AML12 Mouse Hepatocytes Research Articles

Involvement of BRD4 in Alcoholic Liver Injury: Autophagy Modulation via Regulation of the SIRT1/Beclin1 Axis

Alcoholic liver disease (ALD) caused by chronic alcohol abuse involves complex processes from steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma, posing a global health issue. Bromodomain protein 4 (BRD4) typically serves as a “reader” modulating the functions of transcription factors involved in various biological processes and disease progression. However, the specific mechanisms underlying alcoholic liver injury remain unclear. In this study, we detected aberrant BRD4 expression in the alcohol-induced ALD mouse model of chronic and binge ethanol feeding developed by the National Institute on Alcohol Abuse and Alcoholism, consistent with the in vitro results in Aml-12 mouse hepatocytes. Blocking and inhibiting BRD4 restored the impaired autophagic flux and lysosomal functions in alcohol-treated Aml-12 cells, whereas BRD4 overexpression reduced the expression levels of autophagy marker and lysosomal genes. Furthermore, mouse BRD4 knockdown, mediated by a short hairpin RNA carried by the adeno-associated virus serotype 8, significantly attenuated the alcohol-induced hepatocyte damage, including lipid deposition and inflammatory cell infiltration. Mechanistically, BRD4 overexpression in alcoholic liver injury inhibited the expression of sirtuin (SIRT)1 in Aml-12 cells. Chromatin immunoprecipitation and dual-luciferase reporter assays revealed that BRD4 functions as a transcription factor and suppressor, actively binding to the SIRT1 promoter region and inhibiting its transcription. SIRT1 activated autophagy, which was suppressed in alcoholic liver injury via Beclin1 deacetylation. In conclusion, our study revealed that BRD4 negatively regulated the SIRT1/Beclin1 axis and that its deficiency alleviated alcohol-induced liver injury in mice, thus providing a new strategy for ALD treatment.

Targeting BRD4 mitigates hepatocellular lipotoxicity by suppressing the NLRP3 inflammasome activation and GSDMD-mediated hepatocyte pyroptosis

Nod-like receptor family pyrin-containing protein 3 (NLRP3) inflammasome plays a pathologic role in metabolic dysfunction-associated steatohepatitis (MASH), but the molecular mechanism regulating the NLRP3 inflammasome activation in hepatocellular lipotoxicity remains largely unknown. Bromodomain-containing protein 4 (BRD4) has emerged as a key epigenetic reader of acetylated lysine residues in enhancer regions that control the transcription of key genes. The aim of this study is to investigate if and how BRD4 regulated the NLRP3 inflammasome activation and pyroptosis in MASH. Using the AML12 and primary mouse hepatocytes stimulated by palmitic acid (PA) as an in vitro model of hepatocellular lipotoxicity, we found that targeting BRD4 by genetic knockdown or a selective BRD4 inhibitor MS417 protected against hepatosteatosis; and this protective effect was attributed to inhibiting the activation of NLRP3 inflammasome and reducing the expression of Caspase-1, gasdermin D (GSDMD), interleukin (IL)-1β and IL-6. Moreover, BRD4 inhibition limited the voltage-dependent anion channel-1 (VDAC1) expression and oligomerization in PA-treated AML12 hepatocytes, thereby suppressing the NLRP3 inflammasome activation. Additionally, the expression of BRD4 enhanced in MASH livers of humans. Mechanistically, BRD4 was upregulated during hepatocellular lipotoxicity that in turn modulated the active epigenetic mark H3K27ac at the promoter regions of the Vdac and Gsdmd genes, thereby enhancing the expression of VDAC and GSDMD. Altogether, our data provide novel insights into epigenetic mechanisms underlying BRD4 activating the NLRP3 inflammasome and promoting GSDMD-mediated pyroptosis in hepatocellular lipotoxicity. Thus, BRD4 might serve as a novel therapeutic target for the treatment of MASH.Graphic abstract

Fermented Syzygium samarangense leaves mitigate oxidative stress-triggered hepatotoxicity through Nrf2 activation and epigenetic regulation

The liver, which is a vital organ for metabolism and detoxification, is susceptible to damage caused by oxidative stress. Excessive consumption of acetaminophen (APAP), a commonly used analgesic ingredient, can cause liver injury due to oxidative stress. The aim of this study is to examine the protective effects and mechanisms of fermented Syzygium samarangense (wax apple) leaves (FWAL) against damage caused by APAP in mouse AML-12 hepatocytes. Ethanolic and water extracts of FWAL (FWAL-EE and FWAL-WE) were prepared, and both contained significant phenolic compounds such as gallic acid, EGCG, ellagic acid, myricitrin, and myricetin, with potent DPPH scavenging activity. FWAL-EE demonstrated superior effectiveness in lowering the death ratio, LDH leakage, and oxidative stress in APAP-treated AML-12 cells. The protective effects of FWAL-EE included the downregulation of inflammatory markers TNF-α and cox-2 mRNA, as well as the upregulation of Nrf2, HO-1, and NQO1 proteins in APAP-treated hepatocytes. Additionally, FWAL-EE reduced the expression of epigenetic enzymes HDACs 1, 2, 3, and 4, and DNMTs 1 and 3b while causing an increase in unmethylated DNA levels in the Nrf2 promoter region. Furthermore, EGCG, a prominent active compound in FWAL, protected AML-12 hepatocytes from APAP-induced damage by activating Nrf2 and NQO1, while also inhibiting epigenetic DNMTs and HDACs. These results suggested that FWAL-EE may effectively prevent APAP-induced liver damage by enhancing the activation of the Nrf2 antioxidant system through epigenetic regulation. FWAL-EE has potential as a nutraceutical for liver protection.

Liraglutide ameliorates hepatic steatosis via retinoic acid receptor-related orphan receptor α-mediated autophagy pathway.

Liraglutide, an analog of human glucagon-like peptide-1 (GLP-1), has been found to improve hepatic steatosis in clinical practice. However, the underlying mechanism remains to be fully defined. Increasing evidence suggests that retinoic acid receptor-related orphan receptor α (RORα) is involved in hepatic lipid accumulation. In the current study, we investigated whether the ameliorating impact of liraglutide on lipid-induced hepatic steatosis is dependent on RORα activity and examined the underlying mechanisms. Cre-loxP-mediated, liver-specific Rorα knockout (Rora LKO) mice, and littermate controls with a Roraloxp/loxp genotype were established. The effects of liraglutide on lipid accumulation were evaluated in mice challenged with a high-fat diet (HFD) for 12 weeks. Moreover, mouse AML12 hepatocytes expressing small interfering RNA (siRNA) of Rora were exposed to palmitic acid to explore the pharmacological mechanism of liraglutide. The results showed that liraglutide treatment significantly alleviated HFD-induced liver steatosis, marked by reduced liver weight and triglyceride accumulation, improved glucose tolerance and serum levels of lipid profiles and aminotransferase. Consistently, liraglutide also ameliorated lipid deposits in a steatotic hepatocyte model in vitro. In addition, liraglutide treatment reversed the HFD-induced downregulation of Rora expression and autophagic activity in mouse liver tissues. However, the beneficial effect of liraglutide on hepatic steatosis was not observed in Rora LKO mice. Mechanistically, the ablation of Rorα in hepatocytes diminished liraglutide-induced autophagosome formation and the fusion of autophagosomes and lysosomes, resulting in weakened autophagic flux activation. Thus, our findings suggest that RORα is essential for the beneficial impact of liraglutide on lipid deposition in hepatocytes and regulates autophagic activity in the underlying mechanism.

Fluorofenidone protects against acute liver failure in mice by regulating MKK4/JNK pathway

AimsAcute liver failure (ALF) is a life-threatening disease characterized by abrupt and extensive hepatic necrosis and apoptosis, resulting in high mortality. The approved drug, N-acetylcysteine (NAC), is only effective for acetaminophen (APAP)-associated ALF at the early stage. Thus, we investigate whether fluorofenidone (AKF-PD), a novel antifibrosis pyridone agent, protects against ALF in mice and explore its underlying mechanisms. MethodsALF mouse models were established using APAP or lipopolysaccharide/D-galactosamine (LPS/D-Gal). Anisomycin and SP600125 were used as JNK activator and inhibitor, respectively, and NAC served as a positive control. Mouse hepatic cell line AML12 and primary mouse hepatocytes were used for in vitro studies. ResultsAKF-PD pretreatment alleviated APAP-induced ALF with decreased necrosis, apoptosis, reactive oxygen species (ROS) markers, and mitochondrial permeability transition in liver. Additionally, AKF-PD alleviated mitochondrial ROS stimulated by APAP in AML12 cells. RNA-sequencing in the liver and subsequent gene set enrichment analysis showed that AKF-PD significantly impacted MAPK and IL-17 pathway. In vitro and in vivo studies demonstrated that AKF-PD inhibited APAP-induced phosphorylation of MKK4/JNK, while SP600125 only inhibited JNK phosphorylation. The protective effect of AKF-PD was abolished by anisomycin. Similarly, AKF-PD pretreatment abolished hepatotoxicity caused by LPS/D-Gal, decreased ROS levels, and diminished inflammation. Furthermore, unlike NAC, AKF-PD, inhibited the phosphorylation of MKK4 and JNK upon pretreatment, and improved survival in cases of LPS/D-Gal-induced mortality with delayed dosing. ConclusionsIn summary, AKF-PD can protect against ALF caused by APAP or LPS/D-Gal, in part, via regulating MKK4/JNK pathway. AKF-PD might be a novel candidate drug for ALF.

Identification of Ubr1 as an amino acid sensor of steatosis in liver and muscle.

Malnutrition is implicated in human metabolic disorders, including hepatic steatosis and myosteatosis. The corresponding nutrient signals and sensors as well as signalling pathways have not yet been well studied. This study aimed to unravel the nutrient-sensing mechanisms in the pathogenesis of steatosis. Plin2, a lipid droplet (LD) protein-inhibiting lipolysis, is associated with steatosis in liver and muscle. Taking advantage of the Gal4-UAS system, we used the Drosophila melanogaster wing imaginal disc as an in vivo model to study the regulation of Plin2 proteostasis and LD homeostasis. Drosophila Schneider 2 (S2) cells were used for western blotting, immunoprecipitation assays, amino acid-binding assays and ubiquitination assays to further investigate the regulatory mechanisms of Plin2 in response to nutrient signals. Mouse AML12 hepatocytes, human JHH-7 and SNU-475 hepatoma cells were used for immunofluorescence, western blotting and immunoprecipitation to demonstrate that the mode of Plin2 regulation is evolutionarily conserved. In addition, we purified proteins from HEK293 cells and reconstituted in vitro cell-free systems in amino acid-binding assays, pulldown assays and ubiquitination assays to directly demonstrate the molecular mechanism by which Ubr1 senses amino acids to regulate Plin2 proteostasis. As a lipolysis inhibitor, Plin2 was significantly elevated in liver (P<0.05) and muscle (P<0.05) in patients with steatosis. Consistently, we found that the ubiquitin moiety can be conjugated to any Lys residue in Plin2, ensuring robust clearance of Plin2 by protein degradation. We further demonstrated that the E3 ubiquitin ligase Ubr1 targets Plin2 for degradation in an amino acid-dependent manner. Ubr1 uses two canonical substrate-binding pockets, independent of each other, to bind basic and bulky hydrophobic amino acids, respectively. Mechanistically, amino acid binding allosterically activates Ubr1 by alleviating Ubr1's auto-inhibition. In the absence of amino acids, or when the amino acid-binding capacity of Ubr1 is diminished, Ubr1-mediated Plin2 degradation is inactivated, leading to steatosis. We identified Ubr1 as an amino acid sensor regulating Plin2 proteostasis, bridging the knowledge gap between steatosis and nutrient sensing. Our work may provide new strategies for the prevention and treatment of steatosis.

Alleviation of Hyperuricemia by Strictinin in AML12 Mouse Hepatocytes Treated with Xanthine and in Mice Treated with Potassium Oxonate

Hyperuricemia, an abnormally high level of blood uric acid, is a major risk factor for gout. Although xanthine oxidase inhibitors were clinically used to lower blood uric acid level, the concerned side effects restricted their utilization. In this study, strictinin, an abundant polyphenol in Pu'er tea, was evaluated for its preventive effects on hyperuricemia. The results showed that the xanthine oxidase activity, uric acid production, and inflammation in AML12 mouse hepatocytes treated with xanthine were significantly reduced by the supplementation of strictinin. Detailed analyses revealed that strictinin inhibited xanthine-induced NLRP3 inflammasome activation. Consistently, the elevated blood uric acid level and the enhanced xanthine oxidase activity in mice treated with potassium oxonate were effectively diminished by strictinin supplementation. Moreover, for the first time, strictinin was found to promote healthy gut microbiota. Overall, strictinin possesses a great potential to be utilized as a functional ingredient for the prevention of hyperuricemia.

Water extract of lotus leaves has hepatoprotective activity by enhancing Nrf2- and epigenetics-mediated cellular antioxidant capacity in mouse hepatocytes

• The main phenolics in LL-WE were EGCG, rutin, gallic acid, myricetin, quercetin, ellagic acid, etc. • LL-WE reduced the damage of mouse AML-12 hepatocytes caused by APAP-induced oxidative stress. • LL-WE promoted glutathione synthesis and activated the Nrf2 pathway. • LL-WE upregulated Nrf2-mediated antioxidant enzymes and downregulated HDACs and DNMTs. • LL-WE inhibited the in vitro activity of CpG methyltransferase (M.SssI). The liver is an important organ in the human body; however, studies have shown that the excessive intake of acetaminophen (APAP), the primary ingredient in marketing pain relievers, may cause oxidative damage to liver cells. Here we found that lotus leaf water extract (LL-WE) enriched with various phenolic compounds can prevent mice AML-12 hepatocytes from APAP-induced injury, and trigger the Nrf2 pathway in HepG2-C8 cells with ARE-luciferase plasmid. LL-WE effectively induced Nrf2 nuclear translocation and glutathione synthesis, increased gene expression of antioxidant enzymes (HO-1, NQO1, and UGT1A), and reduced protein levels of epigenetic modification enzymes (HDACs and DNMTs) in AML-12 cells. LL-WE also inhibited the activity of M.SssI CpG methyltransferase in vitro . These results suggest that LL-WE can be a potential hepatoprotective agent to reduce oxidative stress-induced liver damage by activating the Nrf2 pathway and downregulating epigenetic modification enzymes.

The Relationship between Prohibitin 1 Expression, Hepatotoxicity Induced by Acetaminophen, and Hepatoprotection by S-Adenosylmethionine in AML12 Cells.

Prohibitin 1 (Phb1) is a pleiotropic protein, located mainly in the mitochondrial inner membrane and involved in the regulation of cell proliferation and the stabilization of mitochondrial protein. Acetaminophen (APAP) is one of the most commonly used over-the-counter analgesics worldwide. However, at high dose, the accumulation of N-acetyl-p-benzoquinone imine (NAPQI) can lead to APAP-induced hepatotoxicity. In this study, we sought to understand the regulation of mRNA expression in relation to APAP and GSH metabolism by Phb1 in normal mouse AML12 hepatocytes. We used two different Phb1 silencing levels: high-efficiency (HE, >90%) and low-efficiency (LE, 50-60%). In addition, the siRNA-transfected cells were further pretreated with 0.5 mM of S-adenosylmethionine (SAMe) for 24 h before treatment with APAP at different doses (1-2 mM) for 24 h. The expression of APAP metabolism-related and antioxidant genes such as Cyp2e1 and Ugt1a1 were increased during SAMe pretreatment. Moreover, SAMe increased intracellular GSH concentration and it was maintained after APAP treatment. To sum up, Phb1 silencing and APAP treatment impaired the metabolism of APAP in hepatocytes, and SAMe exerted a protective effect against hepatotoxicity by upregulating antioxidant genes.

Pyruvate Upregulates Hepatic FGF21 Expression by Activating PDE and Inhibiting cAMP–Epac–CREB Signaling Pathway

Fibroblast growth factor 21 (FGF21) functions as a polypeptide hormone to regulate glucose and lipid metabolism, and its expression is regulated by cellular metabolic stress. Pyruvate is an important intermediate metabolite that acts as a key hub for cellular fuel metabolism. However, the effect of pyruvate on hepatic FGF21 expression and secretion remains unknown. Herein, we examined the gene expression and protein levels of FGF21 in human hepatoma HepG2 cells and mouse AML12 hepatocytes in vitro, as well as in mice in vivo. In HepG2 and AML12 cells, pyruvate at concentrations above 0.1 mM significantly increased FGF21 expression and secretion. The increase in cellular cAMP levels by adenylyl cyclase activation, phosphodiesterase (PDE) inhibition and 8-Bromo-cAMP administration significantly restrained pyruvate-stimulated FGF21 expression. Pyruvate significantly increased PDE activities, reduced cAMP levels and decreased CREB phosphorylation. The inhibition of exchange protein directed activated by cAMP (Epac) and cAMP response element binding protein (CREB) upregulated FGF21 expression, upon which pyruvate no longer increased FGF21 expression. The increase in plasma pyruvate levels in mice induced by the intraperitoneal injection of pyruvate significantly increased FGF21 gene expression and PDE activity with a reduction in cAMP levels and CREB phosphorylation in the mouse liver compared with the control. In conclusion, pyruvate activates PDEs to reduce cAMP and then inhibits the cAMP–Epac–CREB signaling pathway to upregulate FGF21 expression in hepatocytes.

3,4-dihydroxyphenylethyl alcohol glycoside reduces acetaminophen-induced acute liver failure in mice by inhibiting hepatocyte ferroptosis and pyroptosis.

APAP is one of the most commonly used antipyretic and pain medications, but excessive use can cause liver toxicity and damage. 3,4-dihydroxyphenylethyl alcohol glycoside (DAG) is a component isolated from Sargentodoxa cuneata known to have anti-apoptotic, anti-oxidation and anti-inflammatory effects. However, the effects of DAG on acute liver failure (ALF) are largely unknown. The purpose of this study is to study the protective effects and mechanism of DAG on APAP-induced ALF in mice. We established an ALF model in adult male pathogen-free C57BL/6 mice treated with APAP (300 mg/kg) by intraperitoneal injection and resolved by 24 h. Hematoxylin and eosin (HE) staining was used to evaluate the pathological changes in mouse liver tissue. The infiltration of neutrophils in liver tissue and reactive oxygen species (ROS) in AML12 cells were analyzed by flow cytometry. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), glutathione (GSH), malondialdehyde (MDA), catalase (CAT), and superoxide dismutase (SOD) were analyzed using relevant kits. Our results show that DAG reduced APAP-induced serum ALT and AST levels, histopathological changes, liver neutrophil infiltration and proinflammatory cytokines production, also attenuated the accumulation of MDA and the exhaustion of GSH, CAT and SOD. In vitro experiment indicated that DAG dose-dependently inhibited APAP-induced the levels of pro-inflammatory factors (IL-1β and IL18), and reactive oxygen species (ROS) and preventing GSH depletion in mouse AML12 hepatocytes. More interestingly, DAG inhibited the expression of ERK, HO-1, NLRP3, Caspase1 (p20) and Gasdermin-D and upregulated the expression of GPX4 in liver tissues and AML12hepatocytes. Therefore, our results indicate that DAG may act as a potential agent to treat ALF induced by APAP by inhibiting hepatocyte ferroptosis and pyroptosis.

Inonotus obliquus aqueous extract prevents histopathological alterations in liver induced by environmental toxicant Microcystin

Environmental toxicants like microcystins are known to adversely impact liver physiology and lead to the increased risk for abnormal liver function and even liver carcinoma. Chaga mushroom (Inonotus obliquus) is reported for various properties mainly antibacterial, antiallergic, anti-inflammatory, antioxidant, and anticancer properties. This study was aimed to assess the effect microcystin (MC-LR) on histopathology of liver in mice and a preventive measure by using aqueous extract of Inonotus obliquus (IOAE). Adult Balb/c mice were administered with MC-LR at 20 ​μg/kg body weight, per day, intraperitoneal (i.p.) for 4 weeks. IOAE was treated to one group of MC-LR mice at 200 ​mg/kg body weight, per oral, for 4 weeks. Histological staining for liver structural details and biochemical assays for functions were assessed. The results of the study showed that MC-LR drastically reduced the body weight of mice which were restored close to the range of control by IOAE treatment. MC-LR exposed mice showed 1.9, 1.7 and 2.2-fold increase in the levels of SGOT, SGPT and LDH which were restored by IOAE treatment as compared to control (one-fold). MC-LR exposed mice showed reduced level of GSH (19.83 ​± ​3.3 ​μM) which were regained by IOAE treatment (50.83 ​± ​3.0 ​μM). Similar observations were noted for catalase activity. Histological examinations show that MC-LR exposed degenerative changes in the liver sections which were restored by IOAE supplementation. The immunofluorescence analysis of caspase-3 counterstained with DAPI showed that MC-LR led to the increased expression of caspase-3 which were comparatively reduced by IOAE treatment. The cell viability decreased on increasing the concentration of MC-LR with 5% cell viability at concentration of 10 ​μg MC-LR/mL as that of control 100% Cell viability. The IC50 was calculated to be 3.6 ​μg/ml, indicating that MC-LR is chronic toxic to AML12 mouse hepatocytes. The molecular docking interaction of NF-κB-NIK with ergosterol peroxidase showed binding interaction between the two and showed the plausible molecular basis for the effects of IOAE in MC-LR induced liver injury. Collectively, this study revealed the deleterious effects of MC-LR on liver through generation of oxidative stress and activation of caspase-3, which were prevented by treatment with IOAE.

In Vitro and In Vivo Protective Effects of Lentil (Lens culinaris) Extract against Oxidative Stress-Induced Hepatotoxicity

Excessive oxidative stress plays a role in hepatotoxicity and the pathogenesis of hepatic diseases. In our previous study, the phenolic extract of beluga lentil (BLE) showed the most potent in vitro antioxidant activity among extracts of four common varieties of lentils; thus, we hypothesized that BLE might protect liver cells against oxidative stress-induced cytotoxicity. BLE was evaluated for its protective effects against oxidative stress-induced hepatotoxicity in AML12 mouse hepatocytes and BALB/c mice. H2O2 treatment caused a marked decrease in cell viability; however, pretreatment with BLE (25–100 μg/mL) for 24 h significantly preserved the viability of H2O2-treated cells up to about 50% at 100 μg/mL. As expected, BLE dramatically reduced intracellular reactive oxygen species (ROS) levels in a dose-dependent manner in H2O2-treated cells. Further mechanistic studies demonstrated that BLE reduced cellular ROS levels, partly by increasing expression of antioxidant genes. Furthermore, pretreatment with BLE (400 mg/kg) for 2 weeks significantly reduced serum levels of alanine transaminase and triglyceride by about 49% and 40%, respectively, and increased the expression and activity of glutathione peroxidase in CCl4-treated BALB/c mice. These results suggest that BLE protects liver cells against oxidative stress, partly by inducing cellular antioxidant system; thus, it represents a potential source of nutraceuticals with hepatoprotective effects.

Mmu-miR-199a-5p regulates CYP2B10 through repression of E4BP4 in mouse AML-12 hepatocytes

miR-199a-5p is an important regulator of many biological processes. However, whether and how CYP enzymes are regulated by miR-199a-5p are unknown. Here, we aimed to investigate the potential role of mmu-miR-199a-5p in regulating CYP2 enzymes. Regulatory effects of mmu-miR-199a-5p on CYP expression were assessed in mouse AML-12 hepatocytes. The metabolic activity of CYP2B10 was probed using cyclophosphamide (CPA) as a specific substrate. The regulatory mechanism was investigated using combined luciferase reporter assays and chromatin immunoprecipitation. Of several important drug-metabolizing CYPs, mmu-miR-199a-5p significantly increased the mRNA levels of Cyp2a10, Cyp2c29, and Cyp2j5 in AML-12 cells with Cyp2a10 altered the most. Consistently, mmu-miR-199a-5p enhanced the expression of CYP2B10 protein and cellular metabolism of CPA. Based on database analysis, Cyp2b10 was not a direct target gene of mmu-miR-199a-5p. Thus, a mediator is necessary for the miRNA regulation of CYP2B10. We found that E4BP4 repressed Cyp2b10 transcription and expression through specific binding to a D-box element in the gene promoter. Moreover, mmu-miR-199a-5p inhibited the expression of E4bp4 at the posttranscriptional level by directly targeting the 59–65 nt segment in its 3′-UTR. In conclusion, mmu-miR-199a-5p positively regulates CYP2B10 expression through inhibiting its repressor E4BP4. Our findings may provide an increased understanding of the complex regulatory pathways for CYP2B10.

Role of STAT3 in hepatocyte regeneration after acetaminophen-induced hepatocellular injury in mice

Objective To investigate the role of STAT3 in hepatocyte proliferation after acetaminophen (APAP)-induced hepatocellular injury in mice. Methods Normal mouse AML12 hepatocytes were cultured in vitro and were stimulated by APAP (1, 2.5, 5, 10, and 20 mmol/L) for 12, 24 or 48 hours, and the hepatocytes treated with an equal volume of phosphate buffered saline were established as control group. After the optimal stimulation concentration and duration of action were screened out, AML12 hepatocytes were treated with AG490 (10, 50, and 100 μmol/L). The CCK-8 assay was used to measure the viability of AML12 hepatocytes; RT-PCR was used to measure the mRNA expression levels of PCNA, CyclinD1, and Ki67 in AML12 hepatocytes, and Western blot was used to measure the protein expression levels of STAT3, p-STAT3, PCNA, and CyclinD1. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. Results After 24 and 48 hours of APAP treatment, compared with the control group, all concentration groups had a significant reduction in the viability of AML12 hepatocytes (all P Conclusion STAT3 participates in hepatocyte proliferation after APAP-induced hepatocyte injury in mice, while AG490, as an STAT3 inhibitor, can inhibit hepatocyte proliferation after APAP-induced hepatocyte injury by inhibiting the phosphorylation of STAT3.

Lipid lowering effects in hepatocytes of cassane-type diterpenoids from the seeds of Caesalpinia minax Hance

Abstract Five new cassane-type diterpenoids, including 16-O-ethyl caesalpin A (1), 16-O-ethyl caesalpin B (2), norcaesalpinin ME (3), 6α-hydroxynorcaesalpinin A (4), and 17-acetylcaesalmin Z (5), were isolated from the seeds of Caesalpinia minax Hance, along with twelve known compounds. The structures of the new compounds were established by means of spectroscopic technique (NMR, HRESIMS and IR). The absolute configurations of the new compounds were determined by their ECD spectra. In palmitic acid/oleic acid-treated AML12 mouse hepatocytes, compound 5 greatly reduced intracellular lipid accumulation, as well as total triglyceride and cholesterol contents, which could be further developed to treat fatty liver disease.

Deletion of Von Willebrand A Domain Containing Protein (VWA8) raises activity of mitochondrial electron transport chain complexes in hepatocytes

VWA8 (Von Willebrand A Domain Containing Protein 8) is a AAA+ ATPase that is localized to the mitochondrial matrix and is widely expressed in highly energetic tissues. Originally found to be higher in abundance in livers of mice fed a high fat diet, deletion of the VWA8 gene in differentiated mouse AML12 hepatocytes unexpectedly produced a phenotype of higher mitochondrial and nonmitochondrial oxidative metabolism, higher ROS (reactive oxygen species) production mainly from NADPH oxidases, and increased HNF4a expression. The purposes of this study were first, to determine whether higher mitochondrial oxidative capacity in VWA8 null hepatocytes is the product of higher capacity in all aspects of the electron transport chain and oxidative phosphorylation, and second, the density of cristae in mitochondria and mitochondrial content was measured to determine if higher mitochondrial oxidative capacity is accompanied by greater cristae area and mitochondrial abundance. Electron transport chain complexes I, II, III, and IV activities all were higher in hepatocytes in which the VWA8 gene had been deleted using CRISPR/Cas9. A comparison of abundance of proteins in electron transport chain complexes I, III and ATP synthase previously determined using an unbiased proteomics approach in hepatocytes in which VWA8 had been deleted showed agreement with the activity assays. Mitochondrial cristae, the site where electron transport chain complexes are located, were quantified using electron microscopy and stereology. Cristae density, per mitochondrial area, was almost two-fold higher in the VWA8 null cells (P < 0.01), and mitochondrial area was two-fold higher in the VWA8 null cells (P < 0.05). The results of this study allow us to conclude that despite sustained, higher ROS production in VWA8 null cells, a global mitochondrial compensatory response was maintained, resulting in overall higher mitochondrial oxidative capacity.

Oxytocin promotes hepatic regeneration in elderly mice

SummaryLiver aging impairs the ability of hepatocyte regeneration. Recent studies have found that oxytocin (OT) plays an important role in promoting tissue repair and maintaining differentiation and regeneration of stem cells. Here, we reported that OT receptors, which are specifically located in hepatocytes, decrease with aging in human and mice. Interestingly, the level of serum OT also decline with age. Notably, OT promotes hepatocyte regeneration only in aged mice but not in young mice in vitro and in vivo. Further studies reveal that OT promotes autophagy in either AML12 mouse hepatocytes or aged mice after partial hepatectomy or with CCl4-induced acute liver injury. In conclusion, OT promotes liver regeneration, especially in aged mice, which may be achieved by promoting autophagy. All these results support the possibility of OT and its analog being a potent anti-aging drug and promote liver rejuvenation.

Mechanism of a methylxanthine drug theophylline-induced Ca2+ signaling and cytotoxicity in AML12 mouse hepatocytes.

Theophylline is a methylxanthine drug used in therapy for respiratory diseases. However, the impact of theophylline on Ca2+ signaling has not been explored in liver cells. This study examined whether theophylline affected Ca2+ homeostasis and its related cytotoxicity in AML12 mouse hepatocytes. Cell viability was measured by the cell viability reagent (WST-1). Cytosolic Ca2+ concentration ([Ca2+]i) was measured by the Ca2+-sensitive fluorescent dye fura-2. Theophylline (25-125μM) induced [Ca2+]i rises and cause cytotoxicity in AML12 cells. This cytotoxic response was reversed by chelation of cytosolic Ca2+ with BAPTA/AM. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished theophylline-induced [Ca2+]i rises. Conversely, treatment with theophylline also abolished thapsigargin-induced [Ca2+]i rises. However, inhibition of PLC failed to alter theophylline-evoked [Ca2+]i rises. In Ca2+-containing medium, modulators of store-operated Ca2+ channels inhibited 30% of the [Ca2+]i rises, whereas the PKC modulators had no effect. Furthermore, theophylline-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. Together, in AML12 cells, theophylline caused Ca2+-associated cytotoxicity and induced Ca2+ entry through PLC-independent Ca2+ release from the endoplasmic reticulum and PKC-insensitive store-operated Ca2+ channels. BAPTA-AM with its protective effects may be a potential compound for prevention of theophylline-induced cytotoxicity.

LncRNA NEAT1/microRNA-129-5p/SOCS2 axis regulates liver fibrosis in alcoholic steatohepatitis

BackgroundLong non-coding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to play an essential role in non-alcoholic fatty liver disease. However, the role of NEAT1 in regulation of alcoholic steatohepatitis (ASH) remains largely unknown. This study aims to explore the role of NEAT1 in ASH by mediating microRNA-129-5p (miR-129-5p) targeting suppressor of cytokine signaling 2 (SOCS2).MethodsNEAT1, miR-129-5p and SOCS2 expression in serum of ASH patients were assessed. In the in vitro cellular experiment, we transfected siRNAs, oligonucleotides or plasmids into ethanol-induced AML-12 mouse hepatocytes to alter NEAT1 and miR-129-5p expression, and inflammatory factors and lipid content were determined. In the in vivo animal experiment, we injected lentiviruses carrying siRNAs, oligonucleotides or plasmids onto ASH mice (ASH induced by feeding mice a Lieber-DeCarli ethanol diet) to alter NEAT1 and miR-129-5p expression through the tail vein. Serum liver function, blood lipids and inflammatory factors were detected; liver histopathology, liver cell apoptosis, and fibrosis were observed. The relationship between NEAT1 and miR-129-5p, or between miR-129-5p and SOCS2 was verified.ResultsMiR-129-5p was reduced while NEAT1 and SOCS2 were elevated in ASH. Inhibited NEAT1 or elevated miR-129-5p suppressed the elevated lipid metabolism and restrained inflammation reaction in ethanol-stimulated AML-12 cells. The promoted miR-129-5p and inhibited NEAT1 could improve the liver function and repress blood lipid, inflammation reaction, hepatocyte apoptosis and liver fibrosis in ethanol-induced ASH mice. Furthermore, NEAT1 could negatively regulate miR-129-5p to target SOCS2.ConclusionWe have found that the inhibited NEAT1 could suppress liver fibrosis in ASH mice by promoting miR-129-5p and restraining SOCS2, thereby decelerating the development of ASH.