Ethanol-induced Oxidative Stress Research Articles

Leonurine Protects Hepatocytes from Ethanol-Induced Oxidative Stress via JAK2-STAT3 Pathway

Leonurine Protects Hepatocytes from Ethanol-Induced Oxidative Stress via JAK2-STAT3 Pathway

Novel functional food from an invasive species Polygonum cuspidatum: safety evaluation, chemical composition, and hepatoprotective effects

Abstract Accidentally, we found that the shoots of Polygonum cuspidatum (SPC) have been consumed for centuries as a traditional vegetable in the Shennongjia region of China. Local residents believe that SPC has biological effects such as antibacterial, anti-aging, and antioxidant. To provide scientific support for the use of SPC as a functional food, SPC was evaluated in terms of safety, chemical composition, and antioxidant activity both in vivo and in vitro. In the first, SPC exhibited no adverse cytotoxic effects or acute toxicity in mice. Then the chemical composition of SPC was determined by ultrahigh performance liquid chromatography–electrospray ioniza­tion–quadrupole time of flight tandem mass spectrometry (UHPLC-ESI-QTOF-MS/MS). Twenty-two compounds were identified from the SPC extracts, including phenolic, flavonoid, stilbene, and anthraquinone. Finally, an acute ethanol-induced oxidative stress model in mice showed hepatoprotective effects. In brief, our study indicated that SPC is a safe, multifunctional food with antioxidant and hepatoprotective activities. Importantly, the consumption of SPC as a functional food provides a novel strategy for the efficient utilization of the invasive plant.

Suppression of colonic oxidative stress caused by chronic ethanol administration and attenuation of ethanol-induced colitis and gut leakiness by oral administration of sesaminol in mice.

Chronic consumption of excess ethanol is one of the major risk factors for colorectal cancer (CRC), and the pathogenesis of ethanol-related CRC (ER-CRC) involves ethanol-induced oxidative-stress and inflammation in the colon and rectum, as well as gut leakiness. In this study, we hypothesised that oral administration of sesaminol, a sesame lignan, lowers the risk of ER-CRC because we found that it is a strong antioxidant with very low prooxidant activity. This hypothesis was examined using a mouse model, in which 2.0% v/v ethanol was administered ad libitum for 2 weeks with or without oral gavage with sesaminol (2.5 mg per day). Oral sesaminol administration suppressed the ethanol-induced colonic lesions and the ethanol-induced elevation of the colonic levels of oxidative stress markers (8-hydroxy-2'-deoxyguanosine, malondialdehyde, and 4-hydroxyalkenals). It consistently suppressed the chronic ethanol-induced expressions of cytochrome P450-2E1 and inducible nitric oxide synthase and upregulated heme oxygenase-1 expression, probably via the nuclear factor erythroid-derived 2-like 2 pathway in the mouse colon. Oral sesaminol administration also suppressed the chronic ethanol-induced elevation of colonic inflammation marker levels, such as those of tumour necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1, probably via the nuclear factor-kappa B pathway. Moreover, it prevented the chronic ethanol-induced gut leakiness by restoring tight junction proteins, giving rise to lower plasma endotoxin levels compared with those of ethanol-administered mice. All of these results suggest that dietary supplementation of sesaminol may lower the risk of ER-CRC by suppressing each of the above-mentioned steps in ER-CRC pathogenesis.

Methylene Blue Protects Against Acute Ethanol-Induced Oxidative Stress and Organ Damage

Ethanol (EtOH) intake is an important global health problem which affects many organs such as the brain, liver and stomach. The aim of the study was to examine the effect of the redox dye methylene blue (MethyB) on oxidative stress and histologic damage to the liver, gastric mucosa, and brain induced high dose ethanol (EtOH). Male rats were treated with EtOH (2 ml/rat, 96%) via intragastric route (for two consecutive days). MethyB (20 or 40 mg/kg, intraperitoneally) was given immediately after EtOH administration. The control group received saline. Rats were euthanized three hours after the last treatment. Brain and liver levels of malondialdehyde (MDA), reduced glutathione (GSH), and paraoxonase-1 (PON-1) as well as brain 5-lipoxygenase (5-LOX) and butyrylcholinesterase (BChE) were determined. Histopathological assessment of brain, liver and gastric damage was done. Results indicated that compared to saline treated animals, EtOH caused significant increase in MDA, along with decreased GSH and PON-1 activity in brain and liver. Additionally, it significantly increased 5-LOX and decreased brain BChE activity. The EtOH group showed the presence of dead and red neurons, and damage of glial cells. The liver exhibited vacuolar degeneration, apoptotic hepatocytes and foci of necrosis. The gastric mucosa showed areas of tissue damage, mucosal atrophy, and loss of normal architecture of glandular cells. The EtOH induced biochemical and histopathological alterations were alleviated after treatment with MethyB at a dose-dependent manner. These results demonstrate that MethyB is able to protect against from acute effects of EtOH on brain, liver and gastric tissue via an antioxidant action. MethyB might be of value in reducing tissue injury in acute EtOH intoxication.

Isolation, Characterization and Neuroprotective Activity of Folecitin: An In Vivo Study.

Neurodegenerative diseases (NDs) extend the global health burden. Consumption of alcohol as well as maternal exposure to ethanol can damage several neuronal functions and cause cognition and behavioral abnormalities. Ethanol induces oxidative stress that is linked to the development of NDs. Treatment options for NDs are yet scarce, and natural product-based treatments could facilitate ND management since plants possess plenty of bioactive metabolites, including flavonoids, which typically demonstrate antioxidant and anti-inflammatory properties. Hypericum oblongifolium is an important traditional medicinal plant used for hepatitis, gastric ulcer, external wounds, and other gastrointestinal disorders. However, it also possesses multiple bioactive compounds and antioxidant properties, but the evaluation of isolated pure compounds for neuroprotective efficacy has not been done yet. Therefore, in the current study, we aim to isolate and characterize the bioactive flavonoid folecitin and evaluate its neuroprotective activity against ethanol-induced oxidative-stress-mediated neurodegeneration in the hippocampus of postnatal day 7 (PND-7) rat pups. A single dose of ethanol (5 g/kg body weight) was intraperitoneally administered after the birth of rat pups on PND-7. This caused oxidative stress accompanied by the activation of phosphorylated-c-Jun N-terminal kinase (p-JNK), nod-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), and cysteine-aspartic acid protease-1 (caspase-1) proteins to form a complex called the NLRP3-inflammasome, which converts pro-interleukin 1 beta (IL-1B) to activate IL-1B and induce widespread neuroinflammation and neurodegeneration. In contrast, co-administration of folecitin (30 mg/kg body weight) reduced ethanol-induced oxidative stress, inhibited p-JNK, and deactivated the NLRP3-inflammasome complex. Furthermore, folecitin administration reduced neuroinflammatory and neurodegenerative protein markers, including decreased caspase-3, BCL-2-associated X protein (BAX), B cell CLL/lymphoma 2 (BCL-2), and poly (ADP-ribose) polymerase-1 (PARP-1) expression in the immature rat brain. These findings conclude that folecitin is a flavone compound, and it might be a novel, natural and safe agent to curb oxidative stress and its downstream harmful effects, including inflammasome activation, neuroinflammation, and neurodegeneration. Further evaluation in a dose-dependent manner would be worth it in order to find a suitable dose regimen for NDs.

Aversive Learning Deficits and Depressive-Like Behaviors Are Accompanied by an Increase in Oxidative Stress in a Rat Model of Fetal Alcohol Spectrum Disorders: The Protective Effect of Rapamycin.

Fetal alcohol spectrum disorders (FASDs) are one of the most common consequences of ethanol exposure during pregnancy. In adulthood, these disorders can be manifested by learning and memory deficits and depressive-like behavior. Ethanol-induced oxidative stress may be one of the factors that induces FASD development. The mammalian target of the Rapamycin (mTOR) signaling pathway that acts via two distinct multiprotein complexes, mTORC1 and mTORC2, can affect oxidative stress. We investigated whether mTOR-dependent or mTOR-independent mechanisms are engaged in this phenomenon. Thus, Rapamycin—a selective inhibitor of mTORC1, Torin-2—a non-selective mTORC1/mTORC2 inhibitor, and FK-506—a drug that impacts oxidative stress in an mTOR-independent manner were used. Behavioral tests were performed in adult (PND60-65) rats using a passive avoidance (PA) task (aversive learning and memory) and forced swimming test (FST) (depressive-like behaviors). In addition, the biochemical parameters of oxidative stress, such as lipid peroxidation (LPO), as well as apurinic/apyrimidinic (AP)-sites were determined in the hippocampus and prefrontal cortex in adult (PND65) rats. The rat FASD model was induced by intragastric ethanol (5 g/kg/day) administration at postnatal day (PND)4–9 (an equivalent to the third trimester of human pregnancy). All substances (3 mg/kg) were given 30 min before ethanol. Our results show that neonatal ethanol exposure leads to deficits in context-dependent fear learning and depressive-like behavior in adult rats that were associated with increased oxidative stress parameters in the hippocampus and prefrontal cortex. Because these effects were completely reversed by Rapamycin, an mTORC1 inhibitor, this outcome suggests its usefulness as a preventive therapy in disorders connected with prenatal ethanol exposure.

Inducible nitric oxide synthase (iNOS) mediates ethanol-induced redox imbalance and upregulation of inflammatory cytokines in the kidney.

Overexpression of the inducible isoform of the enzyme nitric oxide synthase (iNOS) has been associated to pathological processes in the kidney. Ethanol consumption induces the renal expression of iNOS; however, the contribution of this enzyme to the deleterious effects of ethanol in the kidney remains elusive. We examined whether iNOS plays a role in the renal dysfunction and oxidative stress induced by ethanol consumption. With this purpose, male C57BL/6 wild-type (WT) or iNOS-deficient (iNOS-/-) mice were treated with ethanol (20% v/v) for 10 weeks. Treatment with ethanol increased the expression of Nox4 as well as the concentration of thiobarbituric acid reactive substances and the levels of tumor necrosis factor α in the renal cortex of WT but not iNOS-/- mice. Augmented serum levels of creatinine and increased systolic blood pressure were found in WT and iNOS-/- mice treated with ethanol. WT mice treated with ethanol showed increased production of reactive oxygen species and myeloperoxidase activity, but these responses were attenuated in iNOS-/- mice. We concluded that iNOS played a role in ethanol-induced oxidative stress and pro-inflammatory cytokine production in the kidney. These are mechanisms that may contribute to the renal toxicity induced by ethanol.

Metformin and Probiotics Interplay in Amelioration of Ethanol-Induced Oxidative Stress and Inflammatory Response in an In Vitro and In Vivo Model of Hepatic Injury.

Alcohol-induced liver injury implicates inflammation and oxidative stress as important mediators. Despite rigorous research, there is still no Food and Drug Administration (FDA) approved therapies for any stage of alcoholic liver disease (ALD). Interestingly, metformin (Met) and several probiotic strains possess the potential of inhibiting alcoholic liver injury. Therefore, we investigated the effectiveness of combination therapy using a mixture of eight strains of lactic acid-producing bacteria, commercialized as Visbiome® (V) and Met in preventing the ethanol-induced hepatic injury using in vitro and in vivo models. Human HepG2 cells and male Wistar rats were exposed to ethanol and simultaneously treated with probiotic V or Met alone as well as in combination. Endoplasmic reticulum (ER) stress markers, inflammatory markers, lipid metabolism, reactive oxygen species (ROS) production, and oxidative stress were evaluated, using qRT-PCR, Oil red O staining, fluorimetry, and HPLC. In vitro, probiotic V and Met in combination prevented ethanol-induced cellular injury, ER stress, oxidative stress, and regulated lipid metabolism as well as inflammatory response in HepG2 cells. Probiotic V and Met also promoted macrophage polarization towards the M2 phenotype in ethanol-exposed RAW 264.7 macrophage cells. In vivo, combined administration of probiotic V and Met ameliorated the histopathological changes, inflammatory response, hepatic markers (liver enzymes), and lipid metabolism induced by ethanol. It also improved the antioxidant markers (HO-1 and Nrf-2), as seen by their protein levels in both HepG2 cells as well as liver tissue using ELISA. Hence, probiotic V may act, in addition to the Met, as an effective preventive treatment against ethanol-induced hepatic injury.

Metabolic mechanism of Prunella vulgaris in treatment of ethanol-induced oxidative stress in rats based on metabonomics

Prunella vulgaris(PV) is an edible and traditional medicinal herb which has a wide range application in fighting inflammation and oxidative stress, and protecting liver. Now it has been used to treat various types of liver diseases and has significant clinical efficacy. This study aims to investigate the effects of PV on ethanol-induced oxidative stress injury in rats and its metabolic mechanism. The rats were divided into control group, model group, PV group, and VC group. The liver protection of PV was identified by measuring pharmacological indexes such as antioxidant and anti-inflammatory activity. The metabolic mechanism of long-term ethanol exposure and the metabolic regulation mechanism of PV treatment were studied by LS-MS metabonomics. The pharmacological investigation indicated that ethanol could significantly decrease the contents of SOD, GSH-Px, CAT and other antioxidant enzymes in liver and increase the content of MDA. At the same time, PV could significantly reduce the contents of inflammatory factors(TNF-α, IL-6 and IL-1β) and liver function markers(ALT, AST, ALP) in serum. What's more, long-term ethanol exposure could significantly cause liver injury, while PV could protect liver. Metabolomics based on multiple statistical analyses showed that long-term ethanol exposure could cause significant metabolic disorder, and fatty acids, phospholipids, carnitines and sterols were the main biomarkers. Meanwhile, pathway analysis and enrichment analysis showed that the β oxidation of branched fatty acids was the main influencing pathway. Also, PV could improve metabolic disorder of liver injury induced by ethanol, and amino acids, fatty acids, and phospholi-pids were the main biomarkers in PV treatment. Metabolic pathway analysis showed that PV mainly regulated metabolic disorder of ethanol-induced liver injury through phenylalanine, tyrosine and tryptophan biosynthetic pathways. This study could provide a new perspective on the hepatoprotective effect of natural medicines, such as PV.

Evidence Based of Effects and Protection Mechanism of Lycium Barbarum Polysaccharide on Ethanol-Induced Liver Injury

Excessive ethanol intakes have been reported to be linked to liver diseases such as hepatitis and cancerrelated diseases of ethanol. Excessive ethanol can lead to the accumulation of reactive oxygen species(ROS) and to the depletion of glutathione (GSH) in the liver. Ethanol-induced oxidative stress substantiallyincreases peroxidation of the lipid and decreases GSH levels in human hepatocyte and animal models.Ethanol contributes to sterol regulating element-binding protein activation (SREBP)-1 by regulating maingenes such as Acetyl-CoA carboxylase (ACC) and Stearoyl-CoA desaturase 1(SCD1) to facilitate lipidaccumulation in the liver. The liquid fraction derived from wolfberry is lycium barbarum polysaccharide(LBP). In many animal disease models, it is an effective antioxidant and tissue protective agent. Researchershave shown hepato-protective functions in acute hepatic injury, NAFLDs, drugs-induced liver injury andhepatocellularcarcinoma models. The protection effect of L. barbarum polysaccharides (LBP) in this studyon ethanol-induced injury in human hepatocytes was explored. In addition, the influence of LBP on oxidativestress protein activity was examined.

Synthesis and Biological Evaluation of Benzimidazole Derivatives as Potential Neuroprotective Agents in an Ethanol-Induced Rodent Model.

Alzheimer's disease (AD) is the most devastating and progressive neurodegenerative disease in middle to elder aged people, which can be exacerbated by lifestyle factors. Recent longitudinal studies demonstrated that alcohol consumption exacerbates memory impairments in adults. However, the underlying mechanism of alcohol-induced memory impairment is still elusive. The increased cellular manifestation of reactive oxygen species (ROS) and the production of numerous proinflammatory markers play a critical role in the neurodegeneration and pathogenesis of AD. Therefore, reducing neurodegeneration by decreasing oxidative stress and neuroinflammation may provide a potential therapeutic roadmap for the treatment of AD. In this study, eight new benzimidazole acetamide derivatives (FP1, FP2, FP5-FP10) were synthesized and characterized to investigate its neuroprotective effects in ethanol-induced neurodegeneration in a rat model. Further, three derivatives (FP1, FP7, and FP8) were selected for in vivo molecular analysis based on preliminary in vitro antioxidant screening assay. Molecular docking analysis was performed to assess the affinity of synthesized benzimidazole acetamide derivatives against selected proinflammatory targets (TNF-α, IL-6). Biochemical analysis revealed elevated expression of neuroinflammatory markers (TNF-α, NF-κB, IL-6, NLRP3), increased cellular oxidative stress, and reduced antioxidant enzymes in ethanol-exposed rats brain. Notably, pretreatment with new benzimidazole acetamide derivatives (FP1, FP7, and FP8) significantly modulated the ethanol-induced memory deficits, oxidative stress, and proinflammatory markers (TNF-α, NF-κB, IL-6, NLRP3) in the cortex. The multipurpose nature of acetamide containing benzimidazole nucleus and its versatile affinity toward numerous receptors highlight its multistep targeting potential. These results indicated the neuroprotective potential of benzimidazole acetamide derivatives (FP1, FP7, and FP8) as novel therapeutic candidates in ethanol-induced neurodegeneration which may partially be due to inhibition of the neuroinflammatory-oxidative stress vicious cycle.

The three-spot seahorse-derived peptide PAGPRGPA attenuates ethanol-induced oxidative stress in LO2 cells through MAPKs, the Keap1/Nrf2 signalling pathway and amino acid metabolism.

Alcoholic liver diseases (ALDs) impose a substantial health burden on many countries. Bioactive peptides isolated from people, marine organisms, animals and plants have shown hepatoprotective effects on animal and hepatocyte models. In this study, an LO2 cell model of ethanol-induced liver injury in vitro was constructed. We investigated the hepatoprotective effects of the three-spot seahorse bioactive peptide (SBP) PAGPRGPA (Pro-Ala-Gly-Pro-Arg-Gly-Pro-Ala; 721.39 Da) and characterised the underlying metabolic pathways and biomarkers through a nontargeted metabolomics approach. We found that ethanol-induced oxidative stress impaired the cellular antioxidant system, leading to an imbalance in cellular homeostasis. However, SBP with a certain antioxidant activity inhibited reactive oxygen species (ROS) production, excessive intracellular Ca2+ level and abnormal apoptosis. It also restored the superoxide dismutase (SOD) and glutathione (GSH) levels and attenuated ethanol-induced oxidative damage and inflammation. SBP suppressed the activation of mitogen-activated protein kinase (MAPK) in ethanol-stimulated LO2 cells. It also regulated the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling pathway to protect LO2 cells from oxidative damage by promoting the expression of antioxidant enzymes, such as heme oxygenase-1 (HO-1). Furthermore, the metabolomics approach demonstrated nine different biomarkers and six metabolic pathways. In summary, the hepatoprotective mechanisms of SBP in vitro, which can be attributed to the upregulation of antioxidant substances and amino acid metabolism, attenuate ethanol-induced oxidative stress.

Echinacoside ameliorates alcohol-induced oxidative stress and hepatic steatosis by affecting SREBP1c/FASN pathway via PPARα

Alcoholic liver disease (ALD) is one of the most common health problems for drinkers, especially in men. Echinacoside (ECH), a natural phenylethanoid glycoside welcomed by the market, has been shown to have a variety of biological activities, such as neuroprotective, anti-fatigue, anti-diabetes and so on. Here, the protective effect and the underlying mechanism of ECH on ethanol-induced liver injuries were studied.In vitro, the HepG2 cells were treated with ECH prior to ethanol. In vivo, C57BL/6 J mice were fed a Lieber-DeCarli ethanol liquid diet and gave with or without 100 mg/kg ECH for 10 days. Our experiments showed that ECH significantly enhanced the levels of antioxidants and reduced the level of ROS, thus attenuating ethanol-induced oxidative stress. Besides, ECH attenuated lipid accumulation caused by ethanol, as evidenced by oil-red O staining, histological examination and the quantification of TG and TC. Finally, ECH increased the level of PPAR-α, and reduced the levels of SREBP-1c and FASN. When PPAR-α inhibitor was introduced in the system, the effects of ECH on SREBP-1c and FASN were reversed. Taken together, our study suggest that ECH can protect against ethanol-induced liver injuries via alleviating oxidative stress and hepatic steatosis by affecting SREBP-1c/FASN pathway via PPAR-α.

Protective effects of rare earth lanthanum on acute ethanol-induced oxidative stress in mice via Keap 1/Nrf2/p62 activation

With the widespread application of rare earth elements (REEs) in environment safety, food and medicine, they accumulate in the ecosystem and different human organs where REEs exert certain biological effects. Low dose REEs are proved to perform antioxidant effects, while high concentration can cause oxidative stress. However, scant information about rational doses and underlying mechanism of REEs as oxidants/antioxidants were illustrated. To elucidate these problems, here we performed a study that the ICR mice were received 0.1, 0.2, 1.0, 2.0 and 20.0 mg/kg lanthanum nitrate (La(NO3)3) by gavage for 30 days, and then were given 12 mL/kg ethanol once to undergo acute ethanol-induced oxidative stress. The antioxidant enzymes, antioxidants, peroxides and related proteins in Keap 1/Nrf2/p62 signaling pathway were measured. The results showed that La(NO3)3 inhibited hepatic morphological alternations by histopathological examination. Meanwhile, elevated superoxide dismutase (SOD) and glutathione (GSH), coupled with decreased alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) and protein carbonyl (PC) were observed in serum and liver tissues of mice by enzyme-linked immunosorbent assay test. Furthermore, western blot analysis demonstrated that oxidative stress was alleviated due to enhanced NF-E2-related factor 2 (Nrf2) and phosphorylated p62 expressions as well as lower Kelch-like ECH-associated protein-1 (Keap 1), followed by the activation of heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO-1) and glutamate cysteine ligase, catalytic (GCLC) proteins. Our findings clearly highlighted that La(NO3)3 could restore the redox homeostasis disrupted by ethanol through provoking Keap 1/Nrf2/p62 signaling pathway, and the optimal dosages were 1.0 and 2.0 mg/kg.

Aqueous extract of Polygonatum sibiricum ameliorates ethanol-induced mice liver injury via regulation of the Nrf2/ARE pathway.

This study investigates the hepatoprotective effect of the aqueous extract of Polygonatum sibiricum (AEPS) against ethanol-induced oxidative stress and explores underlying mechanisms. AEPS was administered by gavage to ICR mice for 30days. The experimental mice were fed a 5% (v/v) ethanol on last 10days and followed by a single megadose of ethanol (5g/kg) to induce ethanol-induced liver injury. Pretreatment with AEPS significantly suppressed the ethanol-induced elevation of aminotransferase activities, total bilirubin (TBIL) level, triglyceride level, and alleviated liver histopathological lesions. Meanwhile, AEPS reduced the level of oxidative stress in the liver and significantly suppressed the mRNA levels of NOX1, p67phox, gp91phox, and CYP2E1. Additionally, AEPS significantly increased the mRNA and protein levels of Nrf2 and its downstream antioxidant genes and promoted the nuclear translocation of Nrf2 in mice liver. Therefore, AEPS can effectively reduce ethanol-induced liver injury via regulation of the Nrf2/ARE pathway. PRACTICAL APPLICATIONS: Alcohol abuse and alcoholism have become a serious public health problem worldwide. Since liver is the major organ of alcohol metabolism, the most impactful damage of alcohol occurs in the liver. Polygonatum sibiricum is a traditional Chinese galenical and it also can be used as food ingredients. Most studies have reported that polysaccharide, flavonoids and saponins are the main bioactive compounds in Polygonatum sibiricum which play important roles in anti-oxidation. AEPS is the aqueous extract of Polygonatum sibiricum and AEPS can protect the mice liver against ethanol-induced oxidative damage. Thus it can be potential antioxidants to product hepatoprotective food and the study also provides a theoretical basis for the development and application of food about Polygonatum sibiricum.

Soluble Epoxide Hydrolase Hepatic Deficiency Ameliorates Alcohol-Associated Liver Disease

Background & AimsAlcohol-associated liver disease (ALD) is a significant cause of liver-related morbidity and mortality worldwide and with limited therapies. Soluble epoxide hydrolase (sEH; Ephx2) is a largely cytosolic enzyme that is highly expressed in the liver and is implicated in hepatic function, but its role in ALD is mostly unexplored.MethodsTo decipher the role of hepatic sEH in ALD, we generated mice with liver-specific sEH disruption (Alb-Cre; Ephx2fl/fl). Alb-Cre; Ephx2fl/fl and control (Ephx2fl/fl) mice were subjected to an ethanol challenge using the chronic plus binge model of ALD and hepatic injury, inflammation, and steatosis were evaluated under pair-fed and ethanol-fed states. In addition, we investigated the capacity of pharmacologic inhibition of sEH in the chronic plus binge mouse model.ResultsWe observed an increase of hepatic sEH in mice upon ethanol consumption, suggesting that dysregulated hepatic sEH expression might be involved in ALD. Alb-Cre; Ephx2fl/fl mice presented efficient deletion of hepatic sEH with corresponding attenuation in sEH activity and alteration in the lipid epoxide/diol ratio. Consistently, hepatic sEH deficiency ameliorated ethanol-induced hepatic injury, inflammation, and steatosis. In addition, targeted metabolomics identified lipid mediators that were impacted significantly by hepatic sEH deficiency. Moreover, hepatic sEH deficiency was associated with a significant attenuation of ethanol-induced hepatic endoplasmic reticulum and oxidative stress. Notably, pharmacologic inhibition of sEH recapitulated the effects of hepatic sEH deficiency and abrogated injury, inflammation, and steatosis caused by ethanol feeding.ConclusionsThese findings elucidated a role for sEH in ALD and validated a pharmacologic inhibitor of this enzyme in a preclinical mouse model as a potential therapeutic approach.

Inhibition of alcohol-induced inflammation and oxidative stress by astaxanthin is mediated by its opposite actions in the regulation of sirtuin 1 and histone deacetylase 4 in macrophages

We previously demonstrated that astaxanthin (ASTX), a xanthophyll carotenoid, repressed ethanol-induced inflammation and oxidative stress in macrophages. We explored the role of sirtuin 1 (SIRT1) and histone deacetylase 4 (HDAC4) in the inhibitory effect of ASTX on inflammation and oxidative stress in macrophages exposed to ethanol. Ethanol decreased mRNA and protein of SIRT1 while increasing those of HDAC4, which was attenuated by ASTX in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). Inhibition of SIRT1 expression or activity augmented ethanol-induced Hdac4 expression, but SIRT1 activation elicited the opposite effect. Consistently, Hdac4 knockdown increased Sirt1 expression with decreases in ethanol-induced inflammatory gene expression, but its overexpression resulted in the opposite effects. Furthermore, BMDMs from mice with macrophage specific-deletion of Hdac4 (Hdac4MKO) showed significant decreases in ethanol-induced inflammatory genes and ROS accumulation but an increase in Sirt1 expression. Macrophage specific deletion of Hdac4 or ASTX abolished the changes in genes for mitochondrial biogenesis and glycolysis by ethanol. Ethanol increased mitochondrial respiration, ATP production, and proton leak, but decreased maximal respiration and spare respiratory capacity, all of which were abolished by ASTX in RAW 264.7 macrophages. The ethanol-induced alterations in mitochondrial respiration were abrogated in Hdac4MKO BMDMs. In conclusion, the anti-inflammatory and antioxidant properties of ASTX in ethanol-treated macrophages may be mediated, at least partly, by its opposite effect on SIRT1 and HDAC4 to empower SIRT1 to counteract ethanol-induced activation of HDAC4.

Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.

During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.

Ethanol-Induced Oxidative Stress Modifies Inflammation and Angiogenesis Biomarkers in Retinal Pigment Epithelial Cells (ARPE-19): Role of CYP2E1 and its Inhibition by Antioxidants.

The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.

Hepatic protein-tyrosine phosphatase 1B disruption and pharmacological inhibition attenuate ethanol-induced oxidative stress and ameliorate alcoholic liver disease in mice

Alcoholic liver disease (ALD) is a major health problem and a significant cause of liver-related death. Currently, the mainstay for ALD therapy is alcohol abstinence highlighting the need to develop pharmacotherapeutic approaches. Protein-tyrosine phosphatase 1B (PTP1B) is an established regulator of hepatic functions, but its role in ALD is mostly unexplored. In this study, we used mice with liver-specific PTP1B disruption as well as pharmacological inhibition to investigate the in vivo function of this phosphatase in ALD. We report upregulation of hepatic PTP1B in the chronic plus binge mouse model and, importantly, in liver biopsies of alcoholic hepatitis patients. Also, mice with hepatic PTP1B disruption attenuated ethanol-induced injury, inflammation, and steatosis compared with ethanol-fed control animals. Moreover, PTP1B deficiency was associated with decreased ethanol-induced oxidative stress in vivo and ex vivo. Further, pharmacological modulation of oxidative balance in hepatocytes identified diminished oxidative stress as a contributor to the salutary effects of PTP1B deficiency. Notably, PTP1B pharmacological inhibition elicited beneficial effects and mitigated hepatic injury, inflammation, and steatosis caused by ethanol feeding. In summary, these findings causally link hepatic PTP1B and ALD and define a potential therapeutic target for the management of this disease.