Understanding the molecular crossroads in acute liver failure: A pathway to new therapies.

In this editorial we comment on the article published in a recent issue of the World Journal of Gastroenterology. Acute liver failure (ALF) is a critical condition characterized by rapid hepatocellular injury and organ dysfunction, and it often necessitates liver transplant to ensure patient survival. Recent research has elucidated the involvement of distinct cell death pathways, namely ferroptosis and pyroptosis, in the pathogenesis of ALF. Ferroptosis is driven by iron-dependent lipid peroxidation, whereas pyroptosis is an inflammatory form of cell death; both pathways contribute to hepatocyte death and exacerbate tissue damage. This comprehensive review explores the interplay between ferroptosis and pyroptosis in ALF, highlighting the role of key regulators such as silent information regulator sirtuin 1. Insights from clinical and preclinical studies provide valuable perspectives on the dysregulation of cell death pathways in ALF and the therapeutic potential of targeting these pathways. Collaboration across multiple disciplines is essential for translating the experimental insights into effective treatments for this life-threatening condition.

Histopathological impact of SARS-CoV-2 on the liver: Cellular damage and long-term complications.

Coronavirus disease 2019 (COVID-19), caused by the highly pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), primarily impacts the respiratory tract and can lead to severe outcomes such as acute respiratory distress syndrome, multiple organ failure, and death. Despite extensive studies on the pathogenicity of SARS-CoV-2, its impact on the hepatobiliary system remains unclear. While liver injury is commonly indicated by reduced albumin and elevated bilirubin and transaminase levels, the exact source of this damage is not fully understood. Proposed mechanisms for injury include direct cytotoxicity, collateral damage from inflammation, drug-induced liver injury, and ischemia/hypoxia. However, evidence often relies on blood tests with liver enzyme abnormalities. In this comprehensive review, we focused solely on the different histopathological manifestations of liver injury in COVID-19 patients, drawing from liver biopsies, complete autopsies, and in vitro liver analyses. We present evidence of the direct impact of SARS-CoV-2 on the liver, substantiated by in vitro observations of viral entry mechanisms and the actual presence of viral particles in liver samples resulting in a variety of cellular changes, including mitochondrial swelling, endoplasmic reticulum dilatation, and hepatocyte apoptosis. Additionally, we describe the diverse liver pathology observed during COVID-19 infection, encompassing necrosis, steatosis, cholestasis, and lobular inflammation. We also discuss the emergence of long-term complications, notably COVID-19-related secondary sclerosing cholangitis. Recognizing the histopathological liver changes occurring during COVID-19 infection is pivotal for improving patient recovery and guiding decision-making.

Disturbed intracellular folate homeostasis impairs autophagic flux and increases hepatocytic lipid accumulation.

BACKGROUND: Metabolic associated fatty liver disease (MAFLD), a prevalent liver disorder affecting one-third of the global population, encompasses a spectrum ranging from fatty liver to severe hepatic steatosis. Both genetic and lifestyle factors, particularly diet and nutrition, contribute to its etiology. Folate deficiency, a frequently encountered type of malnutrition, has been associated with the pathogenesis of MAFLD and shown to impact lipid deposition. However, the underlying mechanisms of this relationship remain incompletely understood. We investigated the impact of disturbed folate-mediated one-carbon metabolism (OCM) on hepatic lipid metabolism both in vitro using human hepatoma cells and in vivo using transgenic fluorescent zebrafish displaying extent-, stage-, and duration-controllable folate deficiency upon induction. RESULTS: Disturbed folate-mediated one-carbon metabolism, either by inducing folate deficiency or adding anti-folate drug, compromises autophagy and causes lipid accumulation in liver cells. Disturbed folate status down-regulates cathepsin L, a key enzyme involved in autophagy, through inhibiting mTOR signaling. Interfered mitochondrial biology, including mitochondria relocation and increased fusion-fission dynamics, also occurs in folate-deficient hepatocytes. Folate supplementation effectively mitigated the impaired autophagy and lipid accumulation caused by the inhibition of cathepsin L activity, even when the inhibition was not directly related to folate deficiency. CONCLUSIONS: Disruption of folate-mediated OCM diminishes cathepsin L expression and impedes autophagy via mTOR signaling, leading to lipid accumulation within hepatocytes. These findings underscore the crucial role of folate in modulating autophagic processes and regulating lipid metabolism in the liver.

[Characteristics of liver volume and pathological changes with different stages of liver fibrosis in chronic liver disease].

Objective: To measure the overall and lobulated volume of the liver with different degrees of liver fibrosis and to further observe pathological changes such as liver microvasculature, hepatocyte apoptosis, and regeneration in order to understand the macroscopic volume changes of the liver during liver fibrosis and its relationship with liver tissue microscopic pathology in patients with chronic liver disease. Methods: 53 patients with chronic hepatitis B, alcoholic fatty liver disease, autoimmune liver disease, nonalcoholic fatty liver disease, and drug-induced chronic liver disease who underwent both liver biopsy tissue and abdominal magnetic resonance imaging were collected. Patients were divided into early (F1-2), middle (F3-4), and late (F5-6) in accordance with the Ishak fibrosis stage and Masson stain. The liver and spleen volumes were measured using ITK-SNAP software. CD31 immunohistochemical staining was used to reflect intrahepatic angiogenesis. Ki67 and HNF-4α multiplex immunohistochemical staining were used to reflect hepatocyte regeneration. GS staining was used to determine parenchymal extinction lesions. TUNEL staining was used to observe hepatocyte apoptosis. Spearman correlation analysis was used to analyze the relationship between liver volume changes and liver histopathological changes. Results: As liver fibrosis progressed, the total liver volume and right lobe liver volume gradually decreased (P<0.05), while the spleen volume gradually increased (P<0.05). The expression of CD31 and GS gradually increased (P<0.05), and the expression of Ki67 first increased and then decreased (P<0.05). The positivity rate of CD31 was negatively correlated with the right lobe liver volume (r=-0.609, P<0.001) and the total liver volume (r=-0.363, P=0.017). The positivity rate of Ki67 was positively correlated with the right lobe liver volume (r=0.423, P=0.018), while the positivity rate of apoptotic cells was significantly negatively correlated with the total liver volume (r=-0.860, P<0.001). The positivity rate of GS was negatively correlated with the right lobe liver volume (r=-0.440, P=0.002), and the number of PELs was negatively correlated with RV (r=-0.476, P=0.013). The CD31 positive staining area was negatively correlated with the Ki67 positive staining area(r=-0.511, P=0.009). Conclusion: As liver fibrosis progresses, patients with chronic liver disease have a depletion in total liver volume and right lobe liver volume, and this is mainly in correlation with fewer liver cells and liver tissue microvasculature disorders.

Functions and Clinical Relevance of Liver-Derived Immunoglobulins.

Liver is the largest internal organ of the body with vital functions. In addition to its endocrine and exocrine activities, liver also plays a pivotal role in the immune system, including haematopoietic functions. Liver parenchymal cells, which are epithelial cells, have been found to possess innate immune functions by expressing pattern-recognition receptors (PRRs), producing complement components, and secreting cytokines. Intriguingly, in recent years, it has been discovered that liver epithelial cells also produce immunoglobulins (Igs), which have long been thought to be produced exclusively by B cells. Notably, even liver epithelial cells from B lymphocyte-deficient mice, including SCID mice and µMT mice, could also produce Igs. Compelling evidence has revealed both the physiological and pathological functions of liver-derived Igs. For instance, liver epithelial cells-derived IgM can serve as a source of natural and specific antibodies that contribute to innate immune responses, while liver-produced IgG can act as a growth factor to promote cell proliferation and survival in normal hepatocytes and hepatocarcinoma. Similar to that in B cells, the toll-like receptor 9 (TLR9)-MyD88 signaling pathway is also actively involved in promoting liver epithelial cells to secrete IgM. Liver-derived Igs could potentially serve as biomarkers, prognostic indicators, and therapeutic targets in the clinical setting, particularly for liver cancers and liver injury. Nevertheless, despite significant advances, much remains unknown about the mechanisms governing Ig transcription in liver cells, as well as the detailed functions of liver-derived Igs and their involvement in diseases and adaptive immunity. Further studies are still needed to reveal these underlying, undefined issues related to the role of liver-derived Igs in both immunity and diseases.

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)-1ß 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.

Activation of AMPD2 drives metabolic dysregulation and liver disease in mice with hereditary fructose intolerance.

Hereditary fructose intolerance (HFI) is a painful and potentially lethal genetic disease caused by a mutation in aldolase B resulting in accumulation of fructose-1-phosphate (F1P). No cure exists for HFI and treatment is limited to avoid exposure to fructose and sugar. Using aldolase B deficient mice, here we identify a yet unrecognized metabolic event activated in HFI and associated with the progression of the disease. Besides the accumulation of F1P, here we show that the activation of the purine degradation pathway is a common feature in aldolase B deficient mice exposed to fructose. The purine degradation pathway is a metabolic route initiated by adenosine monophosphate deaminase 2 (AMPD2) that regulates overall energy balance. We demonstrate that very low amounts of fructose are sufficient to activate AMPD2 in these mice via a phosphate trap. While blocking AMPD2 do not impact F1P accumulation and the risk of hypoglycemia, its deletion in hepatocytes markedly improves the metabolic dysregulation induced by fructose and corrects fat and glycogen storage while significantly increasing the voluntary tolerance of these mice to fructose. In summary, we provide evidence for a critical pathway activated in HFI that could be targeted to improve the metabolic consequences associated with fructose consumption.

Transcriptional Control: A Directional Sign at the Crossroads of Adult Hepatic Progenitor Cells' Fates.

Hepatic progenitor cells (HPCs) have a bidirectional potential to differentiate into hepatocytes and bile duct epithelial cells and constitute a second barrier to liver regeneration in the adult liver. They are usually located in the Hering duct in the portal vein region where various cells, extracellular matrix, cytokines, and communication signals together constitute the niche of HPCs in homeostasis to maintain cellular plasticity. In various types of liver injury, different cellular signaling streams crosstalk with each other and point to the inducible transcription factor set, including FoxA1/2/3, YB-1, Foxl1, Sox9, HNF4α, HNF1α, and HNF1ß. These transcription factors exert different functions by binding to specific target genes, and their products often interact with each other, with diverse cascades of regulation in different molecular events that are essential for homeostatic regulation, self-renewal, proliferation, and selective differentiation of HPCs. Furthermore, the tumor predisposition of adult HPCs is found to be significantly increased under transcriptional factor dysregulation in transcriptional analysis, and the altered initial commitment of the differentiation pathway of HPCs may be one of the sources of intrahepatic tumors. Related transcription factors such as HNF4α and HNF1 are expected to be future targets for tumor treatment.

High dietary Fructose Drives Metabolic Dysfunction-Associated Steatotic Liver Disease via Activating ubiquitin-specific peptidase 2/11ß-hydroxysteroid dehydrogenase type 1 Pathway in Mice.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver-related morbidity and mortality. Though high fructose intake is acknowledged as a metabolic hazard, its role in the etiology of MASLD requires further clarification. Here, we demonstrated that high dietary fructose drives MASLD development and promotes MASLD progression in mice, and identified Usp2 as a fructose-responsive gene in the liver. Elevated USP2 levels were detected in the hepatocytes of MASLD mice; a similar increase was observed following fructose exposure in primary hepatocytes and mouse AML12 cells. Notably, hepatocytes overexpressing USP2 presented with exaggerated lipid accumulation and metabolic inflammation when exposed to fructose. Conversely, USP2 knockdown mitigated these fructose-induced changes. Furthermore, USP2 was found to activate the C/EBPα/11ß-HSD1 signaling, which further impacted the equilibrium of cortisol and cortisone in the circulation of mice. Collectively, our findings revealed the role of dietary fructose in MASLD pathogenesis and identified the USP2-mediated C/EBPα/ 11ß-HSD1 signaling as a potential target for the management of MASLD.

HMGB1-Mediated Cell Death-A Crucial Element in Post-Hepatectomy Liver Failure.

Liver resection (LR) is the primary treatment for hepatic tumors, yet posthepatectomy liver failure (PHLF) remains a significant concern. While the precise etiology of PHLF remains elusive, dysregulated inflammatory processes are pivotal. Therefore, we explored the theragnostic potential of extracellular high-mobility-group-box protein 1 (HMGB1), a key damage-associated molecular pattern (DAMP) released by hepatocytes, in liver recovery post LR in patients and animal models. Plasma from 96 LR patients and liver tissues from a subset of 24 LR patients were analyzed for HMGB1 levels, and associations with PHLF and liver injury markers were assessed. In a murine LR model, the HMGB1 inhibitor glycyrrhizin, was administered to assess its impact on liver regeneration. Furthermore, plasma levels of keratin-18 (K18) and cleaved cytokeratin-18 (ccK18) were quantified to assess suitability as predictive biomarkers for PHLF. Patients experiencing PHLF exhibited elevated levels of intrahepatic and circulating HMGB1, correlating with markers of liver injury. In a murine LR model, inhibition of HMGB1 improved liver function, reduced steatosis, enhanced regeneration and decreased hepatic cell death. Elevated levels of hepatic cell death markers K18 and ccK18 were detected in patients with PHLF and correlations with levels of circulating HMGB1 was observed. Our study underscores the therapeutic and predictive potential of HMGB1 in PHLF mitigation. Elevated HMGB1, K18, and ccK18 levels correlate with patient outcomes, highlighting their predictive significance. Targeting HMGB1 enhances liver regeneration in murine LR models, emphasizing its role in potential intervention and prediction strategies for liver surgery.

Olprinone, a Selective Phosphodiesterase III Inhibitor, Has Protective Effects in a Septic Rat Model after Partial Hepatectomy and Primary Rat Hepatocyte.

Olprinone (OLP) is a selective inhibitor of phosphodiesterase III and is used clinically in patients with heart failure and those undergoing cardiac surgery; however, little is known about the effects of OLP on hepatoprotection. The purpose of this study aimed to determine whether OLP has protective effects in in vivo and in vitro rat models of endotoxin-induced liver injury after hepatectomy and to clarify the mechanisms of action of OLP. In the in vivo model, rats underwent 70% partial hepatectomy and lipopolysaccharide treatment (PH/LPS). OLP administration increased survival by 85.7% and decreased tumor necrosis factor-α, C-X-C motif chemokine ligand 1, and inducible nitric oxide synthase (iNOS) mRNA expression in the livers of rats treated with PH/LPS. OLP also suppressed nuclear translocation and/or DNA binding ability of nuclear factor kappa B (NF-κB). Pathological liver damage induced by PH/LPS was alleviated and neutrophil infiltration was reduced by OLP. Primary cultured rat hepatocytes treated with the pro-inflammatory cytokine interleukin-1ß (IL-1ß) were used as a model of in vitro liver injury. Co-treatment with OLP inhibited dose-dependently IL-1ß-stimulated iNOS induction and NF-κB activation. Our results demonstrate that OLP may partially inhibit the induction of several inflammatory mediators through the suppression of NF-κB and thus prevent liver injury induced by endotoxin after liver resection.

A PNPLA3-Deficient iPSC-Derived Hepatocyte Screen Identifies Pathways to Potentially Reduce Steatosis in Metabolic Dysfunction-Associated Fatty Liver Disease.

The incidence of nonalcoholic fatty liver disease (NAFLD), or metabolic dysfunction-associated fatty liver disease (MAFLD), is increasing in adults and children. Unfortunately, effective pharmacological treatments remain unavailable. Single nucleotide polymorphisms (SNPs) in the patatin-like phospholipase domain-containing protein (PNPLA3 I148M) have the most significant genetic association with the disease at all stages of its progression. A roadblock to identifying potential treatments for PNPLA3-induced NAFLD is the lack of a human cell platform that recapitulates the PNPLA3 I148M-mediated onset of lipid accumulation. Hepatocyte-like cells were generated from PNPLA3-/- and PNPLA3I148M/M-induced pluripotent stem cells (iPSCs). Lipid levels were measured by staining with BODIPY 493/503 and were found to increase in PNPLA3 variant iPSC-derived hepatocytes. A small-molecule screen identified multiple compounds that target Src/PI3K/Akt signaling and could eradicate lipid accumulation in these cells. We found that drugs currently in clinical trials for cancer treatment that target the same pathways also reduced lipid accumulation in PNPLA3 variant cells.

Baicalin protects against hepatocyte injury caused by aflatoxin B1 via the TP53-related ferroptosis Pathway.

OBJECTIVE: Baicalin has antioxidative, antiviral, and anti-inflammatory properties. However, its ability to alleviate oxidative stress (OS) and DNA damage in liver cells exposed to aflatoxin B1 (AFB1), a highly hepatotoxic compound, remains uncertain. In this study, the protective effects of baicalin on AFB1-induced hepatocyte injury and the mechanisms underlying those effects were investigated. METHODS: Stable cell lines expressing CYP3A4 were established using lentiviral vectors to assess oxidative stress levels by conducting assays to determine the content of reactive oxygen species (ROS), malondialdehyde (MDA), and superoxide dismutase (SOD). Additionally, DNA damage was evaluated by 8-hydroxy-2-deoxyguanosine (8-OHdG) and comet assays. Transcriptome sequencing, molecular docking, and in vitro experiments were conducted to determine the mechanisms underlying the effects of baicalin on AFB1-induced hepatocyte injury. In vivo, a rat model of hepatocyte injury induced by AFB1 was used to evaluate the effects of baicalin. RESULTS: In vitro, baicalin significantly attenuated AFB1-induced injury caused due to OS, as determined by a decrease in ROS, MDA, and SOD levels. Baicalin also considerably decreased AFB1-induced DNA damage in hepatocytes. This protective effect of baicalin was found to be closely associated with the TP53-mediated ferroptosis pathway. To elaborate, baicalin physically interacts with P53, leading to the suppression of the expression of GPX4 and SLC7A11, which in turn inhibits ferroptosis. In vivo findings showed that baicalin decreased DNA damage and ferroptosis in AFB1-treated rat liver tissues, as determined by a decrease in the expression of γ-H2AX and an increase in GPX4 and SLC7A11 levels. Overexpression of TP53 weakened the protective effects of baicalin. CONCLUSIONS: Baicalin can alleviate AFB1-induced OS and DNA damage in liver cells via the TP53-mediated ferroptosis pathway. In this study, a theoretical foundation was established for the use of baicalin in protecting the liver from the toxic effects of AFB1.

Primary Duck Hepatocyte Culture and Duck Hepatitis B Virus Infection Model.

Duck hepatitis B virus (DHBV) is an avian member of the hepatotropic DNA viruses, or hepadnaviridae. It shares with the human hepatitis B virus (HBV) a similar genomic organization and replication strategy via reverse transcription, but is simpler than HBV in lacking the X gene and in expressing just two coterminal envelope proteins: Large (L) and small (S). DHBV has been extensively used as a convenient and valuable animal model for study of the hepadnaviral life cycle, and for drug screening in vitro but also in vivo. Ducks and primary duck hepatocytes (PDHs) are inexpensive, easily accessible, and readily infected with DHBV. The high levels of genome replication and protein expression in duck liver and PDHs also facilitate monitoring of viral life cycle using conventional molecular biology techniques such as Southern blot for replicative DNA and covalently closed circular DNA (cccDNA), Northern blot for viral RNAs, and Western blot for viral proteins.

Stem Cell-Derived Hepatocyte-Like Cells for Hepatitis B Virus Infection.

Hepatitis B, the leading cause of liver diseases worldwide, is a result of infection with hepatitis B virus (HBV). Due to its obligate intracellular life cycle, culture systems for efficient HBV replication are vital. Although basic and translational research on HBV has been performed for many years, conventional hepatocellular culture systems are not optimal. These studies have greatly benefited from recent improvements in cell culture models based on stem cell technology for HBV replication and infection studies. Here we describe a protocol for the differentiation of human stem cell-derived hepatocyte-like cells (HLCs) and subsequent HBV infection. HLCs are capable of expressing hepatocyte markers and host factors important for hepatic function maintenance. These cells fully support HBV infection and virus-host interactions. Stem cell-derived HLCs provide a new tool for antiviral drug screening and development.

A Particle Gel Assay for Detection of Intracellular Hepatitis B Virus Subviral Particles in Cultured Cells.

Chronic hepatitis B virus (HBV) infection is due to the failure of host immune system to resolve the viral infection. Accordingly, restoration or reconstitution of a functional antiviral immune response to HBV is essential to achieve durable control of HBV replication leading to a functional cure of chronic hepatitis B (CHB). Noninfectious subviral particles (SVPs), comprised of HBV surface antigen (HBsAg), are the predominant viral products secreted by HBV-infected hepatocytes. The high levels of SVPs in the circulation induce immune tolerance and contribute to the establishment of chronic HBV infection. The current standard-of-care medications for CHB efficiently suppress HBV replication but fail to reduce the levels of HBsAg in majority of treated patients. Further understanding the mechanisms underlying SVP morphogenesis, secretion and regulation by viral and host cellular factors are critical for the discovery of therapeutics that can inhibit SVP production and/or induce the degradation of HBV envelope proteins. We describe herein a protocol for intracellular SVP detection by a native agarose gel electrophoresis-based particle gel assy. The method is suitable for quantitative detection of intracellular HBV SVPs and can be applied in dissecting the molecular mechanism of SVP morphogenesis and the discovery of antiviral agents targeting SVP formation in hepatocytes.

Detection of Hepatitis B Virus Covalently Closed Circular DNA and Intermediates in Its Formation.

Hepatitis B virus (HBV) infection remains a global public health issue, and approximately 294 million individuals worldwide are chronically infected with HBV. Approved antivirals rarely cure chronic HBV infection due to their inability to eliminate the HBV covalently closed circular DNA (cccDNA), the viral episome, in the nucleus of infected hepatocytes. The persistence of cccDNA underlies the chronic nature of HBV infection and the frequent relapse after the cessation of antiviral treatment. However, drug development targeting cccDNA formation and maintenance is hindered by the lack of sufficient biological knowledge on cccDNA, and of its reliable detection due to its low abundance and the presence of high levels of HBV DNA species similar to cccDNA. Here, we describe a Southern blot method for reliably detecting the HBV cccDNA even in the presence of high levels of plasmid DNA and other HBV DNA species, based on the efficient removal of plasmid DNA and all DNA species with free 3' ends. This approach also allows the detection of certain potential intermediates during cccDNA formation.

Modeling HBV Infection and Therapy in Immunodeficient NOD-Rag1-/-IL2RgammaC-null (NRG) Fumarylacetoacetate Hydrolase (FAH) Knockout Mice with Human Chimeric Liver.

Chimeric mouse models with a humanized liver (Hu-HEP mice) provide a unique tool to study human hepatotropic virus diseases, including viral infection, viral pathogenesis, and anti-viral therapy. Here, we describe a detailed protocol for studying hepatitis B infection in NRG-derived fumarylacetoacetate hydrolase (FAH) knockout mice repopulated with human hepatocytes (FRG-Hu HEP mice). The procedures include (1) maintenance and genotyping of the FRG mice, (2) intrasplenic injection of primary human hepatocytes (PHH), (3) 2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) drug reduction cycling to improve human hepatocyte repopulation, (4) human albumin detection, and (5) HBV infection and detection. The method is simple and allows for highly reproducible generation of FRG-Hu HEP mice for HBV infection and therapy investigations.

Melatonin mitigated methotrexate-induced hepatotoxicity through interrelated biological processes.

BACKGROUND: Hepatotoxicity associated with methotrexate (MTX) is mainly due to disruption of redox balance and development of oxidative injury to hepatocytes. Melatonin (MLT) is a potent antioxidant and regulates wide range of biological functions, processes and utilized as adjuvant for number of medical applications. The current study investigated the mitigating effect of MLT on the MTX-induced hepatotoxicity. METHODS AND RESULTS: Adult male rats received MLT (25 mg/kg, orally) for seven days flowed by single injection of MTX (20 mg/kg, ip) then treat with MLT continued for additional 7 days. The present result showed MLT treatment mitigated histopathological changes in the liver that associated with normalization of ALT and AST activity as well as bilirubin, albumin and alfa-fetoprotein levels in serum of MLT + MTX-treated rat to comparable control level. MLT treatment significantly reduced MDA content and myeloperoxidase activity while enhanced the activity of superoxide dismutase, catalase and glutathione content in the liver indicating the empowerment of the antioxidant status. Amelioration of MLT-induced oxidative stress resulted in a reduction in the inflammatory response due to antioxidant restoration and inhibited apoptosis indicated by downregulation of caspase-3 expression. The replenishment of antioxidant content powers the defense system of the hepatocytes. As a result, apoptosis is reduced which might be due to the ability of MLT protect DNA integrity thus maintaining hepatocyte functions and structure. Consequently, liver histology was protected. CONCLUSIONS: In summary, MLT modulates liver function and structure by orchestrating linked processes, including redox balance, inflammatory response, suppression of caspase-3, and DNA damage.

Drp1 Aggravates Copper Nanoparticle-Induced ER-Phagy by Disturbing Mitochondria-Associated Membranes in Chicken Hepatocytes.

As an efficient alternative copper (Cu) source, copper nanoparticles (nano-Cu) have been widely supplemented into animal-producing food. Therefore, it is necessary to assess the effect of nano-Cu exposure on the biological health risk. Recently, the toxic effects of nano-Cu have been confirmed but the underlying mechanism remains unclear. This study reveals the impact of nano-Cu on endoplasmic reticulum autophagy (ER-phagy) in chicken hepatocytes and further identifies Drp1 and its downstream gene FAM134B as crucial regulators of nano-Cu-induced hepatotoxicity. Nano-Cu exposure can induce Cu ion overaccumulation and pathological injury in the liver, trigger excessive mitochondrial fission and mitochondria-associated membrane (MAM) integrity damage, and activate ER-phagy in vivo and in vitro. Interestingly, the knockdown of Drp1 markedly decreases the expression of FAM134B induced by nano-Cu. Furthermore, the expression levels of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I induced by nano-Cu exposure are decreased by inhibiting the expression of Drp1. Simultaneously, the inhibition of FAM134B effectively alleviates nano-Cu-induced ER-phagy by downregulating the expression of ATL3, CCPG1, SEC62, TEX264, and LC3II/LC3I. Overall, these results suggest that Drp1-mediated impairment of MAM integrity leads to ER-phagy as a novel molecular mechanism involved in the regulation of nano-Cu-induced hepatotoxicity. These findings provide new ideas for future research on the mechanism of nano-Cu-induced hepatotoxicity.