In vivo Bruton's tyrosine kinase inhibition attenuates alcohol-associated liver disease by regulating CD84-mediated granulopoiesis.

Severe alcohol-associated hepatitis (AH) is a life-threatening form of alcohol-associated liver disease. Liver neutrophil infiltration is a hallmark of AH, yet the effects of alcohol on neutrophil functions remain elusive. Identifying therapeutic targets to reduce neutrophil-mediated liver damage is essential. Bruton's tyrosine kinase (BTK) plays an important role in neutrophil development and function; however, the role of BTK in AH is unknown. Using RNA sequencing of circulating neutrophils, we found an increase in Btk expression (P = 0.05) and phosphorylated BTK (pBTK) in patients with AH compared with healthy controls. In vitro, physiologically relevant doses of alcohol resulted in a rapid, TLR4-mediated induction of pBTK in neutrophils. In a preclinical model of AH, administration of a small-molecule BTK inhibitor (evobrutinib) or myeloid-specific Btk knockout decreased proinflammatory cytokines and attenuated neutrophil-mediated liver damage. We found that pBTK was essential for alcohol-induced bone marrow granulopoiesis and liver neutrophil infiltration. In vivo, BTK inhibition or myeloid-specific Btk knockout reduced granulopoiesis, circulating neutrophils, liver neutrophil infiltration, and liver damage in a mouse model of AH. Mechanistically, using liquid chromatography-tandem mass spectrometry, we identified CD84 as a kinase target of BTK, which is involved in granulopoiesis. In vitro, CD84 promoted alcohol-induced interleukin-1ß and tumor necrosis factor-α in primary human neutrophils, which was inhibited by CD84-blocking antibody treatment. Our findings define the role of BTK and CD84 in regulating neutrophil inflammation and granulopoiesis, with potential therapeutic implications in AH.

IFN-ß Overexpressing Adipose-Derived Mesenchymal Stem Cells Mitigate Alcohol-Induced Liver Damage and Gut Permeability.

Alcoholic liver disease (ALD) is a form of hepatic inflammation. ALD is mediated by gut leakiness. This study evaluates the anti-inflammatory effects of ASCs overexpressing interferon-beta (ASC-IFN-ß) on binge alcohol-induced liver injury and intestinal permeability. In vitro, ASCs were transfected with a non-viral vector carrying the human IFN-ß gene, which promoted hepatocyte growth factor (HGF) secretion in the cells. To assess the potential effects of ASC-IFN-ß, C57BL/6 mice were treated with three oral doses of binge alcohol and were administered intraperitoneal injections of ASC-IFN-ß. Mice treated with binge alcohol and administered ASC-IFN-ß showed reduced liver injury and inflammation compared to those administered a control ASC. Analysis of intestinal tissue from ethanol-treated mice administered ASC-IFN-ß also indicated decreased inflammation. Additionally, fecal albumin, blood endotoxin, and bacterial colony levels were reduced, indicating less gut leakiness in the binge alcohol-exposed mice. Treatment with HGF, but not IFN-ß or TRAIL, mitigated the ethanol-induced down-regulation of cell death and permeability in Caco-2 cells. These results demonstrate that ASCs transfected with a non-viral vector to induce IFN-ß overexpression have protective effects against binge alcohol-mediated liver injury and gut leakiness via HGF.

Enhanced In Vitro Recapitulation of In Vivo Liver Regeneration by Co-Culturing Hepatocyte Organoids with Adipose-Derived Mesenchymal Stem Cells, Alleviating Steatosis and Apoptosis in Acute Alcoholic Liver Injury.

Hepatocyte organoids (HOs) have superior hepatic functions to cholangiocyte-derived organoids but suffer from shorter lifespans. To counteract this, we co-cultured pig HOs with adipose-derived mesenchymal stem cells (A-MSCs) and performed transcriptome analysis. The results revealed that A-MSCs enhanced the collagen synthesis pathways, which are crucial for maintaining the three-dimensional structure and extracellular matrix synthesis of the organoids. A-MSCs also increased the expression of liver progenitor cell markers (KRT7, SPP1, LGR5+, and TERT). To explore HOs as a liver disease model, we exposed them to alcohol to create an alcoholic liver injury (ALI) model. The co-culture of HOs with A-MSCs inhibited the apoptosis of hepatocytes and reduced lipid accumulation of HOs. Furthermore, varying ethanol concentrations (0-400 mM) and single-versus-daily exposure to HOs showed that daily exposure significantly increased the level of PLIN2, a lipid storage marker, while decreasing CYP2E1 and increasing CYP1A2 levels, suggesting that CYP1A2 may play a critical role in alcohol detoxification during short-term exposure. Moreover, daily alcohol exposure led to excessive lipid accumulation and nuclear fragmentation in HOs cultured alone. These findings indicate that HOs mimic in vivo liver regeneration, establishing them as a valuable model for studying liver diseases, such as ALI.

Alcohol- and Low-Iron Induced Changes in Antioxidant and Energy Metabolism Associated with Protein Lys Acetylation.

Understanding the role of iron in ethanol-derived hepatic stress could help elucidate the efficacy of dietary or clinical interventions designed to minimize liver damage from chronic alcohol consumption. We hypothesized that normal levels of iron are involved in ethanol-derived liver damage and reduced dietary iron intake would lower the damage caused by ethanol. We used a pair-fed mouse model utilizing basal Lieber-DeCarli liquid diets for 22 weeks to test this hypothesis. In our mouse model, chronic ethanol exposure led to mild hepatic stress possibly characteristic of early-stage alcoholic liver disease, seen as increases in liver-to-body weight ratios. Dietary iron restriction caused a slight decrease in non-heme iron and ferritin (FeRL) expression while it increased transferrin receptor 1 (TfR1) expression without changing ferroportin 1 (FPN1) expression. It also elevated protein lysine acetylation to a more significant level than in ethanol-fed mice under normal dietary iron conditions. Interestingly, iron restriction led to an additional reduction in nicotinamide adenine dinucleotide (NAD+) and NADH levels. Consistent with this observation, the major mitochondrial NAD+-dependent deacetylase, NAD-dependent deacetylase sirtuin-3 (SIRT3), expression was significantly reduced causing increased protein lysine acetylation in ethanol-fed mice at normal and low-iron conditions. In addition, the detection of superoxide dismutase 1 and 2 levels (SOD1 and SOD2) and oxidative phosphorylation (OXPHOS) complex activities allowed us to evaluate the changes in antioxidant and energy metabolism regulated by ethanol consumption at normal and low-iron conditions. We observed that the ethanol-fed mice had mild liver damage associated with reduced energy and antioxidant metabolism. On the other hand, iron restriction may exacerbate certain activities of ethanol further, such as increased protein lysine acetylation and reduced antioxidant metabolism. This metabolic change may prove a barrier to the effectiveness of dietary reduction of iron intake as a preventative measure in chronic alcohol consumption.

Effects of elafibranor on liver fibrosis and gut barrier function in a mouse model of alcohol-associated liver disease.

BACKGROUND: Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality, but there are no therapeutic targets and modalities to prevent ALD-related liver fibrosis. Peroxisome proliferator activated receptor (PPAR) α and δ play a key role in lipid metabolism and intestinal barrier homeostasis, which are major contributors to the pathological progression of ALD. Meanwhile, elafibranor (EFN), which is a dual PPARα and PPARδ agonist, has reached a phase III clinical trial for the treatment of metabolic dysfunction-associated steatotic liver disease and primary biliary cholangitis. However, the benefits of EFN for ALD treatment is unknown. AIM: To evaluate the inhibitory effects of EFN on liver fibrosis and gut-intestinal barrier dysfunction in an ALD mouse model. METHODS: ALD-related liver fibrosis was induced in female C57BL/6J mice by feeding a 2.5% ethanol (EtOH)-containing Lieber-DeCarli liquid diet and intraperitoneally injecting carbon tetrachloride thrice weekly (1 mL/kg) for 8 weeks. EFN (3 and 10 mg/kg/day) was orally administered during the experimental period. Histological and molecular analyses were performed to assess the effect of EFN on steatohepatitis, fibrosis, and intestinal barrier integrity. The EFN effects on HepG2 lipotoxicity and Caco-2 barrier function were evaluated by cell-based assays. RESULTS: The hepatic steatosis, apoptosis, and fibrosis in the ALD mice model were significantly attenuated by EFN treatment. EFN promoted lipolysis and ß-oxidation and enhanced autophagic and antioxidant capacities in EtOH-stimulated HepG2 cells, primarily through PPARα activation. Moreover, EFN inhibited the Kupffer cell-mediated inflammatory response, with blunted hepatic exposure to lipopolysaccharide (LPS) and toll like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling. EFN improved intestinal hyperpermeability by restoring tight junction proteins and autophagy and by inhibiting apoptosis and proinflammatory responses. The protective effect on intestinal barrier function in the EtOH-stimulated Caco-2 cells was predominantly mediated by PPARδ activation. CONCLUSION: EFN reduced ALD-related fibrosis by inhibiting lipid accumulation and apoptosis, enhancing hepatocyte autophagic and antioxidant capacities, and suppressing LPS/TLR4/NF-κB-mediated inflammatory responses by restoring intestinal barrier function.

Apolipoprotein H deficiency exacerbates alcohol-induced liver injury via gut Dysbiosis and altered bile acid metabolism.

BACKGROUND: APOH plays an essential role in lipid metabolism and the transport of lipids in the circulation. Previous studies have shown that APOH deficiency causes fatty liver and gut microbiota dysbiosis in mouse models. However, the role and potential mechanisms of APOH deficiency in the pathogenesis of alcoholic liver disease remain unclear. METHODS: C57BL/6 WT and ApoH-/- mice were used to construct the binge-on-chronic alcohol feeding model. Mouse liver transcriptome, targeted bile acid metabolome, and 16S gut bacterial taxa were assayed and analyzed. Open-source human liver transcriptome dataset was analyzed. RESULTS: ApoH-/- mice fed with alcohol showed severe hepatic steatosis. Liver RNAseq and RT-qPCR data indicated that APOH deficiency predominantly impacts hepatic lipid metabolism by disrupting de novo lipogenesis, cholesterol processing, and bile acid metabolism. A targeted bile acid metabolomics assay indicated significant changes in bile acid composition, including increased percentages of TCA in the liver and DCA in the gut of alcohol-fed ApoH-/- mice. The concentrations of CA, NorCA, and HCA in the liver were higher in ApoH-/- mice on an ethanol diet compared to the control mice (p < 0.05). Additionally, APOH deficiency altered the composition of gut flora, which correlated with changes in the liver bile acid composition in the ethanol-feeding mouse model. Finally, open-source transcript-level data from human ALD livers highlighted a remarkable link between APOH downregulation and steatohepatitis, as well as bile acid metabolism. CONCLUSION: APOH deficiency aggravates alcohol induced hepatic steatosis through the disruption of gut microbiota homeostasis and bile acid metabolism in mice.

Neutrophil PAD4 Expression and Its Pivotal Role in Assessment of Alcohol-Related Liver Disease.

Neutrophils release neutrophil extracellular traps (NETs) as a defense strategy in response to broad-spectrum infections and sterile triggers. NETs consist of a DNA scaffold decorated with antimicrobial peptides (AMPs) and enzymatically active proteases, including peptidyl arginine deiminase type 4 (PAD4). Susceptibility to infections and inflammatory dysregulation are hallmarks of alcohol-related liver disease (ALD). Sixty-two patients with ALD were prospectively recruited, and they were followed for 90 days. Twenty-four healthy volunteers served as the control group. PAD4 concentrations were quantified using immunoenzymatic ELISAs. Correlation coefficients between PAD4 blood concentrations and markers of systemic inflammation; liver dysfunction severity scores; and ALD complications were calculated. The receiver operating curves (ROCs) and their areas under the curve (AUCs) were checked in order to assess the accuracy of PAD4 expression in predicting the degree of liver failure and the development of ALD complications. Systemic concentrations of PAD4 were significantly increased in the patients with ALD in comparison with controls. PAD4 levels correlated with the standard markers of inflammation and revealed a good predictive AUC (0.76) for survival in the whole ALD group. PAD4 seems to be an inflammatory mediator and may be potentially applied as a predictor of patient survival in ALD.

Alpha-Asarone attenuates alcohol-induced hepatotoxicity in a murine model by ameliorating oxidative stress, inflammation, and modulating apoptotic-Autophagic cell death.

Alcoholic liver disease (ALD) is a major cause of chronic liver injury characterized by steatosis, inflammation, and fibrosis. This study explored the hepatoprotective mechanisms of alpha-asarone in a mouse model of chronic-binge alcohol feeding. Adult male mice were randomized into control, alcohol, and alcohol plus alpha-asarone groups. Serum aminotransferases and histopathology assessed liver injury. Oxidative stress was evaluated via malondialdehyde content, glutathione, superoxide dismutase, and catalase activities. Pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 were quantified by ELISA. P53-mediated apoptosis was determined by immunohistochemistry. Key autophagy markers phospho-AMPK, AMPK, Beclin-1, LC3-I/LC3-II ratio, and LC3 were examined by immunoblotting. Alcohol administration increased serum ALT, AST and ALP, indicating hepatocellular damage. This liver dysfunction was associated with increased oxidative stress, inflammation, p53 expression and altered autophagy. Alpha-asarone treatment significantly decreased ALT, AST and ALP levels and improved histological architecture versus alcohol alone. Alpha-asarone also mitigated oxidative stress, reduced TNF-α, IL-1ß and IL-6 levels, ameliorated p53 overexpression and favorably modulated autophagy markers. Our findings demonstrate that alpha-asarone confers protective effects against ALD by enhancing antioxidant defenses, suppressing hepatic inflammation, regulating apoptotic signaling, and restoring autophagic flux. This preclinical study provides compelling evidence for the therapeutic potential of alpha-asarone in attenuating alcohol-induced liver injury and warrants further evaluation as a pharmacotherapy for ALD.

PSTPIP2 protects against alcoholic liver injury and invokes STAT3-mediated suppression of apoptosis.

Alcoholic liver injury (ALI) stands as a prevalent affliction within the spectrum of complex liver diseases. Prolonged and excessive alcohol consumption can pave the way for liver fibrosis, cirrhosis, and even hepatocellular carcinoma. Recent findings have unveiled the protective role of proline serine-threonine phosphatase interacting protein 2 (PSTPIP2) in combating liver ailments. However, the role of PSTPIP2 in ALI remains mostly unknown. This study aimed to determine the expression profile of PSTPIP2 in ALI and to uncover the mechanism through which PSTPIP2 affects the survival and apoptosis of hepatocytes in ALI, using both ethyl alcohol (EtOH)-fed mice and an EtOH-induced AML-12 cell model. We observed a consistent decrease in PSTPIP2 expression both in vivo and in vitro. Functionally, we assessed the impact of PSTPIP2 overexpression on ALI by administering adeno-associated virus 9 (AAV9)-PSTPIP2 into mice. The results demonstrated that augmenting PSTPIP2 expression significantly shielded against liver parenchymal distortion and curbed caspase-dependent hepatocyte apoptosis in EtOH-induced ALI mice. Furthermore, enforcing PSTPIP2 expression reduced hepatocyte apoptosis in a stable PSTPIP2-overexpressing AML-12 cell line established through lentivirus-PSTPIP2 transfection in vitro. Mechanistically, this study also identified signal transducer and activator of transcription 3 (STAT3) as a direct signaling pathway regulated by PSTPIP2 in ALI. In conclusion, our findings provide compelling evidence that PSTPIP2 has a regulatory role in hepatocyte apoptosis via the STAT3 pathway in ALI, suggesting PSTPIP2 as a promising therapeutic target for ALI.

MLKL deficiency alleviates acute alcoholic liver injury via inhibition of NLRP3 inflammasome.

Mixed lineage kinase domain-like protein (MLKL) is identified as the terminal executor of necroptosis. However, its role in acute alcoholic liver injury remains unclear. This study elucidates that MLKL can contribute to acute alcoholic liver injury independently of necroptosis. Although the expression of MLKL was upregulated, no significant increase in its phosphorylation or membrane translocation was observed in the liver tissues of mice treated with ethanol. This finding confirms that alcohol intake does not induce necroptosis in mouse liver tissue. Additionally, the deletion of Mlkl resulted in the downregulation of NLRP3 expression, which subsequently inhibited the activation of the NLRP3 inflammasome and the ensuing inflammatory response, thereby effectively mitigating liver injury induced by acute alcohol consumption. The knockout of Nlrp3 did not affect the expression of MLKL, further confirming that MLKL acts upstream of NLRP3. Mechanistically, inhibiting the nuclear translocation of MLKL reduced the nuclear entry of p65, the principal transcriptional regulator of NLRP3, thereby limiting the transcription of Nlrp3 mRNA and subsequent NLRP3 expression. Overall, this study unveils a novel mechanism of MLKL regulates the activation of NLRP3 inflammasomes in a necroptosis independent way in acute alcoholic liver injury.

Metallofullerenol alleviates alcoholic liver damage via ROS clearance under static magnetic and electric fields.

Alcoholic liver disease (ALD) is a common chronic redox disease caused by increased alcohol consumption. Abstinence is a major challenge for people with alcohol dependence, and approved drugs have limited efficacy. Therefore, this study aimed to explore a new treatment strategy for ALD using ferroferric oxide endohedral fullerenol (Fe3O4@C60(OH)n) in combination with static magnetic and electric fields (sBE). The primary hepatocytes of 8-9-week-old female BALB/c mice were used to evaluate the efficacy of the proposed combination treatment. A mouse chronic binge ethanol feeding model was established to determine the alleviatory effect of Fe3O4@C60(OH)n on liver injury under sBE exposure. Furthermore, the ability of Fe3O4@C60(OH)n to eliminate •OH was evaluated. Alcohol-induced hepatocyte and mitochondrial damage were reversed in vitro. Additionally, the combination therapy reduced liver damage, alleviated oxidative stress by improving antioxidant levels, and effectively inhibited liver lipid accumulation in animal experiments. Here, we used a combination of magnetic derivatives of fullerenol and sBE to further improve the ROS clearance rate, thereby alleviating ALD. The developed combination treatment may effectively improve alcohol-induced liver damage and maintain redox balance without apparent toxicity, thereby enhancing therapy aimed at ALD and other redox diseases.

Fucoidan from Apostichopus japonicus ameliorates alcoholic liver disease by regulating gut-liver axis homeostasis.

Long-term and excessive alcohol consumption can lead to the development of alcoholic liver disease (ALD), characterized by oxidative damage, intestinal barrier injury, and disruption of intestinal microbiota. In this study, we extracted fucoidan (Aj-FUC) from Apostichopus japonicus using enzymatic methods and characterized its structure. The ALD model was established in male Balb/c mice using 56° Baijiu, with silymarin as a positive control. Mice were orally administered 100 mg/kg·bw and 300 mg/kg·bw of Aj-FUC for 28 days to evaluate its effects on liver injury in ALD mice and explore its potential role in modulating the gut-liver axis. The results showed significant improvements in histopathological changes and liver disease in the Aj-FUC group. Aj-FUC treatment significantly increased the levels of glutathione (GSH) and glutathione peroxidase (GSH-Px) while weakly reduced the elevation of malondialdehyde (MDA) induced by ALD. It also regulated the Nrf2/HO-1 signaling pathway, collectively alleviating hepatic oxidative stress. Aj-FUC intervention upregulated the expression of ZO-1 and Occludin, thus contributing to repair the intestinal barrier. Additionally, Aj-FUC increased the content of short-chain fatty acids (SCFAs) and regulated the imbalance in gut microbiota. These results suggested that Aj-FUC alleviates ALD by modulating the gut-liver axis homeostasis. It may prove to be a useful dietary supplement in the treatment of alcoholic liver damage.

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.

Cyanidin-3-O-glucoside alleviates ethanol-induced liver injury by promoting mitophagy in a Gao-binge mouse model of alcohol-associated liver disease.

BACKGROUND: Alcohol-associated liver disease (ALD) is a leading cause of liver disease-related deaths worldwide. Unfortunately, approved medications for the treatment of this condition are quite limited. One promising candidate is the anthocyanin, Cyanidin-3-O-glucoside (C3G), which has been reported to protect mice against hepatic lipid accumulation, as well as fibrosis in different animal models. However, the specific effects and mechanisms of C3G on ALD remain to be investigated. EXPERIMENTAL APPROACH: In this report, a Gao-binge mouse model of ALD was used to investigate the effects of C3G on ethanol-induced liver injury. The mechanisms of these C3G effects were assessed using AML12 hepatocytes. RESULTS: C3G administration ameliorated ethanol-induced liver injury by suppressing hepatic oxidative stress, as well as through reducing hepatic lipid accumulation and inflammation. Mechanistically, C3G activated the AMPK pathway and enhanced mitophagy to eliminate damaged mitochondria, thus reducing mitochondria-derived reactive oxidative species in ethanol-challenged hepatocytes. CONCLUSIONS: The results of this study indicate that mitophagy plays a potentially important role underlying the hepatoprotective action of C3G, as demonstrated in a Gao-binge mouse model of ALD. Accordingly, C3G may serve as a promising, new therapeutic drug candidate for use in ALD.

Alcohol-Associated Liver Disease Outcomes: Critical Mechanisms of Liver Injury Progression.

Alcohol-associated liver disease (ALD) is a substantial cause of morbidity and mortality worldwide and represents a spectrum of liver injury beginning with hepatic steatosis (fatty liver) progressing to inflammation and culminating in cirrhosis. Multiple factors contribute to ALD progression and disease severity. Here, we overview several crucial mechanisms related to ALD end-stage outcome development, such as epigenetic changes, cell death, hemolysis, hepatic stellate cells activation, and hepatic fatty acid binding protein 4. Additionally, in this review, we also present two clinically relevant models using human precision-cut liver slices and hepatic organoids to examine ALD pathogenesis and progression.

Loss of GCN5L1 exacerbates damage in alcoholic liver disease through ferroptosis activation.

BACKGROUND AND AIMS: Iron overload, oxidative stress and ferroptosis are associated with liver injury in alcohol-associated liver disease (ALD), however, the crosstalk among these regulatory pathways in ALD development is unclear. METHODS: ALD mouse model and general control of amino acid synthesis 5 like 1 (GCN5L1) liver knockout mice were generated to investigate the role of GCN5L1 in ALD development. Proteomic screening tests were performed to identify the key factors mediating GCN5L1 loss-induced ALD. RESULTS: Gene Expression Omnibus data set analysis indicates that GCN5L1 expression is negatively associated with ALD progression. GCN5L1 hepatic knockout mice develop severe liver injury and lipid accumulation when fed an alcohol diet. Screening tests identified that GCN5L1 targeted the mitochondrial iron transporter CISD1 to regulate mitochondrial iron homeostasis in ethanol-induced ferroptosis. GCN5L1-modulated CISD1 acetylation and activity were crucial for iron accumulation and ferroptosis in response to alcohol exposure. CONCLUSION: Pharmaceutical modulation of CISD1 activity is critical for cellular iron homeostasis and ethanol-induced ferroptosis. The GCN5L1/CISD1 axis is crucial for oxidative stress and ethanol-induced ferroptosis in ALD and is a promising avenue for novel therapeutic strategies.

Pharmacological inhibition of the Src homology phosphatase 2 confers partial protection in a mouse model of alcohol-associated liver disease.

Alcohol-associated liver disease (ALD) is a leading factor of liver-related death worldwide. ALD has various manifestations that include steatosis, hepatitis, and cirrhosis and is currently without approved pharmacotherapies. The Src homology phosphatase 2 (Shp2) is a drug target in some cancers due to its positive regulation of Ras-mitogen-activated protein kinase signaling and cell proliferation. Shp2 pharmacological inhibition yields beneficial outcomes in animal disease models, but its impact on ALD remains unexplored. This study aims to investigate the effects of Shp2 inhibition and its validity using a preclinical mouse model of ALD. We report that the administration of SHP099, a potent and selective allosteric inhibitor of Shp2, partially ameliorated ethanol-induced hepatic injury, inflammation, and steatosis in mice. Additionally, Shp2 inhibition was associated with reduced ethanol-evoked activation of extracellular signal-regulated kinase (ERK), oxidative, and endoplasmic reticulum (ER) stress in the liver. Besides the liver, excessive alcohol consumption induces multi-organ injury and dysfunction, including the intestine. Notably, Shp2 inhibition diminished ethanol-induced intestinal inflammation and permeability, abrogated the reduction in tight junction protein expression, and the activation of ERK and stress signaling in the ileum. Collectively, Shp2 pharmacological inhibition mitigates the deleterious effects of ethanol in the liver and intestine in a mouse model of ALD. Given the multifactorial aspects underlying ALD pathogenesis, additional studies are needed to decipher the utility of Shp2 inhibition alone or as a component in a multitherapeutic regimen to combat this deadly malady.

Role of ghrelin hormone in the development of alcohol-associated liver disease.

Fatty liver is the earliest response of the liver to excessive alcohol consumption. Previously we identified that chronic alcohol administration increases levels of stomach-derived hormone, ghrelin, which by reducing circulating insulin levels, ultimately contributes to the development of alcohol-associated liver disease (ALD). In addition, ghrelin directly promotes fat accumulation in hepatocytes by enhancing de novo lipogenesis. Other than promoting ALD, ghrelin is known to increase alcohol craving and intake. In this study, we used a ghrelin receptor (GHSR) knockout (KO) rat model to characterize the specific contribution of ghrelin in the development of ALD with emphasis on energy homeostasis. Male Wistar wild type (WT) and GHSR-KO rats were pair-fed the Lieber-DeCarli control or ethanol diet for 6 weeks. At the end of the feeding period, glucose tolerance test was conducted, and tissue samples were collected. We observed reduced alcohol intake by GHSR-KOs compared to a previous study where WT rats were fed ethanol diet ad libitum. Further, when the WTs were pair-fed to GHSR-KOs, the KO rats exhibited resistance to develop ALD through improving insulin secretion/sensitivity to reduce adipose lipolysis and hepatic fatty acid uptake/synthesis and increase fatty acid oxidation. Furthermore, proteomic data revealed that ethanol-fed KO exhibit less alcohol-induced mitochondrial dysfunction and oxidative stress than WT rats. Proteomic data also confirmed that the ethanol-fed KOs are insulin sensitive and are resistant to hepatic steatosis development compared to WT rats. Together, these data confirm that inhibiting ghrelin action prevent alcohol-induced liver and adipose dysfunction independent of reducing alcohol intake.

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.