Search Results (3,486)

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a continuum of hepatic pathological manifestations of the metabolic syndrome. Pathogenesis is not clearly understood despite recent progress, but Kupffer cells and bone marrow-derived macrophages (BMDMs) have a fundamental role. In this review, the multiple pathophysiological aspects of MASLD are presented, including genetics, insulin resistance, lipotoxicity, and inflammation. The participation of innate and adaptive immunity, as well as the implications of the recently described trained immunity, is presented. The interplay of the liver with the gut microbiota is also analyzed. A recent adipocentric theory and the various mechanisms of hepatocyte death are also described. The fundamental role of Kupffer cells and other liver macrophages is discussed in detail, including their extreme phenotypic plasticity in both the normal and the MASLD liver. The functional differentiation between pro-inflammatory and anti-inflammatory subpopulations and their protective or detrimental involvement is further described, including the participation of Kupffer cells and BMDMs in all aspects of MASLD pathogenesis. The role of macrophages in the development of advanced MASLD, including fibrosis and hepatocellular carcinoma, is analyzed and the lack of explanation for the transition from MASLD to MASH is recognized. Finally, current modalities of drug treatment are briefly presented and the effects of different drugs on macrophage polarization and functions are discussed. Full article

Background/Objectives: Advanced 3D cell culture techniques enhance the physiological relevance of in vitro models, while supporting the 3Rs principles (Reduction, Refinement, and Replacement) of animal experimentation. In this context, 3D collagen-based systems mimic key extracellular matrix properties, enabling more accurate cellular organization and phenotype. However, changes in culture dimensionality can affect RT-qPCR reference gene stability, underscoring the need for careful validation when combining 2D and 3D systems. Methods: AML12 cells were cultured for 7 days under different 2D and collagen-based 3D conditions. The expression stability of nine candidate housekeeping genes was systematically evaluated using established algorithms (BestKeeper, NormFinder, geNorm, RefFinder, and ΔCt method), followed by inter-group statistical and correlation analyses of raw Ct values. Albumin gene expression was used as a target gene. Results: Although all candidate genes initially met acceptable variability thresholds, a stepwise, exclusion-based analysis revealed distinct performance differences. Hprt, Ppia, and Actb emerged as the most stable, showing no intra-group variability or interaction with Albumin expression. Nevertheless, Ywhaz and Rplp0, despite their high stability, were compromised by significant correlation with Albumin. Furthermore, Ywhaz showed significant downregulation under 3D culture conditions. B2M, Gapdh, 18S, and Hmbs exhibited increased variability, likely reflecting metabolic and microenvironmental heterogeneity associated with prolonged 2D cultivation of AML12 cells. Conclusions: Overall, this study highlights the importance of context-dependent, exclusion-based reference gene validation when comparing 2D and 3D models, and demonstrates a new approach for reliable gene expression normalization in complex in vitro culture systems. Full article

Background: Intestinal failure-associated liver disease (IFALD) is a serious complication in patients receiving parenteral nutrition, often exacerbated by inflammation, lipid overload, and oxidative stress. Nobiletin (NOB), a polymethoxylated flavone, is known for its anti-inflammatory and lipid-regulating properties. Methods: We employed an in vitro model using THLE-2 human hepatocytes and primary human cholangiocytes exposed to Intralipid (INT) and lipopolysaccharide (LPS) to simulate IFALD conditions. NOB was tested at non-toxic concentrations (10 and 25 µM) to assess its protective effects. MTT viability assays, multiplex bead-based immunoassays (MAGPIX), RT-qPCR, and Western blotting were used to evaluate changes in inflammation markers, gene expression, and protein signaling. Moreover, ALT and AST activities were used to assess hepatocellular injury. Results: NOB maintained high cell viability in THLE-2 hepatocytes and cholangiocytes, confirming its low cytotoxicity. NOB normalized ALT and AST activities in both tested cell lines, but the effect reached statistical significance only for ALT in cholangiocytes. Under IFALD-like conditions (LPS+INT), NOB significantly preserved metabolic activity in both cell types. In THLE-2 and cholangiocytes, NOB markedly reduced the phosphorylation of pro-inflammatory proteins JNK, NF-κB, and STAT3, indicating a broad inhibition of inflammatory signaling. Moreover, in THLE-2 cells, NOB upregulated lipid metabolism-related genes (PRKAA2, CYP7A1, and ABCA1) and decreased oxidative stress, thereby enhancing the nuclear translocation of Nrf2 and increasing SOD1 level, which supports the activation of antioxidant defenses. Conclusions: NOB exhibits hepatoprotective properties under IFALD-like conditions in vitro, likely through modulation of inflammation-related signaling and lipid metabolism pathways. Full article

The liver stage of Plasmodium infection represents a critical bottleneck in malaria pathogenesis and a unique interface between parasite development and hepatocyte-intrinsic immunity. Recent evidence suggests that hepatocytes do not eliminate liver-stage parasites through canonical xenophagy, as previously assumed, but instead employ a noncanonical autophagy response known as the conjugation of ATG8 to single membranes (CASM). CASM drives rapid lipidation of LC3 onto the parasitophorous vacuole membrane (PVM) via a V-ATPase-ATG16L1-dependent mechanism, thereby activating the Plasmodium-associated autophagy-related (PAAR) response. This process represents a major hepatocyte-intrinsic mechanism that limits early liver-stage parasite development. Plasmodium liver-stage parasites have evolved specialized strategies to counteract this host defense. The PVM proteins UIS3 and UIS4 enable parasite evasion by sequestering LC3 and remodeling perivacuolar actin, thereby preventing endolysosomal fusion and inhibiting PAAR execution. In parallel, parasites selectively exploit host autophagy components—particularly GABARAP paralogs—to activate TFEB, promoting lysosomal biogenesis and improving access to host-derived nutrients. These interactions highlight autophagy as both a protective and parasite-supportive pathway, depending on the molecular context. Understanding how CASM, PAAR, and parasite evasion mechanisms intersect is crucial for designing pathway-selective interventions that amplify hepatocyte-intrinsic clearance while avoiding the inadvertent enhancement of parasite-supportive autophagy programs. Selective modulation of noncanonical autophagy offers a promising avenue for host-directed therapies that restrict liver-stage development while limiting the emergence of antimalarial resistance. This review synthesizes recent advances in the mechanistic interplay between Plasmodium liver stages and hepatocyte autophagy, identifies major knowledge gaps, and outlines future directions for translating these discoveries into therapeutic innovation. Full article

The mesenchymal–epithelial transition (MET) receptor is a tyrosine kinase activated by its sole known ligand, hepatocyte growth factor (HGF). MET signaling regulates key cellular processes, including proliferation, survival, migration, motility, and angiogenesis. Dysregulation and hyperactivation of this pathway are implicated in multiple malignancies, including lung, breast, colorectal, and gastrointestinal cancers. In non–small cell lung cancer (NSCLC), aberrant activation of the MET proto-oncogene contributes to 1% of known oncogenic drivers and is associated with poor clinical outcomes. Several mechanisms can induce MET hyperactivation, including MET gene amplification, transcriptional upregulation of MET or HGF, MET fusion genes, and MET exon 14 skipping mutations. Furthermore, MET pathway activation represents a frequent mechanism of acquired resistance to EGFR- and ALK-targeted tyrosine kinase inhibitors (TKIs) in EGFR- and ALK-driven NSCLCs. Although MET has long been recognized as a promising therapeutic target in NSCLC, the clinical efficacy of MET-targeted therapies has historically lagged behind that of EGFR and ALK inhibitors. Encouragingly, several MET TKIs such as capmatinib, tepotinib, and savolitinib have been approved for the treatment of MET exon 14 skipping mutations. They have also demonstrated potential in overcoming MET-driven resistance to EGFR TKIs or ALK TKIs. On 14 May 2025, the U.S. Food and Drug Administration granted accelerated approval to telisotuzumab vedotin-tllv for adult patients with locally advanced or metastatic non-squamous NSCLC whose tumors exhibit high c-Met protein overexpression and who have already received prior systemic therapy. In this review, we summarize the structure and physiological role of the MET receptor, the molecular mechanisms underlying aberrant MET activation, its contribution to acquired resistance against targeted therapies, and emerging strategies for effectively targeting MET alterations in NSCLC. Full article

Bupleuri radix has demonstrated therapeutic potential in treating liver disorders, and polysaccharides are one of its main bioactive components; however, the effects of Bupleuri radix polysaccharides (BRP) on metabolic dysfunction-associated steatotic liver disease (MASLD) remain unclear. This study aimed to identify the BRP fractions with anti-MASLD activity and elucidate their underlying mechanisms. We prepared BRP and characterized its physicochemical properties. It markedly alleviated liver injury and restored intestinal barrier function in MASLD. The correlation analysis between transcriptomics and targeted metabolomics showed that BRP restored intestinal acetic acid and propionic acid, with acetic acid activating AMPK and propionic acid promoting cholesterol efflux and metabolism in the liver, thereby reducing lipid accumulation in hepatocytes. Mechanistically, 16S RNA sequencing and diversity analysis indicated that BRP enriched short chain fatty acids (SCFAs)-producing bacteria, such as the genus Muribaculaceae, and inhibited pro-inflammatory microbiota. Interestingly, Paramuribaculum intestinale (P. intestinale), a representative species in the genus Muribaculaceae, synergistically enhanced BRP in improving liver and colonic mucosal damage in MASLD. In conclusion, our findings revealed that BRP improved MASLD by regulating Muribaculaceae-derived SCFAs in the gut–liver axis and could be used in combination with probiotics as a novel therapeutic strategy for MASLD. Full article

The liver is the central organ in metabolism; however, modeling hepatic diseases remains limited by current experimental models. Animal models frequently fail to predict human liver physiology, while primary hepatocytes rapidly dedifferentiate in culture. We performed comprehensive transcriptomic profiling of induced pluripotent stem cells (iPSCs) differentiation into hepatocyte-like cells (HLCs) under two-dimensional (2D) and three-dimensional (3D) culture conditions. RNA sequencing analysis revealed the sequential activation of lineage-specific markers across major developmental stages: definitive endoderm (FOXA2, SOX17, CXCR4, CER1, GATA4), posterior foregut (PROX1, GATA6), and hepatoblasts (HNF4A, AFP). Comparative analysis demonstrated a markedly enhanced hepatic gene expression of 3D organoids, as demonstrated by a 33-fold increase in HNF4A expression and elevated levels of mature hepatocyte markers, including ALB, SERPINA1, and UGT2B15. However, the 3D cultures retained fetal characteristics (290-fold higher AFP expression) and exhibited significantly impaired metabolic function, with CYP3A4 expression levels reduced by 2000-fold compared to the adult human liver. This partial maturation was further supported by a moderate correlation with adult liver tissue (ρ = 0.57). We demonstrated high reproducibility across five biologically distinct iPSCs lines, including those derived from patients with rare monogenic disorders. The establishment of quantitative benchmarks provides a crucial tool for standardizing in vitro liver models. Furthermore, we delineate the specific limitations of the current model, highlighting the need for further protocol optimization to enhance metabolic maturation and P450 enzyme activity. Functional validation of metabolic activity (CYP enzyme assays, albumin secretion) was not performed; therefore, conclusions regarding hepatocyte functionality are based on transcriptomic evidence. Full article

Background/Objectives: Sex-specific differences in the mechanisms of acute liver injury remain poorly understood. CDK5 regulatory subunit-associated protein 3 (CDK5RAP3) is crucial for liver development and endoplasmic reticulum (ER) homeostasis. This study aimed to investigate sex-dependent changes in CDK5RAP3 expression in a carbon tetrachloride (CCl₄)-induced acute liver injury model and to explore the mechanisms underlying differential susceptibility between males and females. Methods: Acute liver injury was induced in male and female mice by CCl₄ administration. Liver injury was evaluated by serum biochemical parameters and histopathological analysis. CDK5RAP3 expression, inflammatory cytokines, and ER stress-related apoptotic markers were assessed. Hepatocyte apoptosis was examined by TUNEL staining. In addition, CDK5RAP3 was conditionally deleted in mouse embryonic fibroblasts (MEFs) using 4-hydroxytamoxifen to assess its direct role in regulating inflammatory and apoptotic responses in vitro. Results: CCl₄ exposure caused liver injury in both sexes, with male mice showing more severe biochemical and histological damage. CDK5RAP3 expression was significantly reduced after CCl₄ treatment, particularly in males. Inflammatory mediators and ER stress-associated apoptotic markers were upregulated, accompanied by increased hepatocyte apoptosis. A similar enhancement of inflammatory and apoptotic signaling was observed in CDK5RAP3-deficient MEFs. Conclusions: Downregulation of CDK5RAP3 is associated with ER stress, inflammation, and apoptosis, contributing to increased susceptibility of male mice to acute liver injury. These findings provide insight into sex-specific mechanisms of hepatic injury and highlight CDK5RAP3 as a potential therapeutic target. Full article

Abnormal lipid metabolism is a key feature of many diseases. Therefore, investigating its underlying mechanisms is of great importance. Recently, blue light has shown promise as a drug-free way to influence energy metabolism, relying on the light-sensitive protein Opsin 3 (Opn3). This study aimed to investigate the effects of blue light irradiation on lipid droplet degradation in cells and its molecular mechanism, while also evaluating its potential antiviral effects. The results demonstrate that exposure to 470–480 nm blue light significantly reduced oleic-acid-induced intracellular lipid droplet accumulation and decreased triglyceride and total cholesterol levels, an effect dependent on the Opn3. It was found that blue light affects the Pparα signaling pathway through Opn3, and, at the same time, blue light and Opn3 promote autophagy mediated by p62 protein, thereby cooperatively regulating lipid droplet degradation. In Opn3 knockout cells, blue-light-induced lipid droplet degradation, nuclear accumulation of Pparα, and autophagic effects were all suppressed. Additionally, the study unexpectedly observed that blue light, via Opn3, significantly suppressed the replication of VSV, H1N1 and EMCV and alleviated virus-induced cell death and inflammatory responses. This study reveals the critical role of the blue light–Opn3-Pparα/p62 axis in regulating lipid droplet degradation in hepatocytes and identifies a novel antiviral function of Opn3-mediated blue light exposure. These findings provide a new theoretical basis and potential targets for innovative therapeutic strategies against metabolic diseases and viral infections. Full article

Acute liver injury (ALI) is a potentially life-threatening condition lacking effective clinical drugs. Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of both inflammation and metabolism. In ALI, HIF-1α expressions are upregulated, but the role of HIF-1α in hepatocytes and whether it can be targeted remain unclear. Herein, clinical samples and ALI murine models including lipopolysaccharide/D-galactosamine (LPS/D-GalN), acetaminophen (APAP), and thioacetamide (TAA) revealed an increase in HIF-1α expression and ferroptosis. Using HIF-1α gain and loss of function mouse and hepatocyte culture models, we demonstrated that HIF-1α upregulation exacerbated liver ferroptosis and injury. Mechanistically, HIF-1α/transferrin receptor protein 1 (TFR1) axis drives hepatic iron overload, promoting ferroptotic cell death and liver injury. In addition, TFR1 inhibition reversed HIF-1α-induced ALI. Importantly, pharmacological inhibition of HIF-1α and TFR1 significantly reduced ferroptosis and mitigated liver injury both in vivo and in vitro. Together, our findings demonstrate the pathological role of hepatic HIF-1α, which may serve as a promising target of therapeutic intervention. Full article

Background/Objectives: Elevated circulating non-esterified fatty acids (NEFAs) are closely associated with hepatic inflammatory injury in dairy cattle, simultaneously with the entry of lipopolysaccharide (LPS) into the liver. This study aimed to investigate the synergistic effects of NEFAs and LPS on pyroptosis in bovine hepatocytes. Methods: Primary bovine hepatocytes were allocated into control, NEFA, NEFA + LPS, NEFA + LPS + Caspase-1 inhibitor, and NEFA + LPS + NLRP3 inhibitor groups. Levels and activation of pyroptosis-related markers (NLRP3, ASC, Caspase-1, GSDMD, IL-18 and IL-1β) were measured. Results: NEFAs alone upregulated these markers in a dose-dependent manner. Compared to NEFAs alone, NEFA + LPS co-treatment significantly enhanced levels of the markers, increased IL-1β secretion, and promoted NLRP3/Caspase-1 co-localization and Caspase-1activity. Notably, these effects of NEFA + LPS were attenuated by the NLRP3 or Caspase-1 inhibitors. Similar results were obtained when repeating the experiments in carcinoma HepG2 cells. Also, a random liver section from the subclinical ketotic cows displayed a higher fluorescence intensity of NLRP3 and Caspase-1 and stronger co-localization than that from a healthy cow. Conclusions: NEFAs and LPS synergistically contribute to pyroptosis in bovine hepatocytes by enhancing NLRP3 inflammasome assembly and subsequent Caspase-1 activation, providing a potential target for mitigating hepatic injury. Full article

The global burden of acute and chronic liver diseases warrants safe and effective regenerative therapies that can complement or defer liver transplantation. Mesenchymal stromal/stem cells (MSCs) have been recognized as versatile biologics that modulate inflammation, reverse fibrosis, and promote hepatic repair predominantly through paracrine signaling. In hepatic milieu, MSCs act on hepatocytes, hepatic stellate cells, endothelial cells, and immune cell subsets through trophic factors and extracellular vesicles (EVs). Despite demonstrating hepatocyte-like differentiation of MSCs, their in vivo efficacy is primarily attributed to micro-environmental reprogramming rather than durable engraftment. This review covers MSC biology, liver regeneration, and cell-based versus EV therapies, including administration, dosing, quality, and safety. Future directions focus on biomarkers, multi-center trials, and engineered MSC/EV platforms for scalable personalized liver regeneration. Full article

Variants of the MYO5B gene, which encodes the molecular motor protein myosin-Vb, have gained prominence as a causative factor in familial intrahepatic cholestasis (FIC). Understanding the disease mechanism is pivotal for therapy development and clinical decision-making. The prevailing theory for the mechanism underlying MYO5B-associated cholestasis implicates faulty trafficking of the ABCB11-encoded bile salt export pump (BSEP) in hepatocytes due to dysfunctional myosin-Vb. This is supported by cell and mouse studies. However, while BSEP localization was abnormal in some patients’ liver biopsies, BSEP appeared normally localized in others, raising questions with regard to the role of BSEP in MYO5B-associated FIC. We present a focused systematic narrative review of all cases of MYO5B variant-associated isolated FIC reported in the MEDLINE database. We assembled a comprehensive patient dataset and assessed clinical features of MYO5B-associated FIC, their relationship with MYO5B genotype, the clinical value and significance of BSEP abnormalities, and the relationship of MYO5B-associated FIC to ABCB11 variant-associated FIC. Our review revealed that aberrant BSEP localization correlated with the absence of one MYO5B allele carrying a truncating nonsense or frameshift variant. Notably, biochemical and clinical parameters including treatment outcome were indistinguishable between patients presenting with normal and aberrant BSEP localization. Further, myosin-Vb and BSEP deficiency-associated FIC patient cohorts showed distinct biochemical and clinical phenotypes, indicating different underlying mechanisms. This suggests that whether or not BSEP localization was abnormal depended on the MYO5B genotype without a predictable effect on clinical parameters and treatment response. Treatment decisions should be guided by clinical parameters rather than by genotype or immunohistochemistry findings. Full article

Hepatitis B virus (HBV) persists in infected hepatocytes through covalently closed circular DNA (cccDNA), a stable episomal form that serves as the transcriptional template for viral replication. Accurate and sensitive quantification of intrahepatic cccDNA is crucial for evaluating antiviral therapies, particularly those targeting a functional cure. Here, we report the development of a novel, cccDNA-specific detection system combining recombinase polymerase amplification (RPA) with CRISPR/Cas12a-based fluorescence detection. We designed and validated CRISPR RNAs (crRNAs) targeting HBV cccDNA-specific regions conserved across genotypes A–D. Reaction conditions for both RPA and Cas12a detection were optimized to enhance sensitivity, specificity, and accuracy. The system reliably detected as few as 10 copies of cccDNA-containing plasmid per reaction and showed no cross-reactivity with non-cccDNA forms in serum or plasma, indicating assay specificity. When applied to liver tissue samples from 10 HBV-infected and 6 non-HBV patients, the RPA-CRISPR/Cas12a assay exhibited a high sensitivity (90%) and a strong correlation with qPCR results (R² = 0.9155), confirming its accuracy. In the conclusion, the RPA-CRISPR/Cas12a system provides a robust, cost-effective, and scalable platform for sensitive and specific quantification of intrahepatic HBV cccDNA. This method holds promises for research and high-throughput therapeutic screening applications targeting cccDNA clearance. Full article

Candida albicans is a pathogenic fungus that expresses a fungal NADPH oxidase known as C. albicans Cfl11, which produces reactive oxygen species (ROS). Secretion of these ROS triggers caspase 3–mediated cell death in hepatocytes, which was attenuated in a mutant with a disrupted CaCFL11 gene (designated Cacfl11Δ mutant). Here, we compared the effects of the C. albicans wild-type strain and the Cacfl11Δ mutant. Our findings revealed that C. albicans reduces the viability of HaCaT keratinocytes in a contact-independent manner. Furthermore, exposure to C. albicans increased intracellular ROS production and caspase 3 activity in HaCaT keratinocytes. These changes were attenuated when HaCaT keratinocytes were exposed to the Cacfl11Δ mutant or when HaCaT keratinocytes were treated with the known antioxidant N-acetylcysteine. Furthermore, wild-type C. albicans, but not the Cacfl11Δ mutant, disrupted transepithelial electrical resistance and modulated the downregulation of the tight-junction genes occludin and junction adhesion molecule 1 in HaCaT keratinocytes. Collectively, these results show that fungal ROS secretion via CaCFL11 is a potent virulence factor in mediating keratinocyte viability and barrier function. Full article