-3β、t-GSK-3β蛋白的表达水平较0h的变化情况。GSK-3β特异性抑制剂Lithium chloride与软脂酸钠共同诱导（抑制剂组）和软脂酸钠诱导（模型组）培养细胞0、12、24、48h，收集细胞，通过Western blot、Annexin V-PE/7-AAD流式细胞术方法，对比相同时间点抑制剂组与模型组p-GSK-3β蛋白表达水平和细胞凋亡率的变化。将沉默效果最佳的GSK-3β shRNA质粒和Control shRNA质粒转染L02和HepG2细胞24h，再予软脂酸钠诱导细胞48h，收集细胞，提取胞浆蛋白和核蛋白，通过Western blot、Annexin V-PE/7-AAD流式细胞术方法，对比GSK-3β shRNA+软脂酸钠组与Control shRNA+软脂酸钠组t-GSK-3β蛋白表达水平及细胞凋亡率的变化。2.抑制GSK-3β蛋白活性和沉默基因表达对软脂酸钠诱导的GSK-3β非UPR通路的影响GSK-3β特异性抑制剂Lithium chloride与软脂酸钠共同诱导（抑制剂组）和软脂酸钠诱导（模型组）培养细胞0、12、24、48h，收集细胞，Western blot方法、Caspase-3分光光度法分别检测磷酸化JNK（p-JNK）、Bax蛋白表达和Caspase-3活性，对比同一时间点抑制剂组与模型组p-JNK、Bax表达水平和Caspase-3活性的变化。将沉默效果最佳的GSK-3β shRNA质粒和Control shRNA质粒转染L02和HepG2细胞24h，再予软脂酸钠诱导细胞48h，收集细胞，提取胞浆蛋白和核蛋白，通过Western blot方法，对比GSK-3β shRNA+软脂酸钠组与Control shRNA+软脂酸钠组p-JNK、Bax、GRP78、磷酸化IRE1（p-IRE1）、p-PERK蛋白表达水平及Caspase-3活性变化。结果：一软脂酸钠诱导L02和HepG2细胞发生脂毒性凋亡1.不同浓度（36、72、108、144、180μmol/L）的软脂酸钠诱导L02和HepG2细胞12、24h，细胞存活率变化不明显；当浓度超过72μmol/L诱导细胞48h，细胞存活率较对照组明显降低，因此在后续实验中选用接近48h的2/3的IC50浓度（108μmol/L）作为软脂酸钠最佳诱导浓度。2.软脂酸钠诱导L02和HepG2细胞12h，细胞内甘油三酯含量和脂质沉积变化不明显；诱导24、48h，细胞内甘油三酯合成增加、脂质明显沉积。3.软脂酸钠诱导L02和HepG2细胞24h，与对照组比较，细胞凋亡率无明显变化；诱导细胞48h，凋亡率较对照组明显增加（P <0.05）、凋亡细胞发生形态变化、凋亡小体形成。二UPR调节软脂酸钠诱导肝细胞脂毒性凋亡的作用和机制1.软脂酸钠诱导L02和HepG2细胞0、12、24、48h，GRP78蛋白的表达量逐渐增加，与对照组比较，差异均具有统计学意义（P <0.05）。2.软脂酸钠诱导L02和HepG2细胞0、12、24、48h，p-PERK、ATF4、CHOP蛋白的表达量均逐渐增加，与对照组比较，差异具有统计学意义（P <0.05）；仅HepG2细胞12h时uXBP-1mRNA水平较0h一过性的升高，在HepG2其它时间点及L02细胞中uXBP-1mRNA水平无明显变化，并且在各时间点均未检测到XBP-ls mRNA表达。3.构建的3组PERK shRNA质粒中，PERK1shRNA质粒的沉默效果最佳。沉默L02和HepG2细胞PERK基因后，与Control shRNA+软脂酸钠组比较，PERK shRNA+软脂酸钠组t-PERK、ATF4和CHOP蛋白表达降低，差异具有统计学意义（P <0.05）。4. PERK基因沉默后，PERK shRNA+软脂酸钠组细胞凋亡率较ControlshRNA+软脂酸钠组降低，差异具有统计学意义（P <0.05）。三GSK-3β（非UPR）调节软脂酸钠诱导肝细胞脂毒性凋亡的作用和可能机制1.随着软脂酸钠诱导L02和HepG2细胞时间（0、12、24、48h）延长，p-GSK-3β表达逐渐减低，间接反应了GSK-3β蛋白的活性逐渐增加，与对照组比较，差异均具有统计学意义（P <0.05）。2.与模型组比较，L02和HepG2细胞抑制剂组p-GSK-3β蛋白表达明显增加，差异具有统计学意义（P <0.05），说明Lithium chloride可以通过增加软脂酸钠诱导肝细胞中p-GSK-3β（Ser9）蛋白的表达从而抑制GSK-3β活性。抑制剂组肝细胞凋亡率较模型组显著降低，差异具有统计学意义（P <0.05）。3.构建的3组GSK-3β shRNA质粒中，GSK-3β1shRNA质粒的沉默效果最佳。沉默L02和HepG2细胞GSK-3β基因后，与Control shRNA+软脂酸钠组比较，GSK-3β shRNA+软脂酸钠组细胞凋亡率明显降低，差异具有统计学意义（P <0.05）。4.随着软脂酸钠诱导L02和HepG2细胞时间（0、12、24、48h）延长，p-JNK和Bax蛋白表达以及Caspase-3活性逐渐增加。抑制剂组p-JNK、Bax蛋白表达水平和Caspase-3活性均较模型组降低。沉默GSK-3β基因后，与Control shRNA+软脂酸钠组比较，GSK-3β shRNA+软脂酸钠组p-JNK、Bax蛋白表达水平和Caspase-3活性降低，以上差异均具有统计学意义（P <0.05）。5.沉默L02和HepG2细胞GSK-3β基因后，Control shRNA+软脂酸钠组GRP78、p-IRE1和p-PERK蛋白表达较Control shRNA组明显上调，差异具有统计学意义（P <0.05）。然而，GSK-3β shRNA+软脂酸钠组与Control shRNA+软脂酸钠组比较，上述蛋白表达无明显变化，差异无统计学意义（P>0.05）。结论：一饱和脂肪酸诱导L02和HepG2细胞发生脂毒性凋亡。二PERK/ATF4/CHOP是介导饱和脂肪酸诱导肝细胞脂毒性凋亡的主要UPR信号通路。三GSK-3β（非UPR）可能通过p-JNK/Bax/Caspase-3途径调节饱和脂肪酸诱导的肝细胞脂毒性凋亡。更多还原

【Abstract】 BACKGROUND AND PURPOSE:Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolicdisorder characterized by fat accumulation in the liver, which is not due toexcessive alcohol consumption and definite factors damaged to liver. Theincidence of NAFLD has increased rapidly. Thus, NAFLD has emerged asa major public health issue in China. Clinically, NAFLD encompasses abroad spectrum of hepatic derangements from steatosis to nonalcoholicsteatohepatitis (NASH). The latter is characterized by hepatic fataccumulation coincident with inflammation, reduced liver function, fibrosis,and eventually liver cirrhosis. Many recent studies have reported thatNASH is a risk factor for cardiovascular disease, such as atherosclerosis,cerebral infarction and myocardial infarction. Therefore, NASH is crucialfor the prognosis of NAFLD. However, to date, the pathogenesis of NASHis still poorly understood. The charactiristic of NASH involves increasedlevels of free fatty acids (FFAs), hepatic fat accumulation and apoptosis of liver cells. The excess of FFAs overwhelms the capacity of the liver tooxidative and esterify FFAs leading to intrahepatic lipid deposition andactivation of inflammatory factors and apoptosis signal pathway, aphenomenon termed lipotoxicity, which includes lipoapoptosis. The latter isa prominent feature of NASH and is associated with severity of the disease.The molecular mechanism responsible for NASH and FFAs-inducedhepatocyte lipoapoptosis remains undefined. Endoplasmic reticulum (ER)is a highly dynamic organelle that synthesizes and processes secretory andtransmembrane proteins. The ER also serves important functions in calciumstorage and signaling, lipid biosynthesis, steroid hormone sythnesis and celldetoxification. A number of biochemical and physiologic stimuli associatedwith liver injury can disrupt ER homeostasis, imposing stress to the ER(ER stress), which includes unfolded protein response (UPR) andnon-unfolded protein response (non-UPR). Disruption of ER homeostasiscauses aberrant accumulation of unfolded or misfolded proteins in the ERlumen, triggering an evolutionarily conserved response, termed the UPR.Proximal sensors of UPR include protein kinase RNA-like ER kinase(PERK), inositol-requiring protein1(IRE1), and activating transcriptionfactor6(ATF6). Non-UPR involves phosphatidylinositide-3-OHkinase/protein kinase B (PI3K/Akt), mitogen-activated proteinkinase/extracellular signal-regulated protein kinases (MEK/ERK),glycogen synthase kinase-3(GSK-3), nuclear transcription factor (NF-κB), Toll-like receptor (TLR), which contributes to the apoptosis, inflammationresponse and immune response，respectively. The mild ER stress serves toovercome the stress stimulus; however, prolonged ER stress will promotecell apoptosis. Although ER stress is related to hepatocytes lipoapoptosis ofNASH, the definite mechanism responsible for the regulation of ER stresson NASH needs to be fully investigated. Furthermore, This study treatedhuman liver L02and HepG2cell lines with sodium palmitate, a saturatedfatty acid, to establish a steatosis model. In this model, we examined theeffects of sodium palmitate on the levels of proteins that are known to beassociated with ER stress, and the regulation of UPR and non-UPR onlipoapoptosis, to provide further mechanistic insights into the coreapoptotic machinery during saturated FFA-mediated lipotoxicity, whichcontributes to clearfy the definite mechanism of NASH.METHORDS:1. Sodium palmitate-induced L02and HepG2cells to lipoapoptosis1） Establishment of the steatosis model of L02and HepG2cells inducedby sodium palmitateThis study treated human liver L02and HepG2cell lines with variousdoses of sodium palmitate, a saturated fatty acid, for12，24and48h. MTTassay was used for cell viability, to adopt the optimal concentration ofsodium palmitate for the following experiments. Measurement ofintracellular triglyceride and oil red O staining was used to detect altered accumulation of lipid droplets and triglyceride in L02and HepG2cells.2） Sodium palmitate induced lipoapoptosis in L02and HepG2cells.Flow cytometry with Annexin V-PE binding and7-amino-actinomycinD (7-AAD) staining and Hoechst33258staining was used to detectapoptosis. Transmission electron microscopy was used to detectmorphological changes.2. The regulation of UPR on sodium palmitate-induced lipoapoptosis inL02and HepG2cells1） The expression levels of ER stress marker and UPR related proteins insodium palmitate-treated L02and HepG2cellsThe total RNAs and proteins were collected in L02and HepG2cellstreated with sodium palmitate for up to48h. Western blot analysis wasused to detect protein expression of glucose-regulated protein78(GRP78),phosphorylated PKR-like ER kinase (p-PERK), activating transcriptionfactor4(ATF4) and C/EBP-homologous protein (CHOP). RT-PCR wasused to detect mRNA expression of unspliced X-box binding protein-1(uXBP-1) and X-box binding protein-1splicing (XBP-1s).2） The effect of PERK gene knockdown on sodium palmitate-inducedPERK/ATF4/CHOP pathway and lipoapoptosisL02and HepG2cells were grown and transiently transfected withPERK shRNA or negative control shRNA from using transfection reagentPoly JetTMaccording to the manufacturer’s instructions. Twenty-four hours after transfection, the medium was changed to regular medium and the cellswere treated with sodium palmitate for an additional48h. Western blotanalysis was used to detect protein expression of total PERK, ATF4andCHOP. Flow cytometry was used to detect apoptosis. The proteinexpression levels and the number of apoptotic cells were comparedbetween the PERK shRNA plus sodium palmitate group and ControlshRNA plus sodium palmitate group.3. The regulation of GSK-3β (non-UPR) on sodium palmitate-inducedlipoapoptosis in L02and HepG2cells1） The expression levels of GSK-3β after sodium palmitate treatment andthe effect of Inhibition of GSK-3β activity or expression on sodiumpalmitate-induced apoptosis in steatotic L02and HepG2cells.L02and HepG2cells were treated with sodium palmitate for up to48h, and then subjected to protein extraction and western blot analyses. Theprotein expression levels of phosphorylated GSK-3β and total GSK-3βwere compared between model group and Control group.L02and HepG2cells were treated with sodium palmitate for up to48h, in the presence or absence of lithium chloride, a GSK-3β inhibitor, andthen subjected to protein extraction, western blot analyses, and flowcytometry with Annexin-PE and7AAD double staining. The proteinexpression levels of phosphorylated GSK-3β and the number of apoptoticcells were compared between inhibitor group and model group at the same time point.The cells were grown and transfected with GSK-3β shRNA ornegative control shRNA for24h, then treated with sodium palmitate for anadditional48h, and subjected to protein extraction, western blot analysesand flow cytometry with Annexin-PE and7AAD double staining. Theprotein expression levels of total GSK-3β and the number of apoptotic cellswere compared between the GSK-3β shRNA plus sodium palmitate groupand Control shRNA plus sodium palmitate group.2） Inhibition of GSK-3β activity or expression on regulation of sodiumpalmitate-induced c-Jun NH2-terminal kinase (JNK) phosphorylation,Bcl-2-associated X protein (Bax) upregulation, Caspase-3activity andactivation of UPR in L02and HepG2cells.L02and HepG2cells were treated with sodium palmitate for up to48h, in the presence or absence of lithium chloride, a GSK-3β inhibitor, andthen subjected to protein extraction, western blot analyses, and caspase-3activity measurements with a caspase-3colorimetric assay kit.The protein expression levels of phosphorylated JNK, Bax and Caspase-3activity were compared between inhibitor group and model group at thesame time point.The cells were grown and transfected with GSK-3β shRNA ornegative control shRNA for24h, then treated with sodium palmitate for anadditional48h, and subjected to protein extraction and western blot analyses. The protein expression levels of phosphorylated JNK, Bax,GRP78, phosphorylated inositol-requiring protein1(IRE1),phosphorylated PERK and Caspase-3activity were compared between theGSK-3β shRNA plus sodium palmitate group and Control shRNA plussodium palmitate group.RESULTS:1. Sodium palmitate-induced L02and HepG2cells to lipoapoptosis1） MTT assay data showed that sodium palmitate had very little effect atlow dose and short exposure time, while higher doses (i.e.,144and180μmol/L sodium palmitate) inhibited the growth of cells by more than50%at48h. Thus, we adopted the lower cytotoxic dose of108μmol/L sodiumpalmitate for the following experiments.2） Oil Red O staining and triglyceride quantification revealed prominentlipid accumulation in the cytoplasm and increased triglyceride levels,respectively.3） There was no significant difference in the number of apoptotic cells inL02and HepG2cells treated with sodium palmitate for24in comparisonwith the control. However, incubation of hepatocytes with108μmol/Lsodium palmitate for48h caused a significant increase in the number ofapoptotic cells, compared to the control (P <0.05). The Hoechst33258stainand electron microscopic revealed that L02and HepG2cells treated withsodium palmitate for48h displayed apoptotic cells with nuclear fragmentation, chromatin condensation and formation of apoptotic bodies,respectively.2. The regulation of UPR on sodium palmitate-induced lipoapoptosis inL02and HepG2cells1） The data showed that GRP78expression was obviously upregulated ina time-dependent manner in L02cells. In contrast, GRP78expressionreached a peak at24h and slightly decreased24h after treatment withsodium palmitate in HepG2cells (P <0.05).2） The data showed that the levels of phosphorylated PERK increased insodium palmitate-treated L02and HepG2cells over time. The same is truefor the expression of the transcription factor ATF4and CHOP (P <0.05).uXBP-1expression was stimulated only after the12h treatment withsodium palmitate, as compared to the control HepG2cells. In L02cells,there was no significant difference in uXBP-1expression in either of thesodium palmitate treatment groups in comparison with the control. Cellstreated with sodium palmitate did not have the spliced version of XBP-1.3） PERK1shRNA plasmid had the optimal silencing effect among thethree constructed PERK shRNA plasmids. In L02and HepG2cells inwhich PERK shRNA had been transiently transfected, PERK proteinexpression was significantly suppressed, as compared to the cells of thenegative control. ShRNA-targeted knockdown of PERK also suppressedATF4and CHOP induction by palmitate sodium (P <0.05). 4） The rate of apoptosis induced by sodium palmitate treatment was alsoreduced by PERK shRNA transfection, compared to the control shRNA (P<0.05).3. The regulation of GSK-3β（non-UPR）on sodium palmitate-inducedlipoapoptosis in L02and HepG2cells1） our data showed that sodium palmitate induced the dephosphorylationof GSK-3β at Ser-9in a time-dependent manner, suggesting GSK-3βactivity progressively increased after sodium palmitate treatment (P <0.05).2） Compared with the model group, the level of p-GSK-3β (Ser9) ininhibitor group had significantly increased (P<0.05). Our data suggestedthat lithium chloride inhibited GSK-3β activity by induction ofphosphorylation of GSK-3β at Ser-9. In addition, lithium chloride reducedsodium palmitate-induced apoptosis in steatotic L02and HepG2cells, ascompared with the model group (P <0.05).3） GSK-3β1shRNA plasmid had the optimal silencing effect among thethree constructed GSK-3β shRNA plasmids. The rate of apoptosis inducedby sodium palmitate treatment was reduced by GSK-3β shRNAtransfection, compared to the control shRNA (P <0.05).4） Our data showed that phosphorylated JNK levels progressivelyincreased in sodium palmitate-treated L02and HepG2cells, while Bax, apro-apoptotic effector downstream of JNK, was also significantlyupregulated at24to48h after sodium palmitate treatment. The same is true for the Caspase-3activity. Inhibition of GSK-3β expression or activitysuppressed sodium palmitate-induced JNK phosphorylation, Baxupregulation and Caspase-3activity (P <0.05).5） Our data showed that treatment of L02and HepG2cells with sodiumpalmitate promoted a significant increase in GRP78expression andphosphorylation of PERK and IRE1(P <0.05), but inhibition of GSK-3βexpression using GSK-3β shRNA transfection did not affect GRP78expression or sodium palmitate-induced phosphorylation of PERK andIRE1(P>0.05).CONCLUSION:1. Saturated fatty acid-induced L02and HepG2cells to lipoapoptosis.2. Saturated fatty acid-induced lipoapoptosis in L02and HepG2wereenacted through the PERK/ATF4/CHOP signaling pathway.3. Saturated fatty acid-induced lipoapoptosis of L02and HepG2cells wasregulated by GSK-3β activation, which may depend on JNKphosphorylation and Bax upregulation.更多还原