EtOH Group Research Articles

Effects of Long-Term Ethanol Consumption on Learning-Memory Functions in Mice and the Mechanisms Involved

To investigate the effect of long-term ethanol consumption on learning-memory functions in mice and the mechanisms involved. Thirty male C57BL6/J mice were randomly assigned to 3 groups, with 10 mice in each group. The three groups included a control group in which the mice were given water ad libitum for 30 days, a long-term ethanol consumption group, or the EtOH group, in which the mice were given 6% (volume fraction) ethanol ad libitum for 30 days, and a long-term alcoholism group, or the EtOH+G group, in which the mice were given 5% (volume fraction) ethanol ad libitum for 30 days plus intermittent intragastric gavage of 20% ethanol at 3.5 g per kilogram body mass once every three days. After 30 days, the learning-memory functions of the mice were evaluated. At the conclusion of the experiment, the brain tissue of the mice was collected in order to examine the oxygen consumption rate (OCR) of mitochondria, the levels of pan-acetylation and protein oxidative stress in the hippocampal tissue, and the expression of sirtuin-3 (SIRT3) in hippocampus. Morris water maze test showed that, compared with those of the control group, the times of crossing the platform and the percentage of platform time in the EtOH group and the EtOH+G group were both lower, and the EtOH+G group had the lowest results ( P<0.05). Western blot results showed that long-term ethanol intake increased the levels of protein oxidative stress and pan-acetylation in the hippocampal tissue and down-regulated SIRT3 expression of hippocampal mitochondria. The results of mitochondrial complex Ⅱ respiration showed that the brain mitochondrial 3-state respiration in the EtOH group and the EtOH+G group was lower than that in the control group ( P<0.05). Compared that with the control group, the mitochondrial maximum respiration in EtOH+G group was decreased ( P<0.05). Both long-term ethanol consumption and long-term alcoholism can reduce learning-memory functions and long-term alcoholism has the greater impact of the two. The potential mechanism may involve the down-regulation of the expression of SIRT3 protein in the hippocampus, which results in an increased level of pan-acetylation and enhanced expression of oxidative stress protein in the hippocampus, affects the mitochondrial functions of the brain, inhibits the oxidative phosphorylation capacity of mitochondrial complex Ⅱ, reduces the ATP energy supply of the brain tissue, and thus affects the learning-memory function.

Lactoferrin Prevents Chronic Alcoholic Injury by Regulating Redox Balance and Lipid Metabolism in Female C57BL/6J Mice.

This study aimed to investigate the preventive effects of lactoferrin (Lf) on chronic alcoholic liver injury (ALI) in female mice. Female C57BL/6J mice were randomly divided into four groups: control group (CON), ethanol administration group (EtOH), low-dose Lf treatment group (LLf), and high-dose Lf group (HLf). In the last three groups, chronic ALI was induced by administering 20% ethanol ad libitum for 12 weeks. Mice in the CON and EtOH groups were fed with AIN-93G diet. Meanwhile, 0.4% and 4% casein in the AIN-93G diet were replaced by Lf as the diets of LLf and HLf groups, respectively. HLf significantly reduced hepatic triglyceride content and improved pathological morphology. HLf could inhibit cytochrome P450 2E1 overexpression and promote alcohol dehydrogenase-1 expression. HLf activated protein kinase B and AMP-activated protein kinase (AMPK), as well as upregulating nuclear-factor-erythroid-2-related factor-2 expression to elevate hepatic antioxidative enzyme activities. AMPK activation also benefited hepatic lipid metabolism. Meanwhile, HLf had no obvious beneficial effects on gut microbiota. In summary, Lf could alleviate chronic ALI in female mice, which was associated with redox balance and lipid metabolism regulation.

Effect of pycnogenol on ethanol-related oxidative retinal injury: an experimental study.

We aimed at determining the protective effects of Pycnogenol on ethanol-induced retinotoxicity in an experimental model. 30 male Wistar albino rats were randomly divided into three groups: an untreated healthy control (HC group), a group in which only ethanol was daily administered for six weeks (EtOH group), and a group in which ethanol + 40 mg/kg Pycnogenol was daily administered orally for six weeks (PEtOH group). The same volume (0.5 ml) of distilled water as solvent was applied in the same manner to the rats in the HC and EtOH groups. With the rats in the PEtOH and EtOH groups, 32% ethanol at a dose of 5 g/kg was administered by oral gavage one hour after the application of pycnogenol or distilled water. At the end of the experimental period, tissue samples were obtained for biochemical examination of malondialdehyde (MDA) and total glutathione (tGSH) levels, and afterwards, the eyes were removed for histopathological examination. Histopathological evaluations in the EtOH group showed significant destruction of retinal tissue with marked edema, decomposition and degeneration in layers, polymorphonuclear cell infiltration, dilatation and congestion in blood vessels. However, it was observed that MDA values increased and tGSH values decreased in the EtOH group. In the PEtOH group, MDA values decreased and GSH values increased. Again, degenerative changes were considerably less in this group. In the light of biochemical markers and histopathological evaluations, it was observed that ethanol exposure caused a significant degeneration in the retinal tissue. It was found that Pycnogenol administration significantly reduced the destructive effects seen histopathologically. Biochemical results also coincided with other findings. It was concluded that ethanol-induced rethytosis can be prevented to a large extent by Pycnogenol administration.

Acute and subacute effects of thymoquinone on acute methanol intoxication: An assessment based on serum TBARS and BDNF levels in rat model

Objectives: Previous studies have shown the role of oxidative stress in methanol (MeOH) neurotoxicity. In clinical practice ethanol (EtOH) was used for the treatment of MeOH intoxication. Treatment with EtOH results in depression of the central nervous system, which may occur even at therapeutic doses. It also induces oxidative stress. Antioxidant and neuroprotective effects of thymoquinone (TQ) are known in different models of neurotoxicity. There are no studies investigating the protective effect of TQ against acute MeOH intoxication. We aimed to evaluate the effect of TQ administration on serum thiobarbituric acid reactive substances (TBARS) and Brain-Derived Neurotrophic Factor (BDNF) levels in rats with experimentally-induced MeOH intoxication. Materials and Methods: Six groups were constituted. Methotrexate (Mtx) treatment (0.3 mg/kg/day) intraperitoneally (i.p.) was given for 7 days to slow down the formate metabolism of all rats except controls in order to create a MeOH intoxication similar to that in humans. On the 8th day of the experiment, 3 g/kg MeOH was injected i.p. in MeOH, EtOH and TQ groups. Four hours after MeOH administration, 0.5 g/kg EtOH was injected i.p. in EtOH group and 30 mg/kg TQ was administered i.p. in TQ1 and TQ2 groups. In addition, a total of 5 doses of 30 mg/kg TQ was injected i.p. 24, 48, 72 and 96 hours after the first dose in TQ2 group. Saline solution was given i.p. in the other groups. Blood samples were obtained for evaluating serum TBARS and BDNF levels. Results: The highest TBARS level was found in MeOH+MTx group and this increase was statistically significant as compared to control and Mtx groups (p&amp;lt;0.001) . A statistically significant reduction was detected in serum TBARS levels in MeOH+Mtx+EtOH, MeOH+Mtx+TQ1 and MeOH+Mtx+TQ2 groups (p&amp;lt;0.001). Maximum serum BDNF level elevation was found in MeOH+Mtx group and this increase was statistically significant as compared to control and Mtx groups (p&amp;lt;0.001). Serum BDNF levels were higher in MeOH+Mtx+EtOH, MeOH+Mtx+TQ1 and MeOH+Mtx+TQ2 groups and the difference was statistically significant (p&amp;lt;0.001). Conclusions: Thymoquinone could suppress proinflammation and lipid peroxidation in MeOH intoxication, lead to rapid toxicity adaptation, and play the role of neuroprotection more effectively than EtOH. These results may suggest that TQ could be used as an alternative treatment option in MeOH intoxication.

Resolvin E1's Antimicrobial Potential Against Aggregatibacter Actinomycetemcomitans.

BackgroundMicroorganisms along with host response play a key role in the development of periodontal and peri-implant infections. Advanced periodontal and peri-implant diseases are most likely associated with bacterial plaques that trigger host immune response and eventually lead to the destruction of the attachment apparatus and bone loss around a tooth or a dental implant. A recent systematic review and meta-analysis revealed that Aggregatibacter actinomycetemcomitans had the highest association with peri-implantitis. Resolvin E1 (RvE1) is part of the specialized pro-resolving lipid mediator family biosynthesized from omega-3, polyunsaturated fatty acids (PUFAs), and eicosapentaenoic acid (EPA). Although RvE1 is an established anti-inflammatory agent, it was found that its application as a treatment or as a preventive drug had an indirect effect on the subgingival microbiota of both rats and rabbits with experimental periodontitis.AimThe aim of this study is to evaluate the direct antimicrobial effect of RvE1 on Aggregatibacter actinomycetemcomitans bacteria.Materials and MethodsThe study comprised three groups that underwent minimum inhibitory concentration (MIC) against Aggregatibacter actinomycetemcomitans. The first group was tested with the RvE1 working concentration of 5 ug/ml, the second group was tested with ethanol (EtOH), 10% as the working concentration, and the final group was diluted in phosphate-buffered saline (PBS) as the positive control. Optical density (OD600) was used for the comparison of bacterial growth among the tested groups. The experiment was conducted in three biological replicates. Data were analyzed using SPSS, and results were analyzed by using one-way analysis of variance (ANOVA) followed by post-hoc Bonferroni using a minimum level of significance (P-value) of 0.05.ResultsMinimum inhibitory concentration was 1.25 μg/ml and 5% for RvE1 and EtOH, respectively. RvE1's mean optical density (OD600) was 0.156 ± 0.021 and was significantly lower compared with all the other groups (P-value < 0.01). The EtOH group (mean OD600 0.178 ± 0.013) and the PBS group (mean OD600 0.1855 ± 0.022) did not reveal a significant difference (P-value = 0.185).ConclusionRvE1 demonstrated significant antimicrobial activity against A. actinomycetemcomitans with an MIC of 1.25 μg/ml. The RvE1 group showed significantly lower bacterial growth compared to the EtOH and PBS groups.

Moringa oleifera mitigates ethanol-induced oxidative stress, fatty degeneration and hepatic steatosis by promoting Nrf2 in mice

BackgroundMoringa oleifera (M. oleifera) is cultivated throughout the world and it is known by numerous regional names and is consumed as medication for various diseases such as hypertension, diabetes, HIV and is potential source of nutrients and natural antioxidants making it among the most useful trees. MethodsWe evaluated the therapeutic potential of M. oleifera on ethanol-induced fatty liver. The mice were treated with 30% ethanol (EtOH) alone or in combination with different concentration of M. oleifera extracts (100, 200 and 400 mg/kg). We performed biochemical estimation for the serum of important liver damage markers such as aspartate aminotransferase (AST), alanine aminotransferase (ALT) and triglyceride (TG). We performed histopathological analysis from the liver tissues of different mice groups. We also performed ELISA assay, western blotting analysis and SPECT imaging to obtain our results. ResultsThe results for serum (AST, p < 0.0001), (ALT, p < 0.0006) and triglyceride (TG, p < 0.0003) were found to be significantly reduced in all doses of M. oleifera extract treatment groups in comparison with the ethanol group. H&E staining analysis and scoring revealed a significant reduction in lipid droplet accumulation and a significant reduction of liver steatosis (p < 0.0001), lobular inflammation (p < 0.0013), ballooning (p < 0.0004) and immunohistochemistry for TNF-α. M. oleifera also ameliorated ethanol-induced oxidative stress evaluated through MDA (p < 0.0001), H2DCFDA, JC-1 staining and a significant down-regulation of CYP2E1 enzyme (p < 0.0001) in the 200 and 400 mg/kg groups in comparison with EtOH groups. M. oleifera extract also boosted the antioxidant response evaluated through total GSH assay (p < 0.0001) and nuclear translocation of Nrf2. Furthermore, we performed SPECT imaging and evaluated the liver uptake value (LUV) to assess the extent of liver damage. LUV was observed to be lower in the ethanol group, whereas LUV was higher in control and M. olifera treated groups. ConclusionIn summary, from this experiment we conclude that M. oleifera extract has the potential to ameliorate ethanol-induced liver damage.

Effects of acute ethanol and/or diazepam exposure on immediate and delayed hippocampal metabolite levels in rats anesthetized with isoflurane.

Alcohol and benzodiazepines are psychoactive substances frequently associated in voluntary drug intoxications that share common mechanisms of action, including facilitation of GABAergic transmission. This study aimed to assess the separate and combined effects of ethanol and diazepam acute exposure on hippocampal metabolite levels, as well as on delayed cognitive performance, in rats anesthetized with isoflurane. Adult male Wistar rats received one intraperitoneal injection containing either saline solution ("CTL" group, N = 15), a 5-mg/kg dose of diazepam ("DIA" group, N = 16), a 2-g/kg dose of ethanol ("EtOH" group, N = 18), or a 5-mg/kg dose of diazepam + a 2-g/kg dose of ethanol ("DIA + EtOH" group, N = 24). The levels of brain metabolites in the hippocampal region were assessed using in vivo magnetic resonance spectroscopy (MRS) before and after injection. Behavioral testing, including working memory and visual recognition memory assessment, was performed at week 3, while a new MRS acquisition was conducted 4 weeks after the injection. In the hour following acute exposure, a decrease in glutamate levels was found in the DIA + EtOH group only. Four weeks after injection, a decrease in GABA and glutamate levels and an increase in NAA levels were found in the EtOH group only. No significant between-group differences were found in the behavioral assessment. While the initial decrease in glutamate levels in the DIA + EtOH group suggests an early potentiation effect between ethanol and diazepam, the long-term modifications found only in the EtOH group suggest a possible downregulation of ethanol's effect by diazepam at 4 weeks.

Betulin Targets Lipin1/2-Meidated P2X7 Receptor as a Therapeutic Approach to Attenuate Lipid Accumulation and Metaflammation.

The present study focused on the potential mechanism of betulin (BT), a pentacyclic triterpenoid isolated from the bark of white birch (Betula pubescens), against chronic alcohol-induced lipid accumulation and metaflammation. AML-12 and RAW 264.7 cells were administered ethanol (EtOH), lipopolysaccharide (LPS) or BT. Male C57BL/6 mice were fed Lieber-DeCarli liquid diets containing 5% EtOH for 4 weeks, followed by single EtOH gavage on the last day and simultaneous treatment with BT (20 or 50 mg/kg) by oral gavage once per day. In vitro, MTT showed that 0-25 mM EtOH and 0-25 μM BT had no toxic effect on AML-12 cells. BT could regulate sterolregulatory-element-binding protein 1 (SREBP1), lipin1/2, P2X7 receptor (P2X7r) and NOD-like receptor family, pyrin domains-containing protein 3 (NLRP3) expressions again EtOH-stimulation. Oil Red O staining also indicated that BT significantly reduced lipid accumulation in EtOH-stimulated AML-12 cells. Lipin1/2 deficiency indicated that BT might mediate lipin1/2 to regulate SREBP1 and P2X7r expression and further alleviate lipid accumulation and inflammation. In vivo, BT significantly alleviated histopathological changes, reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and triglyceride (TG) levels, and regulated lipin1/2, SREBP1, peroxisome proliferator activated receptor α/γ (PPARα/γ) and PGC-1α expression compared with the EtOH group. BT reduced the secretion of inflammatory factors and blocked the P2X7r-NLRP3 signaling pathway. Collectively, BT attenuated lipid accumulation and metaflammation by regulating the lipin1/2-mediated P2X7r signaling pathway.

Allium victorialis L. Extracts Promote Activity of FXR to Ameliorate Alcoholic Liver Disease: Targeting Liver Lipid Deposition and Inflammation

Allium victorialis L. (AVL) is a traditional medicinal plant recorded in the Compendium of Materia Medica (the Ming Dynasty). In general, it is used for hemostasis, analgesia, anti-inflammation, antioxidation, and to especially facilitate hepatoprotective effect. In recent years, it has received more and more attention due to its special nutritional and medicinal value. The present study investigates the effect and potential mechanism of AVL against alcoholic liver disease (ALD). C57BL/6 mice were fed Lieber–DeCarli liquid diet containing 5% ethanol plus a single ethanol gavage (5 g/kg), and followed up with the administration of AVL or silymarin. AML12 cells were stimulated with ethanol and incubated with AVL. AVL significantly reduced serum transaminase and triglycerides in the liver and attenuated histopathological changes caused by ethanol. AVL significantly inhibited SREBP1 and its target genes, regulated lipin 1/2, increased PPARα and its target genes, and decreased PPARγ expression caused by ethanol. In addition, AVL significantly enhanced FXR, LXRs, Sirt1, and AMPK expressions compared with the EtOH group. AVL also inhibited inflammatory factors, NLRP3, and F4/80 and MPO, macrophage and neutrophil markers. In vitro, AVL significantly reduced lipid droplets, lipid metabolism enzymes, and inflammatory factors depending on FXR activation. AVL could ameliorate alcoholic steatohepatitis, lipid deposition and inflammation in ALD by targeting FXR activation.

Prenatal alcohol and cannabis exposure can have opposing and region-specific effects on parvalbumin interneuron numbers in the hippocampus.

We recently showed that alcohol and cannabis can interact prenatally, and in a recent review paper, we identified parvalbumin-positive (PV) interneurons in the hippocampus as a potential point of convergence for these teratogens. A 2 (Ethanol [EtOH], Air)×2 (tetrahydrocannabinol [THC], Vehicle) design was used to expose pregnant Sprague-Dawley rats to either EtOH or air, in addition to either THC or the inhalant vehicle solution, during gestational days 5-20. Immunohistochemistry was performed to detect PV interneurons in 1male and 1 female pup from each litter at postnatal day 70. Significant between-group and subregion-specific effects were found in the dorsal cornu ammonis 1 (CA1)subfield and the ventral dentate gyrus (DG). In the dorsal CA1subfield, there was an increase in the number of PV interneurons in both the EtOH and EtOH +THC groups, but a decrease with THC alone. There were fewer changes in interneuron numbers overall in the DG, though there was a sex difference, with a decrease in the number of PV interneurons in the THC-exposed group in males. There was also a greater cell layer volume in the DG in the EtOH +THC group than the control group, and in the CA1 region in the EtOH group compared to the control and THC groups. Prenatal exposure to alcohol and THC differentially affects parvalbumin-positive interneuron numbers in the hippocampus, indicating that both individual and combined exposure can impact the balance of excitation and inhibition in a structure critically involved in learning and memory processes.

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.

The Correlation of Histopathological Findings with Ultrastructural Changes in Hepatocytes after Yangonin “Ya”-Intoxicated Rats Alone and In Combination with EtOH: Sub-Acute & Sub-Chronic Study

Yangonin “Ya” has been used for centuries as a herbal supplement, for its mood-altering properties. It has been used as a recreation agent, for relaxation, as well as for pain relief. However, hepatotoxicity is a widespread problem associated with medicines in general. Most herbal supplements are metabolized by the liver, and thus the liver represents the target organ. At present, Yangonin toxicity appears to be “idiosyncratic”. Therefore, a study was designed in order to investigate the organelle-based changes in hepatocytes, after treatment with Yangonin alone and in combination with EtOH. Thirty rats were divided into five groups comprising of six animals each. The groups comprised of the control groups {(NCx) & (PCx)}, Yangonin (Ya) group, ethanol (EtOH) group, and the combination of (Ya) and ethanol (EtOH+Ya) group. The experiment was conducted over a period of 14 weeks, as a sub-chronic study. At the end of the 14 th week, mitochondria, peroxisome, rough and smooth endoplasmic reticulum, and nuclei of hepatocytes, were evaluated using a scoring system. The results were compared with histopathological findings, as well. Treatment with Ya significantly induced hepatotoxic scores as compared to the control groups. Organelle injury scores increased significantly with Ya treatment, while rats that received “EtOH+Ya” showed the severest lesions of liver scores such as, severe hepatocellular degeneration, necrosis, and hypertrophy. Ultrastructural and histopathological scores in both groups were in very strong correlation (r = 0.928 for EtOH, r = 0.921 for Ya alone and r = 0.903 for Ya plus EtOH group). In conclusion, ethanol enhanced the sedative and hypnotic activity of Ya, and markedly increased toxicity. Findings based on TEM examination of organelles, supported the histological results as well as tissue lesions/injuries in hepatocytes, a result of hepatotoxin-induced hepatopathy.

Alcohol Worsens Cancer Cachexia and Anabolic Signaling in C26 Tumor Bearing Mice

Cancer cachexia results in a loss of skeletal muscle mass, leading to increased morbidity and mortality. Similarly, chronic alcohol consumption leads to skeletal muscle loss and dysfunction. It is unknown, whether prior alcohol intake or its continued use after cancer establishment, exacerbates cancer cachexia. Anabolic pathways are central to the maintenance of muscle mass and are notoriously decreased by alcohol intake. Among these pathways, the mTORC1 pathway and subsequent rates of protein synthesis are of central importance to the maintenance of muscle health. Purpose To determine if prior or concurrent alcohol consumption worsens skeletal muscle anabolic signaling (i.e. puromycin and mTOR pathway) during cancer cachexia. Methods Male CD2F1 mice (n=42), 12-weeks old, were randomized into 6 groups (n=6-8/gr): control (CON), control-cancer (CON-C), prior EtOH (PRIOR), prior EtOH-cancer (PRIOR-C), continuous EtOH (EtOH), and continuous EtOH w/cancer (EtOH-C). Mice were acclimated to a control liquid diet, after which the EtOH groups transitioned to the Lieber DeCarli EtOH diet at 20% kcal EtOH for 6 weeks. At week 7, PRIOR mice weaned off EtOH and remained on the control diet until sacrifice. Also at week 7, cancer mice were injected subcutaneously on the right flank with C26 colon carcinoma cells (1x106 cells) and consumed their assigned diet for 14 days while tumors developed. After a 4-hour fast, puromycin was injected and 30 minutes later the gastrocnemius (GAS) was excised. GAS protein was isolated and Western Blots performed. Data were analyzed via 1-way ANOVA with post hoc testing when appropriate. Data are presented relative to CON (set to 100%) as mean ± SE, with protein expression normalized either to ponceau staining or total levels of the given protein. Results Body weight at sacrifice was lower in the PRIOR-C and EtOH-C (p<0.05) indicating an alcohol effect. Tumor weight and tibia length were not different between groups. GAS weight was reduced by cancer in all groups, and most notably in PRIOR-C and EtOH-C mice (p<0.001). Protein synthesis, assessed by puromycin, decreased in all cancer groups, with a ~70% reduction in PRIOR-C and ~82% reduction in EtOH-C relative to CON. Similarly, phosphorylation of S6K1 Thr389, rpS6 Ser240/244 and Ser235/236 was significantly reduced in PRIOR-C and EtOH-C relative to CON (p<0.05), indicating a greater reduction in these markers of mTORC1 activity with alcohol intake. Surprisingly, no differences in 4E-BP1 Ser65, mTOR Ser2448, or mTOR Ser2481 were observed across groups. PRAS40 Thr246 was increased by cancer alone as well as in EtOH-C mice (35% vs. CON) (p<0.05). Increases in eEF2 Thr56 were also detected in PRIOR-C (50%) and EtOH-C (83%) (p<0.05), possibly indicating impaired peptide chain elongation. No changes were noted in markers of protein breakdown including ubiquitinated proteins, and autophagic proteins ULK1 Ser757 and LC3B-II/I. Conclusions The intake of alcohol both prior to, and during tumor development lead to greater decreases in GAS weight, protein synthesis and several markers of mTORC1 activity indicating that alcohol significantly worsened and accelerated the development of cancer cachexia.

Effect of low-dose ethanol on NLRP3 inflammasome in diabetes-induced lung injury.

To observe the changes in NLR family pyrin domain containing 3 (NLRP3) inflammasome in a rat model of diabetes-induced lung injury, and investigate the effect of low-dose ethanol on the production of NLRP3 inflammasome. The type I diabetic mellitus (DM) rat model was established, and the rats were divided into four groups: normal control group (CON group), low-dose ethanol group (EtOH group), diabetes group (DM group) and DM+EtOH group. The rats were fed for 6 and 12 weeks, respectively. The ratio of lung wet weight/body weight (lung/body coefficient) was calculated, and the changes of pulmonary morphology and fibrosis were observed by HE and Masson staining. The changes in pulmonary ultra-structure were examined by electron microscopy. The expressions of mitochondrial acetaldehyde dehydrogenase 2 (ALDH2) and NLRP3 inflammasome key factors, NLRP3, ASC and caspase-1 proteins were detected by western blot. Compared with the CON group, the lung/body coefficient was increased (P<0.05), lung fibrosis occurred, ALDH2 protein expression was decreased, and NLRP3, ASC and caspase-1 protein expressions were increased in the DM rats (P<0.05). Compared with the DM group, the lung/body coefficient and fibrosis degree were decreased, ALDH2 protein expression was increased (P<0.05), and NLRP3, ASC and caspase-1 protein expressions were decreased in the DM+EtOH group (P<0.05). Hence, low-dose ethanol increased ALDH2 protein expression and alleviated diabetes-induced lung injury by inhibiting the production of NLRP3 inflammasome.

Curcumin combined with Baicalin attenuated ethanol-induced hepatitis by suppressing p38 MAPK and TSC1/ eIF-2α/ATF4 pathways in rats

Autophagy activation is one of the survival mechanisms of ethanol-induced hepatitis in mammals. Both Curcumin and Baicalin have anti-inflammatory effects, but the mechanism of their combined action in ethanol-induced hepatitis is still unclear. This study aimed to investigate whether Curcumin combined with Baicalin can resist ethanol-induced p38 MAPK-mediated hepatitis via suppressing TSC1/eIF-2α/ATF4 pathway to activate autophagy. Rats were randomly divided into Control group, EtOH group, EtOH + Curcumin group, EtOH + Baicalin group, and EtOH + Curcumin + Baicalin group. The indicators of liver function and inflammation, and the levels of p38 MAPK and autophagy-related proteins and genes were assayed. Our study found that ethanol increased serum AST, TG, T-CHO levels and liver inflammatory cytokines (TNF-α, IL-1β, INF-γ, NO and iNOS) contents, and also reduced the levels of Beclin1 mRNA and protein, LC3B mRNA and LC3BII/I ratio, but up-regulated the levels of p-p38, P62, p-TSC1, p-eIF-2α and p-ATF4 in rats. Curcumin combined with Baicalin decreased the levels of serum AST, TG, T-CHO and liver inflammatory cytokines, down-regulated the expression of p-p38, P62, p-TSC1, p-eIF-2α and p-ATF4, as well as up-regulated the expression of Beclin1 mRNA and protein, LC3B mRNA and LC3BII/I ratio in rats. Besides, combination therapy was superior to monotherapy. Our results indicated that Curcumin combined with Baicalin can improve ethanol-induced hepatitis by inhibiting p38 MAPK and TSC1/eIF-2α/ATF4 pathway, which may be a potential therapeutic strategy for ethanol-induced hepatitis in the future.

Sex- and age-dependent differences in nicotine susceptibility evoked by developmental exposure to tobacco smoke and/or ethanol in mice.

Either tobacco smoking or alcohol consumption during pregnancy sex-selectively increases susceptibility to drugs of abuse later in life. Considering that pregnant smoking women are frequently intermittent consumers of alcoholic beverages, here, we investigated whether a short-term ethanol exposure restricted to the brain growth spurt period when combined with chronic developmental exposure to tobacco smoke aggravates susceptibility to nicotine in adolescent and adult mice. Swiss male and female mice were exposed to tobacco smoke (SMK; research cigarettes 3R4F, whole-body exposure, 8 h/daily) or ambient air during the gestational period and until the tenth postnatal day (PN). Ethanol (ETOH, 2 g/Kg, 25%, i.p.) or saline was injected in the pups every other day from PN2 to PN10. There were no significant differences in cotinine (nicotine metabolite) and ethanol serum levels among SMK, ETOH and SMK + ETOH groups. During adolescence (PN30) and adulthood (PN90), nicotine (NIC, 0.5 mg/Kg) susceptibility was evaluated in the conditioned place preference and open field tests. NIC impact was more evident in females: SMK, ETOH and SMK + ETOH adolescent females were equally more susceptible to nicotine-induced place preference than control animals. At adulthood, SMK and SMK + ETOH adult females exhibited a nicotine-evoked hyperlocomotor profile in the open field, with a stronger effect in the SMK + ETOH group. Our results indicate that ethanol exposure during the brain growth spurt, when combined to developmental exposure to tobacco smoke, increases nicotine susceptibility with stronger effects in adult females. This result represents a worsened outcome from the early developmental dual exposure and may predispose nicotine use/abuse later in life.

Ethanol-mediated alterations in oligodendrocyte differentiation in the developing brain

IntroductionAlterations of white matter integrity and subsequent white matter structural deficits are consistent findings in Fetal Alcohol Syndrome (FAS), but knowledge regarding the molecular mechanisms underlying these abnormalities is incomplete. Experimental rodent models of FAS have shown dysregulation of cytokine expression leading to apoptosis of oligodendrocyte precursor cells (OPCs) and altered oligodendrocyte (OL) differentiation, but whether this is representative of human FAS pathogenesis has not been determined. MethodsFetal brain tissue (12.2–21.4 weeks gestation) from subjects undergoing elective termination of pregnancy was collected according to an IRB-approved protocol. Ethanol (EtOH) exposure status was classified based on a detailed face-to-face questionnaire adapted from the National Institute on Alcohol Abuse and Alcoholism Prenatal Alcohol and Sudden Infant Death Syndrome and Stillbirth (PASS) study. Twenty EtOH-exposed fetuses were compared with 20 gestational age matched controls. Cytokine and OPC marker mRNA expression was quantified by Real-Time Polymerase chain reaction (qRT-PCR). Patterns of protein expression of OPC markers and active Capase-3 were studied by Fluorescence Activated Cell Sorting (FACS). ResultsEtOH exposure was associated with reduced markers of cell viability, OPC differentiation, and OL maturation, while early OL differentiation markers were unchanged or increased. Expression of mRNAs for proteins specific to more mature forms of OL lineage (platelet-derived growth factor α (PDGFRα) and myelin basic protein (MBP) was lower in the EtOH group than in controls. Expression of the multifunctional growth and differentiation-promoting growth factor IGF-1, which is essential for normal development, also was reduced. Reductions were not observed for markers of early stages of OL differentiation, including Nuclear transcription factor NK-2 homeobox locus 2 (Nkx2.2). Expression of mRNAs for the proinflammatory cytokine, tumor necrosis factor-α (TNFα), and several proinflammatory chemokines was higher in the EtOH group compared to controls, including: Growth regulated protein alpha/chemokine (C-X-C motif) ligand 1 (GRO-α/CXCL1), Interleukin 8/chemokine (C-X-C motif) ligand 8 (IL8/CXCL8), Chemokine (C-X-C motif) ligand 6/Granulocyte chemotactic protein 2 (CXCL16/GCP2), epithelial-derived neutrophil-activating protein 78/chemokine (C-X-C motif) ligand 5 (ENA-78/CXCL5), monocyte chemoattractant protein-1 (MCP-1). EtOH exposure also was associated with an increase in the proportion of cells expressing markers of early stage OPCs, such as A2B5 and NG2. Finally, apoptosis (measured by caspase-3 activation) was increased substantially in the EtOH group compared to controls. ConclusionPrenatal EtOH exposure is associated with excessive OL apoptosis and/or delayed OL maturation in human fetal brain. This is accompanied by markedly dysregulated expression of several chemokines and cytokines, in a pattern predictive of increased OL cytotoxicity and reduced OL differentiation. These findings are consistent with findings in animal models of FAS.

Metabolomics Study of Serum from a Chronic Alcohol-Fed Rat Model Following Administration of Defatted Tenebrio molitor Larva Fermentation Extract.

We have previously showed that defatted mealworm fermentation extract (MWF) attenuates alcoholic liver injury by regulating lipid, inflammatory, and antioxidant metabolism in chronic alcohol-fed rats. The current metabolomics study was performed to monitor biochemical events following the administration of MWF (daily for eight weeks) to a rat model of alcoholic liver injury by gas chromatography-tandem mass spectrometry (GC-MS/MS). The levels of 15 amino acids (AAs), 17 organic acids (OAs), and 19 free fatty acids (FFAs) were measured in serum. Analysis of variance (ANOVA), principal component analysis (PCA), and partial least squares discriminant analysis (PLS-DA) were used to compare the levels of 51 metabolites in serum. In particular, 3-hydroxybutyric acid (3-HB), pyroglutamic acid (PG), octadecanoic acid, and docosahexaenoic acid (DHA) were evaluated as high variable importance point (VIP) scores and PCA loading scores as determined by PLS-DA and PCA, and these were significantly higher in the MWF and silymarin groups than in the EtOH group. MWF showed a protective effect from alcohol-induced liver damage by elevating hepatic β-oxidation activity, and serum 3-HB levels were significantly higher in the MWF group than in the EtOH control group. Glycine levels were higher in the MWF group than in the EtOH group, and PG levels (related to glutathione production) were also elevated, indicating a reduction in alcohol-related oxidative stress. In addition, MWF is protected from alcohol-induced inflammation and steatosis by increasing serum DHA, palmitic, and octadecanoic acid levels as compared with the EtOH group. These results suggest that MWF might attenuate alcoholic liver disease, due to its anti-inflammatory and antioxidant effects by up-regulating hepatic β-oxidation activity and down-regulating liver FFA uptake.

New strain of Pediococcus pentosaceus alleviates ethanol-induced liver injury by modulating the gut microbiota and short-chain fatty acid metabolism.

BACKGROUNDIntestinal dysbiosis has been shown to be associated with the pathogenesis of alcoholic liver disease (ALD), which includes changes in the microbiota composition and bacterial overgrowth, but an effective microbe-based therapy is lacking. Pediococcus pentosaceus (P. pentosaceus) CGMCC 7049 is a newly isolated strain of probiotic that has been shown to be resistant to ethanol and bile salts. However, further studies are needed to determine whether P. pentosaceus exerts a protective effect on ALD and to elucidate the potential mechanism.AIMTo evaluate the protective effect of the probiotic P. pentosaceus on ethanol-induced liver injury in mice.METHODSA new ethanol-resistant strain of P. pentosaceus CGMCC 7049 was isolated from healthy adults in our laboratory. The chronic plus binge model of experimental ALD was established to evaluate the protective effects. Twenty-eight C57BL/6 mice were randomly divided into three groups: The control group received a pair-fed control diet and oral gavage with sterile phosphate buffered saline, the EtOH group received a ten-day Lieber-DeCarli diet containing 5% ethanol and oral gavage with phosphate buffered saline, and the P. pentosaceus group received a 5% ethanol Lieber-DeCarli diet but was treated with P. pentosaceus. One dose of isocaloric maltose dextrin or ethanol was administered by oral gavage on day 11, and the mice were sacrificed nine hours later. Blood and tissue samples (liver and gut) were harvested to evaluate gut barrier function and liver injury-related parameters. Fresh cecal contents were collected, gas chromatography–mass spectrometry was used to measure short-chain fatty acid (SCFA) concentrations, and the microbiota composition was analyzed using 16S rRNA gene sequencing.RESULTSThe P. pentosaceus treatment improved ethanol-induced liver injury, with lower alanine aminotransferase, aspartate transaminase and triglyceride levels and decreased neutrophil infiltration. These changes were accompanied by decreased levels of endotoxin and inflammatory cytokines, including interleukin-5, tumor necrosis factor-α, granulocyte colony-stimulating factor, keratinocyte-derived protein chemokine, macrophage inflammatory protein-1α and monocyte chemoattractant protein-1. Ethanol feeding resulted in intestinal dysbiosis and gut barrier disruption, increased relative abundance of potentially pathogenic Escherichia and Staphylococcus, and the depletion of SCFA-producing bacteria, such as Prevotella, Faecalibacterium, and Clostridium. In contrast, P. pentosaceus administration increased the microbial diversity, restored the relative abundance of Lactobacillus, Pediococcus, Prevotella, Clostridium and Akkermansia and increased propionic acid and butyric acid production by modifying SCFA-producing bacteria. Furthermore, the levels of the tight junction protein ZO-1, mucin proteins (mucin [MUC]-1, MUC-2 and MUC-4) and the antimicrobial peptide Reg3β were increased after probiotic supplementation.CONCLUSIONBased on these results, the new strain of P. pentosaceus alleviated ethanol-induced liver injury by reversing gut microbiota dysbiosis, regulating intestinal SCFA metabolism, improving intestinal barrier function, and reducing circulating levels of endotoxin and proinflammatory cytokines and chemokines. Thus, this strain is a potential probiotic treatment for ALD.

High Alcohol-Preferring Mice Show Reaction to Loss of Ethanol Reward Following Repeated Binge Drinking.

Beyond yielding high blood ethanol (EtOH) concentrations (BECs), binge-drinking models allow examination of drinking patterns which may be associated with EtOH's rewarding effects, including front-loading and consummatory successive negative contrast (cSNC), a decrease in intake when only water is available to subjects expecting EtOH. The goals of the current study were to broaden our understanding of these reward-related behaviors during binge EtOH access in high alcohol-preferring (HAP) replicate lines (HAP2 and HAP3) of mice selectively bred to prefer alcohol. We hypothesized that both lines would show evidence of front-loading during binge EtOH access and that we would find a cSNC effect in groups where EtOH was replaced with water, as these results have been shown previously in HAP1 mice. HAP replicate 2 and replicate 3 female and male mice were given 2hours of EtOH or water access in the home cage for 15 consecutive days using "drinking in the dark" (DID) procedures. Mice received the same fluid (either 20% unsweetened EtOH or water) for the first 14days. However, on the 15th day, half of the mice from these 2 groups were provided with the opposite assigned fluid (EtOH groups received water and vice versa). Intake was measured in 1-minute bins using specialized sipper tubes, which allowed within-session analyses of binge-drinking patterns. EtOH front-loading was observed in both replicates. HAP3 mice displayed front-loading on the first day of EtOH access, whereas front-loading developed following alcohol experience in HAP2 mice, which may suggest differences in initial sensitivity to EtOH reward. Consummatory SNC, which manifests as lower water intake in mice expecting EtOH as compared to mice expecting water, was observed in both replicates. These findings increase confidence that defined changes in home cage consummatory behavior are driven by the incentive value of EtOH. The presence of cSNC across HAP replicates indicates that this reaction to loss of reward is genetically mediated, which suggests that there is a biological mechanism that might be targeted.