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: 9568233
• 负责人: BYOUNG-JOON SONG
• 金额: $ 76.61万
• 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/9568233
• 关键词: Acetaldehyde; Acetaminophen; Acetylation; Acetylcysteine; Adipose tissue; Age; Aging; Albumins; Alcoholic Fatty Liver; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcohols; Amino Acids; Amyloid; Animal Model; Animals; Antioxidants; Apoptosis; Apoptotic; Area; Autopsy; Bax protein; Binding; Blood; Brain; C-terminal; CASP3 gene; CYP2E1 gene; Carbon Tetrachloride; Cell Communication; Cell Death; Cells; Chemicals; Cholesterol; Cleaved cell; Cultured Cells; Cytochrome P450; Death Rate; Desmosomes; Diabetes Mellitus; Diet; Disease; Dose; E-Cadherin; Electron Microscopy; Endotoxemia; Endotoxins; Enterocytes; Enzymes; Epithelial Cells; Ethanol; Ethanol Metabolism; Etiology; Event; Excision; Exposure to; Extrahepatic; Fatty Liver; Fatty acid glycerol esters; Fibrosis; Fish Oils; Fructose; Fruit; Functional disorder; Gender; Gene Proteins; Glutathione; Goals; HIV; HIV-1; Hepatic; Hepatocyte; High Fat Diet; Histologic; Histology; Human; Immunoblot Analysis; In Vitro; Indole-3-Carbinol; Infiltration; Inflammation; Inflammatory; Injury; Insulin Resistance; Intestines; JUN gene; Juglans; Knockout Mice; Knowledge; Lipid Peroxidation; Lipid Peroxides; Lipopolysaccharides; Liver; Liver Fibrosis; Liver diseases; MAPK14 gene; Malnutrition; Mammals; Mediating; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mouse Strains; Mus; N-terminal; NADPH Oxidase; NOS2A gene; Natural Products; Necrosis; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Nitrates; Nitrogen; Obesity; Omega-3 Fatty Acids; Oral; Organ; Oxygen; Pathologic; Patients; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Physiological; Play; Poison; Post-Translational Protein Processing; Protein Isoforms; Protein Kinase; Protein Kinase C; Proteins; Proteomics; Rattus; Reactive Oxygen Species; Reperfusion Injury; Reporting; Research Project Grants; Respiratory Chain; Risk Factors; Rodent; Rodent Model; Role; SIRT1 gene; Senile Plaques; Serum; Signal Pathway; Signal Transduction; Smoking; Stress; Supplementation; Tauopathies; Thapsigargin; Tight Junctions; Time; Tissues; Transgenic Organisms; Translational Research; Virus Diseases; Wild Type Mouse; Xanthine Oxidase; acute liver injury; adduct; adenylate kinase; adherent junction; alcohol exposure; aldehyde dehydrogenases; antioxidant enzyme; astrogliosis; base; claudin-1 protein; density; dietary supplements; drug of abuse; endoplasmic reticulum stress; experience; extracellular vesicles; hyperphosphorylated tau; ileum; in vivo; inflammatory marker; inhibitor/antagonist; interest; liver inflammation; liver injury; liver-specific protein; macrophage; member; mitochondrial dysfunction; neuron apoptosis; neuron loss; nitration; non-alcoholic; non-alcoholic fatty liver; nonalcoholic steatohepatitis; occludin; oxidation; permissiveness; plakoglobin; potential biomarker; prevent; problem drinker; protein biomarkers; protein complex; protein kinase P; research study; restoration; stress activated protein kinase; tau Proteins

Members of my lab have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on promoting tissue injury by alcohol and other potentially toxic substances. Alcohol-induced oxidative and nitrative (nitroxidative) stress inactivated the ALDH2 activity, resulting in elevated amounts of acetaldehyde and lipid peroxides. In addition, CYP2E1-mediated nitroxidative stress can stimulate different types of post-translational modification (PTM) of cellular proteins, contributing to mitochondrial dysfunction, endoplasmic reticulum (ER) stress and organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, adduct formation, etc that usually occur after exposure to alcohol and nonalcoholic substances or under pathological conditions. In the past, we showed an important role of CYP2E1 in stimulating various PTMs in rodent tissues and demonstrated their causal roles in tissue injury by evaluating the time-dependent events of PTMs and actual tissue injury in the presence or absence of an antioxidant N-acetylcysteine (NAC) or a specific CYP2E1 inhibitor chlormethiazole (CMZ). However, PTMs and functional alterations of covalently-modified proteins are not restricted to the liver. Our recent results indicate that similar alterations can take place in extra-hepatic tissues such as gut and brain. Therefore, we have also characterized the nitrated and/or p-JNK-target proteins in gut and brain of wild-type (WT) mice, WT rats and HIV-1 transgenic (Tg) rats where elevated levels of nitroxidative stress and p-JNK are observed after exposure to binge alcohol, fructose, or a high fat diet (HFD) containing high cholesterol. We previously reported the critical role of CYP2E1 in binge alcohol-mediated gut leakiness, endotoxemia, and inflammatory liver injury in WT mice and rats but not in Cyp2e1-null mice. We have continuously studied the mechanism of barrier dysfunction by investigating the role of different PTMs of the junctional complex proteins including tight junction (TJ), adherent junction (AJ), and desmosome in alcohol-induced gut leakiness and endotoxemia. Binge alcohol exposure significantly increased the levels of intestinal CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, serum endotoxin and fecal albumin contents, suggesting elevated apoptosis of enterocytes, gut leakiness and endotoxemia. Differential mass-spectral analyses of the TJ-enriched fractions of gut epithelial cells showed that several TJ, AJ and desmosome proteins were decreased in alcohol-exposed rats compared to controls. Immunoblots revealed that the levels of TJ proteins (claudin-1, occludin and ZO-1), AJ proteins (-catenin and E-cadherin) and desmosome plakoglobin were markedly decreased in binge-alcohol exposed rats, WT mice, and autopsied human ileums but not in Cyp2e1-null mice. We are studying the causal role of nitration or phosphorylation of junctional complex proteins in elevated gut leakiness, leading to endotoxemia and liver injury. We recently reported an important role of CYP2E1 in nonalcoholic steatohepatitis (NASH) and aging-related liver inflammation and fibrosis. We continued studying the role of CYP2E1 in liver fibrosis promoted by HFD containing high cholesterol for 24 weeks. Liver histology showed that only WT fed HFD (WT-HFD) developed NASH and fibrosis with elevated levels of fibrosis marker proteins but not in HFD-fed Cyp2e1-null mice. The nitroxidative stress marker iNOS, but not CYP2E1, was significantly elevated only in HFD-fed WT. Electron microscopy and immunoblot analyses showed significantly higher levels of ER stress in HFD-fed WT that also showed the highest levels of serum endotoxin, TLR-4 levels, and inflammatory markers. These results demonstrate that CYP2E1 plays a permissive role for other proteins/genes to stimulate HFD-mediated liver fibrosis. From 2016, we have started a new project to characterize extracellular vesicles (EVs) in circulating blood secreted from the damaged liver in mice and rats after exposure to binge alcohol (ethanol) or acetaminophen (APAP). The amounts of total and liver-specific proteins in circulating EVs from mice exposed to APAP were significantly increased in a dose- and time-dependent manner. Proteomic analysis of EVs revealed that the amounts of liver-specific proteins were increased in APAP-exposed mice compared to those of controls. Consistently, binge ethanol exposure markedly elevated liver-specific proteins in circulating EVs from WT mice and patients with alcoholic hepatitis, compared to their respective controls. The number of EVs and the amounts of EV CYP2E1 and other P450 isoforms were markedly elevated in both alcoholics and alcohol-exposed rats and WT mice, but not in the corresponding Cyp2e1-null mice. The amounts of EV CYP2E1 depended on increased oxidative and ER stress, since their levels were decreased by co-treatment with NAC or CMZ, but increased by an ER stress inducer thapsigargin. Moreover, cell death rates were elevated when recipient primary hepatocytes were exposed to exogenous EVs from alcohol-exposed rodents and alcoholics, showing that exogenous EVs from alcohol-exposed mammals are functional in cell communication and can promote cell death by activating the apoptosis signaling pathway. These results demonstrate an important role of CYP2E1 in elevating EV CYP2E1 through increased oxidative and ER stress. Elevated EV-CYP2E1 can be used as a potential biomarker for drug- and alcohol-mediated liver injury. Due to our experience in CYP2E1-related nitroxidative stress, PTMs, and apoptosis signaling pathway in the liver, we have extended our study on the mechanisms of aging-dependent neuronal apoptosis and neurodegeneration in HIV-1 Tg rats. Histological and immunohistochemical analyses showed decreased density of neuronal cells with elevated astrogliosis in 5-month old HIV-1 Tg rats compared to the age- and gender-matched WT. Increased levels of nitroxidative stress marker proteins, such as CYP2E1, iNOS, the stress-activated JNK and p38K, nitrated proteins, hyper-phosphorylated tau, and amyloid plaques, were consistently observed in HIV-1 Tg rats. Moreover, activated p-JNK directly binds tau and phosphorylates multiple amino acids, suggesting an important role of p-JNK in tau hyper-phosphorylation and tauopathy, accompanied with elevated levels of many apoptosis-related proteins, including Bax and cleaved (activated) caspase-3. These results collectively indicate that nitroxidative stress accompanied by activated p-JNK, C-terminal C99 amyloid fragment formation and tau hyper-phosphorylation are responsible for increased neuronal cell death and neurodegeneration in 5-month old HIV-Tg rats. Based on our basic mechanistic studies, we conducted translational research by evaluating the beneficial effects of dietary supplements, including walnut and indole-3-carbinol (I3C), against alcoholic fatty liver disease (AFLD) and NASH in animal models. Daily supplementation with physiologically relevant levels of walnut (20% energy-derived) or I3C (40 mg/kg/day orally) significantly prevented HFD (45% energy-derived) or AFLD by activating sirtuin-1 and AMP-kinase with restoration of antioxidant glutathione, while decreasing CYP2E1, lipid peroxidation, and protein nitration. Walnut or I3C also decreased the elevated levels of activated p-JNK, p-p38K and hepatocyte apoptosis. These hepatic improvements by walnut or I3C also coincided with reduction of HFD- or alcohol-induced inflammation of adipose tissues and macrophage infiltration. Finally, we also plan to study the beneficial effects of n-3 fatty acids on liver steatosis and obesity in rodent models.

Members of my lab have been studying the combined effect of activated ethanol-inducible cytochrome P450-2E1 (CYP2E1), a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an antioxidant enzyme for the removal of toxic acetaldehyde and lipid peroxides, on promoting tissue injury by酒精和其他潜在有毒物质。酒精诱导的氧化和硝酸（氮）应激使ALDH2活性灭活，导致乙醛和脂质过氧化物的量升高。此外，CYP2E1介导的硝基应激可以刺激细胞蛋白的不同类型的翻译后修饰（PTM），导致线粒体功能障碍，内质网应激（ER）应激和器官损伤。这些PTM包括通常在暴露于酒精和非酒精物质后或在病理条件下发生的通常发生的氧化，硝化，硝化，磷酸化，加合物形成等。过去，我们通过评估在存在或不存在抗氧化剂N-乙酰乳突（NAC）或不存在的情况下，通过评估PTM的时间依赖性事件和实际组织损伤，表现出CYP2E1在刺激啮齿动物组织中的各种PTM中的重要作用，并证明了它们在组织损伤中的作用。但是，PTMS和共价修饰蛋白的功能改变不限于肝脏。我们最近的结果表明，在肠道和大脑等肝外组织中可能会发生类似的改变。因此，我们还表征了野生型（WT）小鼠，WT大鼠和HIV-1转基因（TG）大鼠肠道和大脑中的硝化和/或P-JNK-TARGET蛋白，其中氮氧化应激水平升高，在膨胀酒精，脂肪量较高后，观察到降低的硝基氧化应激水平和P-JNK水平（较高的饮食）。 我们先前报道了CYP2E1在WT小鼠和大鼠中CYP2E1在炎症饮酒介导的肠道泄漏，内毒素血症和炎症性肝损伤中的关键作用，但在CYP2E1-NULL小鼠中却没有。我们通过研究了连接性复合蛋白的不同PTM的作用，包括紧密连接（TJ），粘附连接（AJ）和脱糖体在酒精诱导的肠肠漏和内毒素中，我们一直在研究屏障功能障碍的机制。暴饮暴食显着提高了肠道CYP2E1，iNOS，硝化蛋白，与凋亡相关的标记蛋白，血清内毒素和粪便白蛋白含量的水平，这表明肠细胞凋亡升高，肠泄漏和内毒素血症。肠道上皮细胞的TJ富集级分的差异质谱分析表明，与对照组相比，酒精暴露大鼠的几种TJ，AJ和脱骨蛋白减少了。免疫印迹表明，TJ蛋白（Claudin-1，occludin和ZO-1），AJ蛋白（-Catenin和e-钙粘蛋白）和脱骨蛋白的水平显着降低，暴露的大鼠，WT小鼠，WT小鼠，WT小鼠，以及自动人类Ileopsied ilepopsive andopsied Muthops Indops Inepopsied Muthops Inepied in cipy2e1-null Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull Inull MICE。我们正在研究连接性复合蛋白在升高的肠道泄漏中的硝化或磷酸化的因果作用，从而导致内毒素血症和肝损伤。 我们最近报道了CYP2E1在非酒精性脂肪性肝炎（NASH）以及与衰老相关的肝脏炎症和纤维化中的重要作用。我们继续研究CYP2E1在含有高胆固醇的HFD促进的肝纤维化中的作用24周。肝组织学表明，只有WT喂养的HFD（WT-HFD）出现了NASH和纤维化，纤维化标记蛋白水平升高，而在HFD喂养的CYP2E1-NULL小鼠中则没有。仅在HFD喂养的WT中，硝基氧化应力标记INOS而非CYP2E1显着升高。电子显微镜和免疫印迹分析显示，在HFD喂养的WT中，ER应激水平明显更高，这也显示出最高水平的血清内毒素，TLR-4水平和炎症标记。这些结果表明，CYP2E1对其他蛋白质/基因刺激HFD介导的肝纤维化起着允许的作用。 从2016年开始，我们开始了一个新项目，以表征细胞外囊泡（EV）在暴露于暴饮暴食（乙醇）或对乙酰氨基酚（APAP）后，在小鼠和大鼠受损的肝脏循环血液中的表征。暴露于APAP的小鼠中循环EV中的总和肝特异性蛋白的量显着增加，剂量和时间依赖性方式。电动汽车的蛋白质组学分析表明，与对照组相比，APAP暴露的小鼠中肝特异性蛋白的量增加。一致地，与各自的对照相比，狂风乙醇暴露在WT小鼠和酒精性肝炎患者的循环EV中显着升高。在酒精和酒精暴露的大鼠和WT小鼠中，EV CYP2E1和其他P450同工型的EV CYP2E1和其他P450同工型的数量显着升高，但在相应的CYP2E1-NULL小鼠中却没有明显升高。 EV CYP2E1的量取决于氧化和ER应激的增加，因为它们的水平通过与NAC或CMZ共同处理而降低，但通过ER应力诱导剂Thapsigargin增加。此外，当受体原发性肝细胞暴露于酒精暴露的啮齿动物和酒精中毒的外源性电动汽车时，细胞死亡率升高，表明来自酒精暴露的哺乳动物的外源性电动汽车在细胞通信中起作用，并可以通过激活凋亡信号通路来促进细胞死亡。这些结果表明，CYP2E1在通过增加的氧化和ER应力来提升EV CYP2E1方面的重要作用。 EV-CYP2E1升高可用作药物和酒精介导的肝损伤的潜在生物标志物。 由于我们在肝脏中与CYP2E1相关的硝基应激，PTM和凋亡信号通路方面的经验，因此我们扩展了HIV-1 TG大鼠衰老依赖性神经元细胞凋亡和神经减退的机制的研究。组织学和免疫组织化学分析表明，与年龄和性别匹配的WT相比，5个月大的HIV-1 TG大鼠的神经元细胞的密度降低，星形胶质细胞的密度升高。在HIV-1 TG大鼠中始终观察到硝基氧化应激标志物蛋白（例如CYP2E1，INOS，INOS，INOS，应激激活的JNK和P38K，硝化蛋白，硝化蛋白，高磷酸化的TAU和淀粉样蛋白酶）的水平。此外，活化的P-JNK直接结合tau并磷酸化多种氨基酸，这表明p-jnk在tau高磷酸化和陶氏病中的重要作用，伴随着许多与凋亡相关的蛋白的升高，包括BAX和裂解（激活的）Caspase-3。这些结果共同表明，氮氧化应激伴随着活化的P-JNK，C末端C99淀粉样蛋白片段形成和Tau高磷酸化，导致5个月大的HIV HIV-TG大鼠的神经元细胞死亡和神经变性增加。 基于我们的基本机械研究，我们通过评估包括饮食补充剂的有益作用，包括核桃和吲哚-3-carbinol（I3C），对动物模型中的酒精脂肪肝疾病（AFLD）进行了转化研究。 Daily supplementation with physiologically relevant levels of walnut (20% energy-derived) or I3C (40 mg/kg/day orally) significantly prevented HFD (45% energy-derived) or AFLD by activating sirtuin-1 and AMP-kinase with restoration of antioxidant glutathione, while decreasing CYP2E1, lipid peroxidation, and protein nitration.核桃或I3C还降低了活化的P-JNK，P-P38K和肝细胞凋亡的升高。核桃或I3C的这些肝脏改善也与减少了HFD或酒精诱导的脂肪组织和巨噬细胞浸润的炎症相吻合。最后，我们还计划研究啮齿动物模型中N-3脂肪酸对肝脏脂肪变性和肥胖症的有益作用。