【作者】 王建青；

【导师】 李俊；

【作者基本信息】 安徽医科大学 ， 药理学， 2012， 博士

【摘要】 肝脏是体内最大的生物代谢器官，也是外源性物质及其代谢产物攻击的主要靶器官。机体短期接触较大剂量外源性物质可引起急性肝损伤，在外来因素长期作用下，如果不能完全修复，可发展成慢性肝损伤。肝纤维化是各种慢性肝损伤的共有病理变化，其持续发展为肝硬化可引起肝功能衰竭甚至肝细胞癌，已成为一个危害人类生命健康的世界性问题。但目前急慢性肝损伤作用的发病机制尚未完全阐明，仍未发现十分确定、特效的药物，因此，深入研究急慢性肝损伤的发病机制，为临床寻找理想的抗肝损伤药物，尤其是抗纤维化的药物，具有重要的理论和临床意义。最近研究发现内质网应激（endoplasmic reticulum stress, ER Stress）在许多肝脏疾病中发挥了重要作用。内质网是广泛存在于真核细胞中的一种重要的细胞器，负责蛋白质合成、折叠、组装和运输等。在外来因素刺激作用下，细胞处于应激状态，引起未折叠蛋白反应，它本是机体的一种自我保护机制，但当应激持续发生时，则会激活脂肪生成、炎症和凋亡等途径，引发一系列的病理性反应。最近的一些研究也显示内质网应激在急慢性肝损伤形成过程中发挥了一定在作用，与肝纤维化的形成密切相关。然而，内质网应激参与急慢性肝损伤，尤其是肝纤维化的确切机制尚未完全阐明。四氯化碳（carbon tetrachloride, CCl₄）诱导的急慢性肝损伤模型是研究肝损伤机制的经典模型之一。本研究首先观察了内质网应激相关蛋白在CCl₄诱导小鼠急性肝损伤中的变化，在此基础上进一步研究了内质网应激在肝纤维化中的作用及部分机制，主要内容概括如下：1ER stress在CCl₄诱导小鼠急性肝损伤中的作用1.1CCl₄诱导小鼠急性肝损伤为探讨ER stress在小鼠急性肝损伤中的作用，本研究在经腹腔单次注射CCl₄（0.3ml/kg BW）后不同时点（0,2,6,12,24和72h）分别剖杀小鼠，称量体重和肝脏质量，留取血液和肝脏组织。结果发现：与正常对照组相比，CCl₄在处理后12h小鼠肝重与肝指数明显增加，以72h最显著；CCl₄处理后2h即引起ALT明显升高，24h达最高峰，升高约千倍，72h接近对照组正常值。病理组织学检查显示CCl₄处理6h后，引起明显的气球样变和点状坏死；12h进展为块状坏死；至24h坏死最显著，肝细胞呈大片坏死；72h坏死区域有大量的炎性细胞浸润。1.2CCl₄诱导急性肝损伤小鼠肝脏ER Stress效应为观察急性肝损伤小鼠肝脏ER Stress效应，采用免疫组化技术检测急性肝损伤小鼠肝脏GRP78分布的动态变化。结果发现：与正常对照组相比，在CCl₄处理后2h，在少量肝细胞中出现GRP78棕黄色特异性染色，6h肝细胞特异性染色呈明显的带状分布，12h肝细胞出现区域性特异性染色，24h肝细胞特异性染色分布最广且最深，72h肝细胞GRP78特异性染色明显减弱。采用Western blotting技术检测GRP78蛋白表达以及下游IRE1α、eIF2α蛋白磷酸化水平和ATF6核蛋白水平的动态变化。结果发现：与正常对照组相比，在CCl₄处理后6h肝脏组织GRP78显著升高，在12h达到高峰，并持续至24h，在72h恢复至正常对照水平；在CCl₄处理后2h，肝脏组织IRE1α蛋白磷酸化水平显著升高，至12h肝脏组织IRE1α蛋白磷酸化水平仍明显升高；在CCl₄处理后12h，肝脏组织eIF2α蛋白磷酸化水平明显升高，并持续至72h，以24h磷酸化水平最显著；在CCl₄处理后6h肝脏组织ATF6核蛋白水平显著升高，在24h升高最为显著，在72h恢复至正常对照水平。上述结果提示CCl₄诱导的小鼠急性肝损伤模型中发生ER Stress效应。1.3急性肝损伤小鼠肝细胞凋亡的动态变化为观察急性肝损伤小鼠肝细胞凋亡的动态变化，采用TUNEL技术进行检测。结果发现：与正常对照组相比，在CCl₄处理后2至12h，仅有少量肝脏细胞发生凋亡，在24h有大量区域性肝细胞发生凋亡，72h与正常对照组无明显差异。上述结果提示CCl₄诱导急性肝损伤模型小鼠在CCl₄处理后24h发生大量区域性肝细胞凋亡。1.4内质网应激抑制剂PBA对急性肝损伤小鼠肝细胞凋亡的影响为进一步验证CCl₄诱导急性肝损伤模型小鼠发生肝细胞凋亡是否由内质网应激介导，观察内质网抑制剂PBA对急性肝损伤小鼠肝细胞凋亡的影响，在经腹腔注射给予CCl₄（0.30ml/kg BW）前24h、12h和在CCl₄处理后12h分别经腹腔注射给予150mg/kg PBA进行干预，在CCl₄处理后24h剖杀小鼠，采用TUNEL技术检测肝细胞凋亡。结果发现：与模型组相比，PBA可明显抑制CCl₄诱导的肝细胞凋亡。以上结果提示内质网应激在CCl₄诱导急性肝损伤中发挥了重要作用，可能与其介导的肝细胞凋亡有关。2CCl₄诱导小鼠肝纤维化与ER stress效应2.1CCl₄诱导小鼠肝纤维化模型的建立为建立CCl₄诱导的小鼠肝纤维化模型，每周2次经腹腔注射给予CCl₄0.15ml/kg（按10%溶于橄榄油中），共持续8周。结果发现：与正常对照组比较，CCl₄诱导的小鼠血清丙氨酸氨基转移酶（alanine aminotransferase, ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase, AST）、碱性磷酸酶（alkaline phosphatase，ALP）水平和总胆红素（Total bilirubin，TBIL）、直接胆红素（Direct Bilirubin，DBIL）和总胆汁酸（total biliary acid，TBA）含量以及反应肝纤维化程度的组织羟脯氨酸（Hydroxyproline，Hyp）含量均显著增高；病理组织学结果HE染色提示模型组小鼠肝脏中重度坏死，在坏死组织周围有大量的炎症细胞浸润，胶原染色提示肝组织有大量胶原沉积，并分割肝组织形成假小叶趋势。以上结果提示CCl₄诱导的小鼠肝纤维化模型建立成功。2.2CCl₄诱导肝纤维化小鼠肝脏ER Stress效应为观察CCl₄诱导小鼠肝纤维化模型中，是否存在ER Stress效应，采用Westernblotting技术检测ER stress效应标志性蛋白GRP78蛋白表达以及下游IRE1α、eIF2α磷酸化水平和ATF6核蛋白水平。结果发现：与正常对照组相比，CCl₄诱导肝纤维化模型小鼠肝脏GRP78蛋白表达显著上调， IRE1α、eIF2α磷酸化水平以及ATF6核蛋白水平显著增加。以上结果提示CCl₄诱导的小鼠肝纤维化模型发生ER stress效应。3内质网应激抑制剂PBA对CCl₄诱导的小鼠肝纤维化的影响为进一步探讨ER stress在CCl₄诱导肝纤维化形成过程中的作用，采用内质网应激抑制剂PBA进行干预，观察ER stress效应被抑制后，对CCl₄诱导小鼠肝纤维化的影响，PBA+CCl₄干预组，在每周2次经腹腔注射给予0.15ml/kg CCl₄的同时，每天两次经腹腔注射给予150mg/kg PBA，共持续8周。结果发现：与肝纤维化模型组相比，PBA干预显著降低小鼠肝重和肝指数，并明显降低血清DBIL、TBA水平和肝组织Hyp含量；病理学组织结果，PBA干预明显减轻肝脏组织炎症细胞数量与肝脏坏死程度，改善肝组织结构，减少胶原沉积。α-SMA是肝星状细胞激活的标志，采用免疫组化技术和Western blotting技术分析α-SMA蛋白表达。结果发现：与模型组比较，PBA干预显著降低小鼠肝脏α-SMA的蛋白表达。采用实时定量RT-PCR技术检测肝纤维化关键性细胞因子TGF-β1mRNA水平，结果发现：与模型组相比较，PBA干预显著降低TGF-β1mRNA水平。采用实时定量RT-PCR技术检测肝脏胶原标志物Col1a1和Col1a2mRNA水平以及胶原降解相关蛋白基质金属蛋白酶（matrix metalloproteinases，MMPs）中Mmp2和Mmp9mRNA、金属蛋白酶组织抑制因子（tissue inhibitor ofmetallopeptidases，TIMPs）中Timp1和Timp2mRNA水平。结果发现，与正常对照组相比，肝纤维化模型组小鼠Col1a1、Col1a2、Mmp2、Mmp9、Timp1、Timp2mRNA水平显著增加；与模型组相比，PBA干预显著抑制Colla1、Colla2、Mmp2和Timp1mRNA水平。以上结果提示：内质网应激可能与增加小鼠肝脏胶原纤维表达有关。为进一步验证ER stress在CCl₄诱导肝纤维化中的作用，采用内质网应激抑制剂PBA进行干预，观察对肝纤维化小鼠的ER stress效应的影响，采用Western blotting技术检测GRP78蛋白表达、IRE1α、eIF2α磷酸化水平和ATF6核蛋白水平。结果发现：与模型组相比，PBA干预明显抑制CCl₄诱导的肝纤维化小鼠肝脏GRP78蛋白表达的上调作用，降低IRE1α、eIF2α磷酸化水及核蛋白ATF6水平。以上结果进一步提示PBA可以抑制CCl₄诱导的肝脏内质网应激和未折叠蛋白反应，进一步证明内质网应激在肝纤维化的形成过程中发挥了重要的作用。4ER stress在CCl₄诱导的小鼠肝纤维化中的作用机制4.1ER stress介导的NF-κB信号通路在CCl₄诱导小鼠肝纤维化中的作用为进一步研究ER stress在CCl₄诱导的小鼠肝纤维化中的作用机制，采用Western blotting和实时定量RT-PCR技术检测CCl₄诱导的肝纤维化模型组以及内质网应激抑制剂干预组对NF-κB信号通路的影响。结果发现：与正常对照组相比，CCl₄诱导的肝纤维化模型小鼠肝脏细胞核转录因子NF-κB p65核蛋白水平显著增加；与模型组相比，PBA干预显著降低细胞核内NF-κB p65核蛋白水平。采用实时定量RT-PCR技术检测NF-κB信号通路下游靶基因TNF-α mRNA水平，结果显示：肝纤维化模型组小鼠肝脏TNF-α mRNA水平明显增高；与模型组相比，PBA干预显著降低TNF-α mRNA水平。以上结果提示ER stress在CCl₄诱导肝纤维化小鼠中的作用机制，可能与其介导的NF-κB信号通路及炎症反应有关。4.2ER stress介导的MAPKs信号通路在CCl₄诱导小鼠肝纤维化中的作用为进一步研究内质网应激在CCl₄诱导的小鼠肝纤维化作用中的机制，采用Western blotting技术检测CCl₄诱导的肝纤维化模型组以及内质网应激抑制剂干预组对MAPKs信号通路的影响。结果发现：与正常对照组相比，CCl₄诱导的肝纤维化模型组小鼠ERK和JNK磷酸化水平显著增加，而p38的磷酸化水平明显降低；与模型组相比，PBA干预显著降低肝脏ERK和JNK的磷酸化水平，对肝脏磷酸化的p38的表达无明显影响。以上结果提示ER stress在CCl₄诱导的小鼠肝纤维化形成过程中的作用机制，可能与其介导的ERK和JNK信号通路有关。以上实验结果综合表明，ER stress在CCl₄诱导的急性肝损伤发挥了重要的作用，可能与其介导的肝细胞凋亡有关；ER stress在CCl₄诱导的慢性肝损伤——肝纤维化过程中也起了十分重要的作用，其作用机制可能与其介导的NF-κB信号通路和JNK、ERK信号通路有关。更为重要的是，ER stress效应被抑制后，对肝纤维化具有一定保护作用，提示内质网应激可为开发防治肝纤维化疾病的药物提供新的作用靶点和思路。更多还原

【Abstract】 The liver is not only an important organ in the metabolism for human, but also atarget organ which is most vulnerable to the damage of numerous extraneous factors. Ashort-term expose of high dose xenobiotics induce acute liver injury, and long-termexpose of xenobiotics induced chronic liver injury. Liver fibrosis is a repair response tochronic liver injury. Liver fibrosis is a slow pathological process and a commonpathological change in chronic liver disease，which has become a worldwide healthproblem. The change of texture in the liver is abnormal hyperplasia and deposition ofliver extracellular matrix （ECM）, which may cause severe liver fibrosis or cirrhosis andeven lead to liver failure if not treated in time. Nevertheless, the molecular mechanismsof acute and chronic liver injury remain poorly understood. Many anti-liver injury drugshave been developed in recent years, but the effects have not been affirmed yet. It is ofsignificance to find an ideal anti-fibrosis drug and understand the pathogenesis of liverfibrosis.The endoplasmic reticulum （ER） stress plays an important role in numerous liverdiseases. ER is the organelle responsible for protein folding and maturation andmaintaining Ca2+homeostasis. Accumulation of unfolded/misfolded proteins andalteration of Ca2+homeostasis in the ER lumen triggers ER stress. ER stress is aself-defence mechanism，but strong and long lasting ER stress leads to irreversible cellinjury. Many liver diseases such as viral hepatitis, alcohol liver disease, drug induced hepatitis, nonalcoholic fatty liver diseases are related to ER stress. A recent reportshowed that bile acids caused hepatic ER stress and Unfolded Protein Reaction （UPR）signaling activation in a model of cholestasis-induced hepatic fibrosis. Nevertheless,whether ER stress is involved in the pathogenesis of acute and chronic liver injury,especially for hepatic fibrogenesis, remain poorly understood.Carbon tetrachloride （CCl₄）-induced mouse models of acute and chronic liverinjury were for study of its molecular mechanism. The aim of this study was to observea single dose and long-term of CCl₄-induced hepatic ER stress and UPR signalingactivation, moreover, investigate the effects of Sodium4-phenylbutyrate （PBA） onCCl₄-induced hepatic fibrogenesis and elucidate its molecular mechanism in mice.1. The roles of ER Stress on a single dose of CCl₄-induced liver injury in mice1.1The effects of a single dose of CCl₄-induced liver injury in miceTo investigate the effects of a single dose of CCl₄-induced liver injury in mice,mice were sacrificed at0,2,6,12,24and72hours after a single intraperitonealinjection of CCl₄（0.30ml/kg BW）. Results showed the absolute and relative liverweights were significantly increased from12h to72h after a single intraperitonealinjection of CCl₄. The lever of serum ALT was slightly increased, beginning as early as2h and remained significantly elevated up to24h after a single dose of CCl₄administration. CCl₄-induced hepatic histopathological damage are analyzed withhematoxylin and eosin （HE） staining. Numerous hepatocytes with necrosis wereobserved at6h after a single injection of CCl₄. Liver zonal necrosis was observed at12h and was significant at24h. Numberous inflammatory cells around the necrotic tissuewere observed in liver sections at72h. These results demonstrated a single dose ofCCl₄-induced liver injury in mice.1.2The effects of a single dose of CCl₄-induced ER Stress in mice To investigate the effects of a single dose of CCl₄-induced ER Stress in mice,western blotting and immunohistochemistry were for the determination of ER Stress.Asingle dose of CCl₄administration significantly increased the level of hepatic GRP78,an ER chaperone, used by western blotting and positive hepatocytes of GRP78determined by immunohistochemistry. In addition, a single dose of CCl₄administrationsignificantly increased the level of hepatic peIF2α and pIRE1α were significantlyincreased in CCl₄-treated mice.1.3The effects of a single dose of CCl₄-induced hepatic apoptosis in miceTo investigate the effects of a single dose of CCl₄-induced hepatic apoptosis inmice, TUNEL was for the determination of hepatic apoptosis. TUNEL+cells weresignificantly increased in liver of mice at24h after a single intraperitoneal injection ofCCl₄.1.4ER stress inhibitor alleviates CCl₄-induced hepatic apoptosis in miceTo investigate the effects of ER stress inhibitor, PBA, on a single dose ofCCl₄-induced hepatic apoptosis in mice, mice received three doses of PBA, two （150mg/kg） intraperitoneally injected12h and24h before CCl₄（0.30ml/kg BW）, the thirdinjected12h after CCl₄. All mice were sacrificed at24h after CCl₄. TUNEL was for thedetermination of hepatic apoptosis. PBA significantly alleviated CCl₄-induced hepaticapoptosis in mice at24h after a single intraperitoneal injection of CCl₄.2. The roles of ER stress on CCl₄-induced liver fibrosis and its molecularmechanism2.1CCl₄administration induces liver fibrosis model in miceMice were intraperitoneally injected with CCl₄（0.15ml/kg BW, twice per week） incombination with PBA （150mg/kg, twice per day） for8weeks. Liver weight wassignificantly increased in mice administered with CCl₄. Results showed long-term CCl₄ administration significantly increased the levels of serum ALT, AST, ALP, TBIL andTBA. Hepatic hydroxyproline content was also significantly increased in miceadministered with CCl₄. Further analysis showed that the levels of serum weresignificantly increased in mice administered with CCl₄. CCl₄-induced hepatichistopathological damage are including numerous inflammatory cells around thenecrotic tissue were observed in liver sections from CCl₄-treated mice using H&Estaining. CCl₄-induced hepatic fibrosis was determined using Sirius red staining. Asexpected, an obvious bridging fibrosis was observed in liver of mice administered withCCl₄. These results demonstrated long-term CCl₄administration induce liver fibrosis inmice.2.2ER stress in CCl₄-induced liver fibrosis model in miceTo investigate long-term CCl₄-induced hepatic ER stress, the effects of CCl₄onhepatic ER stress were analyzed using western blotting. Results showed long-term CCl₄administration significantly increased the level of hepatic GRP78, an ER chaperone. Inaddition, long-term CCl₄administration significantly increased the level of ATF6protein in hepatic nuclear extracts. The levels of hepatic peIF2α and pIRE1α were alsosignificantly increased in CCl₄-treated mice. These results suggested long-term CCl₄induced ER stress.3PBA alleviates CCl₄-induced liver fibrosisTo investigate the effects of PBA, which could act as a chemical chaperone thatinhibits ER stress and the UPR signaling activation, on long-term CCl₄-induced liverfibrosis, mice were intraperitoneally injected with CCl₄（0.15ml/kg BW, twice per week）in combination with PBA （150mg/kg, twice per day） for8weeks. Interestingly, PBAsignificantly alleviated CCl₄-induced elevation of liver weight. The analysis of serumbiological parameters showed that PBA significantly alleviated CCl₄-induced elevationof serum DBIL and TBA levels. The effect of PBA on CCl₄-induced hepatic histopathological damage was determined by HE staining.The results showed that thenumber of inflammatory cells was significantly decreased in liver of mice administeredwith PBA plus CCl₄as compared with CCl₄alone. In addition, the grades of necrosiswere slightly lower in liver of mice administered with PBA plus CCl₄as compared withCCl₄alone. CCl₄-induced hepatic fibrosis was determined using Sirius red staining. Asexpected, PBA significantly alleviated CCl₄-induced hepatic fibrosis. Further analysisshowed that the area of hepatic fibrosis was significantly reduced in mice administeredwith PBA plus CCl₄as compared with CCl₄alone. The effects of PBA on hepatichydroxyproline, an indicator of hepatic fibrosis, were then analyzed. Result showedPBA significantly attenuated CCl₄-induced elevation of hepatic hydroxyproline content.These results showed PBA protects mice from CCl₄-induced liver fibrosis.The effects of PBA on CCl₄-induced hepatic α-SMA, a marker associated withHSC activation, were analyzed by immunohistochemistry and western blotting.Immunohistochemistry showed that the percentage of α-SMA-positive area, α-SMAwas mainly distributed in area of liver with bridging fibrosis, was significantlydecreased in liver of mice administered with PBA plus CCl₄as compared with CCl₄alone. Correspondingly, PBA significantly reduced CCl₄-induced expression of hepaticα-SMA protein. The effects of PBA on CCl₄-induced expression of TGF-β1wereanalyzed by real-time RT-PCR. As expected, long-term CCl₄administrationsignificantly increased the levels of hepatic TGF-β1mRNAs. Interestingly, PBAsignificantly attenuated CCl₄-induced upregulation of hepatic TGF-β1mRNAs. Theseresults suggested the protection of PBA on CCl₄-induced liver fibrosis was associatedwith the inhibition of PBA on CCl₄-induced the activation of HSCs.The effects of PBA on CCl₄-induced expression of hepatic Mmp2and Mmp9wereanalyzed by real-time RT-PCR. Long-term CCl₄administration significantlyupregulated the expression of hepatic Mmp2and Mmp9. PBA significantly attenuated CCl₄-induced upregulation of hepatic Mmp2mRNA. The levels of hepatic Timp1andTimp2mRNA were significantly increased in mice administered with CCl₄. PBAsignificantly attenuated CCl₄-induced upregulation of hepatic Timp1mRNA. ActivatedHSCs produce type I collagen molecules. Thus, the effects of PBA on CCl₄-inducedhepatic mRNA accumulation of collagen1α1（Col1a1） and Col1a2were then analyzed.As expected, the levels of hepatic Col1a1and Col1a2mRNAs were obviouslyincreased in mice administered with CCl₄. PBA significantly attenuated CCl₄-inducedelevation of hepatic Col1a1and Col1a2mRNAs.To investigate the effects of PBA on long-term CCl₄-induced hepatic ER stress, thelevel of hepatic GRP78, eIF2α and IRE1α phosphorylation and nuclear ATF6proteinwere analyzed by western blotting. Interestingly, PBA significantly attenuatedCCl₄-induced elevation of hepatic GRP78and nuclear ATF6protein. In addition, PBAsignificantly inhibited CCl₄-induced hepatic eIF2α and IRE1α phosphorylation. Theseresults suggested PBA alleviated ER stress and UPR signaling activation.4The molecular mechanism of ER stress on CCl₄-induced liver fibrosis in mice4.1PBA alleviates CCl₄-induced activation of hepatic NF-κB signaling pathwayThe effects of PBA on CCl₄-induced hepatic NF-κB activation are analyzed bywestern blotting. Interestingly, PBA significantly inhibited CCl₄-induced nucleartranslocation of NF-κB p65. The effects of PBA on CCl₄-induced expression ofinflammatory cytokines were analyzed by real-time RT-PCR. As expected, long-termCCl₄administration significantly increased the levels of hepatic TNF-α mRNAs.Interestingly, PBA significantly attenuated CCl₄-induced upregulation of hepatic TNF-αmRNAs. These results suggested the protection of PBA on CCl₄-induced liver fibrosiswas mediated by the inhibition of PBA on CCl₄-induced hepatic NF-κB activation andinflammation. 4.2PBA alleviates CCl₄-induced activation of hepatic MAPK signaling pathwayThe effects of PBA on CCl₄-induced hepatic MAPK signaling were then analyzedby western blotting. The levels of hepatic pERK and pJNK were significantly increasedin mice administered with CCl₄. PBA significantly attenuated CCl₄-induced hepaticERK and JNK phosphorylation. Unexpectedly, the level of hepatic pp38wassignificantly decreased in mice administered with CCl₄. PBA had no effect on the levelof hepatic pp38. These results suggested the protection of PBA on CCl₄-induced liverfibrosis was associated with the inhibition of PBA on CCl₄-induced hepatic ERK andJNK phosphorylation.In summary, the present study demonstrates that a single dose of CCl₄administration induce liver injury, hepatic ER stress and UPR signaling activation withhepatic apoptosis. Long-term CCl₄administration also induced hepatic ER stress andUPR signaling activation, which may play an important role on CCl₄-induced HSCactivation and subsequent hepatic fibrosis. PBA, an ER chemical chaperone, inhibitsCCl₄-induced hepatic ER stress and UPR signaling activation. In addition, PBAalleviates CCl₄-induced inflammation through inhibiting activation of NF-κB and JNK,ERK signaling. Importantly, PBA effectively protects against CCl₄-induced HSCactivation and hepatic fibrosis. Therefore, ER stress is involved in CCl₄-induced acuteand chronic liver injury, and liver fibrosis partly mediated through activation of NF-κBand MAPK signaling.更多还原