Attenuation of alcohol-induced hepatocyte damage by ginsenoside Rg1 evaluated using atomic force microscopy.

Alcoholic liver disease is an important cause of death worldwide. Hepatocyte apoptosis is commonly observed in alcoholic liver disease. In this study, we investigated the effect of ginsenoside Rg1 (G-Rg1), an organic component of ginseng, on the alcohol-induced morphological and biophysical properties of hepatocytes. Human hepatocytes (HL-7702) were treated in vitro with alcohol and G-Rg1. The cell morphology was observed using scanning electron microscopy. Cell height, roughness, adhesion, and elastic modulus were detected using atomic force microscopy. We found that alcohol significantly induced hepatocyte apoptosis, whereas G-Rg1 attenuated the alcohol-induced hepatocyte damage. Scanning electron microscopy revealed that alcohol-induced significant morphological changes in hepatocytes, including decreased cell contraction, roundness, and pseudopods, whereas G-Rg1 inhibited these negative changes. Atomic force microscopy revealed that alcohol increased the cell height and decreased the adhesion and elastic modulus of hepatocytes. Following treatment with G-Rg1, the cell height, adhesion, and elastic modulus of alcohol-injured hepatocytes were all similar to those of normal cells. Thus, G-Rg1 can attenuate the alcohol-induced damage to hepatocytes by modulating the morphology and biomechanics of the cells. RESEARCH HIGHLIGHTS: In this study, the morphological characteristics of hepatocytes were observed using SEM. The changes in hepatocyte three-dimensional images and biomechanical action caused by alcohol and G-Rg1 were examined at the nanoscale using AFM under near-physiological conditions. Alcohol-induced hepatocytes showed abnormal morphology and biophysical properties. G-Rg1 attenuated the alcohol-induced damage to hepatocytes by modulating the morphology and biomechanics of the cells.

Sodium Butyrate Protects Against Ethanol-Induced Toxicity in SH-SY5Y Cell Line.

Alcohol use disorder (AUD), brought about by excessive alcohol use, is associated with damages to several organs including the brain. Chronic excessive use of alcohol can compromise intestinal integrity, leading to changes in gut microbiota (GM) composition known as dysbiosis. Dysbiosis, by disruption of the gut-brain axis (GBA), further exacerbates the deleterious effects of alcohol. One of the fermentation by-products of GM is butyrate (BUT), a short-chain fatty acid (SCFA) that plays an important role in maintaining homeostasis of the GBA. Alcohol metabolism results in formation of acetaldehyde, a highly reactive compound that reacts with dopamine in the brain to form toxic adducts such as salsolinol. Recent studies indicate potential neuro-protective effects of BUT against various toxicants including salsolinol. Here, we sought to investigate whether BUT can also protect against alcohol toxicity. Pretreatment of neuroblastoma-derived SH-SY5Y cells with 500 mM ethanol (ETOH) for 24 h resulted in approximately 40% reduction in cell viability, which was totally blocked by 10 µM of either BUT or AR 420,626 (AR), a selective fatty acid 3 receptor (FA3R) agonist. The neuro-protective effects of both BUT and AR were significantly (80%) attenuated by beta-hydroxy butyrate (BHB), a selective FA3R antagonist. Interestingly, combination of BUT and AR resulted in synergistic protection against ETOH, which was totally blocked by BHB. These findings suggest potential utility of butyrate and/or FA3R agonists against ETOH-induced toxicity.

Lactobacillus plantarum ZS62 Alleviates Alcohol-Induced Gastric Injury in Mice via an Anti-Oxidative Mechanism.

AIM: Gastric mucosal injury is a typical characteristic of gastric diseases. The prevalence of gastric mucosal injury caused by alcohol has been on the rise, which has been considered a serious problem. The purpose of this study is to explore the protective effect on gastric injury of Lactobacillus plantarum ZS62 (LP-ZS62) isolated from naturally fermented yak yoghurt. METHODS: We established a gastric injury model through alcohol and evaluated the protective effect of LP-ZS62 on gastric injury in mice. The injury to the gastric mucosa, histopathological sections, related biochemical indicators, and related genes were examined to evaluate the protective effect of LP-ZS62. RESULTS: LP-ZS62 effectively alleviated alcohol-induced gastric injury according to visual observations of gastric tissue and pathological tissue sections. The experimental results revealed that LP-ZS62 decreased malondialdehyde (MDA) level, and elevated superoxide dismutase (SOD) and glutathione (GSH) levels in gastric tissues. Additionally, LP-ZS62 increased glutathione peroxidase (GSH-Px), prostaglandin E2 (PGE2), and somatostatin (SS) levels. LP-ZS62 also decreased inflammatory cytokines interleukin (IL)-1ß, tumor necrosis factor-α (TNF-α) and IL-6 levels, and increased the anti-inflammatory cytokine IL-10 level. The quantitative polymerase chain reaction results showed that LP-ZS62 upregulated mRNA expression of nuclear factor E2-related factor 2 (Nrf2), copper/zinc superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT), gamma-glutamylcysteine synthetase (GSH1), and glutathione peroxidase (GSH-Px). CONCLUSION: This study confirmed that LP-ZS62 alleviated alcohol-induced gastric injury by regulating antioxidant capacity. Therefore, LP-ZS62 could be developed as a probiotic product to treat alcoholic gastric injury.

Optimization of Extraction and Purification of Polysaccharides from Veronicastrum axillare, and Evaluation of Their Biological Activities.

Veronicastrum axillare polysaccharides (VAP) were isolated by cellulase-assisted digestion. The optimum conditions (2 % cellulase, 47 °C for 2.5 h, then, 95 °C for 2.5 h, pH 4.1, solid/liquid ratio 1 : 7.6) were identified by a combination of single factor optimization and response surface DOE (design of experiment) methods, and achieved a yield of 4.7 %. Treatment with 1 % TCA for 10 min, then, 2 % DEAE-cellulose removed protein and colored impurities. Purified VAP retained most of the radical-scavenging activities and GES-1 cell protection capability in vitro, indicating VAP were the key active components of V. axillare. Some molecular features were identified by FT-IR and NMR analyses. The molecular weight was estimated from DOSY NMR experiments to be around 21 kDa. There were 6.3 % uronic acid residues in the VAP. The constituent sugars after TFA hydrolysis were identified by HPLC to include glucose, arabinose, rhamnose, galactose, and xylose in a molar ratio of 405 : 259 : 82 : 42 : 1.

Impact of Caffeine on Ethanol-Induced Stimulation and Sensitization: Changes in ERK and DARPP-32 Phosphorylation in Nucleus Accumbens.

BACKGROUND: Caffeine is frequently consumed with ethanol to reduce the impairing effects induced by ethanol, including psychomotor slowing or incoordination. Both drugs modulate dopamine (DA)-related markers in accumbens (Acb), and Acb DA is involved in voluntary locomotion and locomotor sensitization. The present study determined whether caffeine can affect locomotion induced by acute and repeated ethanol administration in adult male CD-1 mice. METHODS: Acute administration of caffeine (7.5 to 30.0 mg/kg) was evaluated for its effects on acute ethanol-induced (1.5 to 3.5 g/kg) changes in open-field horizontal locomotion, supported rearing, and rearing not supported by the wall. DA receptor-dependent phosphorylation markers were assessed: extracellular signal-regulated kinase (pERK), and dopamine-and cAMP-regulated phosphoprotein Mr32kDa phosphorylated at threonine 75 site (pDARPP-32-Thr75) in Acb core and shell. Acutely administered caffeine was also evaluated in ethanol-sensitized (1.5 g/kg) mice. RESULTS: Acute ethanol decreased both types of rearing. Caffeine increased supported rearing but did not block ethanol -induced decreases in rearing. Both substances increased horizontal locomotion in a biphasic manner, and caffeine potentiated ethanol-induced locomotion. Although ethanol administered repeatedly induced sensitization of locomotion and unsupported rearing, acute administration of caffeine to ethanol-sensitized mice in an ethanol-free state resulted in blunted stimulant effects compared with those seen in ethanol-naïve mice. Ethanol increased pERK immunoreactivity in both subregions of the Acb, but coadministration with caffeine blunted this increase. There were no effects on pDARPP-32(Thr75) immunoreactivity. CONCLUSIONS: The present results demonstrated that, after the first administration, caffeine potentiated the stimulating actions of ethanol, but did not counteract its suppressant or ataxic effects. Moreover, our results show that caffeine has less activating effects in ethanol-sensitized animals.

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.

Celastrol Dilates and Counteracts Ethanol-Induced Constriction of Cerebral Arteries.

The increasing recognition of the role played by cerebral artery dysfunction in brain disorders has fueled the search for new cerebrovascular dilators. Celastrol, a natural triterpene undergoing clinical trials for treating obesity, exerts neuroprotection, which was linked to its antioxidant/anti-inflammatory activities. We previously showed that celastrol fit pharmacophore criteria for activating calcium- and voltage-gated potassium channels of large conductance (BK channels) made of subunits cloned from cerebrovascular smooth muscle (SM). These recombinant BK channels expressed in a heterologous system were activated by celastrol. Activation of native SM BK channels is well known to evoke cerebral artery dilation. Current data demonstrate that celastrol (1-100 µM) dilates de-endothelialized, ex vivo pressurized middle cerebral arteries (MCAs) from rats, with EC50 = 45 µM and maximal effective concentration (Emax)= 100 µM and with MCA diameter reaching a 10% increase over vehicle-containing, time-matched values (P < 0.05). A similar vasodilatory efficacy is achieved when celastrol is probed on MCA segments with intact endothelium. Selective BK blocking with 1 µM paxilline blunts celastrol vasodilation. Similar blunting is achieved with 0.8 mM 4-aminopirydine, which blocks voltage-gated K+ channels other than BK. Using an in vivo rat cranial window, we further demonstrate that intracarotid injections of 45 µM celastrol into pial arteries branching from MCA mimics celastrol ex vivo action. MCA constriction by ethanol concentrations reached in blood during moderate-heavy alcohol drinking (50 mM), which involves SM BK inhibition, is both prevented and reverted by celastrol. We conclude that celastrol could be an effective cerebrovascular dilator and antagonist of alcohol-induced cerebrovascular constriction, with its efficacy being uncompromised by conditions that disrupt endothelial and/or BK function. SIGNIFICANCE STATEMENT: Our study demonstrates for the first time that celastrol significantly dilates rat cerebral arteries both ex vivo and in vivo and both prevents and reverses ethanol-induced cerebral artery constriction. Celastrol actions are endothelium-independent but mediated through voltage-gated (KV) and calcium- and voltage-gated potassium channel of large conductance (BK) K+ channels. This makes celastrol an appealing new agent to evoke cerebrovascular dilation under conditions in which endothelial and/or BK channel function are impaired.

Protective effects of quercetin, polydatin, and folic acid and their mixtures in a zebrafish (Danio rerio) fetal alcohol spectrum disorder model.

Protective effects of quercetin (QUE), polydatin (POL), and folic acid (FA) and their mixtures were tested using zebrafish to model fetal alcohol spectrum disorder in this study. Zebrafish embryos were exposed to 150 mM ethanol for 6 or 22 h and co-treated with QUE, POL, FA, and their mixtures (37.5-100.0 µM). Epiboly progression, teratogenic effects, and behavior were evaluated. Ethanol exposure reduced epiboly, and FA and QUE protected against these ethanol-induced defects. POL did not reduce epiboly defects. The mixture QUE + FA showed a possible antagonistic effect. The observed teratogenic effects were similar in all ethanol exposed groups. QUE, FA and QUE + POL reduced the percentage of affected animals, but treatments did not eliminate teratogenic effects. Behavioral measurements were divided into small (between 4 and 8 mm/s) and high swimming activity (>8 mm/s). All experimental groups displayed a reduction in small swimming activity as compared to control and ethanol groups when exposed to bright light. Additionally, larvae exposed to ethanol were more inhibited than control, not showing a habituation period (after 60 min of experiment) in high swimming activity. Chemical treatments like QUE and POL reduced behavioral defects induced by ethanol exposure. In conclusion, this study presents new evidence that QUE, POL, FA and their mixtures partially protected epiboly, teratogenic, and behavioral defects induced by ethanol exposure. QUE, FA and QUE + POL were more effective in reducing these defects than the other studied compounds and mixtures.

A possible neuroprotective property of ethanol and/or NeuroAiD on the modulation of cognitive function.

Cognitive impairments and poor performance on tasks needing behavioral flexibility are observable in chronic alcohol exposure. NeuroAid decreases cognitive deficits and improves functional outcomes by restoring neuronal circuits. The aim of the current study was to assess the hypothesis that ethanol exposure would induce neurobehavioral defects which may be reversed by the neuroprotective property of NeuroAid. Adult male Wistar rats were treated with saline, ethanol (0.2 g/kg), NeuroAid (0.8 g/kg) and ethanol (0.2 g/kg) + NeuroAid (0.8 g/kg). Then, behavioral tests were performed using the Y-maze apparatus, hot-plate and tail-flick apparatuses, locomotion apparatus as well as the loss of righting reflex (LORR) and hanging protocols (performance in a wire hanging test). Our results indicated that intraperitoneal (i.p.) administration of ethanol alone and administration of ethanol along with NeuroAid for one week reversed ethanol-induced spatial memory deficits in rats (P < 0.01). Interestingly, treatment with ethanol (0.2 g/kg) for one week induced nociception (P < 0.01). Moreover, one week administration of ethanol (0.2 g/kg) along with NeuroAid (0.8 g/kg) increased latency to LORR (P < 0.001) while four weeks administration of ethanol (0.2 g/kg) along with NeuroAid (0.8 g/kg) decreased sleep time (P < 0.01). In addition, a single administration of all drugs did not alter locomotor activity (P > 0.05) and hanging (P > 0.05). Improvement of behavioral tasks after one-week i.p. administration of ethanol and/or NeuroAid in comparison with a single administration of ethanol and/or NeuroAid may be due to the neuroprotective property of ethanol and/or NeuroAiD.

Extraction, purification, and determination of the gastroprotective activity of glucomannan from Bletilla striata.

In this study, a water-soluble polysaccharide (BSP) was extracted and purified from pseudobulb of Bletilla striata. The preliminary structure and gastroprotective activity of BSP were analyzed. Results indicate that BSP is a glucomannan with a molar ratio of 7.45:2.55 (Man:Glc), and its molecular weight is approximately 1.7 × 105 Da. BSP displayed outstanding protective action against ethanol-induced GES-1 cell injury in vitro, as well as, excellent gastroprotective activity in vivo. Especially, a high-dose of BSP (100 mg/kg) could reduce the ulcer index of the gastric mucosa and increase the percentage of ulcer inhibition, which possibly caused by enhancing the antioxidant capacity and inhibiting the apoptotic pathway in gastric tissue. Interestingly, BSP exhibited a comparative gastroprotective activity to that of positive control (omeprazole). In summary, our results indicated that BSP could be considered as a potential supplement for the prevention of gastric injury.

Role of betaine in inhibiting the induction of RNA Pol III gene transcription and cell growth caused by alcohol.

Alcohol has been classified as carcinogenic to humans by the International Agency for Research on Cancer (IARC). Studies have demonstrated that alcohol intake increases the risk of breast cancer, and alcohol also stimulates breast cancer cell growth. Deregulation of Pol III genes is tightly associated with tumour development. Transcription factor II-B (TFIIB)-related factor 1 (Brf1) is a transcription factor that specifically regulates Pol III gene transcription. Our in vivo and in vitro studies have indicated that alcohol enhances the transcription of Pol III genes to cause an alteration of cellular phenotypes, which is closely related with human breast cancer. Betaine is a vegetable alkaloid and has antitumor functions. Most reports about betaine show that the consumption level of betaine is inversely associated with a risk of breast cancer. Although different mechanisms of betaine against tumour have been investigated, nothing has been reported on the effect of betaine on the deregulation of Brf1 and Pol III genes. In this study, we determine the role of betaine in breast cancer cell growth and colony formation and explore its mechanism. Our results indicate that alcohol increases the rates of growth and colony formation of breast cancer cells, whereas betaine is able to significantly inhibit the effects of alcohol on these cell phenotypes. Betaine decreases the induction of Brf1 expression and Pol III gene transcription caused by ethanol to reduce the rates of cell growth and colony formation. Together, these studies provide novel insights into the role of betaine in alcohol-caused breast cancer cell growth and deregulation of Brf1 and Pol III genes. These results suggest that betaine consumption is able to prevent alcohol-associated human cancer development.

Recombinant Penicillium oxalicum 16 ß-Glucosidase 1 Displays Comprehensive Inhibitory Resistance to Several Lignocellulose Pretreatment Products, Ethanol, and Salt.

ß-Glucosidase (BGL) is a rate-limiting enzyme of lignocellulose hydrolysis for second-generation bioethanol production, but its inhibition by lignocellulose pretreatment products, ethanol, and salt is apparent. Here, the recombinant Penicillium oxalicum 16 BGL 1 (rPO16BGL1) from Pichia pastoris GS115 kept complete activity at 0.2-1.4 mg/mL furan derivatives and phenolic compounds, 50 mg/mL sodium chloride (potassium chloride), or 100 mg/mL ethanol at 40 °C. rPO16BGL1 retained above 50% residual activity at 30 mg/mL organic acid sodium, and 60% residual activity at 40 °C with 300 mg/mL ethanol. Sodium chloride and potassium chloride had a complicated effect on rPO16BGL1, which resulted in activation or inhibition. The inhibition kinetics of the enzyme reaction demonstrated that organic acids and organic acid sodium were non-competitive inhibitors and that ethanol was a competitive inhibitor at < 1.5 mg/mL salicin. Moreover, substrate inhibition of the enzyme was found at > 2 mg/mL salicin, and the Km/KI and Km/KSI average values revealed that the inhibitory strength was ranked as salicin-organic acids > organic acids > salicin-organic acid sodium salt > organic acid sodium salt > salicin > salicin-KCl > salicin-NaCl > salicin-ethanol > ethanol.

Contrasting Roles of Cannabidiol as an Insecticide and Rescuing Agent for Ethanol-induced Death in the Tobacco Hornworm Manduca sexta.

Cannabis sativa, also known as marijuana or hemp, produces a non-psychoactive compound cannabidiol (CBD). To investigate the defensive role of CBD, a feeding preference assay was performed with tobacco hornworm Manduca sexta. The larvae clearly show feeding preference towards the Cannabis tissue containing low CBD over high CBD. While the larva avoided the high CBD diet, we investigated detrimental effects of CBD in the insects' diet. Contrasted to the performance on low CBD-infused artificial diet (AD), larvae reared on the high CBD diet suffer significantly reduced growth and increased mortality. Through testing different carriers, we found that the increase of EtOH in the diet is negatively correlated with insect development and behaviors. Notably, CBD treatment significantly improved ethanol-intoxicated larval survival rate by 40% and also improved diet searching activity, resulting in increased diet consumption. Electrophysiology results revealed that the CBD-treated ganglia had delayed but much larger response with electric stimuli in comparison to the larvae reared on AD only and EtOH-added diet. Our results show CBDs' defensive role against pest insects, which suggests its possible use as an insecticide. We also provide evidence that CBD alleviates alcohol-induced stress; consequently, improving the performance and viability of M. sexta larvae.

Ethanol Exposure Impairs AMPK Signaling and Phagocytosis in Human Alveolar Macrophages: Role of Ethanol Metabolism.

BACKGROUND: Chronic alcohol consumption impairs alveolar macrophage's (AM) function and increases risk for developing lung infection and pneumonia. However, the mechanism and metabolic basis of alcohol-induced AM dysfunction leading to lung infection are not well defined, but may include altered ethanol (EtOH) and reactive oxygen species metabolism and cellular energetics. Therefore, oxidative stress, endoplasmic reticulum (ER) stress, the formation of fatty acid ethyl esters [FAEEs, nonoxidative metabolites of EtOH], AMP-activated protein kinase (AMPK) signaling, and phagocytic function were examined in freshly isolated AM incubated with EtOH. METHODS: AMs separated from bronchoalveolar lavage fluid samples obtained from normal volunteers were incubated with EtOH for 24 hours. AMPK signaling and ER stress were assessed using Western blotting, FAEEs by GC-MS, oxidative stress by immunofluorescence using antibodies to 4-hydroxynonenal, and phagocytosis by latex beads. Oxidative stress was also measured in EtOH-treated AMs with/without AMPK activator [5-aminoimidazole-4-carboxamide ribonucleotide (AICAR)] or inhibitor (Compound C), and in AMs incubated with FAEEs. mRNA expression for interleukins (IL-6 and IL-8), monocyte chemoattractant protein (MCP)-1, and transforming growth factor (TGF)-ß was measured in AM treated with EtOH or FAEEs using RT-PCR. RESULTS: EtOH exposure to AM increased oxidative stress, ER stress, and synthesis of FAEEs, decreased phosphorylated AMPK, and impaired phagocytosis. Attenuation or exacerbation of EtOH-induced oxidative stress by AICAR or Compound C, respectively, suggests a link between AMPK signaling, EtOH metabolism, and related oxidative stress. The formation of FAEEs may contribute to EtOH-induced oxidative stress as FAEEs also produced concentration-dependent oxidative stress. An increased mRNA expression of IL-6, IL-8, and MCP-1 by FAEEs is key finding to suggest a metabolic basis of EtOH-induced inflammatory response. CONCLUSIONS: EtOH-induced impaired phagocytosis, oxidative stress, ER stress, and dysregulated AMPK signaling are plausibly associated with the formation of FAEEs and may participate in the pathogenesis of nonspecific pulmonary inflammation.

Altered amygdala DNA methylation mechanisms after adolescent alcohol exposure contribute to adult anxiety and alcohol drinking.

Binge drinking during adolescence increases the risk for neuropsychiatric disorders including alcoholism in adulthood. DNA methylation in post-mitotic neurons is an important epigenetic modification that plays a crucial role in neurodevelopment. We examined the effects of intermittent ethanol exposure during adolescence on adult behavior and whether DNA methylation changes provide a plausible explanation for the lasting effects of this developmental insult. One hour after last adolescent intermittent ethanol (AIE), growth arrest and DNA damage inducible protein 45 (Gadd45a, Gadd45b, and Gadd45g) mRNA expression was increased and DNA methyltransferase (DNMT) activity and Dnmt3b expression was decreased in the amygdala as compared to adolescent intermittent saline (AIS) rats. However, AIE rats 24 h after last exposure displayed increased DNMT activity but normalized Gadd45 and Dnmt3b mRNA expression compared to AIS rats. In adulthood, rats exposed to AIE show increased Dnmt3b mRNA expression and DNMT activity, along with decreased Gadd45g mRNA expression in the amygdala. DNA methylation of neuropeptide Y (Npy) and brain-derived neurotrophic factor (Bdnf) exon IV is increased in the AIE adult amygdala compared to AIS adult rats. Treatment with the DNMT inhibitor 5-azacytidine (5-azaC) at adulthood normalizes the AIE-induced DNA hypermethylation of Npy and Bdnf exon IV with concomitant reversal of AIE-induced anxiety-like and alcohol-drinking behaviors. These results suggest that binge-like ethanol exposure during adolescence leads to dysregulation in DNA methylation mechanisms in the amygdala which may contribute to behavioral phenotypes of anxiety and alcohol use in adulthood.

The citrus flavonoids hesperidin and naringin alleviate alcohol-induced behavioural alterations and developmental defects in zebrafish larvae.

This study aimed to investigate the protective effects of two citrus flavonoids, hesperidin and naringin, on alcohol-induced thigmotaxis behavioural and developmental defects in zebrafish. The results of behavioural assays indicated that acute exposure to 1% alcohol could induce thigmotaxis behaviour in zebrafish larvae at 3 and 5 days post-fertilization (dpf), whereas hesperidin and naringin could inhibit the thigmotaxis behaviour in zebrafish larvae. Moreover, developmental administration of hesperidin and naringin reduced the levels of morphological defects and apoptosis induced by developmental alcohol exposure. In conclusion, this study suggests that the citrus flavonoids hesperidin and naringin could reduce the effects of alcohol damage on embryonic development and neurobehaviour in zebrafish larvae, which would provide useful knowledge for assessing the underlying mechanism of citrus flavonoids and their protective effects.

In Utero Exposure to Alcohol Impairs Reactivity of Cerebral Arterioles and Increases Susceptibility of the Brain to Damage Following Ischemia/Reperfusion in Adulthood.

BACKGROUND: Maternal consumption of alcohol produces abnormalities in the developing fetus and can contribute to an increased incidence of many cardiovascular-related diseases. The first goal of this study was to determine whether in utero exposure to alcohol influences reactivity of cerebral arterioles in adult (12 to 15 weeks old) rats. The second goal of this study was to examine whether in utero exposure to alcohol increased the susceptibility of the brain to damage following an ischemic event in adult rats. METHODS: We fed Sprague Dawley dams a liquid diet with or without alcohol (3% ethanol) for the duration of their pregnancy (21 to 23 days). In the first series of studies, we examined reactivity of cerebral arterioles to endothelial nitric oxide synthase (eNOS)- (adenosine diphosphate [ADP]) and neuronal nitric oxide synthase (nNOS)-dependent N-methyl-D-aspartate (NMDA, and NOS-independent agonists in adult rats before and during application of l-NMMA. In another series of studies, we examined infarct volume following middle cerebral artery occlusion in adult offspring exposed to alcohol in utero. In both series of studies, we also determined the role for an increase in oxidative stress by feeding dams apocynin for the duration of their pregnancy. RESULTS: We found that in utero exposure to alcohol reduced responses of cerebral arterioles to ADP and NMDA, but not to nitroglycerin in adult rats. In addition, treatment of the dams with apocynin prevented this impairment in cerebral vascular function. We also found that in utero exposure to alcohol worsened brain damage following ischemia/reperfusion in adult rats and that treatment of dams with apocynin prevented this increase in brain damage following ischemia/reperfusion. CONCLUSIONS: We suggest that our findings may have important implications for the pathogenesis of brain abnormalities associated with fetal alcohol exposure.

Agmatine Inhibits Behavioral Sensitization to Ethanol Through Imidazoline Receptors.

BACKGROUND: Locomotor sensitization to repeated ethanol (EtOH) administration is proposed to play a role in early and recurring steps of addiction. The present study was designed to examine the effect of agmatine on EtOH-induced locomotor sensitization in mice. METHODS: Mice received daily single intraperitoneal injection of EtOH (2.5 g/kg, 20 v/v) for 7 consecutive days. Following a 3-day EtOH-free phase, the mice were challenged with EtOH on day 11 with a single injection of EtOH. Agmatine (10 to 40 µg/mouse), endogenous agmatine enhancers (l-arginine [80 µg/mouse], arcaine [50 µg/mouse], aminoguanidine [25 µg/mouse]), and imidazoline receptor agonist/antagonists were injected (intracerebroventricular [i.c.v.]) either daily before the injection of EtOH during the 7-day development phase or on days 8, 9, and 10 (EtOH-free phase). The horizontal locomotor activity was determined on days 1, 3, 5, 7, and 11. RESULTS: Agmatine (20 to 40 µg/mouse) administration for 7 days (development phase) significantly attenuated the locomotor sensitization response of EtOH challenge on day 11. Further, the agmatine administered only during EtOH-free period (days 8, 9, and 10) also inhibited the enhanced locomotor activity on the 11th day to EtOH challenge as compared to control mice indicating blockade of expression of sensitization. Daily treatment (i.c.v.) with endogenous agmatine enhancers like l-arginine (80 µg/mouse) or arcaine (50 µg/mouse) and aminoguanidine (25 µg/mouse) restrained the development as well as expression of sensitization to EtOH. Imidazoline I1 receptor agonist, moxonidine, and I2 agonist, 2-BFI, not only decreased the development and expression of locomotor sensitization but also potentiated the effect of agmatine when employed in combination. Importantly, I1 receptor antagonist, efaroxan, and I2 antagonist, idazoxan, blocked the effect of agmatine, revealing the involvement of imidazoline receptors in agmatine-mediated inhibition of EtOH sensitization. CONCLUSIONS: Inhibition of EtOH sensitization by agmatine is mediated through imidazoline receptors and project agmatine and imidazoline agents in the pharmacotherapy of alcohol addiction.

Glucagon-like peptide-1 receptors within the nucleus of the solitary tract regulate alcohol-mediated behaviors in rodents.

The ability of glucagon-like peptide-1 (GLP-1) to reduce food intake involves activation of GLP-1 receptors (GLP-1R) in the nucleus of the solitary tract (NTS). It has also been demonstrated that systemic administration of GLP-1R agonists attenuates alcohol-mediated behaviors via, to date, unknown mechanisms. Therefore, we evaluated the effects of NTS-GLP-1R activation by exendin-4 (Ex4) on alcohol-induced locomotor stimulation, accumbal dopamine release and memory of alcohol reward in the conditioned place preference (CPP) model in mice. Moreover, the ability of Ex4 infusion into the NTS on alcohol intake was explored in rats. Ex4 into the NTS inhibits the acute effects of alcohol as measured by alcohol-induced locomotor stimulation, accumbal dopamine release and the memory consolidation of alcohol reward in the CPP paradigm. In addition, NTS-Ex4 dose-dependently decreases alcohol intake in rats consuming alcohol for 12 weeks. Pharmacological suppression of GLP-1R in the NTS prevents the ability of systemic Ex4 to block the alcohol-induced locomotor stimulation in mice. These data add a functional role of GLP-1R within the NTS, involving alcohol-related behaviors. In addition, they may provide insight into the GLP-1R containing brain areas that modulate the ability of GLP-1R agonists to reduce alcohol reinforcement. Collectively, this further supports GLP-1R as potential treatment targets for alcohol use disorder.

Oral administration of lutein attenuates ethanol-induced memory deficit in rats by restoration of acetylcholinesterase activity.

The excessive consumption of alcohol affects the central nervous system, resulting in memory and learning deficits. Lutein is a carotenoid known for its antioxidant properties, which can be able to prevent neurodegenerative diseases and cognitive deficits. In the present study, we evaluated the effect of lutein on ethanol-induced memory deficits in the object recognition task in adult rats, as well as the possible involvement of oxidative stress and cholinergic system. Wistar rats were randomly divided into two groups receiving lutein (50 mg/kg) or olive oil (1 mL/kg) by oral gavage once daily for 14 days. On day 8 each group was divided again into two groups receiving either ethanol (3 g/kg) or saline by oral gavage once daily for 7 days. After the last administration, the animals were submitted on the object recognition task 24 h later (on days 15, 16 and 17). After the behavioral test, the hippocampus and cerebral cortex were removed for the determination of oxidative stress indicators (superoxide dismutase, thiobarbituric acid reactive substances, and non-protein thiol) and acetylcholinesterase activity. Ethanol administration induced a memory deficit and increased acetylcholinesterase activity, however, it did not alter the parameters of oxidative stress, evaluated in the cortex and hippocampus. Oral administration of lutein (50 mg/kg during 14 days) attenuated memory deficit and the increase of acetylcholinesterase activity induced by ethanol. These results provide evidence that lutein is an alternative treatment for ethanol-induced memory deficit, and suggest the involvement of cholinergic system.