Sex-Specific Effect of Ethanol on Colon Content Lipidome in a Mice Model Using Nontargeted LC/MS.

Consumption of alcohol has widespread effects on the human body. The organs that are most significantly impacted are the liver and digestive system. When alcohol is consumed, it is absorbed in the intestines and processed by the liver. However, excessive alcohol use may affect gut epithelial integrity, microbiome composition, and lipid metabolism. Despite past studies investigating the effect of ethanol on hepatic lipid metabolism, the focus on colonic lipid metabolism has not been well explored. In this study, we investigated the sex-specific effect of ethanol on the colonic content lipidome in a mouse model using nontargeted liquid chromatography-mass spectrometry. Comprehensive lipidome analysis of colonic flush samples was performed using ethanol-fed (EF) and pair-fed (PF) mice of each sex. Partial least-squares discriminant analysis revealed that ethanol altered colonic lipid composition largely in male mice compared with female mice. A significant increase in free fatty acids, ceramides, and hexosylceramides and decreased phosphatidylglycerols (PG) was observed in the EF group compared to the PF group in male mice. Phosphatidylethanolamine (PE) levels were increased significantly in the EF group of both sexes compared to the PF group. The volcanic plot shows that PG (O-15:1/15:0) and PE (O-18:2/15:0) are common markers that are increased in both sexes of the EF group. In addition, decreased fatty acid esters of hydroxy fatty acids (FAHFA) were observed specifically in the EF group of female mice. Overall, a significant variation in the mice colonic content lipidome between the EF and PF groups was observed. Target pathways, such as sphingolipid metabolism in males, FAHFA in females, and PE metabolism in both sexes, were suggested. This study provides new insight into the sex-dependent lipid change associated with alcohol-induced gut-microbiota dysfunction and its potential health impacts.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

Introduction: The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Methods: Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Results: Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. Conclusion: These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

TRPV6 deficiency attenuates stress and corticosterone-mediated exacerbation of alcohol-induced gut barrier dysfunction and systemic inflammation.

Introduction: Chronic stress is co-morbid with alcohol use disorder that feedback on one another, thus impeding recovery from both disorders. Stress and the stress hormone corticosterone aggravate alcohol-induced intestinal permeability and liver damage. However, the mechanisms involved in compounding tissue injury by stress/corticosterone and alcohol are poorly defined. Here we explored the involvement of the TRPV6 channel in stress (or corticosterone) 3and alcohol-induced intestinal epithelial permeability, microbiota dysbiosis, and systemic inflammation. Methods: Chronic alcohol feeding was performed on adult wild-type and Trpv6-/- mice with or without corticosterone treatment or chronic restraint stress (CRS). The barrier function was determined by evaluating inulin permeability in vivo and assessing tight junction (TJ) and adherens junction (AJ) integrity by immunofluorescence microscopy. The gut microbiota composition was evaluated by 16S rRNA sequencing and metagenomic analyses. Systemic responses were assessed by evaluating endotoxemia, systemic inflammation, and liver damage. Results: Corticosterone and CRS disrupted TJ and AJ, increased intestinal mucosal permeability, and caused endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. Corticosterone and CRS synergistically potentiated the alcohol-induced breakdown of intestinal epithelial junctions, mucosal barrier impairment, endotoxemia, systemic inflammation, and liver damage in wild-type but not Trpv6-/- mice. TRPV6 deficiency also blocked the effects of CRS and CRS-mediated potentiation of alcohol-induced dysbiosis of gut microbiota. Conclusions: These findings indicate an essential role of TRPV6 in stress, corticosterone, and alcohol-induced intestinal permeability, microbiota dysbiosis, endotoxemia, systemic inflammation, and liver injury. This study identifies TRPV6 as a potential therapeutic target for developing treatment strategies for stress and alcohol-associated comorbidity.

Sex-Specific Whole-Transcriptome Analysis in the Cerebral Cortex of FAE Offspring.

Fetal alcohol spectrum disorders (FASDs) are associated with systemic inflammation and neurodevelopmental abnormalities. Several candidate genes were found to be associated with fetal alcohol exposure (FAE)-associated behaviors, but a sex-specific complete transcriptomic analysis was not performed at the adult stage. Recent studies have shown that they are regulated at the developmental stage. However, the sex-specific role of RNA in FAE offspring brain development and function has not been studied yet. Here, we carried out the first systematic RNA profiling by utilizing a high-throughput transcriptomic (RNA-seq) approach in response to FAE in the brain cortex of male and female offspring at adulthood (P60). Our RNA-seq data analysis suggests that the changes in RNA expression in response to FAE are marked sex-specific. We show that the genes Muc3a, Pttg1, Rec8, Clcnka, Capn11, and pnp2 exhibit significantly higher expression in the male offspring than in the female offspring at P60. FAE female mouse brain sequencing data also show an increased expression of Eno1, Tpm3, and Pcdhb2 compared to male offspring. We performed a pathway analysis using a commercial software package (Ingenuity Pathway Analysis). We found that the sex-specific top regulator genes (Rictor, Gaba, Fmri, Mlxipl) are highly associated with eIF2 (translation initiation), synaptogenesis (the formation of synapses between neurons in the nervous system), sirtuin (metabolic regulation), and estrogen receptor (involved in obesity, aging, and cancer) signaling. Taken together, our transcriptomic results demonstrate that FAE differentially alters RNA expression in the adult brain in a sex-specific manner.

Photonics probing of pup brain tissue and molecular-specific nuclear nanostructure alterations due to fetal alcoholism via light scattering/localization approaches.

SIGNIFICANCE: Light is a good probe for studying the nanoscale-level structural or molecular-specific structural properties of brain cells/tissue due to stress, alcohol, or any other abnormalities. Chronic alcoholism during pregnancy, i.e., fetal alcoholism, being teratogenic, results in fetal alcohol syndrome, and other neurological disorders. Understanding the nano-to-submicron scale spatial structural properties of pup brain cells/tissues using light/photonic probes could provide a plethora of information in understanding the effects of fetal alcoholism. AIM: Using both light scattering and light localization techniques to probe alterations in nano- to-submicron scale mass density or refractive index fluctuations in brain cells/tissues of mice pups, exposed to fetal alcoholism. APPROACH: We use the mesoscopic physics-based dual spectroscopic imaging techniques, partial wave spectroscopy (PWS) and molecular-specific inverse participation ratio (IPR) using confocal imaging, to quantify structural alterations in brain tissues and chromatin/histone in brain cells, respectively, in 60 days postnatal mice pup brain, exposed to fetal alcoholism. RESULTS: The finer focusing PWS analysis on tissues shows an increase in the degree of structural disorder strength in the pup brain tissues. Furthermore, results of the molecular-specific light localization IPR technique show an increase in the degree of spatial molecular mass density structural disorder in DNA and a decrease in the degree in histone. CONCLUSIONS: In particular, we characterize the spatial pup brain structures from the molecular to tissue levels and address the plausible reasons for such as mass density fluctuations in fetal alcoholism.

TRPV6 channel mediates alcohol-induced gut barrier dysfunction and systemic response.

Intestinal epithelial tight junction disruption is a primary contributing factor in alcohol-associated endotoxemia, systemic inflammation, and multiple organ damage. Ethanol and acetaldehyde disrupt tight junctions by elevating intracellular Ca2+. Here we identify TRPV6, a Ca2+-permeable channel, as responsible for alcohol-induced elevation of intracellular Ca2+, intestinal barrier dysfunction, and systemic inflammation. Ethanol and acetaldehyde elicit TRPV6 ionic currents in Caco-2 cells. Studies in Caco-2 cell monolayers and mouse intestinal organoids show that TRPV6 deficiency or inhibition attenuates ethanol- and acetaldehyde-induced Ca2+ influx, tight junction disruption, and barrier dysfunction. Moreover, Trpv6-/- mice are resistant to alcohol-induced intestinal barrier dysfunction. Photoaffinity labeling of 3-azibutanol identifies a histidine as a potential alcohol-binding site in TRPV6. The substitution of this histidine, and a nearby arginine, reduces ethanol-activated currents. Our findings reveal that TRPV6 is required for alcohol-induced gut barrier dysfunction and inflammation. Molecules that decrease TRPV6 function have the potential to attenuate alcohol-associated tissue injury.

Prevention and mitigation of alcohol-induced neuroinflammation by Lactobacillus plantarum by an EGF receptor-dependent mechanism.

Neuroinflammation is implicated in the pathogenesis of alcohol use disorders. We investigated the role of Gut-Brain interactions in alcohol-induced neuroinflammation by probiotic-mediated manipulation of intestinal dysbiosis in mice. Chronic ethanol feeding induced dysbiosis, as evidenced by an increase in Firmicutes/Bacteroidetes ratio and depletion of Lactobacillus species in the colon. Ethanol increased the levels of IL-1ß, IL-6, and TNFα in plasma and the mRNA for IL-1ß, IL-6, TNFα, and MCP1 genes in the cerebral cortex and hippocampus. Ethanol feeding increased inulin flux from the circulation into different brain regions, accompanied by the increase in TLR4 mRNA levels in the cerebral cortex and hippocampus. The immunofluorescence confocal microscopy showed that ethanol elevates the expression of microglial activation marker TMEM119 in the cerebral cortex. Feeding L. plantarum suppressed the ethanol-induced dysbiosis to some extent, as evidenced by attenuation of ethanol effects on Firmicutes/Bacteroidetes ratio and abundance of Lactobacillus spp. L. plantarum blocked ethanol-induced elevation of plasma cytokines, inulin permeability to the brain, mRNA for TLR4, IL-1ß, IL-6, TNFα, and MCP1 in brain regions, and the expression of TMEM119 in the cerebral cortex. The L. plantarum effect was absent in mice that express a dominant-negative EGFR, suggesting that the EGFR receptor plays an essential role in the protective effect of L. plantarum against ethanol-induced neuroinflammation. L. plantarum, when administered after chronic ethanol-induced injury, rescued the ethanol-induced systemic inflammation and neuroinflammation. This study demonstrates that L. plantarum in the gut prevents and mitigates ethanol-induced neuroinflammation by an EGFR-dependent mechanism.

Photonic technique to study the effects of probiotics on chronic alcoholic brain cells by quantifying their molecular specific structural alterations via confocal imaging.

Molecular specific photonics localization method, the inverse participation ratio (IPR) technique, is a powerful procedure to probe the nano- to submicron scales structural alterations in cells/tissues in their abnormalities due to chronic alcoholism using confocal imaging. Chronic alcoholism introduces abnormalities in brain cells/tissue at the nanoscale level that results in behavioural and psychological disorders which are not well understood. On the other hand, probiotics such as Lactobacillus plantarum enhances brain functions in chronic alcoholism. Using the IPR technique, we probe the molecular specific spatial structural alterations in glial brain cells astrocytes and microglia, as well as in chromatins in the nuclei of cortex brain cells, with or without probiotic treatments in chronic alcoholism. The results show chronic alcoholism alone harms brain cells and the probiotic treatment in chronic alcoholism reverses alcoholic damage in the brain cells/tissues toward normalcy.

Central role of intestinal epithelial glucocorticoid receptor in alcohol- and corticosterone-induced gut permeability and systemic response.

Corticosterone, the stress hormone, exacerbates alcohol-associated tissue injury, but the mechanism involved is unknown. We examined the role of the glucocorticoid receptor (GR) in corticosterone-mediated potentiation of alcohol-induced gut barrier dysfunction and systemic response. Hepatocyte-specific GR-deficient (GRΔHC ) and intestinal epithelial-specific GR-deficient (GRΔIEC ) mice were fed ethanol, combined with corticosterone treatment. Intestinal epithelial tight junction integrity, mucosal barrier function, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation were assessed. Corticosterone potentiated ethanol-induced epithelial tight junction disruption, mucosal permeability, and inflammatory response in GRΔHC mouse colon; these effects of ethanol and corticosterone were absent in GRΔIEC mice. Gut microbiota compositions in ethanol-fed GRΔHC and GRΔIEC mice were similar to each other. However, corticosterone treatment in ethanol-fed mice shifted the microbiota composition to distinctly different directions in GRΔHC and GRΔIEC mice. Ethanol and corticosterone synergistically elevated the abundance of Enterobacteriaceae and Escherichia coli and reduced the abundance of Lactobacillus in GRΔHC mice but not in GRΔIEC mice. In GRΔHC mice, corticosterone potentiated ethanol-induced endotoxemia and systemic inflammation, but these effects were absent in GRΔIEC mice. Interestingly, ethanol-induced liver damage and its potentiation by corticosterone were observed in GRΔHC mice but not in GRΔIEC mice. GRΔIEC mice were also resistant to ethanol- and corticosterone-induced inflammatory response in the hypothalamus. These data indicate that the intestinal epithelial GR plays a central role in alcohol- and corticosterone-induced gut barrier dysfunction, microbiota dysbiosis, endotoxemia, systemic inflammation, liver damage, and neuroinflammation. This study identifies a novel target for potential therapeutic for alcohol-associated tissue injury.

Lactobacillus casei and Epidermal Growth Factor Prevent Osmotic Stress-Induced Tight Junction Disruption in Caco-2 Cell Monolayers.

Osmotic stress plays a crucial role in the pathogenesis of many gastrointestinal diseases. Lactobacillus casei and epidermal growth factor (EGF) effects on the osmotic stress-induced epithelial junctional disruption and barrier dysfunction were investigated. Caco-2 cell monolayers were exposed to osmotic stress in the presence or absence of L. casei or EGF, and the barrier function was evaluated by measuring inulin permeability. Tight junction (TJ) and adherens junction integrity were assessed by immunofluorescence confocal microscopy. The role of signaling molecules in the L. casei and EGF effects was determined by using selective inhibitors. Data show that pretreatment of cell monolayers with L. casei or EGF attenuates osmotic stress-induced TJ and adherens junction disruption and barrier dysfunction. EGF also blocked osmotic stress-induced actin cytoskeleton remodeling. U0126 (MEK1/2 inhibitor), the MAP kinase inhibitor, blocked EGF-mediated epithelial protection from osmotic stress. In contrast, the L. casei-mediated epithelial protection from osmotic stress was unaffected by U0126, AG1478 (EGFR tyrosine kinase inhibitor), SP600125 (JNK1/2 inhibitor), or SB202190 (P38 MAP kinase inhibitor). On the other hand, Ro-32-0432 (PKC inhibitor) blocked the L. casei-mediated prevention of osmotic stress-induced TJ disruption and barrier dysfunction. The combination of EGF and L. casei is more potent in protecting the barrier function from osmotic stress. These findings suggest that L. casei and EGF ameliorate osmotic stress-induced disruption of apical junctional complexes and barrier dysfunction in the intestinal epithelium by distinct signaling mechanisms.

Alterations in the Gut-Microbial-Inflammasome-Brain Axis in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder characterized by memory loss and cognitive decline, is a major cause of death and disability among the older population. Despite decades of scientific research, the underlying etiological triggers are unknown. Recent studies suggested that gut microbiota can influence AD progression; however, potential mechanisms linking the gut microbiota with AD pathogenesis remain obscure. In the present study, we provided a potential mechanistic link between dysbiotic gut microbiota and neuroinflammation associated with AD progression. Using a mouse model of AD, we discovered that unfavorable gut microbiota are correlated with abnormally elevated expression of gut NLRP3 and lead to peripheral inflammasome activation, which in turn exacerbates AD-associated neuroinflammation. To this end, we observe significantly altered gut microbiota compositions in young and old 5xFAD mice compared to age-matched non-transgenic mice. Moreover, 5xFAD mice demonstrated compromised gut barrier function as evident from the loss of tight junction and adherens junction proteins compared to non-transgenic mice. Concurrently, we observed increased expression of NLRP3 inflammasome and IL-1ß production in the 5xFAD gut. Consistent with our hypothesis, increased gut-microbial-inflammasome activation is positively correlated with enhanced astrogliosis and microglial activation, along with higher expression of NLRP3 inflammasome and IL-1ß production in the brains of 5xFAD mice. These data indicate that the elevated expression of gut-microbial-inflammasome components may be an important trigger for subsequent downstream activation of inflammatory and potentially cytotoxic mediators, and gastrointestinal NLRP3 may promote NLRP3 inflammasome-mediated neuroinflammation. Thus, modulation of the gut microbiota may be a potential strategy for the treatment of AD-related neurological disorders in genetically susceptible hosts.

Chronic stress and corticosterone exacerbate alcohol-induced tissue injury in the gut-liver-brain axis.

Alcohol use disorders are associated with altered stress responses, but the impact of stress or stress hormones on alcohol-associated tissue injury remain unknown. We evaluated the effects of chronic restraint stress on alcohol-induced gut barrier dysfunction and liver damage in mice. To determine whether corticosterone is the stress hormone associated with the stress-induced effects, we evaluated the effect of chronic corticosterone treatment on alcoholic tissue injury at the Gut-Liver-Brain (GLB) axis. Chronic restraint stress synergized alcohol-induced epithelial tight junction disruption and mucosal barrier dysfunction in the mouse intestine. These effects of stress on the gut were reproduced by corticosterone treatment. Corticosterone synergized alcohol-induced expression of inflammatory cytokines and chemokines in the colonic mucosa, and it potentiated the alcohol-induced endotoxemia and systemic inflammation. Corticosterone also potentiated alcohol-induced liver damage and neuroinflammation. Metagenomic analyses of 16S RNA from fecal samples indicated that corticosterone modulates alcohol-induced changes in the diversity and abundance of gut microbiota. In Caco-2 cell monolayers, corticosterone dose-dependently potentiated ethanol and acetaldehyde-induced tight junction disruption and barrier dysfunction. These data indicate that chronic stress and corticosterone exacerbate alcohol-induced mucosal barrier dysfunction, endotoxemia, and systemic alcohol responses. Corticosterone-mediated promotion of alcohol-induced intestinal epithelial barrier dysfunction and modulation of gut microbiota may play a crucial role in the mechanism of stress-induced promotion of alcohol-associated tissue injury at the GLB axis.

TEM study of chronic alcoholism effects on early carcinogenesis by probing the nanoscale structural alterations of cell nuclei.

Nanoscale structural alteration in the nuclei of cells with the progression of carcinogenesis is due to the rearrangements of the basic building blocks in the cell such as DNA, RNA, lipids, etc. Although epigenetic modifications underlie the development of cancer, exposure to carcinogenic chemicals such as alcohol also enhances the development of cancer. We report the effects of chronic alcoholism on early-carcinogenesis based on changes in the degree of nanoscale structural alterations (L d) in nuclei. For this, transmission electron microscopy (TEM) imaging of the nuclei of colonic cells is performed for the following four mouse models: control mice; chronic alcoholic mice treated with ethanol (i.e., EtOH mice); mice treated with colonic carcinogen azoxymethane (AOM) and dextran sulfate sodium (DSS) that induced colitis (i.e., AOM + DSS mice); and chronic alcoholic or EtOH treated mice, together with AOM and DSS treatment (i.e., AOM + DSS + EtOH mice). The disordered optical lattices are constructed from their respective TEM images of thin colonic cell nuclei and the L d values are calculated using the inverse participation ratio (IPR) technique from the spatially localized eigenfunctions of these lattices. Results show no significant difference in the average L d value of the colon cell nuclei of alcohol treated mice relative to its control [i.e., L d(C) ∼ L d(EtOH)]; however, an increase in the L d value of alcohol treated precancerous cells [i.e., L d(AOM + DSS + EtOH) > L d(AOM + DSS)], indicating that alcohol accelerates the early carcinogenic process.

LPAR2 receptor activation attenuates radiation-induced disruption of apical junctional complexes and mucosal barrier dysfunction in mouse colon.

The tight junction (TJ) and barrier function of colonic epithelium is highly sensitive to ionizing radiation. We evaluated the effect of lysophosphatidic acid (LPA) and its analog, Radioprotein-1, on γ-radiation-induced colonic epithelial barrier dysfunction using Caco-2 and m-ICC12 cell monolayers in vitro and mice in vivo. Mice were subjected to either total body irradiation (TBI) or partial body irradiation (PBI-BM5). Intestinal barrier function was assessed by analyzing immunofluorescence localization of TJ proteins, mucosal inulin permeability, and plasma lipopolysaccharide (LPS) levels. Oxidative stress was analyzed by measuring protein thiol oxidation and antioxidant mRNA. In Caco-2 and m-ICC12 cell monolayers, LPA attenuated radiation-induced redistribution of TJ proteins, which was blocked by a Rho-kinase inhibitor. In mice, TBI and PBI-BM5 disrupted colonic epithelial tight junction and adherens junction, increased mucosal permeability, and elevated plasma LPS; TJ disruption by TBI was more severe in Lpar2-/- mice compared to wild-type mice. RP1, administered before or after irradiation, alleviated TBI and PBI-BM5-induced TJ disruption, barrier dysfunction, and endotoxemia accompanied by protein thiol oxidation and downregulation of antioxidant gene expression, cofilin activation, and remodeling of the actin cytoskeleton. These data demonstrate that LPAR2 receptor activation prevents and mitigates γ-irradiation-induced colonic mucosal barrier dysfunction and endotoxemia.

Plasma exosomes exacerbate alcohol- and acetaminophen-induced toxicity via CYP2E1 pathway.

Cellular CYP2E1 is well-known to mediate alcohol- (ALC) and acetaminophen- (APAP) induced toxicity in hepatic and extra-hepatic cells. Although exosomes have been gaining importance in understanding mechanism of intra- and inter-cellular communication, the functional role of drug metabolizing cytochrome P450 (CYP) enzymes in human plasma exosomes are yet to be explored. In our previous study, we reported that human plasma-derived exosomes contain substantial level of functional CYP2E1. In the current project, we investigated the potential role of plasma exosomal CYP2E1 in mediating ALC- and APAP-induced toxicity. We treated hepatic and extra-hepatic (monocytic) cells with exosomes ± ALC/APAP. We observed that the plasma exosomes containing CYP2E1 cargo further exacerbate ALC- and APAP-induced toxicity in both hepatic and monocytic cells. Further, both exosomes- and ALC/APAP-induced toxicity was reduced/abolished by a selective inhibitor of CYP2E1 enzyme activity (diallyl ether). However, only ALC-, but not exosome-induced toxicity was reduced/abolished by CYP2E1 siRNA. These findings suggest that ALC/APAP-induced toxicity in the presence of exosomes are mediated, at least in part, by CYP2E1 enzyme. To validate these in vitro findings, we characterized plasma exosomal contents in a binge-drinking animal model and their effect on ALC/APAP-induced toxicity in monocytic cells. Our results showed that ALC exposure caused a significant induction of the plasma exosomal CYP2E1 level in a binge drinking murine model. These exosomes containing increased levels of CYP2E1 caused significant toxicity in monocytic cells compared to exosomes derived from control mice. Overall, our results showed an important role of exosomal CYP2E1 in exacerbating ALC- and APAP-induced toxicity. The study is significant in terms of understanding the role of exosomal CYP2E1 in cell-cell interactions, and their effects on drug-induced toxicity.

EGF receptor plays a role in the mechanism of glutamine-mediated prevention of alcohol-induced gut barrier dysfunction and liver injury.

Recent study indicated that glutamine prevents alcoholic tissue injury in mouse gut and liver. Here we investigated the potential role of Epidermal Growth Factor Receptor (EGFR) in glutamine-mediated prevention of ethanol-induced colonic barrier dysfunction, endotoxemia and liver damage. Wild-type and EGFR*Tg transgenic (expressing dominant negative EGFR) mice were fed 1-6% ethanol in Lieber-DeCarli diet. Gut permeability was measured by vascular-to-luminal flux of FITC-inulin, and junctional integrity assessed by confocal microscopy. Liver injury was evaluated by plasma transaminases, histopathology and triglyceride analyses. Glutamine effect on acetaldehyde-induced tight junction disruption was investigated in Caco-2 cell monolayers. Doxycycline-induced expression of EGFR* blocked glutamine-mediated prevention of ethanol-induced disruption of colonic epithelial tight junction, mucosal permeability and endotoxemia. Ethanol activated cofilin and disrupted actin cytoskeleton, which was blocked by glutamine in an EGFR-dependent mechanism. Ethanol down-regulated antioxidant gene expression and up-regulated cytokine and chemokine gene expression, which were blocked by glutamine in wild-type mice in the presence or absence of doxycycline, but not in EGFR*Tg mice in the presence of doxycycline. Histopathology, plasma transaminases, triglyceride and expression of chemokine and antioxidant genes indicated ethanol-induced liver damage, which were blocked by glutamine in an EGFR-dependent mechanism. Src kinase activity and extracellular ligand binding domain of EGFR are required for glutamine-mediated protection of barrier function in Caco-2 cell monolayers. Glutamine released metalloproteinases into the medium, and metalloproteinase inhibitors blocked glutamine-mediated protection of barrier function. Results demonstrate that EGFR plays an important role in glutamine-mediated prevention of alcoholic gut permeability, endotoxemia and liver damage.

Optical study of stress hormone-induced nanoscale structural alteration in brain using partial wave spectroscopic microscopy.

Chronic stress affects nano to microscale structures of the brain cells/tissues due to suppression of neural growths and reconnections, hence the neuronal activities. This results in depression, memory loss and even death of the brain cells. Our recently developed novel optical technique, partial wave spectroscopic microscopy has nanoscale sensitivity, and hence, can detect nanoscale changes in brain tissues due to stress. In this study, we applied this technique to quantify the stress related structural changes in the corticosterone-treated mouse model of stress. Our results show that brains from corticosterone-treated mice showed higher nanoscale structural disorder in the hippocampal region as compared to the brain from normal (vehicle) mice. The increase in structural alteration correlates with the duration of the stress. We further quantified the relative changes and the spatial localization of these changes in this mouse model and found out that the maximum changes occurred nearly symmetrically in both regions of the hippocampus. The mRNA for stress-related genes, brain-derived neurotrophic factor and tyrosine kinase-coupled receptor were also significantly reduced in the hippocampus of corticosterone-treated mice compared to that in control mice. These results indicate that chronic corticosterone treatment induces nanoscale structural alterations in mouse brain that corresponds to changes in stress-related gene expression.

Human Defensin-5 Blocks Ethanol and Colitis-Induced Dysbiosis, Tight Junction Disruption and Inflammation in Mouse Intestine.

Alcohol consumption has been shown to cause dysbiosis, but the mechanism involved in it is unknown. Recurrent colitis is known to induce expression of α-defensins in the colon, but the effect of alcohol consumption on it is not known. We investigated the effect of ethanol on α-defensin expression in the small intestine and colitis-induced expression in colon in mice. Furthermore, we evaluated the effect of human defensin-5 (HD5) on ethanol and colitis-induced gut barrier dysfunction and mucosal damage. Recurrent colitis was induced by feeding dextran sulfate sodium (DSS), 3 cycles of 5-days each with 15 days intervals, followed by 30-days remission. Ethanol was fed during the intervals and recovery in a liquid diet with or without HD5. Expression of α-defensins, tight junction (TJ) integrity and cytokine/chemokine expression were analyzed. Chronic ethanol feeding reduced α-defensin expression in the small intestine and colitis-induced defensin expression in the colon. HD5 attenuated the growth of enterotoxigenic Bacteriodes fragilis and E. coli, but had no effect on non-toxigenic Bacteriodes fragilis or probiotics, the Lactobacilli. Ethanol and colitis elevated Enterobacteriaceae, Firmicutes and Firmicutes to Bacteriodetes ratio in colonic mucosa. HD5 feeding attenuated ethanol and colitis-induced dysbiosis, disruption of intestinal epithelial TJ, mucosal inflammation, expression of pro-inflammatory cytokines and chemokines in the small intestine and colon, and endotoxemia. These results demonstrate that ethanol suppresses intestinal α-defensin expression, leading to dysbiosis, barrier dysfunction, inflammation and endotoxemia. HD5 feeding attenuates intestinal injury caused by ethanol and colitis, indicating that defensin expression is a potential target for treatment of alcoholic tissue injury and colitis.

Deletion of TLR-4 attenuates fetal alcohol exposure-induced gene expression and social interaction deficits.

Fetal alcohol spectrum disorders (FASD) are associated with social interaction behavior and gastrointestinal (GI) abnormalities. These abnormal behaviors and GI abnormalities overlap with autism spectrum disorder (ASD). We investigated the effect of fetal alcohol exposure (FAE) on social interaction deficits (hallmark of autism) in mice. Evidence indicates that exogenous lipopolysaccharide (LPS) administration during gestation induces autism-like behavior in the offspring. LPS regulates the expression of genes underlying differentiation, immune function, myelination, and synaptogenesis in fetal brain by the LPS receptor, TLR-4-dependent mechanism. In this study, we evaluated the role of TLR-4 in FAE-induced social behavior deficit. WT and TLR4-/- pregnant mice were fed Lieber-DeCarli liquid diet with or without ethanol. The control group was pair-fed with an isocaloric diet. Social behavior was tested in the adult offspring at postnatal day 60. Frontal cortex mRNA expression of autistic candidate genes (Ube3a, Gabrb3, Mecp2) and inflammatory cytokine genes (IL-1ß, IL-6, TNF-α) were measured by RT-qPCR. Adult male offspring of ethanol-fed WT dams showed low birth weight compared to offspring of pair-fed WT dams. However, their body weights at adulthood were greater compared to the body weights of offspring of pair-fed WT dams. There were no body weight differences in offspring of TLR4-/- dams. Social interaction deficit was observed only in male offspring of ethanol-fed WT dams, but it was not observed in both male and female offspring of ethanol-fed TLR4-/- dams. Expressions of autism candidate genes, Gabrb3 and Ube3a, were elevated, while that of the Mecp2 gene was suppressed in the frontal cortex of male, but not female, offspring of ethanol-fed WT mice. The expressions of inflammatory cytokine genes, IL-1ß, IL-6, and TNF-α, were also significantly increased in the frontal cortex of male, but not female, offspring of ethanol-fed dams. The changes in the expression of autistic and cytokine genes were unaffected in the offspring of ethanol-fed TLR4-/- dams. These data also indicate that TLR4 mediates FAE-induced changes in social interactions and gene expression in brain, suggesting that ethanol-induced LPS absorption from the maternal gut may be involved in gene expression changes in the fetal brain.

Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.

Pathogenesis of alcohol-related diseases such as alcoholic hepatitis involves gut barrier dysfunction, endotoxemia, and toxin-mediated cellular injury. Here we show that Lactobacillus plantarum not only blocks but also mitigates ethanol (EtOH)-induced gut and liver damage in mice. L. plantarum blocks EtOH-induced protein thiol oxidation, and down-regulation of antioxidant gene expression in colon L. plantarum also blocks EtOH-induced expression of TNF-α, IL-1ß, IL-6, monocyte chemotactic protein 1 ( MCP1), C-X-C motif chemokine ligand ( CXCL)1, and CXCL2 genes in colon. Epidermal growth factor receptor (EGFR) signaling mediates the L. plantarum-mediated protection of tight junctions (TJs) and barrier function from acetaldehyde, the EtOH metabolite, in Caco-2 cell monolayers. In mice, doxycycline-mediated expression of dominant negative EGFR blocks L. plantarum-mediated prevention of EtOH-induced TJ disruption, mucosal barrier dysfunction, oxidative stress, and inflammatory response in colon. L. plantarum blocks EtOH-induced endotoxemia as well as EtOH-induced pathologic lesions, triglyceride deposition, oxidative stress, and inflammatory responses in the liver by an EGFR-dependent mechanism. L. plantarum treatment after injury accelerated recovery from EtOH-induced TJ, barrier dysfunction, oxidative stress, and inflammatory response in colon, endotoxemia, and liver damage. Results demonstrate that L. plantarum has both preventive and therapeutic values in treatment of alcohol-induced tissue injury, particularly in alcoholic hepatitis.-Shukla, P. K., Meena, A. S., Manda, B., Gomes-Solecki, M., Dietrich, P., Dragatsis, I., Rao, R. Lactobacillus plantarum prevents and mitigates alcohol-induced disruption of colonic epithelial tight junctions, endotoxemia, and liver damage by an EGF receptor-dependent mechanism.