Neuroprotective Effect of Sterculia setigera Leaves Hydroethanolic Extract.

Plants are a valuable source of information for pharmacological research and new drug discovery. The present study aimed to evaluate the neuroprotective potential of the leaves of the medicinal plant Sterculia setigera. In vitro, the effect of Sterculia setigera leaves dry hydroethanolic extract (SSE) was tested on cultured cerebellar granule neurons (CGN) survival when exposed to hydrogen peroxide (H2O2) or 6-hydroxydopamine (6-OHDA), using the viability probe fluorescein diacetate (FDA), a lactate dehydrogenase (LDH) activity assay, an immunocytochemical staining against Gap 43, and the quantification of the expression of genes involved in apoptosis, necrosis, or oxidative stress. In vivo, the effect of intraperitoneal (ip) injection of SSE was assessed on the developing brain of 8-day-old Wistar rats exposed to ethanol neurotoxicity by measuring caspase-3 activity on cerebellum homogenates, the expression of some genes in tissue extracts, the thickness of cerebellar cortical layers and motor coordination. In vitro, SSE protected CGN against H2O2 and 6-OHDA-induced cell death at a dose of 10 µg/mL, inhibited the expression of genes Casp3 and Bad, and upregulated the expression of Cat and Gpx7. In vivo, SSE significantly blocked the deleterious effect of ethanol by reducing the activity of caspase-3, inhibiting the expression of Bax and Tp53, preventing the reduction of the thickness of the internal granule cell layer of the cerebellar cortex, and restoring motor functions. Sterculia setigera exerts neuroactive functions as claimed by traditional medicine and should be a good candidate for the development of a neuroprotective treatment against neurodegenerative diseases.

Multiomics Analysis of PCB126's Effect on a Mouse Chronic-Binge Alcohol Feeding Model.

BACKGROUND: Environmental pollutants, including polychlorinated biphenyls (PCBs) have been implicated in the pathogenesis of liver disease. Our group recently demonstrated that PCB126 promoted steatosis, hepatomegaly, and modulated intermediary metabolism in a rodent model of alcohol-associated liver disease (ALD). OBJECTIVE: To better understand how PCB126 promoted ALD in our previous model, the current study adopts multiple omics approaches to elucidate potential mechanistic hypotheses. METHODS: Briefly, male C57BL/6J mice were exposed to 0.2mg/kg polychlorinated biphenyl (PCB) 126 or corn oil vehicle prior to ethanol (EtOH) or control diet feeding in the chronic-binge alcohol feeding model. Liver tissues were collected and prepared for mRNA sequencing, phosphoproteomics, and inductively coupled plasma mass spectrometry for metals quantification. RESULTS: Principal component analysis showed that PCB126 uniquely modified the transcriptome in EtOH-fed mice. EtOH feeding alone resulted in >4,000 differentially expressed genes (DEGs), and PCB126 exposure resulted in more DEGs in the EtOH-fed group (907 DEGs) in comparison with the pair-fed group (503 DEGs). Top 20 significant gene ontology (GO) biological processes included "peptidyl tyrosine modifications," whereas top 25 significantly decreasing GO molecular functions included "metal/ion/zinc binding." Quantitative, label-free phosphoproteomics and western blot analysis revealed no major significant PCB126 effects on total phosphorylated tyrosine residues in EtOH-fed mice. Quantified hepatic essential metal levels were primarily significantly lower in EtOH-fed mice. PCB126-exposed mice had significantly lower magnesium, cobalt, and zinc levels in EtOH-fed mice. DISCUSSION: Previous work has demonstrated that PCB126 is a modifying factor in metabolic dysfunction-associated steatotic liver disease (MASLD), and our current work suggests that pollutants also modify ALD. PCB126 may, in part, be contributing to the malnutrition aspect of ALD, where metal deficiency is known to contribute and worsen prognosis. https://doi.org/10.1289/EHP14132.

Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

[Yeast extract improves the inflammatory response of HepG2 cells induced by ethyl alcohol and lipopolysaccharide].

OBJECTIVE: To explore the ameliorative effect of yeast extract(YE) on the inflammatory response of human hepatoma cells(HepG2) induced by ethyl alcohol(EtOH) and lipopolysaccharide(LPS), and further explore the potential molecular mechanism based on Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB) signaling pathway. METHODS: HepG2 cells were induced by 50 mmol/L EtOH and 1 µg/mL LPS combined with YE intervention. The expression level of inflammatory cytokines was detected by ELISA. The expression level of TLR4 and the nuclear translocation of NF-κB were detected by immunofluorescence staining. The expression levels of TLR4, NF-κB, phospho-NF-κB-P65(P-NF-κB-p65), nucleus-phospho-NF-κB-p65(N-P-NF-κB-p65), tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1ß(IL-1ß) were detected by Western blot. RESULTS: Compared with the control group, the cells in EtOH+LPS group produced a large number of inflammatory factors and had a significant inflammatory response. YE intervention significantly alleviated EtOH+LPS induced hepatocyte inflammatory response. Further molecular mechanism studies showed that YE significantly reduced TLR4 expression level and inhibited NF-κB nuclear translocation in hepatocytes. CONCLUSION: YE can effectively inhibit the inflammatory response of HepG2 cells induced by EtOH and LPS, and its molecular mechanism may be related to the down-regulation of TLR4/NF-κB pathway.

Phosphoglycerate mutase 5 aggravates alcoholic liver disease through disrupting VDAC-1-dependent mitochondrial integrity.

Alcoholic liver disease (ALD) poses a substantial global health challenge, with its pathogenesis deeply rooted in mitochondrial dysfunction. Our study explores the pivotal roles of Phosphoglycerate mutase family member 5 (Pgam5) and Voltage-Dependent Anion Channel 1 (VDAC1) in the progression of ALD, providing novel insights into their interplay and impact on mitochondrial integrity. We demonstrate that Pgam5 silencing preserves hepatocyte viability and attenuates ethanol-induced apoptosis, underscoring its detrimental role in exacerbating hepatocyte dysfunction. Pgam5's influence extends to the regulation of VDAC1 oligomerization, a key process in mitochondrial permeability transition pore (mPTP) opening, mitochondrial swelling, and apoptosis initiation. Notably, the inhibition of VDAC1 oligomerization through Pgam5 silencing or pharmacological intervention (VBIT-12) significantly preserves mitochondrial function, evident in the maintenance of mitochondrial membrane potential and reduced reactive oxygen species (ROS) production. In vivo experiments using hepatocyte-specific Pgam5 knockout (Pgam5hKO) and control mice reveal that Pgam5 deficiency mitigates ethanol-induced liver histopathology, inflammation, lipid peroxidation, and metabolic disorder, further supporting its role in ALD progression. Our findings highlight the critical involvement of Pgam5 and VDAC1 in mitochondrial dysfunction in ALD, suggesting potential therapeutic targets. While promising, these findings necessitate further research, including human studies, to validate their clinical applicability and explore broader implications in liver diseases. Overall, our study provides a significant advancement in understanding ALD pathophysiology, paving the way for novel therapeutic strategies targeting mitochondrial pathways in ALD.

Fish oil ameliorates ethanol-induced gastric injury in rat by modulating gene related to apoptosis.

Gastric ulcers are a type of digestive disease that can severely affect a person's quality of life. Our study aimed to investigate the effects of fish oil on ethanol-induced gastric ulcers in rats, with the purpose of providing more comprehensive information on the topic. The study looked at various factors such as gastric ulcer index, and nitric oxide (NO) levels in stomach tissue. To investigate apoptosis, the mRNA levels of Bax, Bcl-2, and Caspase 3 were analyzed. The results showed that fish oil can reduce gastric acidity and the gastric ulcer index in cases of ethanol-induced gastric ulcers. It was found that fish oil can increase NO levels and improve the anti-apoptotic system by increasing the expression of Bcl-2 while decreasing the expression of Bax and Caspase 3. In general, the study demonstrates that fish oil can protect the stomach from ethanol-induced damage by reducing the apoptosis pathway via nitric oxide.

Activation of STING signaling aggravates chronic alcohol exposure-induced cognitive impairment by increasing neuroinflammation and mitochondrial apoptosis.

AIMS: Chronic alcohol exposure leads to persistent neurological disorders, which are mainly attributed to neuroinflammation and apoptosis. Stimulator of IFN genes (STING) is essential in the cytosolic DNA sensing pathway and is involved in inflammation and cellular death processes. This study was to examine the expression pattern and biological functions of STING signaling in alcohol use disorder (AUD). METHODS: Cell-free DNA was extracted from human and mouse plasma. C57BL/6J mice were given alcohol by gavage for 28 days, and behavior tests were used to determine their mood and cognition. Cultured cells were treated with ethanol for 24 hours. The STING agonist DMXAA, STING inhibitor C-176, and STING-siRNA were used to intervene the STING. qPCR, western blot, and immunofluorescence staining were used to assess STING signaling, inflammation, and apoptosis. RESULTS: Circulating cell-free mitochondrial DNA (mtDNA) was increased in individuals with AUD and mice chronically exposed to alcohol. Upregulation of STING signaling under alcohol exposure led to inflammatory responses in BV2 cells and mitochondrial apoptosis in PC12 cells. DMXAA exacerbated alcohol-induced cognitive impairment and increased the activation of microglia, neuroinflammation, and apoptosis in the medial prefrontal cortex (mPFC), while C-176 exerted neuroprotection. CONCLUSION: Activation of STING signaling played an essential role in alcohol-induced inflammation and mitochondrial apoptosis in the mPFC. This study identifies STING as a promising therapeutic target for AUD.

Hepatoprotective Effect of Allium ochotense Extracts on Chronic Alcohol-Induced Fatty Liver and Hepatic Inflammation in C57BL/6 Mice.

This study was performed to investigate the protective effects of Allium ochotense on fatty liver and hepatitis in chronic alcohol-induced hepatotoxicity. The physiological compounds of a mixture of aqueous and 60% ethanol (2:8, w/w) extracts of A. ochotense (EA) were identified as kestose, raffinose, kaempferol and quercetin glucoside, and kaempferol di-glucoside by UPLC Q-TOF MSE. The EA regulated the levels of lipid metabolism-related biomarkers such as total cholesterol, triglyceride, low-density lipoprotein (LDL), and high-density lipoprotein (HDL)-cholesterol in serum. Also, EA ameliorated the levels of liver toxicity-related biomarkers such as glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), and total bilirubin in serum. EA improved the antioxidant system by reducing malondialdehyde contents and increasing superoxide dismutase (SOD) levels and reduced glutathione content. EA improved the alcohol metabolizing enzymes such as alcohol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P450 2E1 (CYP2E1). Treatment with EA alleviated lipid accumulation-related protein expression by improving phosphorylation of AMP-activated protein kinase (p-AMPK) expression levels. Especially, EA reduced inflammatory response by regulating the toll-like receptor-4/nuclear factor kappa-light-chain-enhancer of activated B cells (TLR-4/NF-κB) signaling pathway. EA showed an anti-apoptotic effect by regulating the expression levels of B-cell lymphoma 2 (BCl-2), BCl-2-associated X protein (BAX), and caspase 3. Treatment with EA also ameliorated liver fibrosis via inhibition of transforming growth factor-beta 1/suppressor of mothers against decapentaplegic (TGF-ß1/Smad) pathway and alpha-smooth muscle actin (α-SMA). Therefore, these results suggest that EA might be a potential prophylactic agent for the treatment of alcoholic liver disease.

Ethanol-Induced Hepatic Ferroptosis Is Mediated by PERK-Dependent MAMs Formation: Preventive Role of Quercetin.

SCOPE: Iron deposition is frequently observed in alcoholic liver disease (ALD), which indicates a potential role of ferroptosis in its development. This study aims to explore the effects of quercetin on ferroptosis in ALD and elucidates the underlying mechanism involving the formation of mitochondria-associated endoplasmic reticulum membranes (MAMs) mediated by protein kinase RNA-like endoplasmic reticulum kinase (PERK). METHODS AND RESULTS: C57BL/6J mice are fed either a regular or an ethanol-containing liquid diet (with 28% energy form ethanol) with or without quercetin supplementation (100 mg kg-1 BW) for 12 weeks. Ethanol feeding or treatment induced ferroptosis in mice and AML12 cells, which is associated with increased MAMs formation and PERK expression within MAMs. Quercetin attenuates these changes and protects against ethanol-induced liver injury. The antiferroptotic effect of quercetin is abolished by ferroptosis inducers, but mimicked by ferroptosis inhibitors and PERK knockdown. The study demonstrates that PERK structure, rather than its kinase activity (transfected with the K618A site mutation that inhibits kinase activity-ΔK plasmid or protein C terminal knockout-ΔC plasmid of PERK), mediates the enhanced MAMs formation and ferroptosis during the ethanol exposure. CONCLUSION: Quercetin ameliorates ethanol-induced liver injury by inhibiting ferroptosis via modulating PERK-dependent MAMs formation.

Protective effect of Enterococcus faecium against ethanol-induced gastric injury via extracellular vesicles.

Recently, Enterococcus has been shown to have gastric protective functions, and the mechanisms by which Enterococcus modulates gastric function are still being investigated. Herein, we investigated how Enterococcus faecium (Efm) and E. faecium-derived extracellular vesicles (EVs) (EfmEVs) exert protective effect against ethanol-induced gastric injury by investigating the effect of EfmEVs on gastric mucosal ulcer scoring, histological lesion, mucosal glycoprotein production, acidity, anti-oxidative function, and inflammatory responses in rat. Pretreatment with Efm showed significant reduction of ethanol-induced gastric injury, as evidenced by the lowering of ulcer index, histological lesion, gastric pH, and inflammatory responses and the enhancement of mucosal glycoprotein production and anti-oxidative function. Further functional studies on three bioactive components [inactivated Efm, EfmEVs (EVs), and EV-free supernatants] of the bacterial culture showed that EVs are mostly responsible for the gastroprotective effect. Moreover, EV secretion is beneficial for the gastroprotective effect of Efm. Hence, EVs mediated the protective effect of Efm against ethanol-induced gastric injury by lowering inflammatory responses and enhancing anti-oxidative function and may be a potent anti-inflammatory and anti-oxidative strategy to alleviate hyperinflammatory gastrointestinal tract conditions.IMPORTANCEThis study indicated that Enterococcus faecium provided a protective effect against rat gastric injury, which involved improvement of the mucosal glycoprotein production, anti-oxidative function, and inflammatory responses. Furthermore, we confirmed that three bioactive components (inactivated Efm, extracellular vesicles, and EV-free supernatants) of E. faecium culture also contributed to the gastroprotective effect. Importantly, E. faecium-derived EVs showed an effective impact for the gastroprotective effect.

Ethanol binge drinking exposure during adolescence displays long-lasting motor dysfunction related to cerebellar neurostructural damage even after long-term withdrawal in female Wistar rats.

Ethanol is one of the psychoactive substances most used by young individuals, usually in an intermittent and episodic manner, also called binge drinking. In the adolescent period, brain structures undergo neuromaturation, which increases the vulnerability to psychotropic substances. Our previous studies have revealed that ethanol binge drinking during adolescence elicits neurobehavioral alterations associated with brain damage. Thus, we explored the persistence of motor function impairment and cerebellum damage in the context of ethanol withdrawal periods (emerging adulthood and adult life) in adolescent female rats. Female Wistar rats (35 days old) received orally 4 cycles of ethanol (3.0 g/kg/day) or distilled water in 3 days on-4 days off paradigm (35th until 58th day of life). Motor behavioral tests (open field, grip strength, beam walking, and rotarod tests) and histological assays (Purkinje's cell density and NeuN-positive cells) were assessed on the 1-, 30-, and 60-days of binge alcohol exposure withdrawal. Our findings demonstrate that the adolescent binge drinking exposure paradigm induced cerebellar cell loss in all stages evaluated, measured through the reduction of Purkinje's cell density and granular layer neurons. The cerebellar tissue alterations were accompanied by behavioral impairments. In the early withdrawal, the reduction of spontaneous movement, incoordination, and unbalance was seen. However, the grip strength reduction was found at long-term withdrawal (60 days of abstinence). The cerebellum morphological changes and the motor alterations persisted until adulthood. These data suggest that binge drinking exposure during adolescence causes motor function impairment associated with cerebellum damage, even following a prolonged withdrawal, in adult life.

Protective effects of an alcoholic extract of Kaempferia galanga L. rhizome on ethanol-induced gastric ulcer in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: The rhizome of Kaempferia galanga L., a medicinal and edible Plant, was widely distributed in many Asian and African counties. It has been traditionally used to treat gastroenteritis, hypertension, rheumatism and asthma. However, there is a lack of modern pharmacology studies regarding its anti-gastric ulcer activity. AIM OF THE STUDY: The objective of this study is to investigate the protective effects of an extract from K. galanga L. rhizome (Kge) and its active components kaempferol and luteolin on ethanol-induced gastric ulcer. MATERIALS AND METHODS: The kge was prepared by ultrasonic-assisted extraction, and the contents of kaempferol and luteolin were determined by HPLC. The mice were randomly divided into seven groups: blank control (0.5 % CMC-Na; 0.1 mL/10 g), untreatment (0.5 % CMC-Na; 0.1 mL/10 g), Kge (100, 200 and 400 mg/kg), kaempferol (100 mg/kg) and luteolin (100 mg/kg) groups. The mice were treated intragastrically once daily for 7 days. At 1 h post the last administration, the mice in all groups except the blank control group were intragastrically administrated with anhydrous alcohol (0.1 mL/10 g) once to induce gastric ulcer. Then, fasting was continued for 1 h, followed by sample collection for evaluation by enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction assay. RESULTS: The contents of kaempferol and luteolin in Kge were determined as 3713 µg/g and 2510 µg/g, respectively. Alcohol induced severely damages with edema, inflammatory cell infiltration and bleeding, and the ulcer index was 17.63 %. After pre-treatment with Kge (100, 200 and 400 mg/kg), kaempferol and luteolin, the pathological lesions were obviously alleviated and ulcer indices were reduced to 13.42 %, 11.65 %, 6.54 %, 3.58 % and 3.85 %, respectively. In untreated group, the contents of Ca2+, myeloperoxidase, malondialdehyde, NO, cyclic adenosine monophosphate and histamine were significantly increased, while the contents of hexosamine, superoxide dismutase, glutathione peroxidase, and prostaglandin E2 were significantly decreased; the transcriptional levels of IL-1α, IL-1ß, IL-6, calcitonin gene related peptide, substance P, M3 muscarinic acetylcholine receptor, histamine H2 receptor, cholecystokinin 2 receptor and H+/K+ ATPase were significantly increased when compared with the blank control group. After pre-treatment, all of these changes were alleviated, even returned to normal levels. Kge exhibited anti-gastric ulcer activity and the high dose of Kge (400 mg/kg) exhibited comparable activity to that of kaempferol and luteolin. CONCLUSION: The study showed that K. galanga L., kaempferol, and luteolin have protective effects against ethanol-induced gastric ulcers. This is achieved by regulating the mucosal barrier, oxidative stress, and gastric regulatory mediators, as well as inhibiting the TRPV1 signaling pathway and gastric acid secretion, ultimately reducing the gastric ulcer index.

Hepatocyte-specific mitogen-activated protein kinase phosphatase 1 in sexual dimorphism and susceptibility to alcohol induced liver injury.

Background: It is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury. Methods: C57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ "f/f" male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student's t-test. Results: ASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes. Conclusion: Hepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Identification of a novel FOXO3 agonist that protects against alcohol induced liver injury.

Alcohol-related liver disease (ALD) is a global healthcare concern which caused by excessive alcohol consumption with limited treatment options. The pathogenesis of ALD is complex and involves in hepatocyte damage, hepatic inflammation, increased gut permeability and microbiome dysbiosis. FOXO3 is a well-recognized transcription factor which associated with longevity via promoting antioxidant stress response, preventing senescence and cell death, and inhibiting inflammation. We and many others have reported that FOXO3-/- mice develop more severe liver injury in response to alcohol. In the present study, we aimed to develop compounds that activate FOXO3 and further investigate their effects in alcohol induced liver injury. Through virtual screening, we discovered series of small molecular compounds that showed high affinity to FOXO3. We confirmed effects of compounds on FOXO3 target gene expression, as well as antioxidant and anti-apoptotic effects in vitro. Subsequently we evaluated the protective efficacy of compounds in alcohol induced liver injury in vivo. As a result, the leading compound we identified, 214991, activated downstream target genes expression of FOXO3, inhibited intracellular ROS accumulation and cell apoptosis induced by H2O2 and sorafenib. By using Lieber-DeCarli alcohol feeding mouse model, 214991 showed protective effects against alcohol-induced liver inflammation, macrophage and neutrophil infiltration, and steatosis. These findings not only reinforce the potential of FOXO3 as a valuable target for therapeutic intervention of ALD, but also suggested that compound 214991 as a promising candidate for the development of innovative therapeutic strategies of ALD.

Mutation of novel ethanol-responsive lncRNA Gm41261 impacts ethanol-related behavioral responses in mice.

Chronic alcohol exposure results in widespread dysregulation of gene expression that contributes to the pathogenesis of Alcohol Use Disorder (AUD). Long noncoding RNAs are key regulators of the transcriptome that we hypothesize coordinate alcohol-induced transcriptome dysregulation and contribute to AUD. Based on RNA-Sequencing data of human prefrontal cortex, basolateral amygdala and nucleus accumbens of AUD versus non-AUD brain, the human LINC01265 and its predicted murine homolog Gm41261 (i.e., TX2) were selected for functional interrogation. We tested the hypothesis that TX2 contributes to ethanol drinking and behavioral responses to ethanol. CRISPR/Cas9 mutagenesis was used to create a TX2 mutant mouse line in which 306 base-pairs were deleted from the locus. RNA analysis revealed that an abnormal TX2 transcript was produced at an unchanged level in mutant animals. Behaviorally, mutant mice had reduced ethanol, gaboxadol and zolpidem-induced loss of the righting response and reduced tolerance to ethanol in both sexes. In addition, a male-specific reduction in two-bottle choice every-other-day ethanol drinking was observed. Male TX2 mutants exhibited evidence of enhanced GABA release and altered GABAA receptor subunit composition in neurons of the nucleus accumbens shell. In C57BL6/J mice, TX2 within the cortex was cytoplasmic and largely present in Rbfox3+ neurons and IBA1+ microglia, but not in Olig2+ oligodendrocytes or in the majority of GFAP+ astrocytes. These data support the hypothesis that TX2 mutagenesis and dysregulation impacts ethanol drinking behavior and ethanol-induced behavioral responses in mice, likely through alterations in the GABAergic system.

Bulbils of Aerial Yam Attenuate Ethanol-Induced Hepatotoxicity in HepG2 Cells through Inhibition of Oxidative Stress by Activation of the Nuclear Factor Erythroid-2-Related Factor 2 Signaling Pathway.

Bulbil of yam (BY) extract contains various active compounds possessing many pharmacological properties. However, little is known about the effect and underlying mechanism of BY extract on ethanol-induced liver damage. The present study explored the beneficial potential of BY extract on ethanol-induced hepatotoxicity. To evaluate its effectiveness, ethanol-induced HepG2 liver cells were pretreated with BY extract. BY extract effectively rescued cells from ethanol treatment through inhibition of apoptotic cell death as well as inhibiting expression of mitogen-activated protein kinase (MAPK) proteins as stress inducers. BY extract increased the expression of typical antioxidants. Furthermore, BY extract significantly inhibited mitochondrial dysfunction and endoplasmic reticulum (ER) stress, which are major ROS-inducing factors. Finally, as an underlying mechanism of the protective effects of BY extract on ethanol-induced liver damage, it activated Nrf2 protein through translocation from the cytosol to the nucleus, which in turn activated its target oxidative stress suppressor genes. Collectively, our findings demonstrate that BY extract has potential antioxidative effects in ethanol-induced liver cells and contributes to the establishment of a treatment strategy for alcohol-derived liver injuries.

Hydroethanolic extract of Gentiana kurroo Royle rhizome ameliorates ethanol-induced liver injury by reducing oxidative stress, inflammation and fibrogenesis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Gentiana kurroo Royle is a medicinal plant mentioned as Traymana in Ayurveda. In the folklore, it is used to cure fever, stomach ache, skin diseases and liver disorders. However, limited reports are available on the therapeutic potential of Gentiana kurroo Royle against alcohol-induced liver damage. AIM OF THE STUDY: To assess the effectiveness of the hydroethanolic extract of Gentiana kurroo Royle rhizome (GKRE) against alcohol-induced liver injury and explore the mechanism of action. MATERIALS AND METHODS: GKRE was characterized using UHPLC-QTOF-MS/MS. The binding affinity of the identified compound was studied in silico. In vitro studies were performed in the Huh-7 cell line. An acute oral toxicity study (2 g/kg BW) of GKRE was done in rats following OECD 420 guidelines. In the efficacy study, rats were treated with 50% ethanol (5 mL/kg BW, orally) for 4 weeks, followed by a single intraperitoneal dose of CCl4 (30%; 1 mL/kg BW) to induce liver injury. After 4th week, the rats were treated with GKRE at 100, 200 and 400 mg/kg BW doses for the next fifteen days. The biochemical and antioxidant parameters were analyzed using commercial kits and a biochemistry analyzer. Histopathology, gene and protein expressions were studied using qRT PCR and western blotting. RESULTS: Thirteen compounds were detected in GKRE. Few compounds showed a strong interaction with the fibrotic and inflammatory proteins in silico. GKRE reduced (p < 0.05) the ethanol-induced ROS production and inflammation in Huh-7 cells. The acute oral toxicity study revealed no adverse effect of GKRE in rats at 2 g/kg BW. GKRE improved (p < 0.05) the body and liver weights in ethanol-treated rats. GKRE improved (p < 0.05) the mRNA levels of ADH, SREBP1c and mitochondrial biogenesis genes in the liver tissues. GKRE also improved (p < 0.05) the liver damage markers, lipid peroxidation and levels of antioxidant enzymes in the liver. A reduced severity (p < 0.05) of pathological changes, fibrotic tissue deposition and caspase 3/7 activity were observed in the liver tissues of GKRE-treated rats. Further, GKRE downregulated (p < 0.05) the expression of fibrotic (TGFß, αSMA and SMADs) and inflammatory markers (TNFα, IL6, IL1ß and NFκB) in the liver. CONCLUSION: GKRE showed efficacy against alcohol-induced liver damage by inhibiting oxidative stress, apoptosis, inflammation and fibrogenesis in the liver.

BRCA1 protein dose-dependent risk for embryonic oxidative DNA damage, embryopathies and neurodevelopmental disorders with and without ethanol exposure.

Although widely known as a tumor suppressor, the breast cancer 1 susceptibility protein (BRCA1) is also important in development, where it regulates fetal DNA repair pathways that protect against DNA damage caused by physiological and drug-enhanced levels of reactive oxygen species (ROS). We previously showed that conditional heterozygous (+/-) knockout (cKO) mouse embryos with a minor 28% BRCA1 deficiency developed normally in culture, but when exposed to the ROS-initiating drug, alcohol (ethanol, EtOH), exhibited embryopathies not evident in wild-type (+/+) littermates. Herein, we characterized a directBrca1 +/- knockout (KO) model with a 2-fold greater (58%) reduction in BRCA1 protein vs. the cKO model. We also characterized and compared learning & memory deficits in both the cKO and KO models. Even saline-exposed Brca1 +/- vs. +/+ KO progeny exhibited enhanced oxidative DNA damage and embryopathies in embryo culture and learning & memory deficits in females in vivo, which were not observed in the cKO model, revealing the potential pathogenicity of physiological ROS levels. The embryopathic EtOH concentration for cultured direct KO embryos was half that for cKO embryos, and EtOH affected Brca1 +/+ embryos only in the direct KO model. The spectrum and severity of EtOH embryopathies in culture were greater in both Brca1 +/- vs. +/+ embryos, and direct KO vs. cKO +/- embryos. Motor coordination deficits were evident in both male and female Brca1 +/- KO progeny exposed in utero to EtOH. The results in our direct KO model with a greater BRCA1 deficiency vs. cKO mice provide the first evidence for BRCA1 protein dose-dependent susceptibility to developmental disorders caused by physiological and drug-enhanced oxidative stress.

Apoptosis Due to After-effects of Acute Ethanol Exposure in Brain Cortex: Intrinsic and Extrinsic Signaling Pathways.

Alcohol hangover is the combination of negative mental and physical symptoms which can be experienced after a single episode of alcohol consumption, starting when blood alcohol concentration approaches zero. We previously demonstrated that hangover provokes mitochondrial dysfunction, oxidative stress, imbalance in antioxidant defenses, and impairment in cellular bioenergetics. Chronic and acute ethanol intake induces neuroapoptosis but there are no studies which evaluated apoptosis at alcohol hangover. The aim of the present work was to study alcohol residual effects on intrinsic and extrinsic apoptotic signaling pathways in mice brain cortex. Male Swiss mice received i.p. injection of ethanol (3.8 g/kg) or saline. Six hours after injection, at alcohol hangover onset, mitochondria and tissue lysates were obtained from brain cortex. Results indicated that during alcohol hangover a loss of granularity of mitochondria and a strong increment in mitochondrial permeability were observed, indicating the occurrence of swelling. Alcohol-treated mice showed a significant 35% increase in Bax/Bcl-2 ratio and a 5-fold increase in the ratio level of cytochrome c between mitochondria and cytosol. Caspase 3, 8 and 9 protein expressions were 32%, 33% and 20% respectively enhanced and the activity of caspase 3 and 6 was 30% and 20% increased also due to the hangover condition. Moreover, 38% and 32% increments were found in PARP1 and p53 protein expression respectively and on the contrary, SIRT-1 was almost 50% lower than controls due to the hangover condition. The present work demonstrates that alcohol after-effects could result in the activation of mitochondrial and non-mitochondrial apoptosis pathways.

Simvastatin Significantly Reduced Alcohol-Induced Cardiac Damage in Adolescent Mice.

Alcohol abuse by adolescents is becoming a serious health concern as they often progress to becoming alcoholics later in life which may lead to heart problems. Chronic alcohol use alters the cardiac function and structure, such as haemodynamic changes, weakening and loss of cardiomyocytes, myocardial fibrosis, and inflammation. Simvastatin is a commonly used drug for the treatment and management of various cardiovascular problems but information on its protective effects against alcohol-induced cardiomyocyte hypertrophy, fibrosis, and inflammation is lacking in the literature. Four-week-old male (n = 5) and female (n = 5) C57BL/6 J mice were assigned to each experimental group: (I) NT-no administration of alcohol or Simvastatin; (II) ALC-2.5 g/Kg/day of 20% alcohol via intraperitoneal injection (i.p.); (III) SIM-5 mg/Kg/day of Simvastatin via oral gavage; (iv) ALC + SIM5-5 mg/Kg/day of Simvastatin via oral gavage followed by 2.5 g/Kg/day of 20% alcohol via i.p.; and (v) ALC + SIM15-15 mg/Kg/day Simvastatin via oral gavage followed by 2.5 g/Kg/day of 20% alcohol via i.p. After the 28-day treatment period, the heart was removed and processed for H&E, Masson's trichrome, or TNF-α immunolabelling. The area and diameter of cardiomyocytes were measured on the H&E-stained sections. The distribution of collagen or TNF-α expression was quantified using the deconvolution tool of ImageJ software. The results confirmed alcohol-induced toxicity on the cardiomyocytes and Simvastatin reduced alcohol-induced cardiomyocyte hypertrophy, fibrosis, and inflammation in both sexes. This study demonstrated that Simvastatin, an FDA approved and easily accessible drug, may be beneficial in lowering the prevalence of alcohol-induced cardiovascular diseases (especially in adolescents) which will have a huge financial implication on health systems worldwide.