Alcohol-fed Group Research Articles

Lactiplantibacillus plantarum P101 Alleviated Alcohol-Induced Hepatic Lipid Accumulation in Mice via AMPK Signaling Pathway: Gut Microbiota and Metabolomics Analysis.

Mitigating steatosis is essential for delaying the progression of alcoholic liver disease. The effect and mechanism of Lactiplantibacillus plantarum P101 (LP.P101) on alleviating alcohol-induced hepatic lipid accumulation were investigated in our study. The mouse model was constructed by a short-term (10-day)-plus-binge ethanol feeding and gavaged with 108 CFU/mL of LP.P101 daily. Lipid droplet in the liver was significantly reduced by LP.101 intervention on AMPK activation. However, when AMPK was inhibited by dorsomorphin, the levels of related indicators (ALT, TG, etc.) and the expression levels of AMPK and relevant genes in the liver converged to that of the alcohol-fed group. Compared with the alcohol-fed group, LP.P101 reduced the relative abundance of Firmicutes and increased that of Bacteroidetes. Parabacteroides merdae was negatively correlated with lipid accumulation, and unclassified Negativibacillus was negatively associated with AMPK activation. Importantly, LP.P101 modified the compositions of the serum metabolites. The potential biomarker stercobilinogen was positively correlated with AMPK activation and negatively associated with lipid accumulation. This work confirmed that LP.P101 attenuated alcohol-induced hepatic lipid accumulation in mice through AMPK activation, and the alterations in gut microbiota and metabolites may play a significant role on AMPK activation.

Chronic Alcohol Exposure Causes Myeloid-Biased Hematopoiesis Via Double-Strand RNA Sensors and Type 1 Interferon Response

Excessive alcohol use is well-known to cause cytopenias and bone marrow (BM) suppression. However, the mechanisms by which alcohol suppresses BM are poorly understood. Moreover, even though the prevalence of moderate drinking is high worldwide, the effects of chronic moderate alcohol consumption on the human hematopoietic system remain unknown. To fill this knowledge gap, we sought to investigate the effects of chronic moderate alcohol consumption on human hematopoiesis using cell-based and animal models. To examine the effects of alcohol on hematopoietic cells in vitro, we tested varying doses of alcohol (0-100 mM) on four leukemia cell lines (Jurkat, MOLM13, MV-4-11, and THP1) and measured population doubling over 6 days. Population doubling levels significantly decreased in a dose-dependent manner in all cell lines tested. We also measured mitochondrial (mt) ROS by MitoSOX-Red flow cytometry in Jurkat, which showed a dose-dependent increase of mtROS (trends only at 10, 25 mM; significantly increased at 50, 100 mM alcohol) compared to untreated. These findings confirm that alcohol negatively affects cell growth and increases mtROS in a physiologic range of doses. To determine the effects of alcohol on human hematopoietic stem progenitor cells (HSPCs) in vitro, we treated human BM CD34+ cells with 0 vs. 50 mM alcohol for 48 hours and performed bulk RNA-sequencing in triplicate. Gene Set Enrichment Analysis (GSEA) revealed that multiple pathways related to platelet activation and coagulation were enriched in alcohol-treated HSPCs, suggesting the induction of inflammation and/or platelet-biased differentiation by alcohol. To assess the effects of chronic moderate alcohol consumption on HSPCs in vivo, we employed a xenotransplant model. In brief, human BM CD34+ cells were transplanted to 5-to-6-week-old female NBSGW mice. We confirmed their engraftment by bone marrow aspiration at six weeks post-transplant, and then they were randomly assigned to the alcohol vs. control groups. After 2 weeks of adjustment, mice were fed a liquid diet (their only source of water and food) containing alcohol 5% vol/vol vs. iso-caloric control diet for 8 weeks. At 4 and 8 weeks, the alcohol-fed group showed significant myeloid bias, reaching greater than 3 times higher myeloid-to-lymphoid ratio than the control group at 8 weeks, while overall engraftment levels were not different. We will report the results of the secondary transplantation of this experiment at the annual meeting. After 8-week alcohol feeding, sorted human CD45+CD34+ cells from mouse BM were subject to 10x Genomics Chromium X 3' HT single-cell RNA-sequencing (scRNA-seq). Complementing the flow data, our transcriptomic data portrayed stark differences between the two groups. Over 50% of cells in the alcohol group comprised granulocyte progenitors, with B cell progenitors representing less than 25%. Inversely, control group data revealed approximately 30% granulocyte progenitors and 55% B cell progenitors, confirming the myeloid bias of HSPC caused by alcohol. Examining differentially expressed genes and GSEA highlighted a significant upregulation of genes associated with the response to virus and type 1 interferon (IFN-1) signature across all HSPC types. A closer look into the gene set showed increased expression of genes tied to cytosolic double-strand (ds) RNA sensing pathways and subsequent interferon type 1 response. Specifically, dsRNA sensors like MDA5, RIG1, PKR, and OAS1-3, as well as interferon regulatory or responsive genes, such as IRF7, ISG15, IFI6, IFI44L, MX1, and STAT1, were notably increased in HSCs and most progenitor populations. Unexpectedly, alcohol-exposed HSCs tended to be in the G0/G1 phase rather than the S or M phase compared to their counterpart upon cell cycle analysis using scRNA-seq data. This may be due to the activation of the PKR pathway, a known cell cycle inhibitor in mouse HSCs. In summary, our findings demonstrate that chronic moderate alcohol consumption can significantly reshape the hematopoietic system through myeloid-biased differentiation and potentiate inflammation by activating the cytosolic dsRNA sensing pathways and IFN-1 response.

Butyrate ameliorates chronic alcoholic central nervous damage by suppressing microglia-mediated neuroinflammation and modulating the microbiome-gut-brain axis

BackgroundAlcohol abuse triggers neuroinflammation, leading to neuronal damage and further memory and cognitive impairment. Few satisfactory advances have been made in the management of alcoholic central nervous impairment. Therefore, novel and more practical treatment options are urgently needed. Butyrate, a crucial metabolite of short-chain fatty acids (SCFAs), has been increasingly demonstrated to protect against numerous metabolic diseases. However, the impact of butyrate on chronic alcohol consumption-induced central nervous system (CNS) lesions remains unknown. MethodsIn this study, we assessed the possible effects and underlying mechanisms of butyrate on the attenuation of alcohol-induced CNS injury in mice. Firstly, sixty female C57BL/6 J mice were randomly divided into 4 groups: pair-fed (PF) group (PF/CON), alcohol-fed (AF) group (AF/CON), PF with sodium butyrate (NaB) group (PF/NaB) and AF with NaB group (AF/NaB). Each group was fed a modified Lieber-DeCarli liquid diet with or without alcohol. After six weeks of feeding, the mice were euthanized and the associated indicators were investigated. ResultsAs indicated by the behavioral tests and brain morphology, dietary NaB administration significantly ameliorated aberrant behaviors, including locomotor hypoactivity, anxiety disorder, depressive behavior, impaired learning, spatial recognition memory, and effectively reduced chronic alcoholic central nervous system damage. To further understand the underlying mechanisms, microglia-mediated inflammation and the associated M1/M2 polarization were measured separately. Firstly, pro-inflammatory TNF-α, IL-1β, and IL-6 in brain and peripheral blood circulation were decreased, but IL-10 were increased in the AF/NaB group compared with the AF/CON group. Consistently, the abnormal proportions of activated and resting microglial cells in the hippocampus and cortex regions after excessive alcohol consumption were significantly reduced with NaB treatment. Moreover, the rectification of microglia polarization (M1/M2) imbalance was found after NaB administration via binding GPR109A, up-regulating the expression of PPAR-γ and down-regulating TLR4/NF-κB activation. In addition to the direct suppression of neuroinflammation, intriguingly, dietary NaB intervention remarkably increased the levels of intestinal tight junction protein occludin and gut morphological barrier, attenuated the levels of serum lipopolysaccharide (LPS) and dysbiosis of gut microbiota, suggesting that NaB supplementation effectively improved the integrity and permeability of gut microecology. Finally, the neurotransmitters including differential Tryptophan (Trp) and Kynurenine (Kyn) were found with dietary NaB administration, which showed significantly altered and closely correlated with the gut microbiota composition, demonstrating the complex interactions in the microbiome-gut-brain axis involved in the efficacy of dietary NaB therapy for alcoholic CNS lesions. ConclusionDietary microbial metabolite butyrate supplementation ameliorates chronic alcoholic central nervous damage and improves related memory and cognitive functions through suppressing microglia-mediated neuroinflammation by GPR109A/PPAR-γ/TLR4-NF-κB signaling pathway and modulating microbiota−gut−brain axis.

Absolute quantitative lipidomics reveals lipids profiling in liver of mice with early-stage alcoholic liver disease

BackgroundAlcoholic liver disease (ALD) is one of the most prevalent chronic liver disease worldwide. Alcohol-induced alterations in hepatic lipids play an important role in ALD develpoment and progression. The present study aimed to thoroughly describe the changes of lipid profiling in liver of mice with early-stage alcoholic liver disease.MethodsC57BL/6J male mice aged 7-week were randomized into alcohol-fed (AF) group and pair-fed control group (PF) (n = 10 per group). The early stage of ALD was induced with Lieber-DeCarli liquid diet. The lipids profiling was analyzed by absolute quantitative lipidomics with UHPLC-QTRAP-MS/MS.ResultsAlcohol intake significantly increased the levels of alanine aminotransferase (ALT) in plasma, and tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and triacylglycerols (TAG) levels in liver. Lipidomis analyses showed that 41 TAGs were up-regulated and 8 TAGs were down-regulated in response to alcohol intake. The 8 decreased TAGs were with more double bond, longer carbon chain length and mostly contained docosahexaenoic acid (C22:6n-3) and eicosapentaenoic acid (C20:5n-3), compared with the up-regulated TAGs. Furthermore, the down-regulated TAG(56:9)_FA20:5 was inversely associated with ALT and IL-6 levels. In addition, several altered lysophosphatidylcholines (LPC), lysophosphatidylethanolamines (LPE) and hexosylceramides (HCER) were all significantly decreased in response to alcohol consumption, especially HCer(18:1/22:0), with the top reduction among all the down-regulated lipids.ConclusionsThese findings suggest that not only the up-regulated lipids, alcohol-induced reduction in some specific lipids might also contribute to the ALD development, especially TAG(56:9)_FA20:5 and HCer(18:1/22:0). Their physiological functions and effects on ALD development warrants further investigation.

PDE5 inhibitor sildenafil attenuates cardiac microRNA 214 upregulation and pro-apoptotic signaling after chronic alcohol ingestion in mice.

Abusive chronic alcohol consumption can cause metabolic and functional derangements in the heart and is a risk factor for development of non-ischemic cardiomyopathy. microRNA 214 (miR-214) is a molecular sensor of stress signals that negatively impacts cell survival. Considering cardioprotective and microRNA modulatory effects of sildenafil, a phosphodiesterase 5 (PDE5) inhibitor, we investigated the impact of chronic alcohol consumption on cardiac expression of miR-214 and its anti-apoptotic protein target, Bcl-2 and whether sildenafil attenuates such changes. Adult male FVB mice received unlimited access to either normal liquid diet (control), alcohol diet (35% daily calories intake), or alcohol + sildenafil (1mg/kg/day, p.o.) for 14weeks (n = 6-7/group). The alcohol-fed groups with or without sildenafil had increased total diet consumption and lower body weight as compared with controls. Echocardiography-assessed left ventricular function was unaltered by 14-week alcohol intake. Alcohol-fed group had 2.6-fold increase in miR-214 and significant decrease in Bcl-2 expression, along with enhanced phosphorylation of ERK1/2 and cleavage of PARP (marker of apoptotic DNA damage) in the heart. Co-ingestion with sildenafil blunted the alcohol-induced increase in miR-214, ERK1/2 phosphorylation, and maintained Bcl-2 and decreased PARP cleavage levels. In conclusion, chronic alcohol consumption triggers miR-214-mediated pro-apoptotic signaling in the heart, which was prevented by co-treatment with sildenafil. Thus, PDE5 inhibition may serve as a novel protective strategy against cardiac apoptosis due to chronic alcohol abuse.

Lactobacillus fermentum KP-3-fermented ginseng ameliorates alcohol-induced liver disease in C57BL/6N mice through the AMPK and MAPK pathways.

Panax ginseng was fermented using Lactobacillus fermentum KP-3, and the levels of the minor ginsenosides were measured. Then, the effect of fermented ginseng on alcohol-induced liver injury was investigated. C57BL/6N mice were randomly assigned to 4 groups: pair fed (PF), alcohol fed (AF), alcohol with non-fermented ginseng (AF + NFG) and alcohol with fermented ginseng (AF + FG) groups. After treatment for 8 weeks, fermented ginseng intervention significantly reduced the levels of serum ALT, AST, LPS, TG and TC compared with the AF group. The western-blotting results showed that fermented ginseng activated the adenosine-monophosphate-activated protein kinase (AMPK) pathway to inhibit de novo lipogenesis in the liver and inhibited phosphorylation of p38 through the mitogen-activated protein kinase (MAPK) pathway to alleviate hepatic inflammation, and these effects were superior than those of non-fermented ginseng. Furthermore, fermented ginseng reduced alcohol-induced liver oxidative damage by upregulating the levels of antioxidant enzymes. These findings suggested that the L. fermentum KP-3-fermented ginseng product may be used as a potential dietary nutraceutical for alleviating alcoholic liver injury.

Inulin alleviates inflammation of alcoholic liver disease via SCFAs-inducing suppression of M1 and facilitation of M2 macrophages in mice

BackgroundAlcoholic liver disease (ALD) presents one of the leading causes of cirrhosis worldwide. We have demonstrated that inulin alleviates ALD in mice. However, the exact role of hepatic macrophages in effects of inulin on ALD remains largely unclear. MethodsIn vivo, mice were divided into 4 groups: pair-fed (PF) group (PF/CON), alcohol-fed (AF) group (AF/CON), PF with inulin (INU) group (PF/INU) and AF with INU group (AF/INU). Each group was fed modified Lieber-DeCarli liquid diet with or without alcohol. In vitro, RAW264.7 cell lines were polarized to M1 macrophage (Mψ) or M2 Mψ subsets with lipopolysaccharide (LPS) or interleukin-4 (IL-4) stimulation, respectively. The effects of propionate, butyrate and valeric on macrophage M1/M2 were investigated. ResultsThe contents of propionate, butyrate and valeric were significantly increased in AF/INU group compared with that in the AF/CON group. M1 Mψ, inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α) in AF/INU group were significantly lower than those in AF/CON group. In contrast, M2 Mψ, arginase-1 (Arg-1), and interleukin-10 (IL-10) were notably increased in AF/INU group. In vitro, sodium propionate, sodium butyrate and sodium valerate can suppress M1 Mψ and increase M2 Mψ polarization. ConclusionIn ALD, inulin ameliorates the inflammation via SCFAs-inducing suppression of M1 and facilitation of M2 Mψ, which may potentially contribute to the control of the disease.

Inulin Ameliorates Alcoholic Liver Disease via Suppressing LPS-TLR4-Mψ Axis and Modulating Gut Microbiota in Mice.

Alcoholic liver disease (ALD) represents a chronic liver disorder caused by alcohol abuse. Numerous studies have demonstrated that gut microbiota dysbiosis plays a critical role in ALD pathogenesis. Application of prebiotic, probiotic, and dietary supplementation to the modulation of gut microbiota contributes to a novel approach to the management of ALD. Inulin, a natural prebiotic found in plants, can restore gut dysbiosis in ALD. However, the exact mechanism of dietary inulin in ALD remains largely unknown. Sixty female C57BL/6J mice were randomly divided into 4 groups: pair-fed (PF) group (PF/CON); alcohol-fed (AF) group (AF/CON); PF with inulin (INU) group (PF/INU); and AF with INU group (AF/INU). All mice were fed with isocaloric modified Lieber-DeCarli liquid diets with or without alcohol. After 6weeks of feeding, mice were euthanized and associated indications were investigated. The results showed that chronic ethanol (EtOH) intake led to the loss of body weights, abnormal levels of transaminases, and inflammatory indicators (lipopolysaccharide [LPS], interleukin [IL]-6, IL-10, tumor necrosis factor-α, IL-17A), while inulin administration ameliorated these effects. To further understand the underlying mechanism, we investigated macrophages (Mψs) and gut microbiota in diverse groups. The number of Mψs was reduced after dietary inulin treatment in chronic EtOH exposure. Hepatic Toll-like receptor 4 (TLR4+ ) Mψs in AF/INU group were lower than AF/CON group. 16S rRNA sequencing and analysis of gut microbiota indicated the reduction of Allobaculum, Lactobacillus, and Lactococcus, as well as the increase of Parasutterella in AF group compared with PF control. Increased Allobaculum, Lactobacillus, and Lactococcus but reduced Parasutterella in AF/INU group were confirmed that dietary inulin rectified gut dysbiosis to attenuate ALD. Dietary inulin ameliorates ALD via suppressing LPS-TLR4-Mψ axis and modulating gut microbiota in mice, thus potentially provides theoretical foundation for inulin intervention in the prevention and treatment of ALD.

Increased Autolysis ofμ-Calpain in Skeletal Muscles of Chronic Alcohol-Fed Rats

Proteolysis can proceed via several distinct pathways such as the lysosomal, calcium-dependent, and ubiquitin-proteasome-dependent pathways. Calpains are the main proteases that cleave a large variety of proteins, including the giant sarcomeric proteins, titin and nebulin. Chronic ethanol feeding for 6weeks did not affect the activities of μ-calpain and m-calpain in the m.gastrocnemius. In our research, changes in μ-calpain activity were studied in the m.gastrocnemius and m.soleus of chronically alcohol-fed rats after 6months of alcohol intake. SDS-PAGE analysis was applied to detect changes in titin and nebulin contents. Titin phosphorylation analysis was performed using the fluorescent dye Pro-Q Diamond. Western blotting was used to determine μ-calpain autolysis as well as μ-calpain and calpastatin contents. The titin and nebulin mRNA levels were assessed by real-time PCR. The amounts of the autolysed isoform (78kDa) of full-length μ-calpain (80kDa) increased in the m.gastrocnemius and m.soleus of alcohol-fed rats. The calpastatin content increased in m.gastrocnemius. Decreased intact titin-1 (T1) and increased T2-proteolytic fragment contents were found in the m.gastrocnemius and m.soleus of the alcohol-fed rats. The nebulin content decreased in the rat gastrocnemius muscle of the alcohol-fed group. The phosphorylation levels of T1 and T2 were increased in the m.gastrocnemius and m.soleus, and decreased titin and nebulin mRNA levels were observed in the m.gastrocnemius. The nebulin mRNA level was increased in the soleus muscle of the alcohol-fed rats. In summary, our data suggest that prolonged chronic alcohol consumption for 6months resulted in increased autolysis of μ-calpain in rat skeletal muscles. These changes were accompanied by reduced titin and nebulin contents, titin hyperphosphorylation, and development of hindlimb muscle atrophy in the alcohol-fed rats.

Flaxseed oil ameliorates alcoholic liver disease via anti-inflammation and modulating gut microbiota in mice

BackgroundAlcoholic liver disease (ALD) represents a chronic wide-spectrum of liver injury caused by consistently excessive alcohol intake. Few satisfactory advances have been made in management of ALD. Thus, novel and more practical treatment options are urgently needed. Flaxseed oil (FO) is rich in α-linolenic acid (ALA), a plant-derived n-3 polyunsaturated fatty acids (PUFAs). However, the impact of dietary FO on chronic alcohol consumption remains unknown.MethodsIn this study, we assessed possible effects of dietary FO on attenuation of ALD and associated mechanisms in mice. Firstly, mice were randomly allocated into four groups: pair-fed (PF) with corn oil (CO) group (PF/CO); alcohol-fed (AF) with CO group (AF/CO); PF with FO group (PF/FO); AF with FO group (AF/FO). Each group was fed modified Lieber-DeCarli liquid diets containing isocaloric maltose dextrin a control or alcohol with corn oil and flaxseed oil, respectively. After 6 weeks feeding, mice were euthanized and associated indications were investigated.ResultsBody weight (BW) was significantly elevated in AF/FO group compared with AF/CO group. Dietary FO reduced the abnormal elevated aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in chronic ethanol consumption. Amelioration of these parameters as well as liver injury via HE staining in dietary FO supplementation in ALD demonstrated that dietary FO can effectively benefit for the protection against ALD. To further understand the underlying mechanisms, we investigated the inflammatory cytokine levels and gut microbiota. A series of inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-10, were determined. As a result, TNF-α, IL-1β and IL-6 were decreased in AF/FO group compared with control group; IL-10 showed no significant alteration between AF/CO and AF/FO groups (p > 0.05). Sequencing and analysis of gut microbiota gene indicated that a reduction of Porphyromonadaceae and Parasutterella, as well as an increase in Firmicutes and Parabacteroides, were seen in AF group compared with PF control. Furthermore, dietary FO in ethanol consumption group induced a significant reduction in Proteobacteria and Porphyromonadaceae compared with AF/CO group.ConclusionDietary FO ameliorates alcoholic liver disease via anti-inflammation and modulating gut microbiota, thus can potentially serve as an inexpensive interventions for the prevention and treatment of ALD.

The effect of chronic alcohol consumption on mitochondrial calcium handling in hepatocytes.

The damage to liver mitochondria is universally observed in both humans and animal models after excessive alcohol consumption. Acute alcohol treatment has been shown to stimulate calcium (Ca2+) release from internal stores in hepatocytes. The resultant increase in cytosolic Ca2+ is expected to be accumulated by neighboring mitochondria, which could potentially lead to mitochondrial Ca2+ overload and injury. Our data indicate that total and free mitochondrial matrix Ca2+ levels are, indeed, elevated in hepatocytes isolated from alcohol-fed rats compared with their pair-fed control littermates. In permeabilized hepatocytes, the rates of mitochondrial Ca2+ uptake were substantially increased after chronic alcohol feeding, whereas those of mitochondrial Ca2+ efflux were decreased. The changes in mitochondrial Ca2+ handling could be explained by an up-regulation of the mitochondrial Ca2+ uniporter and loss of a cyclosporin A-sensitive Ca2+ transport pathway. In intact cells, hormone-induced increases in mitochondrial Ca2+ declined at slower rates leading to more prolonged elevations of matrix Ca2+ in the alcohol-fed group compared with controls. Moreover, treatment with submaximal concentrations of Ca2+-mobilizing hormones markedly increased the levels of mitochondrial reactive oxygen species (ROS) in hepatocytes from alcohol-fed rats, but did not affect ROS levels in controls. The changes in mitochondrial Ca2+ handling are expected to buffer and attenuate cytosolic Ca2+ increases induced by acute alcohol exposure or hormone stimulation. However, these alterations in mitochondrial Ca2+ handling may also lead to Ca2+ overload during cytosolic Ca2+ increases, which may stimulate the production of mitochondrial ROS, and thus contribute to alcohol-induced liver injury.

Dissociation between diurnal cycles in locomotor activity, feeding behavior and hepatic PERIOD2 expression in chronic alcohol-fed mice

Chronic alcohol consumption contributes to fatty liver disease. Our studies revealed that the hepatic circadian clock is disturbed in alcohol-induced hepatic steatosis, and effects of chronic alcohol administration upon the clock itself may contribute to steatosis. We extended these findings to explore the effects of chronic alcohol treatment on daily feeding and locomotor activity patterns. Mice were chronically pair-fed ad libitum for 4 weeks using the Lieber-DeCarli liquid diet, with calorie-controlled liquid and standard chow diets as control groups. Locomotor activity, feeding activity, and real-time bioluminescence recording of PERIOD2::LUCIFERASE expression in tissue explants were measured. Mice on liquid control and chow diets exhibited normal profiles of locomotor activity, with a ratio of 22:78% day/night activity and a peak during early night. This pattern was dramatically altered in alcohol-fed mice, marked by a 49:51% ratio and the absence of a distinct peak. While chow-diet fed mice had a normal 24:76% ratio of feeding activity, with a peak in the early night, this pattern was dramatically altered in both liquid-diet groups: mice had a 43:57% ratio, and an absence of a distinct peak. Temporal differences were also observed between the two liquid-diet groups during late day. Cosinor analysis revealed a ∼4–h and ∼6–h shift in the alcohol-fed group feeding and locomotor activity rhythms, respectively. Analysis of hepatic PER2 expression revealed that the molecular clock in alcohol-fed and control liquid-diet mice was shifted by ∼11 h and ∼6 h, respectively. No differences were observed in suprachiasmatic nucleus explants, suggesting that changes in circadian phase in the liver were generated independently from the central clock. These results suggest that chronic alcohol consumption and a liquid diet can differentially modulate the daily rhythmicity of locomotor and feeding behaviors, aspects that might contribute to disturbances in the circadian timing system and development of hepatic steatosis.

Fucoidan from Fucus vesiculosus protects against alcohol-induced liver damage by modulating inflammatory mediators in mice and HepG2 cells.

Fucoidan is an l-fucose-enriched sulfated polysaccharide isolated from brown algae and marine invertebrates. In this study, we investigated the protective effect of fucoidan from Fucus vesiculosus on alcohol-induced murine liver damage. Liver injury was induced by oral administration of 25% alcohol with or without fucoidan (30 mg/kg or 60 mg/kg) for seven days. Alcohol administration increased serum aspartate aminotransferase and alanine aminotransferase levels, but these increases were suppressed by the treatment of fucoidan. Transforming growth factor beta 1 (TGF-β1), a liver fibrosis-inducing factor, was highly expressed in the alcohol-fed group and human hepatoma HepG2 cell; however, the increase in TGF-β1 expression was reduced following fucoidan administration. Treatment with fucoidan was also found to significantly reduce the production of inflammation-promoting cyclooygenase-2 and nitric oxide, while markedly increasing the expression of the hepatoprotective enzyme, hemeoxygenase-1, on murine liver and HepG2 cells. Taken together, the antifibrotic and anti-inflammatory effects of fucoidan on alcohol-induced liver damage may provide valuable insights into developing new therapeutics or interventions.

P-27 * THE EFFECTS OF CHRONIC ALCOHOL CONSUMPTION ON ADIPONECTIN AND INSULIN RESISTANCE

The purpose of this study was to understand the effects of chronic alcohol consumption on diabetes. We used 14-week-old OLETF rats which have type 2 diabetes (T2DM) and non-diabetic control LETO rats. The rats were randomized into four groups. : (1) OLETF-Control(OC, n = 10), (2) OLETF-Alcohol (OE, n = 10), (3) LETO-Control (LC, n = 10), (4) LETO-Alcohol LE, n = 10). Rats in the alcohol-fed group were fed with a liquid alcohol diet over 10 week period. After each period of 5 week and 10 week feeding, we did Intraperitoneal Glucose Tolerance Testing (IP-GTT) and measured the blood adiponectin and insulin levels. The first IP-GTT data was not shown any significant difference between groups. After 10 weeks 30 minute-glucose levels of OE group is significantly higher than OC group. And after 90 minute, the glucose level of OE group was also significantly higher than OC group. The level of insulin was increased in OE group than OC group. (p < 0.05) The level of adiponectin in LE group and OE group was significantly lower than each control group. We suggest heavy alcohol consumption reduced the level of adiponectin which regulate insulin sensitivity. Reduced adiponectin level induced the insulin resistance which is one of the main factors of worsening T2DM in diabetic animal model.

Effects of chronic ethanol consumption on rat upper gastrointestinal system: Functional and histologic findings

The purpose of the present study was to determine the effect of chronic alcohol consumption on reactivity of esophageal tunica muscularis mucosae (TMM) and lower esophageal sphincter (LES) smooth muscle. Six male rats in alcohol-fed group received ethanol (7.2% v/v) in a modified liquid diet for 4 weeks. Two control groups were used; six rats in the standard diet-fed group received rat chow and water for 4 weeks. Six rats in sucrose-fed group were given sucrose and received a liquid diet. The smooth muscle reactivity of TMM and LES strips from ethanol-fed and control animals was evaluated in organ chambers. Also histologic study was undertaken to show effects of chronic alcohol consumption. Maximum contractile responses of TMM to KCl and carbachol were decreased in the ethanol-fed group compared to the control groups. Relaxant responses to serotonin were decreased in the ethanol-fed group compared to the control groups. In TMM, isoproterenol- and papaverine-induced relaxant responses were similar in the ethanol-fed and control groups. In LES smooth muscle, relaxant responses to papaverine or isoproterenol were similar in the control groups and the ethanol-fed group. There was no change in agonist potency among the groups. The relaxation response elicited by nicotine and sodium nitroprusside (SNP) or contractile response elicited by carbachol and 80 mM KCl was decreased with maximum responses and pD2 values, in the ethanol-fed group compared to that of the control groups in LES. Decreased nNOS immunoreactivity in myenteric plexus was found in alcohol-exposed group compared to control groups. Our findings suggest that chronic alcohol consumption impairs relaxant and contractile responses of both TMM and LES smooth muscle and it may contribute to gastroesophageal reflux commonly seen after alcohol binges.

Roles of PYK2 and caveolin-1 in alcoholic cardiomyopathy and the effect of drug intervention

To explore the protein expressions of PYK2 (protein-rich tyrosine kinase-2) and caveolin-1 in alcoholic cardiomyopathy (ACM) dog model and the effect of early drug intervention. A total of 28 adult dogs were randomly divided into 4 groups: (i): alcohol-fed; (ii): alcohol plus valsartan, an angiotensin receptor blocker (ARB); (iii): alcohol plus carnitine; (iv): control group. At Month 6, the cardiac functions of all animals were evaluated by echocardiography. The concentrations of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) in cardiac tissue were detected by chronometry. The protein expressions of collagen types I and III were determined by ELISA (enzyme-linked immunosorbent assay) while those of PYK2 and caveolin-1 evaluated by immunohistochemistry. The internal relationship of PYK2, caveolin-1, MDA and collagen types I & III was analyzed. Compared with the control group, the contents of SOD and GSH-Px significantly decreased in the alcohol, valsartan and carnitine groups while MDA significantly increased in the alcohol and carnitine groups. The relative contents in each group were as follows: MDA [alcohol: (28 ± 5) U/mg Pro; valsartan: (33 ± 13) U/mg Pro; carnitine: (33 ± 10) U/mg Pro], GSH-Px [alcohol: (188 ± 37) U/g Pro; valsartan: (362 ± 29) U/g Pro; carnitine: (282 ± 29) U/g Pro and MDA [alcohol: (19.4 ± 3.0) nmol/mg Pro; carnitine: (10.8 ± 2.1) nmol/mg Pro]. Compared with the control group, the concentration of collagen typeI significantly increased in the alcohol, valsartan and carnitine groups. In the alcohol-fed group, the protein expression of PYK2 gradually increased with the progression of ACM while that of caveolin-1 stayed at a low level. Through the interventions of valsartan and carnitine, the protein expression of PYK2 significantly increased while that of caveolin-1 significantly decreased (all P < 0.01). Alcohol promotes the myocardial oxidative stress and fibrosis through an elevated expression of PYK2 and a lowered expression of caveolin-1. Then a deterioration of cardiac structures and functions occur.

Effects of chronic alcohol consumption on expression levels of APP and Aβ-producing enzymes

Chronic alcohol consumption contributes to numerous diseases, including cancers, cardiovascular diseases, and liver cirrhosis. Epidemiological studies have shown that excessive alcohol consumption is a risk factor for dementia. Along this line, Alzheimer's disease (AD) is the most common form of dementia and is caused by the accumulation of amyloid-β (Aβ plaques in neurons. In this study, we hypothesized that chronic ethanol consumption is associated with pathological processing of APP in AD. To investigate the relationship between chronic alcohol consumption and Aβ production, brain samples from rats fed an alcohol liquid diet for 5 weeks were analyzed. We show that the expression levels of APP, BACE1, and immature nicastrin were increased in the cerebellum, hippocampus, and striatum of the alcohol-fed group compared to the control group. Total nicastrin and PS1 levels were induced in the hippocampus of alcohol-fed rats. These data suggest that the altered expression of APP and Aβ-producing enzymes possibly contributes to the chronic alcohol consumption-mediated pathogenesis of AD.

Role of homocysteine in the pathogenesis of alcoholic cardiomyopathy

To explore the role of homocysteine in the pathogenesis of alcoholic cardiomyopathy. A total of 69 male Wistar rats were randomly assigned into two groups: alcohol-fed group and the control. Cardiac function was assessed by pulse Doppler. Plasma Hcy levels were examined using automatic biochemical instrument (chemiluminescence). The protein expression of MMP-9 was evaluated using immunohistochemical method, and collagen fiber of myocardium was quantitative analyzed by Masson stain. After heavy drinking, the LVEDd of alcohol-fed group were larger than the control group [(7.0±0.6) mm vs (5.0±0.4) mm, P<0.05], the LVEF and FS were lower in the 4th month (52%±8% vs 78%±4%, 31%±3% vs 47%±2%, P<0.05), the data changed more significantly (P<0.01) in the 6th month. The level of plasma Hcy from alcohol-fed group was significantly higher from the 2nd month than that before the experiment [(18.1±3.1) µmol/L vs (9.8±2.1) µmol/L, P<0.01], and it was higher in 4th month than that in 2nd month [(26.3±4.0) µmol/L vs (18.1±3.1) µmol/L, P<0.05], it was highest in 6 months. After 4-month and 6-month drinking, the expression of MMP-9 protein from alcohol group was higher than before the experiment (0.161%±0.019%, 0.263%±0.014% vs 0.050%±0.008%, P<0.01). Masson staining showed myocardial collagen of alcohol group was more after 4-month and 6-month drinking than those before the experiment (10.23%±1.20% vs 0.50%±0.09%; 22.41%±2.57% vs 0.50%±0.09%, P<0.01). Plasma Hcy and cardiac tissue MMP-9 is a significant positive correlation (r=0.848, P<0.01). Long-term and large drink liquor can lead to plasma Hcy levels significantly increased, and participate cardiac remodeling and the pathogenesis of ACM through increasing the expression of myocardial tissue MMP-9 protein.

Up-regulation of MicroRNA-155 in Macrophages Contributes to Increased Tumor Necrosis Factor α (TNFα) Production via Increased mRNA Half-life in Alcoholic Liver Disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcohol-induced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

Exploring the molecular mechanisms underlying the potentiation of exogenous growth hormone on alcohol-induced fatty liver diseases in mice

BackgroundGrowth hormone (GH) is an essential regulator of intrahepatic lipid metabolism by activating multiple complex hepatic signaling cascades. Here, we examined whether chronic exogenous GH administration (via gene therapy) could ameliorate liver steatosis in animal models of alcoholic fatty liver disease (AFLD) and explored the underlying molecular mechanisms.MethodsMale C57BL/6J mice were fed either an alcohol or a control liquid diet with or without GH therapy for 6 weeks. Biochemical parameters, liver histology, oxidative stress markers, and serum high molecular weight (HMW) adiponectin were measured. Quantitative real-time PCR and western blotting were also conducted to determine the underlying molecular mechanism.ResultsSerum HMW adiponectin levels were significantly higher in the GH1-treated control group than in the control group (3.98 ± 0.71 μg/mL vs. 3.07 ± 0.55 μg/mL; P < 0.001). GH1 therapy reversed HMW adiponectin levels to the normal levels in the alcohol-fed group. Alcohol feeding significantly reduced hepatic adipoR2 mRNA expression compared with that in the control group (0.71 ± 0.17 vs. 1.03 ± 0.19; P < 0.001), which was reversed by GH therapy. GH1 therapy also significantly increased the relative mRNA (1.98 ± 0.15 vs. 0.98 ± 0.15) and protein levels of SIRT1 (2.18 ± 0.37 vs. 0.99 ± 0.17) in the alcohol-fed group compared with those in the control group (both, P < 0.001). The alcohol diet decreased the phosphorylated and total protein levels of hepatic AMP-activated kinase-α (AMPKα) (phosphorylated protein: 0.40 ± 0.14 vs. 1.00 ± 0.12; total protein: 0.32 ± 0.12 vs. 1.00 ± 0.14; both, P < 0.001) and peroxisome proliferator activated receptor-α (PPARα) (phosphorylated protein: 0.30 ± 0.09 vs. 1.00 ± 0.09; total protein: 0.27 ± 0.10 vs. 1.00 ± 0.13; both, P < 0.001), which were restored by GH1 therapy. GH therapy also decreased the severity of fatty liver in alcohol-fed mice.ConclusionsGH therapy had positive effects on AFLD and may offer a promising approach to prevent or treat AFLD. These beneficial effects of GH on AFLD were achieved through the activation of the hepatic adiponectin-SIRT1-AMPK and PPARα-AMPK signaling systems.