Preparation of Pueraria lobata Root-Derived Exosome-Like Nanovesicles and Evaluation of Their Effects on Mitigating Alcoholic Intoxication and Promoting Alcohol Metabolism in Mice.

Purpose: Pueraria lobata (P. lobata), a dual-purpose food and medicine, displays limited efficacy in alcohol detoxification and liver protection, with previous research primarily focused on puerarin in its dried roots. In this study, we investigated the potential effects and mechanisms of fresh P. lobata root-derived exosome-like nanovesicles (P-ELNs) for mitigating alcoholic intoxication, promoting alcohol metabolism effects and protecting the liver in C57BL/6J mice. Methods: We isolated P-ELNs from fresh P. lobata root using differential centrifugation and characterized them via transmission electron microscopy, nanoscale particle sizing, ζ potential analysis, and biochemical assays. In Acute Alcoholism (AAI) mice pre-treated with P-ELNs, we evaluated their effects on the timing and duration of the loss of the righting reflex (LORR), liver alcohol metabolism enzymes activity, liver and serum alcohol content, and ferroptosis-related markers. Results: P-ELNs, enriched in proteins, lipids, and small RNAs, exhibited an ideal size (150.7 ± 82.8 nm) and negative surface charge (-31 mV). Pre-treatment with 10 mg/(kg.bw) P-ELNs in both male and female mice significantly prolonged ebriety time, shortened sobriety time, enhanced acetaldehyde dehydrogenase (ALDH) activity while concurrently inhibited alcohol dehydrogenase (ADH) activity, and reduced alcohol content in the liver and serum. Notably, P-ELNs demonstrated more efficacy compared to P-ELNs supernatant fluid (abundant puerarin content), suggesting alternative active components beyond puerarin. Additionally, P-ELNs prevented ferroptosis by inhibiting the reduction of glutathione peroxidase 4 (GPX4) and reduced glutathione (GSH), and suppressing acyl-CoA synthetase long-chain family member 4 (ACSL4) elevation, thereby mitigating pathological liver lipid accumulation. Conclusion: P-ELNs exhibit distinct exosomal characteristics and effectively alleviate alcoholic intoxication, improve alcohol metabolism, suppress ferroptosis, and protect the liver from alcoholic injury. Consequently, P-ELNs hold promise as a therapeutic agent for detoxification, sobriety promotion, and prevention of alcoholic liver injury.

Nrf2 pathway activation promotes the expression of genes related to glutathione metabolism in alcohol-exposed astrocytes.

Introduction: Oxidative and antioxidant pathways play essential roles in the development of alcohol-induced brain injury. The Nrf2 pathway is an endogenous antioxidant response pathway, but there has been little research on the role of Nrf2 in alcohol-related diseases. Thus, we examined the effects of alcohol and an Nrf2 agonist (TBHQ) on astrocyte function, mRNA expression, and metabolite content to further explore the protective mechanisms of Nrf2 agonists in astrocytes following alcohol exposure. Methods: CTX TNA2 astrocytes were cultured with alcohol and TBHQ and then subjected to transcriptome sequencing, LC-MS/MS analysis, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and malondialdehyde (MDA) and superoxide dismutase (SOD) activity assays. Results: Alcohol exposure significantly increased malondialdehyde (MDA) levels while decreasing superoxide dismutase (SOD) levels in astrocytes. Treatment with TBHQ effectively reversed these effects, demonstrating its protective role against oxidative stress induced by alcohol. Transcriptome sequencing and qRT-PCR analysis revealed that TBHQ specifically upregulates genes involved in glutathione metabolism, including a notable increase in the expression of the glutathione S-transferase A5 (GSTA5) gene, which was suppressed by alcohol exposure. Additionally, metabolomic analysis showed that TBHQ regulates key components of ether lipid metabolism in alcohol-exposed astrocytes, with significant reductions in the levels of lysophosphatidylcholine (18:0) (LysoPC (18:0)) and 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, both of which are critical markers in the ether lipid metabolic pathway. Discussion: The findings underscore the role of TBHQ as an Nrf2 agonist in mitigating alcohol-induced oxidative damage in astrocytes by modulating glutathione metabolism and ether lipid metabolism. The regulation of GSTA5 gene expression emerges as a key mechanism through which Nrf2 agonists confer neuroprotection against oxidative stress and lipid oxidation. These insights pave the way for potential therapeutic strategies targeting the Nrf2 pathway to protect astrocytes from alcohol-induced damage.

Ethanol responsive lnc171 promotes migration and invasion of HCC cells via mir-873-5p/ZEB1 axis.

BACKGROUNDS: Long nonconding RNAs (lncRNAs) have been found to be a vital regulatory factor in the development process of human cancer, and could regarded as diagnostic or prognostic biomarkers for human cancers. Here, we aim to confirm the expression and molecular mechanism of RP11-171K16.5 (lnc171) in hepatocellular carcinoma (HCC). METHODS: Screening of differentially expressed lncRNAs by RNA sequencing. Expression level of gene was studied by quantitative real-time PCR (qRT-PCR). The effects of lnc171, mir-873-5p, and ethanol on migration and invasion activity of cells were studied used transwell assay, and luciferase reporter assay was used to confirm the binding site. RESULTS: RNA sequencing showed that lnc171 was markedly up-regulated in HCC. siRNA-mediated knockdown of lnc171 repressed the migration and invasion ability of HCC cells. Bioinformatic analysis, dual luciferase reporter assay, and qRT-PCR indicated that lnc171 interacted with mir-873-5p in HCC cells, and Zin-finger E-box binding homeobox (ZEB1) was a downstream target gene of mir-873-5p. In addition, lnc171 could enhance migration and invasion ability of HCC cells by up-regulating ZEB1 via sponging mir-873-5p. More interestingly, ethanol stimulation could up-regulate the increase of lnc171, thereby regulating the expression of competing endogenous RNA (ceRNA) network factors which lnc171 participated in HCC cells. CONCLUSIONS: Our date demonstrates that lnc171 was a responsive factor of ethanol, and plays a vital role in development of HCC via binding of mir-873-5p.

Loss of glycine receptors in the nucleus accumbens and ethanol reward in an Alzheimer´s Disease mouse model.

Alterations in cognitive and non-cognitive cerebral functions characterize Alzheimer's disease (AD). Cortical and hippocampal impairments related to extracellular accumulation of Aß in AD animal models have been extensively investigated. However, recent reports have also implicated intracellular Aß in limbic regions, such as the nucleus accumbens (nAc). Accumbal neurons express high levels of inhibitory glycine receptors (GlyRs) that are allosterically modulated by ethanol and have a role in controlling its intake. In the present study, we investigated how GlyRs in the 2xTg mice (AD model) affect nAc functions and ethanol intake behavior. Using transgenic and control aged-matched litter mates, we found that the GlyRα2 subunit was significantly decreased in AD mice (6-month-old). We also examined intracellular calcium dynamics using the fluorescent calcium protein reporter GCaMP in slice photometry. We also found that the calcium signal mediated by GlyRs, but not GABAAR, was also reduced in AD neurons. Additionally, ethanol potentiation was significantly decreased in accumbal neurons in the AD mice. Finally, we performed drinking in the dark (DID) experiments and found that 2xTg mice consumed less ethanol on the last day of DID, in agreement with a lower blood ethanol concentration. 2xTg mice also showed lower sucrose consumption, indicating that overall food reward was altered. In conclusion, the data support the role of GlyRs in nAc neuron excitability and a decreased glycinergic activity in the 2xTg mice that might lead to impairment in reward processing at an early stage of the disease.

Sex differences in neuronal activation in the cortex and midbrain during quinine-adulterated alcohol intake.

AIMS: Continued alcohol consumption despite negative consequences is a core symptom of alcohol use disorder. This is modeled in mice by pairing negative stimuli with alcohol, such as adulterating alcohol solution with quinine. Mice consuming alcohol under these conditions are considered to be engaging in aversion-resistant intake. Previously, we have observed sex differences in this behavior, with females more readily expressing aversion-resistant consumption. We also identified three brain regions that exhibited sex differences in neuronal activation during quinine-alcohol drinking: ventromedial prefrontal cortex (vmPFC), posterior insular cortex (PIC), and ventral tegmental area (VTA). Specifically, male mice showed increased activation in vmPFC and PIC, while females exhibited increased activation in VTA. In this study, we aimed to identify what specific type of neurons are activated in these regions during quinine-alcohol drinking. METHOD: We assessed quinine-adulterated alcohol intake using the two-bottle choice procedure. We also utilized RNAscope in situ hybridization in the three brain regions that previously exhibited a sex difference to examine colocalization of Fos, glutamate, GABA, and dopamine. RESULT: Females showed increased aversion-resistant alcohol consumption compared to males. We also found that males had higher colocalization of glutamate and Fos in vmPFC and PIC, while females had greater dopamine and Fos colocalization in the VTA. CONCLUSIONS: Collectively, these experiments suggest that glutamatergic output from the vmPFC and PIC may have a role in suppressing, and dopaminergic activity in the VTA may promote, aversion-resistant alcohol consumption. Future experiments will examine neuronal circuits that contribute to sex differences in aversion resistant consumption.

Evaluating the effects of various ethanolic medicinal plant extracts on metastatic breast cancer proliferation, invasion, and expression of a novel potential drug target; CD82 metastatic suppressor protein, and on in vivo angiogenesis using the ex ovo yolk sac membrane (YSM) assay.

PURPOSE: Breast cancer metastasis relies on cellular invasion and angiogenesis facilitated by the downregulation of metastatic suppressor proteins like Cluster of Differentiation 82 (CD82). Currently, no medicines target multiple systems to prevent metastatic progression through CD82 upregulation. This study screened for plant extracts displaying effects on cell proliferation, invasion, and CD82 expression in breast cancer cells, and in vivo angiogenesis, and further correlated between the biological activities and effect on CD82 expression. METHODS: Seventeen ethanolic plant extracts were screened for their effect on cell proliferation (against MDA-MB-231 and MCF-7 breast cancer and Hek293 kidney cells), cell invasion and effect on CD82 expression in metastatic MDA-MB-231 cells. Selected extracts were further evaluated for in vivo anti-angiogenesis. RESULTS: Extracts displayed varying antiproliferative activity against the different cell lines, and those that showed selectivity indexes (SI) > 0.5 against MDA-MB-231 were selected for anti-invasion evaluation. Buddleja saligna Willd. (BS), Combretum apiculatum Sond. (CA), Foeniculum vulgare, Greyia radlkoferi, Gunnera perpensa and Persicaria senegalensis (Meisn.) Soják (PS) displayed 50% inhibitory concentration (IC50) values of 44.46 ± 3.46, 74.00 ± 4.48, 180.43 ± 4.51, 96.97 ± 2.29, 55.29 ± 9.88 and 243.60 ± 2.69 µg/mL, respectively against MDA-MB-231, and compared to Hek293 showed SI of 0.9, 0.7, 1.4, 1.1, 2.2 and 0.5. Significant invasion inhibition was observed at both 20 and 40 µg/mL for BS (94.10 ± 0.74 and 96.73 ± 0.95%) and CA (87.42 ± 6.54 and 98.24 ± 0.63%), whereas GR (14.91 ± 1.62 and 41 ± 1.78%) and PS (36.58 ± 0.54 and 51.51 ± 0.83%), only showed significant inhibition at 40 µg/mL, and FV (< 5% inhibition) and GP (10 ± 1.03 and 22 ± 1.31%) did not show significant inhibition at both concentrations. Due to the significant anti-invasive activity of BS, CA and PS at 40 µg/mL, these extracts were further evaluated for their potential to stimulate CD82. BS showed significant (p < 0.05) reduction in CD82 at 20 and 40 µg/mL (13.2 ± 2.2% and 20.3 ± 1.5% decrease, respectively), whereas both CA and PS at 20 µg/mL increased (p < 0.05) CD82 expression (16.4 ± 0.8% and 5.4 ± 0.6% increase, respectively), and at 40 µg/mL significantly reduced CD82 expression (23.4 ± 3.1% and 11.2 ± 2.9% decrease, respectively). Using the yolk sac membrane assay, BS (59.52 ± 4.12 and 56.72 ± 3.13% newly formed vessels) and CA (83.33 ± 3.17 and 74.00 ± 2.12%) at both 20 and 40 µg/egg showed significant (p < 0.001) angiogenesis inhibition, with BS showing statistical similar activity to the positive control, combretastatin A4 (10 nmol/egg), whereas PS only displayed significant (p < 0.001) angiogenesis stimulation at 40 µg/egg (120.81 ± 3.34% newly formed vessels). CONCLUSION: BS exhibits antiproliferative, anti-invasive, and anti-angiogenic activity despite inhibiting CD82, suggesting an alternative mode of action. CA at 20 µg/mL shows moderate anti-invasive and anti-angiogenic potential by stimulating CD82, while at 40 µg/mL it still displays these properties but inhibits CD82, suggesting an additional mode of action. PS, with the least antiproliferative activity, stimulates CD82 and inhibits angiogenesis at 20 µg/mL but inhibits CD82 and increases angiogenesis at 40 µg/mL, indicating CD82 targeting as a major mode of action. Future studies should explore breast cancer xenograft models to assess the extracts' impact on CD82 expression and angiogenesis in the tumor microenvironment, along with isolating bioactive compounds from the extracts.

Structural and aggregation changes of silver carp myosin induced with alcohols: Effects of ethanol, 1,2-propanediol, and glycerol.

This study investigated the effects of ethanol, 1,2-propanediol, and glycerol on the structure and aggregation behavior of silver carp (Hypophthalmichthys molitrix) myosin. All alcohols induced extensive alteration in the tertiary structure of myosin. Both ethanol and 1,2-propanediol further promoted an increase in the content of ß-sheets in myosin and induced myosin aggregation. While glycerol had almost no impact on the secondary structure of myosin. Molecular dynamics simulations revealed that increasing the concentration of ethanol and 1,2-propanediol affected the overall structural changes in the myosin heavy chain (MHC), while glycerol exerted a more pronounced effect on the MHC tail when compared to the MHC head. Disruption of the hydration layers induced by ethanol and 1,2-propanediol contributed to local structural changes in myosin. Glycerol at a concentration of 20% induced the formation of a larger hydration layer around the MHC tail, which facilitated the stabilization of the protein structure.

Modeling alcohol-associated liver disease in humans using adipose stromal or stem cell-derived organoids.

Alcohol-associated liver disease (ALD) is a prevalent liver disease, yet research is hampered by the lack of suitable and reliable human ALD models. Herein, we generated human adipose stromal/stem cell (hASC)-derived hepatocellular organoids (hAHOs) and hASC-derived liver organoids (hALOs) in a three-dimensional system using hASC-derived hepatocyte-like cells and endodermal progenitor cells, respectively. The hAHOs were composed of major hepatocytes and cholangiocytes. The hALOs contained hepatocytes and nonparenchymal cells and possessed a more mature liver function than hAHOs. Upon ethanol treatment, both steatosis and inflammation were present in hAHOs and hALOs. The incubation of hALOs with ethanol resulted in increases in the levels of oxidative stress, the endoplasmic reticulum protein thioredoxin domain-containing protein 5 (TXNDC5), the alcohol-metabolizing enzymes ADH1B and ALDH1B1, and extracellular matrix accumulation, similar to those of liver tissues from patients with ALD. These results present a useful approach for understanding the pathogenesis of ALD in humans, thus facilitating the discovery of effective treatments.

Effects of prenatal alcohol exposure on the olfactory system development.

Fetal Alcohol Spectrum Disorders (FASD), resulting from maternal alcohol consumption during pregnancy, are a prominent non-genetic cause of physical disabilities and brain damage in children. Alongside common symptoms like distinct facial features and neurocognitive deficits, sensory anomalies, including olfactory dysfunction, are frequently noted in FASD-afflicted children. However, the precise mechanisms underpinning the olfactory abnormalities induced by prenatal alcohol exposure (PAE) remain elusive. Utilizing rodents as a model organism with varying timing, duration, dosage, and administration routes of alcohol exposure, prior studies have documented impairments in olfactory system development caused by PAE. Many reported a reduction in the olfactory bulb (OB) volume accompanied by reduced OB neuron counts, suggesting the OB is a brain region vulnerable to PAE. In contrast, no significant olfactory system defects were observed in some studies, though subtle alterations might exist. These findings suggest that the timing, duration, and extent of fetal alcohol exposure can yield diverse effects on olfactory system development. To enhance comprehension of PAE-induced olfactory dysfunctions, this review summarizes key findings from previous research on the olfactory systems of offspring prenatally exposed to alcohol.

Mixing energy drinks and alcohol during adolescence impairs brain function: A study of rat hippocampal plasticity.

In the last decades, the consumption of energy drinks has risen dramatically, especially among young people, adolescents and athletes, driven by the constant search for ergogenic effects, such as the increase in physical and cognitive performance. In parallel, mixed consumption of energy drinks and ethanol, under a binge drinking modality, under a binge drinking modality, has similarly grown among adolescents. However, little is known whether the combined consumption of these drinks, during adolescence, may have long-term effects on central function, raising the question of the risks of this habit on brain maturation. Our study was designed to evaluate, by behavioral, electrophysiological and molecular approaches, the long-term effects on hippocampal plasticity of ethanol (EtOH), energy drinks (EDs), or alcohol mixed with energy drinks (AMED) in a rat model of binge-like drinking adolescent administration. The results show that AMED binge-like administration produces adaptive hippocampal changes at the molecular level, associated with electrophysiological and behavioral alterations, which develop during the adolescence and are still detectable in adult animals. Overall, the study indicates that binge-like drinking AMED adolescent exposure represents a habit that may affect permanently hippocampal plasticity.

Virulence factors, antifungal susceptibility and molecular profile in Candida species isolated from the hands of health professionals before and after cleaning with 70% ethyl alcohol-based gel.

Fungal infections in neonatal intensive care units (NICU) are mainly related to Candida species, with high mortality rates. They are predominantly of endogenous origin, however, cross-infection transmitted by healthcare professionals' hands has occurred. The aim of this study was to identify Candida species isolated from the hands of healthcare professionals in a NICU before and after hygiene with 70% ethanol-based gel and evaluate virulence factors DNase, phospholipase, proteinase, hemolysin, biofilm biomass production, and metabolic activity. In vitro antifungal susceptibility testing and similarity by random amplified polymorphic DNA (RAPD) were also performed. C. parapsilosis complex was the most frequent species (57.1%); all isolates presented at least one virulence factor; three isolates (Candida parapsilosis complex) were resistant to amphotericin B, two (Candida famata [currently Debaryomyces hansenii] and Candida guilliermondii [currently Meyerozyma guilliermondii]) was resistant to micafungin, and six (Candida parapsilosis complex, Candida guilliermondii [=Meyerozyma guilliermondii], Candida viswanathi, Candida catenulata [currently Diutina catenulata] and Candida lusitaniae [currently Clavispora lusitaniae]) were resistant to fluconazole. Molecular analysis by RAPD revealed two clusters of identical strains that were in the hands of distinct professionals. Candida spp. were isolated even after hygiene with 70% ethanol-based gel, highlighting the importance of stricter basic measures for hospital infection control to prevent nosocomial transmission.

Alcohol exacerbates psychosocial stress-induced neuropsychiatric symptoms: Attenuation by geraniol.

Adaptation to psychosocial stress is psychologically distressing, initiating/promoting comorbidity with alcohol use disorders. Emerging evidence moreover showed that ethanol (EtOH) exacerbates social-defeat stress (SDS)-induced behavioral impairments, neurobiological sequelae, and poor therapeutic outcomes. Hence, this study investigated the effects of geraniol, an isoprenoid monoterpenoid alcohol with neuroprotective functions on EtOH escalated SDS-induced behavioral impairments, and neurobiological sequelae in mice. Male mice chronically exposed to SDS for 14 days were repeatedly fed with EtOH (2 g/kg, p. o.) from days 8-14. From days 1-14, SDS-EtOH co-exposed mice were concurrently treated with geraniol (25 and 50 mg/kg) or fluoxetine (10 mg/kg) orally. After SDS-EtOH translational interactions, arrays of behavioral tasks were examined, followed by investigations of oxido-inflammatory, neurochemicals levels, monoamine oxidase-B and acetylcholinesterase activities in the striatum, prefrontal-cortex, and hippocampus. The glial fibrillary acid protein (GFAP) expression was also quantified in the prefrontal-cortex immunohistochemically. Adrenal weights, serum glucose and corticosterone concentrations were measured. EtOH exacerbated SDS-induced low-stress resilience, social impairment characterized by anxiety, depression, and memory deficits were attenuated by geraniol (50 and 100 mg/kg) and fluoxetine. In line with this, geraniol increased the levels of dopamine, serotonin, and glutamic-acid decarboxylase enzyme, accompanied by reduced monoamine oxidase-B and acetylcholinesterase activities in the prefrontal-cortex, hippocampus, and striatum. Geraniol inhibited SDS-EtOH-induced adrenal hypertrophy, corticosterone, TNF-α, IL-6 release, malondialdehyde and nitrite levels, with increased antioxidant activities. Immunohistochemical analyses revealed that geraniol enhanced GFAP immunoreactivity in the prefrontal-cortex relative to SDS-EtOH group. We concluded that geraniol ameliorates SDS-EtOH interaction-induced behavioral changes via normalization of neuroimmune-endocrine and neurochemical dysregulations in mice brains.

Potential global cis and trans regulation of lncRNAs in Saccharomyces cerevisiae subjected to ethanol stress.

Long noncoding RNAs (lncRNAs) are regulatory RNAs. Saccharomyces cerevisiae strains transcribe hundreds of lncRNAs. LncRNAs can regulate the expression of adjacent genes (cis-regulation) or distant genes from lncRNAs (trans-regulation). Here, we analyzed the potential global cis and trans-regulation of lncRNAs of yeast subjected to ethanol stress. For potential cis regulation, for BMA641-A and S288C strains, we observed that most lncRNA-neighbor gene pairs increased the expression at a certain point followed by a decrease, and vice versa. Based on the transcriptome profile and triple helix prediction between lncRNAs and promoters of coding genes, we observed nine different ways of potential trans regulation that work in a strain-specific manner. Our data provide an initial landscape of potential cis and trans regulation in yeast, which seems to be strain-specific.

Effect of low ethanol concentration in maturation medium on developmental ability, mitochondria, and gene expression profile in mouse oocytes.

Ethanol affects pre-conceptional oocyte quality in women. In this study, we examined the effect of low ethanol concentrations on mouse oocytes. Oocytes were collected from the ovaries of 9-10 week old mice and allowed to mature in vitro in the presence of low concentrations of ethanol (0.1% and 0.2% v/v) for 24 h. Treatment of oocytes with ethanol (0.2%) during maturation decreased the mitochondrial DNA content and membrane potential compared to that in untreated ones, whereas the ATP content did not differ between the groups. Both 0.1% and 0.2% ethanol reduced the lipid content in the oocytes. In addition, immunostaining revealed that oocytes cultured in maturation medium containing ethanol (0.2%) had reduced levels of global DNA methylation and DNMT3A compared with untreated oocytes, and decreased rate of blastocyst development with low mitochondrial protein levels (TOMM40) in embryo. RNA-sequencing of the ethanol-treated (0.2%) and untreated oocytes revealed that mitochondria were a major target of ethanol. In conclusion, treatment of oocytes with low concentration of ethanol reduces the developmental rate to the blastocyst stage, with a lower total cell number and global DNA methylation. In addition, ethanol affected mitochondrial function and mitochondria-related gene expression.

Cholinergic mu-opioid receptor deletion alters reward preference and aversion-resistance.

The endogenous opioid system has been implicated in alcohol consumption and preference in both humans and animals. The mu opioid receptor (MOR) is expressed on multiple cells in the striatum, however little is known about the contributions of specific MOR populations to alcohol drinking behaviors. The current study used mice with a genetic deletion of MOR in cholinergic cells (ChAT-Cre/Oprm1fl/fl) to examine the role of MORs expressed in cholinergic interneurons (CINs) in home cage self-administration paradigms. Male and female ChAT-Cre/Oprm1fl/fl mice were generated and heterozygous Cre+ (knockout) and Cre- (control) mice were tested for alcohol consumption in two drinking paradigms: limited access "Drinking in the Dark" and intermittent access. Quinine was added to the drinking bottles in the DID experiment to test aversion-resistant, "compulsive" drinking. Nicotine and sucrose drinking were also assessed so comparisons could be made with other rewarding substances. Cholinergic MOR deletion did not influence consumption or preference for ethanol (EtOH) in either drinking task. Differences were observed in aversion-resistance in males with Cre + mice tolerating lower concentrations of quinine than Cre-. In contrast to EtOH, preference for nicotine was reduced following cholinergic MOR deletion while sucrose consumption and preference was increased in Cre+ (vs. Cre-) females. Locomotor activity was also greater in females following the deletion. These results suggest that cholinergic MORs participate in preference for rewarding substances. Further, while they are not required for consumption of alcohol alone, cholinergic MORs may influence the tendency to drink despite negative consequences.

Environmental enrichment enhances ethanol preference over social reward in male swiss mice: Involvement of oxytocin-dopamine interactions.

The impact of environmental enrichment (EE) on natural rewards, including social and appetitive rewards, was investigated in male Swiss mice. EE, known for providing animals with various stimuli, was assessed for its effects on conditioned place preference (CPP) associated with ethanol and social stimuli. We previously demonstrated that EE increased the levels of the prosocial neuropeptide oxytocin (OT) in the hypothalamus and enhanced ethanol rewarding effects via an oxytocinergic mechanism. This study also investigated the impact of EE on social dominance and motivation for rewards, measured OT-mediated phospholipase C (PLC) activity in striatal membranes, and assessed OT expression in the hypothalamus. The role of dopamine in motivating rewards was considered, along with the interaction between OT and D1 receptors (DR) in the nucleus accumbens (NAc). Results showed that EE mice exhibited a preference for ethanol reward over social reward, a pattern replicated by the OT analogue Carbetocin. EE mice demonstrated increased social dominance and reduced motivation for appetitive taste stimuli. Higher OT mRNA levels in the hypothalamus were followed by diminished OT receptor (OTR) signaling activity in the striatum of EE mice. Additionally, EE mice displayed elevated D1R expression, which was attenuated by the OTR antagonist (L-368-889). The findings underscore the reinforcing effect of EE on ethanol and social rewards through an oxytocinergic mechanism. Nonetheless, they suggest that mechanisms other than the prosocial effect of EE may contribute to the ethanol pro-rewarding effect of EE and Carbetocin. They also point towards an OT-dopamine interaction potentially underlying some of these effects.

Alcohol effects on interoception shape expectancies and subjective effects: a registered report using the heart rate discrimination task.

AIMS: Alcohol acutely impacts interoceptive processes, which in turn affect the perception of alcohol effects and the development of alcohol expectancies. However, previous research is limited by the tools used to measure cardiac interoception and subjective alcohol effects. This registered report proposes a re-examination of previous findings using a state-of-the-art measure of interoceptive capacity, the heart rate discrimination task, and measurements of subjective alcohol effects across both ascending and descending limbs. METHODS: In a double-blind, placebo-controlled experiment, n = 36 participants were given 0.4 g/kg of ethanol, and a baseline measure of alcohol expectancies was obtained. Changes in interoceptive capacity after beverage administration, along with measures of light-headedness, mood, and biphasic alcohol effects, were assessed over two sessions. HYPOTHESES: As registered in this secondary data analysis, alcohol was expected to acutely impact different indices of interoceptive capacity, and those changes were hypothesized to correlate with subjective alcohol effects and expectancies. Analyses were conducted only following in-principle acceptance. RESULTS: Alcohol-induced changes in interoceptive capacity predicted the development of light-headedness, stimulation, and negative mood. Changes in interoceptive capacity were also correlated with negative alcohol expectancies, as measured 2 weeks prior to the experiment. These effects were unique to the interoceptive condition, as null effects were observed in an exteroceptive control task. DISCUSSION: This report offers a replication of key previous findings that alcohol impacts interoceptive processes to shape the detection of subjective alcohol effects. We propose that, through repeated drinking occasions, bodily responses feed into the experience of intoxication, shaping future expectancies about alcohol effects.

Ethanol disrupts hepatocellular lipophagy by altering Rab5-centric LD-lysosome trafficking.

BACKGROUND: Previous reports suggest that lipid droplets (LDs) in the hepatocyte can be catabolized by a direct engulfment from nearby endolysosomes (microlipophagy). Further, it is likely that this process is compromised by chronic ethanol (EtOH) exposure leading to hepatic steatosis. This study investigates the hepatocellular machinery supporting microlipophagy and EtOH-induced alterations in this process with a focus on the small, endosome-associated, GTPase Rab5. METHODS AND RESULTS: Here we report that this small Ras-related GTPase is a resident component of LDs, and its activity is important for hepatocellular LD-lysosome proximity and physical interactions. We find that Rab5 siRNA knockdown causes an accumulation of LDs in hepatocytes by inhibiting lysosome dependent LD catabolism. Importantly, Rab5 appears to support this process by mediating the recruitment of early endosomal and or multivesicular body compartments to the LD surface before lysosome fusion. Interestingly, while wild-type or a constituently active GTPase form (Q79L) of Rab5 supports LD-lysosome transport, this process is markedly reduced in cells expressing a GTPase dead (S34N) Rab5 protein or in hepatocytes exposed to chronic EtOH. CONCLUSIONS: These findings support the novel premise of an early endosomal/multivesicular body intermediate compartment on the LD surface that provides a "docking" site for lysosomal trafficking, not unlike the process that occurs during the hepatocellular degradation of endocytosed ligands that is also known to be compromised by EtOH exposure.

Comparative evaluation of ethanol versus citric acid in removal of oil-based calcium hydroxide from the apical third of root canal system.

Objectives: To evaluate the effectiveness of ethanol compared to citric acid in the removal of oil-based calcium hydroxide from the apical third of the root canal system using passive ultrasonic irrigation. METHODS: The in vitro study was conducted from September to October 2021 at the dental clinics of the Aga Khan University Hospital, Karachi, and comprised single-rooted teeth that were selected from institutional bank of extracted teeth. They were randomly divided into group A having 70% ethanol + passive ultrasonic irrigation, group B 10% citric acid + passive ultrasonic irrigation, group C positive controls and group D negative controls. The specimens were sectioned at 1mm and 3mm from the apex and examined under a dental operating microscope. A single examiner scored the specimens on two different occasions. Data was analysed using SPSS 25. RESULTS: Of the 90 teeth, there were 40(44.4%) in each of the 2 experimental groups and 5(5.5%) in each of the 2 control groups. At 3mm apical sections, ethanol was significantly more effective in the removal of oil-based calcium hydroxide (p=0.01). However, at 1mm from the apex, there was no significant difference between the experimental groups (p=0.064). Intragroup comparison showed that for groups A and B, residual medicament at 1mm sections was significantly greater than at 3mm sections (p<0.001, p=0.003). CONCLUSIONS: Neither irrigant showed complete removal at 1mm and 3mm from the apex. However, at 3mm apical sections, 70% ethanol was significantly more effective compared to 10% citric acid.