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: 8148171
• 负责人: BYOUNG-JOON SONG
• 金额: $ 76.76万
• 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/8148171
• 关键词: Apoptosis; Ethanol Metabolism; Signal Transduction

Oxidative stress is one of the major contributing factors in ethanol (alcohol)-mediated cell and tissue damage. 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, and xanthine oxidase. 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 enzyme, on increased oxidative stress and its physiological implications in our experimental models. Despite the well-established causal roles of ROS/RNS in alcohol-induced oxidative injury, the target proteins, that are oxidatively-modified by ROS/RNS, and their functional alterations are poorly understood. To solve these problems, we recently developed a sensitive method of using biotin-N-maleimide (biotin-NM) as a specific probe to identify oxidized and/or S-nitrosylated proteins. We continued our efforts to identify and characterize oxidatively-modified proteins in animal models of alcoholic and nonalcoholic fatty liver diseases without inflammation or with inflammation (AFLD and NAFLD, respectively) to understand the underlying mechanisms of mitochondrial dysfunction, apoptosis and tissue injury. By using our own method, we characterized oxidatively-modified cytosolic proteins in rat livers exposed to MDMA (3,4-methylenedioxymethamphetamine, ecstasy), which is often co-abused in alcoholic individuals. Our results showed that many cytosolic proteins including anti-oxidant enzymes such as superoxide dismutase (SOD1) and peroxiredoxin were oxidized and inactivated after MDMA exposure. Consistent with these results, we observed increased oxidative stress with elevated levels of lipid peroxides, stress-activated protein kinases, and phosphorylated (inactivated) Bcl-2 or Bcl-XL, leading to apoptosis. A manuscript about these results is now under revision in Proteomics. We also investigated the role of CYP2E1 in protein nitration and ubiquitin-dependent degradation during liver toxicity caused by a CYP2E1 substrate acetaminophen (APAP). Markedly increased centrilobular liver necrosis and 3-nitrotyrosine (3-NT) protein-adducts were only observed in APAP-exposed wild-type (WT) mice in a dose- and time-dependent manner but not in Cyp2e1-null mice, confirming a key role for CYP2E1 in causing APAP toxicity. Immunoblot analysis further revealed that immunoprecipitated nitrated proteins were ubiquitinated in APAP-exposed WT mice, supporting the fact that nitrated proteins are more susceptible (than the native proteins) to ubiquitin-dependent degradation, resulting in shorter half-lives. For instance, cytosolic SOD1 was nitrated and ubiquitinated before it was degraded in APAP-exposed WT mice. These results were not observed in Cyp2e1-null mice, suggesting an important role of CYP2E1 in 3-NT formation, protein degradation, and liver damage. Our results also indicate that decreased levels of many proteins in the WT mice (compared with Cyp2e1-null mice) likely contribute to APAP-related toxicity. These results were published in Biochemical Pharmacology. In addition, we studied a mechanism of inhibition of mitochondrial ALDH2 in a model of carbon tetrachloride (CCL4)-induced liver injury. CCL4 administration caused marked liver necrosis, while it elevated oxidative stress and activated c-Jun-N-terminal protein kinase (JNK). However, hepatic ALDH2 activity was potently suppressed in a time-dependent manner after CCL4 injection. Little or no change in the ALDH2 protein level suggested that ALDH2 could be inhibited through covalent modifications such as JNK-mediated phosphorylation. To demonstrate phosphorylation, we compared the isoelectric points (pI) of ALDH2 in CCL4-exposed rats and untreated controls. Immunoblot analysis revealed that immunoreactive ALDH2 bands in CCL4-exposed rats were shifted to acidic pI ranges on 2-D gels. Incubation with alkaline phosphatase significantly restored the suppressed ALDH2 activity with concurrent alkaline pI shifts of ALDH2 spots. Both JNK and activated JNK were translocated to mitochondria following CCL4 exposure. In addition, incubation with catalytically active JNK led to significant inhibition of ALDH2 activity with acidic pI shifts on 2-D gels. Furthermore, immunoprecipitation followed by immunoblot analysis with anti-phospho-Ser-Pro antibody revealed detection of one band in the CCL4-exposed rats but not in controls, suggesting phosphorylation of Ser residue(s) of ALDH2. These results collectively indicate a novel mechanism that CCL4 exposure activates JNK, which translocates to mitochondria and phosphorylates ALDH2, contributing to inhibition of ALDH2 activity accompanied with decreased cellular defense capacity and increased lipid peroxidation. These results were published in Free Radic Biol Med. Although many animal models exist for studying the mechanisms of AFLD and NAFLD, the roles of peroxisomal proliferator-activated receptor alpha (PPARalpha) and CYP2E1 in these areas have not been fully characterized. PPARalpha is a transcription factor involved in controlling the expression of many genes in the fatty acid transport, inflammatory reactions, peroxisomal and mitochondrial fat metabolism. Moreover, the expressed level of PPARalpha in humans is much lower (less than one tenth) than that in rodents, suggesting Ppara-null mice can be used as a good model for studying the mechanisms of AFLD and NAFLD in human conditions. We hypothesized that Ppara-null mice are very sensitive to organ damage compared to WT mice while Cyp2e1-null mice are very resistant to tissue damage caused by ethanol and other potentially toxic agents or diets. Based on this hypothesis, we studied the mechanism of NAFLD in WT mice and Ppara-null mice fed a high fat diet (HFD). Age- and gender-matched WT and Ppara-null mice were fed a liquid HFD (70% energy derived from fat) or a standard liquid diet (STD, 35% energy derived from fat) ad libitum for 3 weeks in a 2 x 2 design. Ppara-null mice fed a HFD exhibited the highest levels of hepatocyte ballooning, steatosis, inflammation, and ultimately NASH activity score among the 4 groups. Elevated levels of CYP2E1, tumor necrosis factor-alpha, and lipid peroxides (e.g., malondialdehyde) were observed in HFD-fed Ppara-null mice. Consequently, protein nitration and oxidation were also increased in Ppara-null mice fed a HFD compared to their WT counterparts. Increased oxidative stress and inflammation were associated with activation of JNK and p38 kinase, contributing to increased hepatocyte apoptosis in Ppara-null mice fed a HFD compared with WT mice. These results demonstrate that inhibition of PPAR functions may increase susceptibility to nonalcoholic steatohepatitis (NASH) in the presence of a HFD. On the other hand, Cyp2e1-null mice fed a HFD were relatively resistant to the development of NAFLD or NASH. A manuscript for these results is now under revision in Journal of Nutrition. Based on our own data recently published and unpublished, we believe that Ppara-null or Cyp2e1-null mice are very useful for studying the mechanisms of AFLD and NAFLD treated with an ethanol-liquid diet or a combination of alcohol and another potentially toxic agent such as nicotine and lipopolysaccharide, simulating human conditions. Upon establishment of AFLD and NAFLD in these mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid (DHA) or S-adenosylmethionine against AFLD and NAFLD in Ppara-null or Cyp2e1-null mice compared to WT mice.

氧化应激是乙醇介导的细胞和组织损伤的主要影响因素之一。暴露于酒精的细胞/组织中的大多数活性氧和氮 (ROS/RNS) 是通过直接抑制线粒体呼吸链以及诱导/激活乙醇诱导型细胞色素 P450 2E1 (CYP2E1)、诱导型一氧化氮合酶 (iNOS)、NADPH 氧化酶和黄嘌呤氧化酶产生的。 我们特别感兴趣的是研究激活的 CYP2E1（一种促氧化酶）和受抑制的线粒体醛脱氢酶（ALDH2）（一种抗氧化酶）对氧化应激增加的综合影响及其在我们的实验模型中的生理意义。 尽管 ROS/RNS 在酒精诱导的氧化损伤中的因果作用已被明确，但被 ROS/RNS 氧化修饰的靶蛋白及其功能改变却知之甚少。为了解决这些问题，我们最近开发了一种使用生物素-N-马来酰亚胺（生物素-NM）作为特异性探针来识别氧化和/或S-亚硝基化蛋白质的灵敏方法。 我们继续努力在无炎症或有炎症的酒精性和非酒精性脂肪性肝病（分别为 AFLD 和 NAFLD）动物模型中鉴定和表征氧化修饰蛋白，以了解线粒体功能障碍、细胞凋亡和组织损伤的潜在机制。 通过使用我们自己的方法，我们对暴露于MDMA（3,4-亚甲二氧基甲基苯丙胺，摇头丸）的大鼠肝脏中氧化修饰的胞质蛋白进行了表征，这种物质经常在酗酒者中共同滥用。我们的结果表明，许多胞浆蛋白，包括抗氧化酶，如超氧化物歧化酶 (SOD1) 和过氧化还原酶，在接触 MDMA 后被氧化并失活。与这些结果一致，我们观察到氧化应激随着脂质过氧化物、应激激活蛋白激酶和磷酸化（失活）Bcl-2 或 Bcl-XL 水平的升高而增加，从而导致细胞凋亡。关于这些结果的手稿目前正在《蛋白质组学》杂志上进行修订。 我们还研究了 CYP2E1 底物对乙酰氨基酚 (APAP) 引起的肝毒性过程中 CYP2E1 在蛋白质硝化和泛素依赖性降解中的作用。仅在暴露于 APAP 的野生型 (WT) 小鼠中以剂量和时间依赖性方式观察到小叶中心肝坏死和 3-硝基酪氨酸 (3-NT) 蛋白加合物显着增加，但在 Cyp2e1 缺失小鼠中未观察到，这证实了 CYP2E1 在引起 APAP 毒性中的关键作用。免疫印迹分析进一步表明，免疫沉淀的硝化蛋白在暴露于 APAP 的 WT 小鼠中被泛素化，这支持了硝化蛋白（比天然蛋白）更容易受到泛素依赖性降解的影响，从而导致半衰期更短的事实。例如，在暴露于 APAP 的 WT 小鼠中，胞质 SOD1 在被降解之前先被硝化和泛素化。这些结果在 Cyp2e1 缺失小鼠中没有观察到，表明 CYP2E1 在 3-NT 形成、蛋白质降解和肝损伤中发挥重要作用。我们的结果还表明，WT 小鼠（与 Cyp2e1 缺失小鼠相比）中许多蛋白质水平的降低可能导致 APAP 相关毒性。这些结果发表在《生化药理学》上。 此外，我们在四氯化碳 (CCL4) 诱导的肝损伤模型中研究了线粒体 ALDH2 的抑制机制。 CCL4 给药导致明显的肝坏死，同时升高氧化应激并激活 c-Jun-N 末端蛋白激酶 (JNK)。然而，注射 CCL4 后，肝脏 ALDH2 活性以时间依赖性方式受到有效抑制。 ALDH2 蛋白水平很少或没有变化表明 ALDH2 可以通过共价修饰（例如 JNK 介导的磷酸化）受到抑制。为了证明磷酸化，我们比较了 CCL4 暴露大鼠和未治疗对照大鼠中 ALDH2 的等电点 (pI)。 免疫印迹分析显示，CCL4 暴露大鼠中的免疫反应性 ALDH2 条带在 2-D 凝胶上转移至酸性 pI 范围。与碱性磷酸酶一起孵育显着恢复了受抑制的 ALDH2 活性，同时 ALDH2 斑点发生碱性 pI 变化。 CCL4 暴露后，JNK 和活化的 JNK 均易位至线粒体。此外，与催化活性 JNK 一起孵育可显着抑制 ALDH2 活性，并在 2-D 凝胶上出现酸性 pI 变化。此外，免疫沉淀随后用抗磷酸-Ser-Pro 抗体进行免疫印迹分析显示，在 CCL4 暴露的大鼠中检测到一条条带，但在对照组中未检测到，这表明 ALDH2 的 Ser 残基发生磷酸化。这些结果共同表明了一种新机制，即 CCL4 暴露激活 JNK，JNK 易位至线粒体并磷酸化 ALDH2，从而抑制 ALDH2 活性，并伴随细胞防御能力下降和脂质过氧化增加。这些结果发表在《Free Radic Biol Med》上。 尽管存在许多用于研究 AFLD 和 NAFLD 机制的动物模型，但过氧化物酶体增殖物激活受体 α (PPARα) 和 CYP2E1 在这些领域的作用尚未得到充分表征。 PPARα 是一种转录因子，参与控制脂肪酸转运、炎症反应、过氧化物酶体和线粒体脂肪代谢中许多基因的表达。 此外，人类中 PPARα 的表达水平比啮齿类动物低得多（不到十分之一），这表明 Ppara 缺失小鼠可以作为研究人类 AFLD 和 NAFLD 机制的良好模型。我们假设与 WT 小鼠相比，Ppara-null 小鼠对器官损伤非常敏感，而 Cyp2e1-null 小鼠对乙醇和其他潜在有毒物质或饮食引起的组织损伤非常有抵抗力。 基于这一假设，我们研究了野生型小鼠和高脂饮食（HFD）喂养的 Ppara-null 小鼠的 NAFLD 机制。年龄和性别匹配的 WT 和 Ppara-null 小鼠以 2 x 2 设计随意喂食液体 HFD（70% 能量源自脂肪）或标准流质饮食（STD，35% 能量源自脂肪）3 周。 饲喂 HFD 的 Ppara-null 小鼠在 4 组中表现出最高水平的肝细胞膨胀、脂肪变性、炎症和最终 NASH 活性评分。 在 HFD 喂养的 Ppara-null 小鼠中观察到 CYP2E1、肿瘤坏死因子-α 和脂质过氧化物（例如丙二醛）水平升高。因此，与 WT 小鼠相比，饲喂 HFD 的 Ppara-null 小鼠的蛋白质硝化和氧化也有所增加。氧化应激和炎症的增加与 JNK 和 p38 激酶的激活有关，与 WT 小鼠相比，饲喂 HFD 的 Ppara-null 小鼠的肝细胞凋亡增加。这些结果表明，在存在 HFD 的情况下，抑制 PPAR 功能可能会增加对非酒精性脂肪性肝炎 (NASH) 的易感性。另一方面，饲喂 HFD 的 Cyp2e1 缺失小鼠对 NAFLD 或 NASH 的发展具有相对抵抗力。这些结果的手稿目前正在《营养杂志》上进行修订。 根据我们最近发表和未发表的数据，我们相信 Ppara-null 或 Cyp2e1-null 小鼠对于研究乙醇液体饮食或酒精与另一种潜在有毒物质（如尼古丁和脂多糖）的组合治疗 AFLD 和 NAFLD 的机制非常有用，模拟人类状况。在这些小鼠品系中建立 AFLD 和 NAFLD 后，我们计划通过评估各种抗氧化剂（包括二十二碳六烯酸（DHA）或 S-腺苷甲硫氨酸）与 WT 小鼠相比，在 Ppara-null 或 Cyp2e1-null 小鼠中对 AFLD 和 NAFLD 的有益作用来进行转化研究。