Alcohol Metabolism, Functional Consequences and Apoptosis Signaling Mechanism

我的实验室成员一直在研究激活乙醇诱导的细胞色素P450-2E1 (CYP2E1)，一种促氧化酶，和抑制线粒体醛脱氢酶（ALDH2），一种去除有毒乙醛和脂质过氧化物的抗氧化酶，对促进酒精和其他潜在有毒物质对组织损伤的联合作用。酒精诱导的氧化和硝化（氮氧化）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介导的氮氧化应激可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PTMs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。在过去，我们通过评估在抗氧化剂n -乙酰半胱氨酸（NAC）或特定CYP2E1抑制剂氯甲基唑（CMZ）存在或不存在的情况下PTMs的时间依赖性事件和实际细胞损伤，并使用WT与年龄和性别匹配的CYP2E1缺失小鼠进行比较，证明了CYP2E1在刺激各种PTMs和氧化组织损伤中的因果作用。我们还观察到，在肝脏和其他肝外组织（如肠道）中也观察到这些ptm和共价修饰蛋白的功能改变。因此，我们还对野生型（WT）小鼠、WT大鼠和HIV-1转基因（Tg）大鼠的肠道中硝化和/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胆固醇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。

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.