Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

酒精代谢、功能后果和细胞凋亡信号机制

• 批准号: 8559246
• 负责人: BYOUNG-JOON SONG
• 金额: $ 82.45万
• 依托单位: NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8559246
• 关键词: Acetaldehyde; Acetaminophen; Acetyl-CoA C-Acetyltransferase; Acetylation; Acetylcysteine; Acute; Affinity; Age; Alcoholic Fatty Liver; Alcohols; Alzheimer' s Disease; Amino Acids; Animal Model; Animals; Antibodies; Antioxidants; Apoptosis; Apoptotic; Area; CCL4 gene; Carbon Tetrachloride; Cell Death; Cell physiology; Cells; Chemicals; Chicago; Coenzyme A; Collaborations; Cultured Cells; Cytochrome P-450 CYP2E1; Data; Development; Diabetes Mellitus; Diet; Docosahexaenoic Acids; Dose; Endotoxins; Energy Supply; Enterobacteriaceae; Enzymes; Ethanol; Ethanol Metabolism; Event; Excision; Experimental Models; Exposure to; Fat-Restricted Diet; Fatty acid glycerol esters; Future; Gel; Gender; Goals; Hepatotoxicity; Human; In Vitro; Inflammation; Inflammatory; Injury; Injury to Liver; Ischemia; JUN gene; Knockout Mice; Knowledge; Korea; Lead; Lipid Peroxides; Liver; Liver diseases; MAPK14 gene; Malnutrition; Malondialdehyde; Mediating; Medical center; Metabolic Pathway; Metals; Methods; Mitochondria; Mitochondrial Proteins; Mitogen-Activated Protein Kinases; Modeling; Modification; Molecular; Mouse Protein; Mouse Strains; Mus; N-terminal; NADPH Oxidase; Necrosis; Neurons; Nitrates; Nitrogen; Obesity; Oral; Organ; Oxidative Stress; Oxygen; Peroxonitrite; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Poison; Post-Translational Protein Processing; Protein Kinase; Protein Kinase Inhibitors; Protein Kinase Protein Phosphorylation; Proteins; Rattus; Reactive Oxygen Species; Reperfusion Therapy; Reporting; Research Personnel; Research Project Grants; Respiratory Chain; Role; Signal Pathway; Signal Transduction; Smoking; Spottings; Steatohepatitis; Stress; Testing; Time; Tissues; Translational Research; Tumor Necrosis Factor-alpha; Ubiquitin; Universities; Virus Diseases; Xanthine Oxidase; alcohol exposure; aldehyde dehydrogenases; base; cell type; design; drug of abuse; enzyme activity; feeding; glycosylation; human NOS2A protein; in vitro Model; in vivo; interest; medical schools; mitochondrial dysfunction; mouse model; nitration; nitrosative stress; non-alcoholic fatty liver; nonalcoholic steatohepatitis; oxidation; prevent; problem drinker; protein kinase inhibitor; pyruvate dehydrogenase; research and development; research study; stress activated protein kinase

Ethanol (alcohol)-mediated cell and tissue damage is partly caused by increased oxidative and nitrosative stress. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells/tissues are being produced through direct inhibition of the mitochondrial respiratory chain and induction/activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), NADPH-oxidase, xanthine oxidase, etc. We are particularly interested in studying the combined effects of activated CYP2E1, a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an anti-oxidant defense enzyme responsible for removal of toxic acetaldehyde and lipid peroxides, on increased oxidative stress and their implications in our experimental models. In the past, we developed a sensitive method to identify oxidatively-modified mitochondrial proteins and demonstrated causal relationship between oxidative modifications and their inactivation in mouse models of alcoholic fatty liver (AFLD) and nonalcoholic fatty liver (NAFLD). During fiscal year 2012, we have established sensitive methods to identify other post-translational modifications such as nitration and phosphorylation of various proteins to investigate their roles in mitochondrial dysfunction and acute liver injury. For studying the functional role of protein nitration, we used a mouse model of acetaminophen (APAP)-induced liver injury because of the well-established role of nitration in liver injury. For evaluating the roles of protein phosphorylation in causing cell/tissue injury, we used a model of acute liver injury by carbon tetrachloride (CCL4). In collaboration with Dr. Bong-Hee Lee at Gachon University Medical School in Korea, we also developed a method to identify the glycosylated proteins and studied their roles in promoting NAFLD and neuronal damage in rats. Furthermore, we collaborated with Dr. Ali Keshavarzian at Rush Medical Center in Chicago to investigate the role of CYP2E1 in exacerbating AFLD through increasing gut leakiness. Many investigators reported pathophysiological implications of protein nitration in promoting various types of cell/tissue injury. Protein nitration is a contributing factor in alcohol- or drug-induced liver injury since iNOS-null mice were markedly protected from these types of liver injury. However, the mechanisms of alcohol- or drug-induced liver injury are poorly understood. In fact, it is largely unknown which cellular (including mitochondrial) proteins are nitrated and how the functions of nitrated proteins are altered and thus contribute to alcohol- or APAP-mediated mitochondrial dysfunction and hepatotoxicity. By using Cyp2e1-null mice, we recently showed that CYP2E1 is involved in promoting protein nitration and ubiquitin-dependent degradation of many proteins (Abdelmegeed et al., 2010). Based on the numerous spots of nitrated proteins displayed on 2-D gels, we hypothesized that many more proteins could be nitrated and that nitrated proteins then contribute to liver injury caused by a single dose (350 mg/kg, ip) of APAP, a CYP2E1 substrate. Thus, we aimed to systematically identify nitrated proteins and investigate their functional roles in APAP-induced liver injury. During this study, we showed that many cytosolic and mitochondrial proteins were rapidly nitrated at 1 or 2 h following APAP exposure. We thus antibody-based affinity-purified nitrated proteins from the mouse livers exposed to APAP for 2 h when liver damage was minimal and determined their identities by mass-spectral analysis. Our data revealed that more than 30 cytosolic and 65 mitochondrial proteins involved in anti-oxidant defense, energy supply, amino acid and fat metabolic pathways were nitrated by APAP exposure. The enzyme activities of a few selected nitrated proteins such as ALDH2, ATP synthase, and 3-ketoacyl-CoA thiolase were suppressed in APAP-exposed mice but restored by co-treatment with a peroxynitrite scavenger N-acetylcysteine, which also prevented APAP-induced protein nitration and liver injury. These results established the causal role of protein nitration in APAP-induced liver injury. We previously reported the critical role of the activated JNK in promoting cell death by phosphorylating critical proteins including pro-apoptotic Bax and mitochondrial ALDH2. To better understand the roles of JNK and its target proteins in regulating mitochondrial function and cell/tissue damage, we have initiated a study to identify and characterize JNK-mediated phosphorylation of many mitochondrial proteins. For specific activation of JNK without activating other mitogen-activated protein kinases (MAPKs), we chose a model of liver injury caused by a single dose (50 mg/kg, ip) of carbon tetrachloride (CCL4), another substrate of CYP2E1. We observed that JNK, activated within 30 min, translocated to mitochondria and that many mitochondrial proteins were rapidly phosphorylated between 1 and 8 h after CCL4 exposure where liver injury was minimal. However, these events were not observed in the corresponding Cyp2e1-null mice. To further characterize their functions, we purified phosphorylated mitochondrial proteins from WT mouse livers exposed to CCL4 for 2 h by using metal-affinity columns. Mass-spectral analysis of purified phospho-proteins revealed that more than 100 mitochondrial proteins were phosphorylated by activated JNK. These proteins include pyruvate dehydrogenase, ATP synthase, ALDH2, etc involved in energy supply and cellular defense, respectively. The activities of these phospho-proteins were markedly suppressed in CCL4-exposed mice but significantly restored in CCL4-exposed mice pretreated with a selective JNK inhibitor SU-3327, which blocked the JNK-mediated protein phosphorylation and prevented liver damage. These results provide direct evidence for critical roles of JNK and protein phosphorylation in promoting CCL4-mediated mitochondrial dysfunction and acute liver injury. We also studied the role of CYP2E1 in the development of inflammatory liver disease e.g., alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) induced by binge alcohol (6 g/kg oral gavage for 3 times at 12 h intervals) and a high fat diet (HFD), respectively. Histological signs of inflammatory liver injury were easily observed in WT mice while the age- and gender-matched Cyp2e1-null mice were protected from alcohol-induced ASH and HFD-mediated NASH (60% energy derived from fat compared to a low fat diet with 10% fat-derived energy for 10 weeks in a 2 x 2 design), respectively. Elevated inflammation scores with increased levels of CYP2E1, tumor necrosis factor-alpha, and lipid peroxides (e.g., malondialdehyde+4-hydroxyalkenals) with suppressed ALDH2 activity were observed in binge alcohol-exposed and HFD-fed WT mice. Consequently, protein nitration, oxidation, and glycosylation were also increased in WT mice fed a HFD or exposed to binge alcohol. Our data also showed that CYP2E1 is important promoting gut leakiness with increased levels of endotoxin and translocation of enterobacteria into the liver in binge alcohol-exposed WT mice. In contrast, all these parameters were significantly reduced or absent in the corresponding Cyp2e1-null mice, suggesting an important role of CYP2E1 in promoting gut leakiness and the development of ASH and NASH. Based on the establishment of AFLD/ASH and NAFLD/NASH in our mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid (DHA) against AFLD/ASH and NAFLD/NASH in Ppara-null or Cyp2e1-null mice compared to the WT mice.

乙醇（酒精）介导的细胞和组织损伤部分是由氧化和亚硝化应激增加引起的。酒精暴露细胞/组织中的大多数活性氧和氮种（ROS/RN）是通过直接抑制线粒体呼吸链以及乙醇诱导的细胞色素p450 2e1（CYP2E1）的诱导/激活而产生的我们特别有兴趣研究一种促氧化酶激活的CYP2E1的综合作用，并抑制了线粒体醛脱氢酶（ALDH2），一种抗氧化剂防御酶，这是一种抗氧化剂防御酶，该酶负责去除有毒乙醛和氧化应激的氧化氧化剂和氧化应激的氧化及其实验，从而消除有毒的乙醛过多氧化剂和氧化应激。 过去，我们开发了一种灵敏的方法来鉴定氧化性修饰的线粒体蛋白，并在酒精脂肪肝（AFLD）和非酒精性脂肪肝肝（NAFLD）的小鼠模型中证明了氧化修饰之间的因果关系及其失活之间的因果关系。在2012财年期间，我们建立了灵敏的方法来识别其他翻译后修饰，例如各种蛋白质的硝化和磷酸化，以研究其在线粒体功能障碍和急性肝损伤中的作用。为了研究蛋白质硝化的功能作用，我们使用了乙酰氨基酚（APAP）诱导的肝损伤的小鼠模型，因为硝化作用在肝损伤中的作用良好。为了评估蛋白质磷酸化在引起细胞/组织损伤中的作用，我们使用了四氯化碳（CCL4）的急性肝损伤模型。与韩国Gachon大学医学院的Bong-Hee Lee博士合作，我们还开发了一种识别糖基化蛋白质的方法，并研究了它们在促进大鼠NAFLD和神经元损害方面的作用。此外，我们与芝加哥Rush Medical Center的Ali Keshavarzian博士合作，调查CYP2E1在加剧AFLD中的作用，通过增加肠道泄漏。 许多研究者报道了蛋白质硝化在促进各种类型的细胞/组织损伤方面的病理生理意义。蛋白质硝化是对酒精或药物诱导的肝损伤的促成因素，因为iNOS-NULL小鼠免受了这些类型的肝损伤的保护。然而，对酒精或药物诱导的肝损伤的机制知之甚少。实际上，在很大程度上未知哪种细胞（包括线粒体）蛋白是硝化的，以及硝酸蛋白的功能如何改变，从而有助于酒精或APAP介导的线粒体功能障碍和肝毒性。通过使用CYP2E1-NULL小鼠，我们最近表明CYP2E1参与了许多蛋白质的蛋白硝化和泛素依赖性降解（Abdelmegeed等，2010）。基于在2-D凝胶上显示的众多硝酸蛋白质，我们假设可以硝化更多蛋白质，然后硝化蛋白会导致由单个剂量（350 mg/kg，ip）aPap的单剂量（350 mg/kg，ip）引起的肝损伤。因此，我们旨在系统地鉴定硝化蛋白并研究其在APAP诱导的肝损伤中的功能。在这项研究中，我们表明在APAP暴露后1或2小时，在1或2 h迅速硝化蛋白。因此，当肝脏损伤最小并通过质谱分析确定其身份时，我们从暴露于APAP的小鼠肝脏中基于抗体的亲和纯化硝化蛋白。我们的数据表明，通过APAP暴露，可以硝化30多个胞质和65个线粒体蛋白参与抗氧化剂防御，能源供应，氨基酸和脂肪代谢途径。在APAP暴露的小鼠中抑制了一些一些选定的硝化蛋白的酶活性，例如ALDH2，ATP合酶和3-酮酰基-COA硫醇酶，但通过与过氧合硝酸盐清除剂N-乙酰甲基甲烷共同治疗，还可以恢复，从而预防APAP诱导的蛋白质nitriver和liver niriver niriver和liver。这些结果确定了蛋白硝化在APAP诱导的肝损伤中的因果作用。 我们先前曾报道过激活的JNK通过磷酸化临界蛋白在内的促进细胞死亡的关键作用，包括促凋亡Bax和线粒体ALDH2。为了更好地了解JNK及其靶蛋白在调节线粒体功能和细胞/组织损伤中的作用，我们开始了一项研究，以识别和表征JNK介导的许多线粒体蛋白的磷酸化。为了在不激活其他有丝分裂原激活的蛋白激酶（MAPKS）的情况下进行特异性激活，我们选择了由单剂量（50 mg/kg，IP）碳四氯化碳（CCL4），另一个CYP2E1底物引起的肝损伤模型。我们观察到JNK在30分钟内激活，转移到线粒体上，并且在CCL4暴露后1至8 h之间，许多线粒体蛋白在肝损伤最小的情况下迅速磷酸化。但是，在相应的CYP2E1-NULL小鼠中未观察到这些事件。为了进一步表征其功能，我们使用金属亲和力柱从暴露于CCL4的WT小鼠肝脏中纯化了磷酸化的线粒体蛋白2小时。纯化的磷酸蛋白的质谱分析表明，通过活化的JNK磷酸化了100多个线粒体蛋白。这些蛋白质分别包括丙酮酸脱氢酶，ATP合酶，ALDH2等，分别参与能源供应和细胞防御。这些磷酸蛋白质的活性在CCL4暴露的小鼠中得到了明显抑制，但在用选择性JNK抑制剂SU-3327预处理的CCL4暴露小鼠中显着恢复，该小鼠阻断了JNK介导的蛋白质磷酸化并防止肝损伤。这些结果为JNK和蛋白质磷酸化在促进CCL4介导的线粒体功能障碍和急性肝损伤中的关键作用提供了直接证据。 我们还研究了CYP2E1在炎症性肝病的发展中的作用，例如酒精性脂肪性肝炎（ASH）和非酒精性脂肪性肝炎（NASH），分别由暴饮暴食（6 g/kg的口服凝视3次，在12 h间隔）和高脂肪饮食（HFD）分别为3次。在WT小鼠中很容易观察到炎症性肝损伤的组织学迹象，而年龄和性别匹配的CYP2E1-NULL小鼠则被保护免受酒精诱导的灰分和HFD介导的NASH的保护（与在2 x 2设计中分别具有10％脂肪源能量的低脂肪饮食相比，从脂肪饮食中获得了60％的能量（与低脂饮食相比，脂肪饮食中的10％脂肪饮食）。在暴饮暴食和HFD喂养的WT小鼠中，观察到炎症得分升高，肿瘤1，肿瘤坏死因子-Alpha和脂质过氧化物（例如，丙二醛+4-羟基烷烃）具有抑制ALDH2活性。因此，在喂养HFD的WT小鼠或暴露于暴饮暴食的WT小鼠中，蛋白质硝化，氧化和糖基化也增加了。我们的数据还表明，CYP2E1是重要的，促进肠道泄漏，内毒素水平升高，肠杆菌易位在暴露于酒精暴露的WT小鼠中进入肝脏。 相反，在相应的CYP2E1-NULL小鼠中，所有这些参数均显着降低或不存在，这表明CYP2E1在促进肠道泄漏和灰烬和NASH的发展中起着重要作用。 基于小鼠菌株中AFLD/ASH和NAFLD/NASH的建立，我们计划通过评估包括Docosahexaenoic（DHA）在内的各种抗氧化剂的有益作用来进行转化研究，与WT小鼠相比，PPARA-NULL或CYP2E-NULL或CYP2E1-NULL小鼠对AFLD/Ash和NAFLD/NASH的有益作用。