Search Results (256)

Exosomes carry bioactive macromolecules driving sepsis pathogenesis, but the mechanisms underlying macrophage-to-hepatocyte communication during systemic inflammation remain poorly understood. We investigated how sepsis-induced macrophage exosomes are involved in remote intercellular crosstalk with hepatic cells via transcription factor-encoding mRNA cargo. Human monocytic THP-1 macrophages were stimulated with lipopolysaccharide (LPS), followed by exosome isolation, recipient cell uptake verification, and high-throughput RNA sequencing cargo analysis. To functionally reconstruct downstream signaling in recipient cells, exosome-enriched MAFB mRNA was transiently overexpressed in a HepG2 cell model, with subsequent expression changes mapped at both the transcript and protein levels using quantitative PCR and Western blot analyses. This ectopic MAFB expression directly upregulates the expression of microRNA-155 (miR-155). Crucially, elevated miR-155 acts as a post-transcriptional repressor that directly targets and downregulates JARID2 and H3F3A mRNAs and their corresponding protein products within the liver cells, orchestrating a “repressor-of-repressors” disinhibition cascade that drives net chromatin remodeling and activation of downstream hepatic target genes. This study demonstrates that sepsis alters exosomal transcription factor mRNA cargo and delineates a mechanistic downstream pathway—MAFB → ↑miR155 → ↓Jarid2 & ↓H3F3A → Chromatin Remodeling → Downstream Hepatic Gene Activation pathway—that provides novel, specific molecular checkpoints for therapeutic intervention in sepsis-induced liver injury. Full article

The adult liver exhibits remarkable regenerative capacity in vivo; however, primary hepatocytes (PHs), the principal functional cells of the liver, swiftly forfeit their proliferative potential and specialized hepatic functions when isolated from their native microenvironment and cultured in vitro. While PHs are the benchmark for studying hepatic physiology, xenobiotic metabolism, and toxicological responses, their rapid dedifferentiation resulting in the loss of hepatic functions significantly limits their further application. Recent studies suggest three converging strategies to address the challenge of long-term maintenance and expansion of PHs. First, defined chemical and growth factor-based protocols can temporarily induce hepatocytes into a proliferative, progenitor-like state, followed by a maturation phase that restores differentiated hepatic functions. In addition, the inhibition of signaling pathways linked to cellular stress responses and identity loss can postpone dedifferentiation and preserve drug-metabolizing activity for prolonged durations, thereby enhancing disease modeling and toxicology studies. Finally, three-dimensional (3D) culture platforms generally improve hepatocyte maturation and functional stability, but these are often not scalable due to matrix dependence, technical complexity, handling requirements, and cost. This review thoroughly examines innovative methodological advancements designed to facilitate the proliferation and prolonged viability of healthy PHs, focusing on their translational relevance and clinical applicability. Full article

Donor liver scarcity is exacerbated by preservation-related injury, particularly ischemia-reperfusion injury (IRI), which reduces organ utilization and increases discard rates. Liver sinusoidal endothelial cells (LSECs) play a critical role in mediating IRI, yet their response to static cold storage (SCS) remains poorly understood. Here, we investigate the impact of SCS on isolated rat LSECs. Isolated rat hepatocytes, stellate cells, Kupffer cells, and LSECs were subjected to up to 3 days of SCS followed by up to 2 days of recovery, with LSECs also receiving apoptosis and/or oxidative stress inhibition. Afterwards, changes in survivability, functionality, and morphology were measured. Additionally, changes in gene, cytokine, and chemokine expression were also measured. We found that SCS reduced cell viability by approximately 40%, accompanied by a 60% reduction in metabolic activity and ATP levels, indicating substantial impairment in cellular energetics. SCS also doubled reactive oxygen species (ROS) production and upregulated oxidative stress and apoptosis-related genes, leading to functional decline in LSECs. Importantly, combined inhibition of apoptosis and oxidative stress improved LSEC viability by 20% and metabolic activity and ATP levels by 30% and 40%, respectively, and reduced ROS production by 50%. These findings highlight LSEC vulnerability to preservation injury and the importance of understanding LSEC-specific injury mechanisms to provide a foundation for the development of endothelial-targeted preservation strategies to significantly improve liver transplantation outcomes, subsequently increasing access to this life-saving treatment. Full article

Background/Objectives: Type 2 diabetes is increasingly recognized as a systemic disorder driven not only by chronic hyperglycemia and insulin resistance, but also by dysregulated interorgan communication. Extracellular vesicles (EVs), including exosomes and microvesicles, have emerged as biologically active carriers of proteins, lipids, and microRNAs capable of modulating gene expression in recipient cells. This narrative review integrates clinical, experimental, and translational evidence on EV-associated microRNAs as candidate biomarkers and potential mediators of diabetic complications, with emphasis on diabetic neuropathy, diabetic kidney disease, diabetic retinopathy, and metabolic dysfunction-associated steatotic liver disease (MASLD). Methods: This review was aligned with the SANRA framework and focused on biological plausibility, evidence from tissue and biofluids, biomarker potential, therapeutic implications, and barriers to clinical translation. Studies were additionally interpreted according to biological matrix, EV-carrier specificity, analytical platform, study design, and level of functional validation. Results: Across complications, EV-associated microRNAs appear to participate in shared pathogenic processes, including oxidative stress, inflammation, endothelial dysfunction, fibrosis, angiogenesis, neurodegeneration, and metabolic memory. In diabetic neuropathy, microRNAs such as miR-146a, miR-155, miR-21-5p, and miR-148a-3p have been linked to neuroinflammation, Schwann-cell dysfunction, axonal injury, and neuropathic pain. In diabetic kidney disease, miR-21, miR-29, miR-30, and miR-126 are implicated in podocyte injury, tubulointerstitial fibrosis, albuminuria, and microvascular dysfunction. In diabetic retinopathy, microRNAs including miR-146a, miR-155, miR-21, miR-126, and miR-200b contribute to neurovascular injury, inflammation, barrier disruption, and angiogenesis. In MASLD associated with diabetes, hepatocyte-derived EVs carrying microRNAs such as miR-1 and miR-126a-3p may link hepatic lipotoxicity to endothelial inflammatory and β-cell dysfunction. Conclusions: Although EV-associated microRNAs offer promising opportunities for biomarker discovery, risk stratification, and targeted therapies, clinical translation remains limited by heterogeneity in EV isolation, microRNA quantification, biological matrices, and outcome definitions. Distinguishing EV-associated miRNAs from total circulating extracellular miRNAs remains essential for biological interpretation. Standardized, longitudinal, and externally validated studies are required before these signals can be implemented as actionable tools in precision diabetes care. Full article

Introduction: Circulating cell-free DNA (cfDNA) has emerged as a promising minimally invasive biomarker in hepatocellular carcinoma (HCC), with potential applications in disease detection and prognostic stratification. This exploratory study aimed to evaluate the relationship between circulating cell-free DNA (cfDNA) concentration, liver dysfunction parameters, and hepatocellular carcinoma stage in an exploratory cohort: we sought to explore the extent to which cfDNA variability may be explained by the underlying liver disease environment and whether cfDNA concentration provides incremental information beyond routinely available markers of liver reserve for the discrimination between early- and late-stage hepatocellular carcinoma. Methods: Sixty-four newly diagnosed HCC patients were included. Clinical, laboratory, and staging data were collected. cfDNA was isolated from plasma, confirmed by on-chip electrophoresis, and quantified by fluorimetry. Logistic regression and ROC curve analyses were performed to assess the ability of several biomarker panels to discriminate early-stage HCC (BCLC 0–A) from intermediate/advanced-stage disease (BCLC B–D). Bootstrap resampling (2000 iterations) evaluated model robustness and coefficient stability. Additional linear regression analyses explored associations between cfDNA concentration and liver dysfunction parameters. Results: Linear regression demonstrated that liver dysfunction parameters explained approximately 53% of cfDNA variability, while HCC stage contributed minimally after adjustment. Models incorporating albumin, bilirubin, and platelet count as individual parameters demonstrated the best discriminatory performance after adjustment for liver disease etiology, achieving AUROCs up to 0.857. The only incremental value that cfDNA concentration added to the panel was an increase to its specificity (from 79.4% to 94.1%), while reducing sensitivity by 13.4%. Bilirubin and platelet count remained the most stable predictors after bootstrap, whereas cfDNA concentration was unstable. Limitations: The study was limited by its small sample size, cross-sectional design, and lack of longitudinal outcome assessment. External validation in larger prospective cohorts is necessary. Conclusion: A substantial proportion of cfDNA concentration variability (approximately 53%) was explained by routinely available liver dysfunction parameters, whereas HCC stage had minimal contribution. These findings suggest that circulating cfDNA concentration in patients with HCC may be influenced to a greater extent by the underlying cirrhotic liver environment and hepatocyte injury than by tumor burden alone. Integrated multimarker panels combining liver reserve parameters and liver disease etiology may be of interest for minimally invasive stratification of HCC. Although cfDNA concentration increased specificity for early- versus advanced-stage disease discrimination, its incremental value was low. Further validation in larger prospective cohorts is required. Full article

Background/Objectives: Exposure to high and sustained levels of non-esterified fatty acids (NEFA) in the peripartal period is the main cause of fatty liver disease in dairy cows. Rumen-protected choline is often fed as part of the nutritional management of peripartal cows, with in vivo and in vitro data indicating positive effects of this nutrient on alleviating liver lipid accumulation. Although hepatic molecular mechanisms associated with choline supply have been studied using a target gene, protein, or metabolite approach, application of high-throughput technologies could vastly enhance fundamental knowledge on the functional role of choline. The main objective was to challenge isolated hepatocytes with a mixture of NEFA and determine proteome- and metabolome-wide effects in response to choline supply. Methods: Three healthy female calves (1 d old, 30–45 kg) were sacrificed to harvest hepatocytes. During a 12 h incubation, isolated hepatocytes were challenged without NEFA (control), 1.2 mM NEFA (c9-18:1, 18:2, 16:0, 18:0, and c9-16:1 at 43.5%, 4.9%, 31.9%, 14.4%, and 5.3% of total NEFA, respectively), or NEFA for 6 h followed by 10 μM choline chloride for another 6 h (NEFA + Chol). iTRAQ labeling-based protein profiling and GC/MS-based metabolomics profiling were used to determine changes in proteins and metabolites. Differentially abundant proteins for each group comparison were determined at a threshold of 1.4-fold change. Differences in metabolite profiles were assessed via pairwise comparisons. A subset of differentially abundant proteins was validated via qRT-PCR and Western blotting. Results: Compared with the control, there were 90 proteins and 22 metabolites in the NEFA group, and 83 proteins and 29 metabolites in the NEFA + Chol. Compared with NEFA, there were 49 proteins and 17 metabolites in the NEFA + Chol group. Greater abundance of hexokinase-1 (HK1), fructose-bisphosphate aldolase (ALDOA), mitochondrial pyruvate carrier 1 (MPC1), and increased concentrations of lactate with high NEFA treatment alone suggested greater glycolytic and TCA cycle activity. Accumulation of triacylglycerol in the NEFA group was associated with lipotoxicity and markers of inflammation, such as greater abundance of prostaglandin reductase 1 (PTGR1), serious cell autophagy processes, such as greater abundance of cell division cycle 42 (CDC42), and NFκB-related proteins. Choline supplementation reduced TAG partly due to greater VLDL secretion driven by greater abundance of diacylglycerol acyltransferase (DGAT1), perilipin 3 (PLIN3), and apolipoprotein C-III (APOC3). In addition, a greater abundance of carnitine O-palmitoyltransferase 1b (CPT1B) with choline suggested enhanced mitochondrial β-oxidation. Activation of the CDC42/JNK pathway and ROS/NFκB axis-related proteins, along with depressed PI3K/AKT/RAC-related proteins, indicated enhanced mitochondrial autophagy in response to NEFA. Conclusions: Overall, data confirmed published effects of choline on TAG accumulation, VLDL secretion, and fatty acid oxidation, while highlighting negative effects of NEFA on the respiratory electron transport chain, autophagy, and inflammatory processes. Full article

Gastrointestinal symptoms (GI) (abdominal pain, vomiting, and diarrhea) during the febrile phase of dengue (less than 5 days from fever onset) might indicate prominent innate immune responses. Serum and feces samples from cases with GI symptoms versus those without GI symptoms (n = 20 per group) were analyzed. From these, only the neutrophil extracellular traps (NETs), serum fibroblast growth factor (FGF) 21, and fecal microbiome analyses, but not the routine parameters, endotoxemia, or serum cytokines, were higher in the GI cases than in the non-GI cases. From the in vitro experiments, both lipopolysaccharide (LPS) and the dengue virus (DENV) upregulated the FGF receptor 1 (FGFR1) and cytokines in hepatocytes (HepG2) and THP-1-differentiated macrophages. Meanwhile, LPS and DENV induced NETs in isolated neutrophils from healthy volunteers. Only the starvation protocol, but not LPS or DENV, enhanced supernatant FGF-21 from hepatocytes. Incubation of recombinant FGF-21 in LPS + DENV-activated cells (hepatocytes, macrophages, and neutrophils) attenuated inflammation, as determined by supernatant cytokines and NETs. Hence, abdominal symptoms in dengue during the febrile phase indicate prominent innate immune responses, as detected by NETs and FGF-21 (an acute-phase protein), implying significant hepatic stress with a possible counteracting anti-inflammation. Full article

Introduction: Temperature is a key environmental factor influencing the physiological and biochemical processes of aquatic organisms, including xenobiotic metabolism. Understanding how temperature modulates the toxicological effects of pollutants such as polycyclic aromatic hydrocarbons (PAHs) is crucial in the context of climate change. Among these compounds, benzo[a]pyrene (BaP) and benzo[a]anthracene (BaA) are priority pollutants in aquatic environments, resulting from incomplete combustion. Their relevance is attributed to persistence and metabolic bioactivation potential. Fish primary hepatocyte cultures represent a relevant in vitro model for studying combined effects of thermal stress and chemical exposures, while supporting the 3Rs principles (Replacement, Reduction, and Refinement). Objective: This study aims to assess temperature-dependent effects of BaP and BaA, and their mixtures in brown trout hepatocytes using cytochrome P450 1A (CYP1A) immunohistochemistry as an indicator of xenobiotic metabolism. Methodology: Primary hepatocytes were isolated using a two-step collagenase perfusion method and cultured in 24-well plates at 18 °C and 22 °C. Cells were exposed for 72 h to supplemented L-15 medium (control) or to 0.1% dimethyl sulfoxide in supplemented L-15 medium (solvent control), as well as to single exposures of 1 and 10 µM of BaP and BaA and to equimolar mixtures of both compounds (1 and 10 µM). Viability was assessed using the lactate dehydrogenase (LDH) assay. CYP1A immunostaining was quantified based on cytoplasmic staining intensity relative to background area. Results: No significant effects on cell viability were observed under any condition. Temperature significantly reduced CYP1A expression in single exposures at 22 °C compared to 18 °C. BaP induced a significant dose-dependent increase, while BaA differed from controls only at 10 µM. In mixtures, only treatment- and dose-dependent effects were observed, with no temperature influence detected. Conclusions: Overall, the data highlight temperature as a key modulator of biochemical responses to PAHs, with single and mixed exposures eliciting distinct effects and suggesting potential synergism in mixtures. Full article

Pancreatic ductal adenocarcinoma frequently metastasises to the liver, although the mechanisms underlying hepatic pre-metastatic niche formation remain unclear. Small extracellular vesicles mediate tumour–host communication and may drive hepatic microenvironment reprogramming. This study investigated the effects of pancreatic ductal adenocarcinoma-derived small extracellular vesicles on extracellular matrix remodelling and epithelial–mesenchymal transition-related plasticity in hepatic cells. Small extracellular vesicles were isolated from pancreatic ductal adenocarcinoma cell lines (MIAPaCa-2, PANC-1) and from the serum of 25 patients, characterized, and administered to hepatic stellate (LX-2) and hepatocyte-like (HEPA-RG) cells. Cell viability and migration were evaluated by functional assays, morphology by scanning electron microscopy, and molecular changes by RT-PCR, Western blotting, and immunofluorescence. In LX-2 cells, small extracellular vesicles exposure increased metabolic activity, adhesion, and migration, while inducing morphological and molecular changes associated with extracellular matrix remodelling, including reduced collagen type I alpha 2 chain, vimentin, and E-cadherin expression. In HEPA-RG cells, viability was minimally affected, whereas migration and EMT-related plasticity were enhanced. Patient-derived small extracellular vesicles induced similar but less pronounced effects. Overall, pancreatic ductal adenocarcinoma-derived small extracellular vesicles induced early hepatic microenvironmental remodelling, supporting a potential role for tumour–liver crosstalk in pre-metastatic niche-associated processes, highlighting tumour–liver crosstalk as a potential therapeutic target. Full article

Heat stress is a major systemic challenge in poultry, but the role of circulating extracellular vesicles (EVs) in liver-directed adaptation remains unclear. This study investigated whether plasma-derived EVs from heat-stressed chickens (HS_EV) mediate hepatoprotective responses under thermal stress. EVs were isolated from the plasma of control and heat-stressed chickens and characterized by morphology, size distribution, and marker expression. Their biodistribution in vivo and uptake by primary hepatocytes in vitro were also evaluated. Hepatocyte injury was induced by heat exposure, and the effects of HS_EV on proliferation, apoptosis, inflammatory cytokine production, transcriptomic reprogramming, and MYD88/NF-κB/MAPK signaling were assessed. Heat stress induced systemic injury in chickens, increased the release of plasma-derived EVs, and promoted their preferential accumulation in the liver. Whole-transcriptome analysis further showed that HS_EV enhanced glutathione metabolism and related metabolic pathways while suppressing apoptosis- and inflammation-related signaling. In primary hepatocytes, HS_EV, but not control EVs, restored proliferative capacity, reduced apoptosis, suppressed the expression and secretion of IL-1β, IL-6, and TNF-α under heat stress, and was associated with attenuation of the MYD88/NF-κB/MAPK axis. These findings suggest that circulating EVs participate in adaptive intercellular communication during heat stress and identify HS_EV as a potential endogenous mediator of hepatoprotection in chickens. Full article

Alcoholic liver disease (ALD) is a prevalent and progressive hepatic disorder driven by chronic excessive alcohol consumption. Leonurine (LH), a bioactive alkaloid isolated from Herba Leonuri, possesses well-documented antioxidant and cytoprotective properties. This study comprehensively investigated the hepatoprotective efficacy of LH against ethanol-induced liver injury and mechanistically dissected its molecular underpinnings. Antioxidant capacity and cytoprotective activity were assessed in ethanol-treated hepatocytes. Network pharmacology and gene expression analysis were performed to identify potential therapeutic targets and signaling pathways. UHPLC–MS/MS-based metabolomics was applied to characterize endogenous metabolic alterations induced by LH. LH demonstrated significant antioxidant activity and was predicted to interact with 44 ALD-related targets. Functional enrichment and gene validation analyses revealed that its protective effects were primarily associated with regulation of the JAK2–STAT3 signaling pathway. Metabolomic profiling identified 48 differential metabolites and 25 significantly affected metabolic pathways. Integrated analysis of metabolites and target genes further supported the JAK–STAT signaling pathway as a central regulatory axis, which was confirmed in cellular experiments. Collectively, these results demonstrate that LH confers hepatoprotection in ALD primarily through modulation of the JAK2–STAT3 signaling pathway, underscoring its translational promise as a mechanism-informed therapeutic candidate. Full article

Insulin resistance is a multifactorial cellular state involving coordinated alterations in protein homeostasis and organelle function; however, its proteome-wide organization and response to bioactive peptides remain incompletely defined. In this study, we employed DIA-based quantitative proteomics to characterize global protein abundance changes associated with insulin resistance in HepG2 cells and to examine proteomic remodeling following treatment with a camel milk-derived peptide (P2). Comparative proteomic profiling revealed that insulin-resistant cells exhibit extensive reorganization of protein networks linked to redox regulation, endoplasmic reticulum protein processing, mitochondrial metabolism, lysosomal function, and extracellular matrix-associated components. Gene Ontology, KEGG pathway, protein domain, and subcellular localization enrichment analyses consistently indicated disruption of organelle-associated proteomic architecture rather than isolated pathway perturbations. Peptide TYYPPQ treatment was associated with selective, rather than global, proteomic shifts, prominently affecting mitochondrial and peroxisome-associated protein groups as well as extracellular and secretory proteins. Enrichment and localization analyses suggest that peptide exposure reshapes organelle-linked protein representation patterns without implying direct activation of signaling pathways or physiological restoration. Collectively, these results define insulin resistance and peptide responsiveness at a systems-level proteomic resolution and establish an organelle-resolved framework for interpreting peptide-induced proteomic remodeling in insulin-resistant hepatocyte models. This dataset provides a foundation for future targeted functional validation of candidate pathways identified through proteomic association. Full article

In this work, Filipendula ulmaria (L.) Maxim, a perennial herbaceous plant from the Rosaceae family, was considered a novel source of obtaining rutin for pharmaceutical purposes. Rutin was extracted from the plant parts collected in the flowering summer period and dried at 40 ± 3 °C. The process was carried out using the ethanol extraction and fractionation of extracted compounds, and it yields the 95 wt% purity crystalline product. The phase composition of the extracted rutin was verified by the XRD analysis and NMR measurements. It was found that 2.85% of rutin could be extracted from Filipendula ulmaria, which is 1.2 times higher than the results of similar studies. The biological activity of the isolated rutin was tested on rats. It was established in vivo that the extracted rutin normalizes blood glucose levels (glucose and glycosylated hemoglobin), insulin resistance (HOMA-IR index) and reduces the severity of dystrophic changes in the liver caused by high-fat and high-carbohydrate diets. The introduction of rutin corrects lipid profile indicators (triglycerides, cholesterol, cholesterol fractions in lipoproteins and atherogenic indices), cytolysis indicators of hepatocytes, and liver steatosis (ALT, AST/ALT, triglycerides). Thus, the novel source of rutin opens the possibility for a wide use of this flavonoid in the food technology and pharmaceutical industry. Full article

Eupalinolide A (EA, Z-configuration) and Eupalinolide B (EB, E-configuration) are cis-trans isomeric sesquiterpenoid monomers isolated from Eupatorium lindleyanum DC. (Asteraceae). Although these compounds display anti-inflammatory and anti-tumor activities, their metabolite profiles and possible hepatotoxicity remain largely unknown. This study aimed to investigate the metabolic profiles of EA and EB in liver microsomes and clarify whether they undergo metabolic activation or detoxification. EA and EB were metabolically profiled in human liver microsomes (HLMs) via UPLC-Q-TOF-MS. A HepG2-HLM co-culture system was used to compare the hepatocyte toxicity of parent compounds and their hydrolysis, oxidation, and hydrolysis–oxidation metabolites, thus evaluating their metabolic detoxification pathways. Sixteen metabolites of EA and 19 of EB were identified, with hydrolysis being the predominant metabolic pathway for both isomers. Both compounds showed low hepatocyte toxicity and underwent metabolic detoxification mainly via hydrolytic and oxidative pathways. Notably, hydrolysis metabolites had significantly lower toxicity than oxidative products in HepG2 cells. These results suggest that EA and EB could present a relatively low risk of in vivo hepatotoxicity, which provides useful information for understanding the metabolic behavior and safety profile of these bioactive sesquiterpenoids. Full article

Hepatitis B virus (HBV) and human T-lymphotropic virus type 1 (HTLV-1) are blood-borne pathogens with overlapping transmission routes, resulting in an increased prevalence of HBV among individuals infected with HTLV-1. Notwithstanding the widespread application of mathematical modeling to the study of each virus in isolation, the within-host dynamics of HBV–HTLV-1 co-infection remain insufficiently characterized. This study introduces a novel within-host co-infection model that characterizes the interactions between HBV and HTLV-1, where HTLV-1 infects CD4⁺ T cells and HBV targets hepatocytes. A comprehensive qualitative analysis yields four threshold parameters ($R_i,i=1,2,3,4$) governing the existence and stability of equilibrium points, with global stability established using Lyapunov functions. Numerical simulations validate the analytical results, and sensitivity analysis identifies parameters that most strongly influence the basic reproduction numbers for HBV ($R_1$) and HTLV-1 ($R_2$) mono-infections. Our results corroborate that, in patients with HBV, the presence of HTLV-1 contributes to an elevated HBV viral load and CD4⁺ T cells play a crucial role in controlling HBV infection. Full article