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.

我的实验室成员一直在研究活化的乙醇诱导细胞色素 P450-2E1 (CYP2E1)（一种促氧化酶）和受抑制的线粒体乙醛脱氢酶 (ALDH2)（一种用于去除有毒乙醛和脂质过氧化物的抗氧化酶）对促进组织损伤的综合作用。 酒精和其他潜在有毒物质。酒精诱导的氧化和硝化（硝基氧化）应激使 ALDH2 活性失活，导致有毒乙醛和脂质过氧化物的积累。此外，CYP2E1介导的硝基氧化应激可以刺激细胞蛋白不同类型的翻译后修饰（PTM），导致线粒体功能障碍、内质网（ER）应激和组织/器官损伤。这些PTM包括氧化、S-亚硝基化、硝化、磷酸化、乙酰化、加合物形成等。所有这些PTM通常在接触酒精和非酒精物质后不久或在病理条件下发生。过去，我们通过评估 PTM 的时间依赖性事件和在存在或不存在抗氧化剂 N-乙酰半胱氨酸 (NAC) 或特定 CYP2E1 抑制剂氯甲噻唑 (CMZ) 的情况下的实际细胞损伤，并使用 WT 与年龄和性别匹配的比较，证明了 CYP2E1 在刺激各种 PTM 和氧化组织损伤中的因果作用 Cyp2e1 缺失小鼠。我们还观察到，这些 PTM 和共价修饰蛋白的功能改变在肝脏和其他肝外组织（如肠道）中观察到。因此，我们还对野生型 (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 靶蛋白进行了表征，这些大鼠在暴露于酗酒、饮用水中的果糖或含有高浓度果糖的西式快餐高脂肪饮食 (FF-HFD) 后，观察到氮氧化应激和 p-JNK 水平升高。 胆固醇。 我们最近报道了与相应的 Cyp2e1 缺失小鼠相比，CYP2E1 在大鼠和 WT 小鼠的酗酒介导的肠屏障功能障碍（肠漏）、内毒素血症和炎症性肝损伤中的关键作用。我们通过研究连接复合蛋白的不同 PTM（包括紧密连接 (TJ)、粘附连接 (AJ) 和桥粒）在酒精诱导的肠道渗漏和内毒素血症中的作用，不断研究肠道上皮屏障功能障碍的机制。暴饮暴食显着降低了肠道TJ/AJ蛋白的水平，同时增加了肠道CYP2E1、iNOS、硝化蛋白、凋亡相关标记蛋白、血清内毒素和粪便白蛋白含量的水平，表明肠道渗漏和内毒素血症升高。差分质谱分析证实了暴饮暴食的大鼠肠道连接复合蛋白数量的减少。基于我们对酗酒介导的肠漏和肠肝轴炎症性肝损伤的研究结果，我们还研究了非酒精物质（如 FF-HFD 和果糖）导致肠漏、内毒素血症和晚期肝病（纤维化）的机制。事实上，我们对暴露于 FF-HFD 或果糖的小鼠和大鼠的机制研究表明，大鼠和 WT 小鼠通过肠道肠细胞凋亡增加和连接复合蛋白硝化而导致肠渗漏、内毒素血症和肝纤维化的机制相似。然而，尽管 Cyp2e1 缺失小鼠对两种非酒精物质造成的肠漏和炎症性肝损伤具有抵抗力，但在后一种模型中 CYP2E1 的水平并未增加。这些结果表明，CYP2E1 在肠道渗漏、内毒素血症和饮用水中的 FF-HFD 和果糖引起的纤维化肝病中具有间接的、许可的作用。我们正在鉴定暴露于酒精或果糖的大鼠肝脏和肠道中的氧化修饰蛋白，以进一步表征它们在促进肠漏和炎症或脂肪肝疾病中的作用。 在2020财年，我们还研究了CYP2E1在硫代乙酰胺（TAA）介导的肝纤维化中的作用。在亚慢性肝纤维化模型中，我们在大鼠每周两次接受 TAA 治疗 4 周后发现肝纤维化。此外，我们观察到暴露于饮用水中果糖8周的大鼠的肝纤维化。基于这些结果，我们正在尝试纯化氧化修饰蛋白并确定其身份，以进一步研究它们在饮用水中 TAA 或果糖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，研究了 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，从而对一些线粒体蛋白的活性产生负面影响，导致 WT 小鼠线粒体功能障碍并最终导致肝细胞死亡。然而，在TAA暴露的Cyp2e1缺失小鼠中几乎没有观察到肝损伤，这证明了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。 基于我们的基本机制研究，我们进行了转化研究，评估了膳食补充剂（包括吲哚-3-甲醇 (I3C) 或石榴 (POM)）对啮齿类动物和 T84 结肠细胞 AFLD 的有益作用。 POM 和 I3C 预处理分别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疾病。 POM 或 I3C 治疗可显着防止暴露于酒精的啮齿动物中肠道 TJ 和 AJ 蛋白数量的减少。在 T48 结肠细胞中也观察到乙醇介导的肠道 TJ 和/或 AJ 蛋白水平降低。在暴露于乙醇的 T84 细胞中，用 POM 的主要成分鞣花酸 (EA) 处理，可以恢复 TJ/AJ 蛋白的损失，并使跨上皮电阻正常化。我们还尝试研究另一种天然化合物对 TAA 介导的肝纤维化的预防作用，并确定其预防肝病的机制。我们与哈佛大学的 Xin Wang 博士合作，证明苦荞提取物可以通过抑制氧化应激和线粒体细胞死亡途径来预防酒精介导的脂肪肝。在本报告中，苦荞提取物可防止长期或急性乙醇治疗的大鼠氧化应激标记物以及 caspase-3 和 -9 活性的增加，同时提高肝脏谷胱甘肽、Bcl-2 和 Bcl-XL 的水平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苦荞提取物的有益作用。 2020 财年，一名实验室成员获得了 NIH ODS 学者奖，以研究生理相关剂量的鞣花酸 (EA)（POM 的主要成分）对酗酒介导的肠漏症和酒精性脂肪肝的有益影响。我们的结果表明，通过组织学、血清标志物测量、免疫印迹和其他生化分析确定，通过口服强饲法进行 EA 预处理可显着预防暴饮暴食引起的肠漏和酒精性脂肪肝。尽管我们的工作进展因 Covid-19 相关的封锁而推迟，但我们正在研究 EA 预处理预防酗酒介导的肠道渗漏和脂肪肝的潜在机制。这项研究可能代表我们转化研究的另一个例子。所有这些体外和体内啮齿动物研究表明，许多具有抗氧化特性的安全膳食补充剂可以减轻酒精相关的脂肪肝损伤。