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: 10267509
• 负责人: BYOUNG-JOON SONG
• 金额: $ 90.73万
• 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/10267509
• 关键词: Acetaldehyde; Acetaminophen; Acetylation; Acetylcysteine; Acute; Aftercare; Age; Aging; Albumins; Alcoholic Fatty Liver; Alcohols; Antioxidants; Apoptosis; Award; BCL2 gene; Basic Science; Biochemical; Brain; Brain Injuries; Buckwheat; CASP3 gene; COVID-19; CYP2E1 gene; Carbon Tetrachloride; Cell Death; Cells; Cessation of life; Cholesterol; Chronic; Cisplatin; Collaborations; Colon; Cultured Cells; Desmosomes; Diabetes Mellitus; Diet; Disease; Dose; Electrical Resistance; Ellagic Acid; Endoplasmic Reticulum; Endotoxemia; Endotoxins; Enterocytes; Enzymes; Epithelial; Epithelial Cells; Epithelium; Ethanol; Ethanol Metabolism; Etiology; Event; Excision; Exposure to; Extrahepatic; Fatty Liver; Fatty acid glycerol esters; Fibrosis; Fructose; Functional disorder; Future; Glutathione; Goals; HIV-1; HepG2; Hepatic; Hepatocyte; High Fat Diet; Histology; In Vitro; Indole-3-Carbinol; Inflammatory; Injury; Intestines; Juglans; Knockout Mice; Lead; Leaky Gut; Lipid Peroxides; Lipopolysaccharides; Liver; Liver Fibrosis; Liver diseases; MAPK8 gene; Malnutrition; Measurement; Mediating; Metabolic syndrome; Methods; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Mouse Strains; Mus; Nerve Degeneration; Nicotine; Nitrates; Obesity; Oral; Organ; Organelles; Oxidative Stress; Pathologic; Pathway interactions; Phosphorylation; Physiological; Poison; Pomegranate; Post-Translational Protein Processing; Preventive; Property; Proteins; Rattus; Reactive Oxygen Species; Reperfusion Injury; Reporting; Research Project Grants; Resistance; Risk Factors; Rodent; Role; Serum; Serum Markers; Signal Pathway; Signal Transduction; Stress; Thioacetamide; Tight Junctions; Time; Tissues; Transgenic Organisms; Translational Research; United States National Institutes of Health; Universities; Virus Diseases; Wild Type Mouse; Work; acute liver injury; adduct; adherent junction; alcohol exposure; alcohol prevention; aldehyde dehydrogenases; animal tissue; antioxidant enzyme; base; cell injury; cytokine; dietary supplements; drinking water; endoplasmic reticulum stress; fast food; gastrointestinal epithelium; gut-liver axis; hepatoma cell; in vivo; inhibitor/antagonist; intestinal barrier; intestinal epithelium; liver injury; member; mitochondrial dysfunction; negative affect; nitration; non-alcoholic; non-alcoholic fatty liver disease; organ injury; oxidation; pre-clinical research; prevent; problem drinker; protein biomarkers; protein complex; research and development; research study; sex; tissue injury; virtual

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 accumulation of toxic 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 tissue/organ damage. These PTMs include oxidation, S-nitrosylation, nitration, phosphorylation, acetylation, adduct formation, etc. All these PTMs generally occur shortly after exposure to alcohol and nonalcoholic substances or under pathological conditions. In the past, we showed a causal role of CYP2E1 in stimulating various PTMs and oxidative tissue injury by evaluating the time-dependent events of PTMs and actual cellular damage in the presence or absence of an antioxidant N-acetylcysteine (NAC) or a specific CYP2E1 inhibitor chlormethiazole (CMZ) and using WT versus the age and sex-matched Cyp2e1-null mice. We also observed that these PTMs and functional alterations of covalently-modified proteins are observed in the liver and other extra-hepatic tissues such as gut. Therefore, we have also characterized the nitrated and/or p-JNK-target proteins in the gut 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 in drinking water, or a western-style fast food-high fat diet (FF-HFD) containing high cholesterol. We recently reported the critical role of CYP2E1 in binge alcohol-mediated intestinal barrier dysfunction (leaky gut), endotoxemia, and inflammatory liver injury in rats and WT mice compared to the corresponding Cyp2e1-null mice. We have continuously studied the mechanism of gut epithelial 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 decreased the levels of gut TJ/AJ proteins while it increased the levels of intestinal CYP2E1, iNOS, nitrated proteins, apoptosis-related marker proteins, serum endotoxin and fecal albumin contents, suggesting elevated gut leakiness and endotoxemia. The decreased amounts of gut junctional complex proteins in binge alcohol-exposed rats were confirmed by differential mass-spectral analyses. Based on our result with binge alcohol-mediated gut leakiness and inflammatory liver injury via gut-liver axis, we have also studied the mechanisms of leaky gut, endotoxemia and advanced liver disease (fibrosis) by nonalcoholic substances such as FF-HFD and fructose. In fact, our mechanistic studies with FF-HFD- or fructose-exposed mice and rats showed similar mechanisms of gut leakiness, endotoxemia and liver fibrosis through increased apoptosis of gut enterocytes and nitration of the junctional complex proteins in rats and WT mice. However, the levels of CYP2E1 were not increased in the latter models, although Cyp2e1-null mice were resistant to leaky gut and inflammatory liver injury by the two nonalcoholic substances. These results suggest an indirect, permissive role of CYP2E1 in gut leakiness, endotoxemia and fibrotic liver disease caused by the FF-HFD and fructose in drinking water. We are in the middle of identifying the oxidatively-modified proteins in alcohol- or fructose-exposed rat livers and intestines to further characterize their roles in promoting leaky gut and inflammatory or fatty liver disease. In FY2020, we have also studied the role of CYP2E1 in thioacetamide (TAA)-mediated liver fibrosis. In a sub-chronic liver fibrosis model, we noticed liver fibrosis after rats were treated with TAA twice a week for 4 weeks. In addition, we observed liver fibrosis in rats exposed to fructose in drinking water for 8 weeks. Based on these results, we are trying to purify the oxidatively-modified proteins and determine their identities to further study their roles in liver fibrosis caused by TAA or fructose in drinking water. Furthermore, we have studied the mechanisms of TAA-mediated acute liver injury by focusing on the role of mitochondrial dysfunction in liver injury. Our results showed that TAA increased the oxidative stress, which negatively affected the activities of a few mitochondrial proteins, leading to mitochondrial dysfunction and eventually death of liver cells in WT mice. However, liver injury was virtually not observed in the TAA-exposed Cyp2e1-null mice, demonstrating the direct role of CYP2E1 in TAA-mediated mitochondrial dysfunction and acute liver injury. Based on our basic mechanistic studies, we have conducted translational research by evaluating the beneficial effects of dietary supplements, including indole-3-carbinol (I3C) or pomegranate (POM), against AFLD in rodents and T84 colon cells. Pre-treatment with POM and I3C prevented alcohol-mediated intestinal barrier dysfunction, endotoxemia and fatty liver disease in rats and mice, respectively. The decreased amounts of gut TJ and AJ proteins in alcohol-exposed rodents were significantly prevented by treatment with POM or I3C. The ethanol-mediated decreased levels of intestinal TJ and/or AJ proteins were also observed in T48 colon cells. Treatment with ellagic acid (EA), a major component of POM, restored the loss of TJ/AJ proteins with normalized trans-epithelial electrical resistance in ethanol-exposed T84 cells. We have also tried to study the preventive effect of another naturally occurring compound against TAA-mediated liver fibrosis and determine its preventive mechanisms against hepatic disease. In collaboration with Dr. Xin Wang at Harvard University, we have demonstrated that Tartary buckwheat extract could prevent alcohol-mediated fatty liver through suppression of oxidative stress and mitochondrial cell death pathway. In this report, Tartary buckwheat extract prevented the increased oxidative stress markers and caspases-3 and -9 activities while it elevated hepatic levels of glutathione, Bcl-2, and Bcl-XL as well as autophagic cell death pathway in rats treated with ethanol chronically or acutely. The beneficial effects of Tartary buckwheat extract were also observed in cultured HepG2 and Huh7 hepatoma cells. In FY2020, one lab member received an NIH ODS Scholar Award to study the beneficial effect of physiologically relevant doses of ellagic acid (EA), a major component of POM, on binge alcohol-mediated leaky gut and alcoholic fatty liver disease. Our results showed that pretreatment with EA via oral gavages significantly prevented binge alcohol-mediated leaky gut and alcoholic fatty liver as determined by histology, measurements of serum markers, immunoblots and other biochemical analyses. Although our work progress was delayed by the Covid-19 related lock-down, we are in the middle of studying the underlying mechanisms by which EA pretreatment prevents binge alcohol-mediated gut leakiness and fatty liver. This study is likely to represent another example of our translational research. All these in vitro and in vivo rodent studies indicate that alcohol-associated fatty liver injury can be alleviated with many safe, dietary supplements with antioxidant properties.

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（硝基氧化）应激使ALDH2活性灭活，导致毒性乙醛和脂质过氧化物的积累。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）应激和组织/器官损伤。这些PTM包括氧化，S-硝基化，硝化，磷酸化，乙酰化，加合物形成等。所有这些PTM通常在暴露于酒精和非酒精性物质或病理条件下不久后不久。过去，我们在存在或不存在抗氧化剂N-乙酰半胱氨酸（NAC）或特定的CYP2E抑制剂chlormethiazole（cmmetermethiazole（Cymetermethiazole）（CMMETHIAZOL）的情况下，在存在或不存在抗氧化剂N-乙酰半胱氨酸（NAC）的情况下，在存在或不存在抗氧化剂N-乙酰半胱氨酸（NAC）的情况下，通过评估PTM的时间依赖性事件以及实际细胞损伤的因果作用，以及实际的细胞损伤。 cyp2e1-null小鼠。我们还观察到，在肝脏和其他肝外组织（如肠道）中观察到这些PTM和共价修饰蛋白的功能改变。因此，我们还表征了野生型（WT）小鼠，WT大鼠和HIV-1转基因（TG）大鼠肠道中的硝化和/或P-JNK-target蛋白，其中硝基氧化应激水平升高，在饮用水中，饮用水量较高后，在暴露于饮用水中的硝基氧化应激水平和P-JNK水平较高（饮用水量很高（均为）饮食（FFF），富含饮食水平（FFF），富含液体，快速饮用水平（fc）。胆固醇。 我们最近报道了与相应的CYP2E1-NULL小鼠相比，我们报道了CYP2E1在大鼠和WT小鼠中的炎症酒精介导的肠道屏障功能障碍（渗漏肠），内毒素血症和炎症性肝损伤中的关键作用。我们通过研究了连接式复合蛋白的不同PTM的作用，包括紧密连接（TJ），依从性连接（AJ）和脱粒体在酒精诱导的肠道泄漏和内毒素中，我们一直在研究肠道上皮屏障功能障碍的机制。暴饮暴食大大降低了肠道TJ/AJ蛋白的水平，同时它增加了肠道CYP2E1，iNOS，硝化蛋白，与凋亡相关的标记蛋白，血清内毒素和粪便蛋白含量，提示肠肠泄漏和内毒素的水平。通过差异质谱分析证实了暴露于酒精暴露大鼠的肠道连接性复合蛋白量减少。基于我们通过肠肝轴暴饮暴食介导的肠道泄漏和炎症性肝损伤的结果，我们还研究了通过非酒精性物质（例如FF-HFD和果糖）的肠道肠道，内毒素血症和晚期肝病（纤维化）的机制。实际上，我们对FF-HFD或果糖暴露的小鼠和大鼠的机理研究表明，通过增加肠道肠细胞的凋亡以及大鼠和WT小鼠中肠道肠细胞的凋亡以及硝化作用，表现出相似的肠渗漏，内毒素血症和肝纤维化的机制。然而，尽管CYP2E1-NULL小鼠对两种非酒精性物质的耐肠道和炎症性肝损伤具有抵抗力，但后者模型中CYP2E1的水平并未升高。这些结果表明，CYP2E1在饮用水中由FF-HFD和果糖引起的肠道泄漏，内毒素血症和纤维化肝脏疾病的间接，允许的作用。我们正处于酒精或果糖暴露的大鼠肝脏中氧化改性的蛋白质以及肠道中的氧化蛋白质，以进一步表征它们在促进漏水和炎症或炎症或脂肪肝病中的作用。 在2020财年，我们还研究了CYP2E1在硫乙酰胺（TAA）介导的肝纤维化中的作用。在亚慢性肝纤维化模型中，我们注意到每周两次用TAA治疗大鼠后肝纤维化4周。此外，我们观察到暴露于饮用水中果糖的大鼠肝纤维化8周。基于这些结果，我们试图净化氧化性改性的蛋白质并确定其身份，以进一步研究其在TAA或果糖在饮用水中引起的肝纤维化中的作用。此外，我们通过关注线粒体功能障碍在肝损伤中的作用来研究TAA介导的急性肝损伤的机制。我们的结果表明，TAA增加了氧化应激，从而对几种线粒体蛋白的活性产生负面影响，从而导致线粒体功能障碍，并最终在WT小鼠中肝细胞死亡。然而，在暴露于TAA的CYP2E1-NULL小鼠中实际上未观察到肝损伤，这表明CYP2E1在TAA介导的线粒体功能障碍和急性肝损伤中的直接作用。 基于我们的基本机理研究，我们通过评估包括啮齿动物和T84结肠细胞中AFLD的饮食补充剂（包括吲哚-3C）或石榴（POM）的有益作用来进行转化研究。 POM和I3C的预处理可预防大鼠和小鼠酒精介导的肠屏障功能障碍，内毒素血症和脂肪肝病。通过使用POM或I3C处理，可以显着防止酗酒啮齿动物中的肠TJ和AJ蛋白减少。在T48结肠细胞中，还观察到乙醇介导的肠TJ和/或AJ蛋白的降低。 POM的主要组成部分椭圆酸（EA）处理TJ/AJ蛋白在乙醇暴露的T84细胞中具有标准化的跨上皮电阻的损失。我们还试图研究另一种天然化合物对TAA介导的肝纤维化的预防作用，并确定其针对肝病的预防机制。与哈佛大学的Xin Wang博士合作，我们已经证明，冬青荞麦提取物可以通过抑制氧化应激和线粒体细胞死亡途径来防止酒精介导的脂肪肝脏。在这份报告中，巨大的荞麦提取物阻止了氧化应激标志物和caspases-3和-9活性增加，同时它升高了用乙醇或急性治疗的大鼠中肝硫酸盐，Bcl-2和Bcl-XL以及自噬细胞死亡途径。在培养的HEPG2和HUH7肝癌细胞中也观察到了荞麦提取物的有益作用。 在2020财年，一位实验室成员获得了NIH ODS学者奖，以研究POM的主要组成部分生理相关剂量的椭圆酸（EA）对暴饮暴食介导的渗漏肠和酒精脂肪肝病的有益作用。我们的结果表明，通过组织学，血清标志物，免疫印迹和其他生化分析确定，对EA进行EA的预处理可显着阻止酒精介导的肠道漏水和酒精脂肪肝。尽管我们的工作进展延迟了Covid-19相关的锁定，但我们仍在研究EA预处理防止暴饮暴力介导的肠道泄漏和脂肪肝的基本机制。这项研究可能代表了我们的翻译研究的另一个例子。所有这些在体外和体内啮齿动物的研究表明，与许多安全的具有抗氧化特性的安全饮食补充剂可以缓解酒精相关的脂肪肝损伤。