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Excessive fructose intake is a risk factor for the development of obesity and its complications. Targeting ketohexokinase (KHK), the first enzyme of fructose metabolism, has been investigated for the management of metabolic dysfunction-associated steatotic liver disease (MASLD). We compared the effects of systemic, small molecule inhibitor of KHK enzymatic activity with hepatocyte-specific, N-acetylgalactosamine siRNA-mediated knockdown of KHK in mice on an HFD. We measured KHK enzymatic activity, extensively quantified glycogen accumulation, performed RNA-Seq analysis, and enumerated hepatic metabolites using mass spectrometry. Both KHK siRNA and KHK inhibitor led to an improvement in liver steatosis; however, via substantially different mechanisms, KHK knockdown decreased the de novo lipogenesis pathway, whereas the inhibitor increased the fatty acid oxidation pathway. Moreover, KHK knockdown completely prevented hepatic fructolysis and improved glucose tolerance. Conversely, the KHK inhibitor only partially reduced fructolysis, but it also targeted triokinase, mediating the third step of fructolysis. This led to the accumulation of fructose-1 phosphate, resulting in glycogen accumulation, hepatomegaly, and impaired glucose tolerance. Overexpression of wild-type, but not kinase-dead, KHK in cultured hepatocytes increased hepatocyte injury and glycogen accumulation after treatment with fructose. The differences between KHK inhibition and knockdown are, in part, explained by the kinase-dependent and -independent effects of KHK on hepatic metabolism.

Liver X receptor-α (LXRα) regulates cellular cholesterol abundance and potently activates hepatic lipogenesis. Here we show that at least 1 in 450 people in the UK Biobank carry functionally impaired mutations in LXRα, which is associated with biochemical evidence of hepatic dysfunction. On a western diet, male and female mice homozygous for a dominant negative mutation in LXRα have elevated liver cholesterol, diffuse cholesterol crystal accumulation and develop severe hepatitis and fibrosis, despite reduced liver triglyceride and no steatosis. This phenotype does not occur on low-cholesterol diets and can be prevented by hepatocyte-specific overexpression of LXRα. LXRα knockout mice exhibit a milder phenotype with regional variation in cholesterol crystal deposition and inflammation inversely correlating with steatosis. In summary, LXRα is necessary for the maintenance of hepatocyte health, likely due to regulation of cellular cholesterol content. The inverse association between steatosis and both inflammation and cholesterol crystallization may represent a protective action of hepatic lipogenesis in the context of excess hepatic cholesterol.

Methionine adenosyltransferase 2 A (MAT2A) and MAT2B are essential for hepatic stellate cells (HSCs) activation. Forkhead box M1 (FOXM1) transgenic mice develop liver inflammation and fibrosis. Here we examine if they crosstalk in male mice. We found FOXM1/MAT2A/2B are upregulated after bile duct ligation (BDL) and carbon tetrachloride (CCl4) treatment in hepatocytes, HSCs and Kupffer cells (KCs). FDI-6, a FOXM1 inhibitor, attenuates the development and reverses the progression of CCl4-induced fibrosis while lowering the expression of FOXM1/MAT2A/2B, which exert reciprocal positive regulation on each other transcriptionally. Knocking down any of them lowers HSCs and KCs activation. Deletion of FOXM1 in hepatocytes, HSCs, and KCs protects from BDL-mediated inflammation and fibrosis comparably. Interestingly, HSCs from Foxm1Hep-/-, hepatocytes from Foxm1HSC-/-, and HSCs and hepatocytes from Foxm1KC-/- have lower FOXM1/MAT2A/2B after BDL. This may be partly due to transfer of extracellular vesicles between different cell types. Altogether, FOXM1/MAT2A/MAT2B axis drives liver inflammation and fibrosis.

Broadening gene therapy applications requires manufacturable vectors that efficiently transduce target cells in humans and preclinical models. Conventional selections of adeno-associated virus (AAV) capsid libraries are inefficient at searching the vast sequence space for the small fraction of vectors possessing multiple traits essential for clinical translation. Here, we present Fit4Function, a generalizable machine learning (ML) approach for systematically engineering multi-trait AAV capsids. By leveraging a capsid library that uniformly samples the manufacturable sequence space, reproducible screening data are generated to train accurate sequence-to-function models. Combining six models, we designed a multi-trait (liver-targeted, manufacturable) capsid library and validated 88% of library variants on all six predetermined criteria. Furthermore, the models, trained only on mouse in vivo and human in vitro Fit4Function data, accurately predicted AAV capsid variant biodistribution in macaque. Top candidates exhibited production yields comparable to AAV9, efficient murine liver transduction, up to 1000-fold greater human hepatocyte transduction, and increased enrichment relative to AAV9 in a screen for liver transduction in macaques. The Fit4Function strategy ultimately makes it possible to predict cross-species traits of peptide-modified AAV capsids and is a critical step toward assembling an ML atlas that predicts AAV capsid performance across dozens of traits.

Currently, there is no effective treatment for obesity and alcohol-associated liver diseases, partially due to the lack of translational human models. Here, we present a protocol to generate 3D human liver spheroids that contain all the liver cell types and mimic "livers in a dish." We describe strategies to induce metabolic and alcohol-associated hepatic steatosis, inflammation, and fibrosis. We outline potential applications, including using human liver spheroids for experimental and translational research and drug screening to identify potential anti-fibrotic therapies.

The hepatocytes within the liver present an immense capacity to adapt to changes in nutrient availability. Here, by using high resolution volume electron microscopy, we map how hepatic subcellular spatial organization is regulated during nutritional fluctuations and as a function of liver zonation. We identify that fasting leads to remodeling of endoplasmic reticulum (ER) architecture in hepatocytes, characterized by the induction of single rough ER sheet around the mitochondria, which becomes larger and flatter. These alterations are enriched in periportal and mid-lobular hepatocytes but not in pericentral hepatocytes. Gain- and loss-of-function in vivo models demonstrate that the Ribosome receptor binding protein1 (RRBP1) is required to enable fasting-induced ER sheet-mitochondria interactions and to regulate hepatic fatty acid oxidation. Endogenous RRBP1 is enriched around periportal and mid-lobular regions of the liver. In obesity, ER-mitochondria interactions are distinct and fasting fails to induce rough ER sheet-mitochondrion interactions. These findings illustrate the importance of a regulated molecular architecture for hepatocyte metabolic flexibility.

Alpha-fetoprotein (AFP) is a glycoprotein that plays an important role in immune regulation with critical involvement in early human development and maintaining the immune balance during pregnancy. Postfetal development, the regulatory mechanisms controlling AFP undergo a shift and AFP gene transcription is suppressed. Instead, these enhancers refocus their activity to maintain albumin gene transcription throughout adulthood. During the postnatal period, AFP expression can increase in the setting of hepatocyte injury, regeneration, and malignant transformation. It is the first oncoprotein discovered and is routinely used as part of a screening strategy for HCC. AFP has been shown to be a powerful prognostic biomarker, and multiple HCC prognosis models confirmed the independent prognostic utility of AFP. AFP is also a useful predictive biomarker for monitoring the treatment response of HCC. In addition to its role as a biomarker, AFP plays important roles in immune modulation to promote tumorigenesis and thus has been investigated as a therapeutic target in HCC. In this review article, we aim to provide an overview of AFP, encompassing the discovery, biological role, and utility as an HCC biomarker in combination with other biomarkers and how it impacts clinical practice and future direction.

Xenobiotics, including drugs, undergo metabolism to facilitate detoxification and excretion. Predicting a compound's metabolic fate before clinical trials is crucial for efficacy and safety. The existing methods rely on in vitro systems and in vivo animal testing. In vitro systems do not replicate the complexity of in vivo systems, and differences in biotransformation pathways between humans and nonclinical species may occur; thus, accurate predictions of human-specific drug metabolism are not always achieved. The aim of this study was to evaluate whether a chimeric mouse with a humanized liver, specifically the PXB-mouse, can mimic human metabolic profiles. PXB-mice have livers engrafted with up to 95% human hepatocytes. The biotransformation of 12 different small-molecule drugs were evaluated in PXB-mice (through analysis of blood and urine) and compared with the metabolism by hepatocytes from humans and mice and, when available, literature reports on human in vivo metabolism. The detected metabolites included major Phase I and II transitions, such as hydroxylation, and N- and O-dealkylation and glucuronidation. The metabolic patterns of the PXB-mice closely matched human in vivo data. It is also worth noting that the human hepatocytes formed most of the circulating metabolites, indicating that hepatocytes provide reliable predictions of human metabolic pathways. Thus, for drugs with human biotransformation pathways that are not observed in nonclinical species, the PXB-mouse model can be valuable in predicting human-specific metabolism. [ABSTRACT FROM AUTHOR]

About 296 million people worldwide are living with chronic hepatitis B viral (HBV) infection, and outcomes to end-stage liver diseases are potentiated by alcohol. HBV replicates in hepatocytes, but other liver non-parenchymal cells can sense the virus. In this study, we aimed to investigate the regulatory effects of macrophages on HBV marker and interferon-stimulated genes (ISGs) expressions in hepatocytes. This study was performed on HBV-replicating HepG2.2.15 cells and human monocyte-derived macrophages (MDMs). We found that exposure of HepG2.2.15 cells to an acetaldehyde-generating system (AGS) increased HBV RNA, HBV DNA, and cccDNA expressions and suppressed the activation of ISGs, APOBEC3G, ISG15, and OAS1. Supernatants collected from IFNα-activated MDMs decreased HBV marker levels and induced ISG activation in AGS-treated and untreated HepG2.215 cells. These effects were reversed by exposure of MDMs to ethanol and mimicked by treatment with exosome release inhibitor GW4869. We conclude that exosome-mediated crosstalk between IFN-activated macrophages and HBV-replicating hepatocytes plays a protective role via the up-regulation of ISGs and suppression of HBV replication. However, ethanol exposure to macrophages breaks this protection. [ABSTRACT FROM AUTHOR]

Acetaminophen is commonly used as a reference hepatotoxin to demonstrate that in vitro human liver platforms can emulate features of clinical drug-induced liver injury. However, the induction of substantial cell death in these models typically requires acetaminophen concentrations (∼10 mM) far higher than blood concentrations of the drug associated with clinical hepatotoxicity (∼1 mM). Using the cytochrome P450 inhibitor 1-aminobenzotriazole, we show that acetaminophen toxicity in cultured human, mouse, and rat hepatocytes is not dependent on N-acetyl-p-benzoquinonimine formation, unlike the in vivo setting. This finding highlights the limitation of using acetaminophen as a reference hepatotoxin for the evaluation of in vitro liver models. Hence, we make recommendations on the selection of reference hepatotoxins for this purpose. [ABSTRACT FROM AUTHOR]

Introduction: Aging is a biological process that increases oxidative damage in the cell. Moringa oleifera (MO) plant has a hepatoprotective effect by inducing the antioxidant defense mechanism. Aim of the Study: To evaluate aging-related impacts on the hepatic structure and function and the possible mitigating role of MO extract, with clarifying the mechanistic role of autophagy in aged rat models. Material and Methods: Twenty four rats, aged 3 months, were assigned into 4 groups as following, group I: adult control, group II: administered MO leaf aqueous extract (50 mg/kg body weight) by nasogastric tube for 4 months, group III: received no treatment till age 20 months, and group IV: received no treatment till age 16 months, and then received MO leaf aqueous extract for 4 months. At the time of sacrificing, serum liver enzymes (aspartate aminotransferase and alanine aminotransferase) were measured and the hepatic specimens were processed for light and electron-microscopic assessment and analysis of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), and mammalian target of rapamycin (mTOR) gene expression. Results: The aged rats revealed significant elevation of liver enzymes and hepatic malondialdehyde as well as reduction of hepatic glutathione content. Aging also inhibited autophagy via upregulation of autophagy inhibitory genes; PI3K, Akt, and mTOR. Immuno-histopathological evaluation of the hepatic tissue of aged rats displayed deteriorated cytoarchitecture in terms of hydropic degeneration, congested veins, and inflammatory infiltrates in addition to increased glial fibrillary acidic protein expression and decreased microtubule-associated protein 1A/1B-light chain 3 (LC3) expression. MO administration to the aged rats promoted significant amelioration of aging-induced hepatic dysfunction and tissue alterations, counteracted oxidative stress and enhanced autophagy via downregulation of PI3K, Akt and mTOR genes Conclusion: MO can reduce aging-induced structural and functional liver damage by combating oxidative stress, and stimulation of autophagy through downregulation of the PI3K/Akt/mTOR signaling pathway. المقدمة: الشيخوخة هي عملية بيولوجية تزيد من الضرر التأكسدي في الخلية. نبات المورينجا أوليفيرا له تأثير وقائي على الكبد عن طريق تحفيز آلية الدفاع المضادة للأكسدة. الهدف من الدراسة: تقييم التأثيرات المرتبطة بالشيخوخة على بنية ووظيفة الكبد والدور المحسن المحتمل لمستخلص المورينجا أوليفيرا ، مع توضيح تأثيرها على الالتهام الذاتي في نماذج الجرذان المسنة. المواد والطرق المستخدمة: تم تقسيم أربعة وعشرين جرذاً، بعمر ثلاثة أشهر، إلى أربع مجموعات على النحو التالي: المجموعة الأولى: مجموعة البالغين الضابطة، والمجموعة الثانية: مجموعة البالغين التي تلقت المستخلص المائي لأوراق المورينجا أوليفيرا) 50 مجم / كجم من وزن الجسم(عن طريق الأنبوب الأنفي المعدي لمدة 4 أشهر، والمجموعة الثالثة: لم تتلق أي علاج حتى سن 20 شهرًا، والمجموعة الرابعة: لم تتلق أي علاج حتى سن 16 شهرًا، ثم تلقوا مستخلص مائي لأوراق نبات المورينجا لمدة 4 أشهر. عند الذبح، تم قياس إنزيمات الكبد)الأسبارتات أمينوترانسفيريز والألانين أمينوترانسفيريز(في الدم وتمت معالجة العينات الكبدية للفحص بالمجهرين الضوئي والإلكتروني وتحليل)mTOR(و ،)akt(و بروتين كيناز ب ،)PI3K(جينات فوسفاتيديل اينوسيتول 3-كيناز النتائج: أظهرت الجرذان المسنة ارتفاعًا ذا دلالة إحصائية في مستويات إنزيمات الكبد والملونديالديهيد في مصل الدم، بالإضافة إلى انخفاض محتوى الجلوتاثيون الكبدي. كما قللت الشيخوخة من الالتهام الذاتي عن طريق زيادة الجينات وقد أظهر تقييم الأنسجة الكبدية للجرذان المسنة بنية خلوية متدهورة. mTOR و Akt و PI3K المثبطة للالتهام الذاتي من حيث التنكس المائي واحتقان الأوردة والتسلل الالتهابي بالإضافة إلى زيادة الإبانة المناعية للبروتين الحمضي الليفي أدى إعطاء المورينجا للجرذان المسنة الى تحسنًا .)LC الدبقي وانخفاض إبانة البروتين المرتبط بالأنابيب الدقيقة) 3 ملحوظًا للخلل الكبدي والتغيرات النسيجية المرتبطة بالشيخوخة ، كما ثبط الإجهاد التأكسدي وحفز الالتهام الذاتي عن. mTOR. و Akt و PI3K طريق تقليل جينات الخلاصة: يمكن أن يقلل مستخلص نبات المورينجا من تلف الكبد التركيبي والوظيفي الناجم عن تقدم العمر عن طريق PI3K / Akt / mTOR مكافحة الإجهاد التأكسدي ، وتحفيز الالتهام الذاتي من خلال تقليل مسار إشارات. [ABSTRACT FROM AUTHOR]

Background: Liver diseases are a global health concern. Many in vitro liver models utilize cryopreserved primary human hepatocytes (PHHs), which commonly undergo post-thaw processing through colloidal silica gradients to remove debris and enrich for a viable PHH population. Post-thaw processing effects on healthy PHHs are partially understood, but the consequences of applying disease-origin PHHs to post-thaw density gradient separation have not been described. Methods: Using the TruVivo® system, diseased, type 2 diabetes mellitus (T2DM), and fibrotic PHHs were cultured for 14 days after initially being subjected to either low-density (permissive) or high-density (selective) gradients using Percoll-based thawing medium. Results: Changes in functionality, including albumin and urea secretion and CYP3A4 activity, were measured in diseased, T2DM, and fibrotic PHHs enriched in low Percoll compared to PHHs enriched in high Percoll. Lipogenesis increased in the PHHs enriched in low Percoll. Higher expression of CK18 and TGF-β, two fibrotic markers, and changes in expression of the macrophage markers CD68 and CD163 were also measured. Conclusions: The use of Percoll for the enrichment of PHHs post-thaw results in differences in attachment and functionality, along with changes in diseased phenotypes, in the TruVivo® system. [ABSTRACT FROM AUTHOR]

Recently approved adeno-associated viral (AAV) vectors for liver monogenic diseases haemophilia A and B are exemplifying the success of liver-directed viral gene therapy. In parallel, additional gene therapy strategies are rapidly emerging to overcome some inherent AAV limitations, such as the non-persistence of the episomal transgene in the rapidly growing liver and immune response. Viral integrating vectors such as in vivo lentiviral gene therapy and non-viral vectors such as lipid nanoparticles encapsulating mRNA (LNP-mRNA) are rapidly being developed, currently at the preclinical and clinical stages, respectively. Macrophages are the first effector cells of the innate immune response triggered by gene therapy vectors. Macrophage uptake and activation following administration of viral gene therapy and LNP have been reported. In this study, we assessed the biodistribution of AAV, lentiviral, and LNP-mRNA gene therapy following the depletion of tissue macrophages by clodronate pre-treatment in neonatal and juvenile mice. Both neonatal and adult clodronate-treated mice showed a significant increase in lentiviral-transduced hepatocytes. In contrast, clodronate pre-treatment did not modify hepatocyte transduction mediated by hepatotropic AAV8 but reduced LNP-mRNA transfection in neonatal and juvenile animals. These results highlight the importance of age-specific responses in the liver and will have translational applications for gene therapy programs. [ABSTRACT FROM AUTHOR]

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Hexavalent chromium (Cr (VI)) is a hazardous heavy metal that induces hepatotoxicity and nephrotoxicity. Thus, this study was planned to explore the ameliorating capacity of Aloe vera leaf gel extract (AV) and their conjugated silver nanoparticles (AVNP) against Cr (VI) induced hepatotoxicity and renal toxicity. The organ indices, level of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, malondialdehyde, total protein, and creatinine in blood serum were measured. The histopathological and micrometric analysis of the hepatic and renal tissue sections were studied. The hepatosomatic index was raised significantly (0.098 ± 0.13 g) in Cr treated group. The blood serum level of AST (484 ± 10.7 U/L), ALT (163 ± 5.5 U/L), ALP (336.7 ± 9.5 U/L), MDA (642.3 ± 28.3 U/L), and creatinine (4.0 ± 0.1 mg/dL) were increased significantly, whereas total protein level was declined (2.8 ± 0.3 g/dL) significantly in Cr exposed group. In the histopathological study, necrosis, disturbed hepatic cords, impaired glomeruli, and Bowman's capsule were noted. Micrometric data from the liver and kidney revealed a significant surge in the size of hepatocytes and their nuclei (1188.2 ± 467.7 µ2 and 456.5 ± 205.6 µ2) and CSA of glomeruli and Bowman's capsule (9051.8 ± 249.8 µ2 and 11,835.5 ± 336.7 µ2) in Cr (VI) exposed group, whereas the brush border (10.2 ± 4.0 µ) size declined significantly. The administration of AV and AVNP reduced the oxidative stress induced by Cr (VI). [ABSTRACT FROM AUTHOR]

The present study was aimed to investigate the impact of malathion on the biochemical and histological parameters of African freshwater catfish, Clarias gariepinus. Ethical permission was taken from the IAEC, and the experiment was conducted according to the CPCSEA guidelines for animal handling. During this study, fish were divided into three groups. The first group served as a control, the second group was treated with a low dose (400µg/L) of malathion, and the third group was treated with a high dose (800µg/L) of malathion for 10 days. The results of the study exhibited alterations in the biochemical parameters and histopathology of the liver. Biochemical constituents such as protein and carbohydrate concentration in the liver of fish treated with 400µg/L and 800µg/L of malathion decreased as compared to the control. The histopathological structure of the liver exposed to 400µg/L malathion for 10 days indicated toxic lesions such as enucleated hepatocytes, degeneration of the central vein, infiltration of erythrocytes, and pycnotic nuclei, whereas the liver exposed to 800µg/L malathion for 10 days showed irregular-shaped hepatocytes, binucleated hepatocytes, melanomacrophage necrosis, reduced central vein, and sinusoidal dilation. Thus, it may be concluded that malathion significantly altered the biochemical constituents of the liver and deteriorated the histological structure, which in turn altered the metabolic functions of the liver. [ABSTRACT FROM AUTHOR]

Introduction: Arylamine N -acetyltransferase 2 (NAT2) expresses a well-defined genetic polymorphism in humans that modifies drug and xenobiotic metabolism. Recent studies and genome wide association studies have reported that genetic variants of NAT2 are associated with differential risks of developing dyslipidemia and cardiometabolic disorders, suggesting a previously unrecognized role of NAT2 in pathophysiology of metabolic disorders. In support of this notion, we recently showed that human NAT2 expression is differentially regulated by glucose and insulin. Moreover, our in silico analysis showed that NAT2 is co-expressed with nuclear receptors enriched in the liver, e.g., NR1H4 (FXR) and NR1I2 (PXR), that have been previously implicated in regulation of hepatic glucose and lipid homeostasis. Identification of transcriptional regulator(s) of human NAT2 would aid in understanding novel functions that it may play in the liver. Thus, the present study was designed to investigate if NAT2 is transcriptionally regulated by hepatic nuclear receptors. Methods: To test this, we treated cryopreserved human hepatocytes with agonists towards four different hepatic transcription factors/nuclear hormone receptors, namely FXR (NR1H4), PXR (NR1I2), LXR (NR1H3), and PPARα (PPARA), and measured their effects on the level of NAT2 mRNA. Results: While the treatment with a FXR, PXR, or LXR agonist (i.e., GW-4064, SR-12813, or GW-3965) significantly induced their respective target genes, treatment with these agonists did not significantly alter the transcript level of NAT2 in human hepatocytes. PPARα agonist, GW-7647, treatment resulted in a statistically significant decrease in the NAT2 transcript level. However, its magnitude was marginal. Conclusion: In summary, hepatic nuclear receptors we examined in the present study (FXR, PXR, LXR, and PPARα) did not significantly alter NAT2 expression in cryopreserved human hepatocytes. Additional studies are needed to identify transcriptional regulators of hepatic NAT2 expression. [ABSTRACT FROM AUTHOR]

The membrane efflux transporter P-glycoprotein (P-gp, [ABCB1, MDR1]) exports a wide range of xenobiotic compounds, resulting in a continuous first line of defense against toxicant accumulation at basal expression levels, and contributing to the multixenobiotic resistance (MXR) phenotype at elevated expression levels. Relatively little information exists on P-gp inhibition in fish by chemosensitizers, compounds which lower toxicity thresholds for harmful P-gp substrates in complex mixtures. The effects of four known mammalian chemosensitizers (cyclosporin A [CsA], quinidine, valspodar [PSC833], and verapamil) on the P-gp-mediated transport of rhodamine 123 (R123) and cortisol in primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes were examined. Competitive accumulation assays using 25 µM R123 or cortisol and varying concentrations of chemosensitizers (0–500 µM) were used. CsA, quinidine, and verapamil inhibited R123 export (IC50 values ± SE: 132 ± 60, 83.3 ± 27.2, and 43.2 ± 13.6 µM, respectively). CsA and valspodar inhibited cortisol export (IC50 values: 294 ± 106 and 92.2 ± 34.9 µM, respectively). In an ATP depletion assay, hepatocytes incubated with all four chemosensitizers resulted in lower free ATP concentrations, suggesting that they act via competitive inhibition. Chemosensitizers that inhibit MXR transporters are an important class of environmental pollutant, and these results show that rainbow trout transporters are inhibited by similar chemosensitizers (and mostly at similar concentrations) as seen in mammals and other fish species. [ABSTRACT FROM AUTHOR]

The study was targeted at the examination of the effect of the specific immunomodulator KIM-M2 on the morphostructure of liver cells of guinea pigs infected with nontuberculosis mycobacteria. The research was carried out in 15 guinea pigs selected by gender at the Diagnostic Research and Biotechnology Laboratory of the Department of Veterinary Medicine of the Omsk Agrarian Scientific Center. All animals were kept in a specialized animal keeping facilities according to standard housing and feeding regime. The experimental animals were subdivided into three groups of five animals in each: group 1 – challenge group (Mycobacterium scrofulaceum), group 2 – experimental group (Mycobacterium scrofulaceum and KIM-M2), group 3 – pure control group (saline solution). On day 30 after the start of the experiment, the animals were removed from the experiment, liver biopsy samples were collected and histologic specimens were prepared according to the classical method. During the experiment, it was found that KIM-M2 had a regenerative effect on the liver tissue of the guinea pigs infected with nontuberculosis mycobacteria, which was associated with 1.5-fold increase in the number of mononuclear hepatocytes, 3-fold increase in binuclear cells and 4.3-fold decrease in anucleate hepatocytes thus indicating the manifestation of compensatory reactions in the organ and increase in the depth of regenerative processes. As for animals in group 1; 1.8and 1.3-fold increase in the area of the nucleus and cytoplasm as compared with the individuals in group 2, and 2.7and 2-fold increase as compared with the animals in the control group, respectively, indicated the launch of the accumulation mechanisms of the potential reparative reserves and increase in their depth in the liver tissues.

Purpose. The aim of the study was to investigate morphological and histological features of the gills, liver and blood of percids ­­­– pikeperch (Sander lucioperca Linnaeus, 1758) and perch (Perca fluviatilis Linnaeus, 1758) from different areas of the Zaporizhzhia Reservoir. Methodology. The study was conducted in two areas of the Zaporizhzhia Reservoir – in the Samara Bay and the lower part of the reservoir near the Viyskove village. The material was collected during research fish surveys in the summer-autumn period of 2024. The object of the study were age-3+ pikeperch and perch. The number of fishes used in the study was 20 specimens of each species. Hematological and histological studies were carried out according to generally accepted methods. The concentration of heavy metals in water samples was determined on a C115-M1 atomic absorption spectrophotometer in mg/L. Findings. According to the content of Cd, Mn, Cu, and Zn in both studied areas of the Zaporizhzhia Reservoir, there was an excess of the MPC for water bodies used for fishery purposes. The difference between the cytometric parameters of fish from different areas of the Zaporizhzhia Reservoir was mostly insignificant (р≥0.05), except for the width of the pikeperch nucleus and the area of ​​the perch erythrocyte nucleus, which were significantly larger in the fish of the Samara Bay (р≤0.05). The results of the study indicate the dependence of changes in the formed elements of the blood on the physiological state of the fish and on their environment. Among the studied fish of the lower part of the reservoir, the largest count of erythrocytes with pathological changes was observed in pikeperch, it was 1.32 times higher than in perch. Among fish of the Samara Bay, the highest count of pathologies in blood cells was also noted for pikeperch and was 1.17 times higher compared to that of perch. The results of these studies indicate an unfavorable ecological condition in both areas of the Zaporizhzhia Reservoir. The histological studies of the fish liver showed a number of pathological phenomena were revealed. The most common pathology of the liver cells of the studied fish from both areas of the reservoir was necrosis in pikeperch from the Samara Bay. Fish in the lower part of the reservoir had significantly fewer pathologies. Histological analysis of pikeperch and perch gills showed a high degree of pathological changes. The majority of pathologies were detected in pikeperch from the Samara Bay, which may indicate an average degree of pollution of the water by heavy metals and oil products. Based on these data, the histo-morphometric parameters of percid tissues can be used in the future to assess the ecological state of the water and the level of human impact of the Zaporizhzhia Reservoir, as well as be used as an additional indicator during the comprehensive assessment of the impact of anthropogenic factors on hydroecosystems. Originality. For the first time, a study of the morphological and histological characteristics of the gills, liver, and blood of percids (pikeperch and perch) from different areas of the Zaporizhzhia Reservoir was conducted. Practical Value. Histo-morphometric parameters of percid tissues can be used in the future to assess the ecological state of water and the level of human impact of the Zaporizhzhia Reservoir, as well as be used as an additional indicator during the comprehensive assessment of the impact of anthropogenic factors on hydroecosystems.

BACKGROUND: The infection of the liver by Plasmodium parasites is an obligatory step leading to malaria disease. Following hepatocyte invasion, parasites differentiate into replicative liver stage schizonts and, in the case of Plasmodium species causing relapsing malaria, into hypnozoites that can lie dormant for extended periods of time before activating. The liver stages of Plasmodium remain elusive because of technical challenges, including low infection rate. This has been hindering experimentations with well-established technologies, such as electron microscopy. A deeper understanding of hypnozoite biology could prove essential in the development of radical cure therapeutics against malaria. RESULTS: The liver stages of the rodent parasite Plasmodium berghei, causing non-relapsing malaria, and the simian parasite Plasmodium cynomolgi, causing relapsing malaria, were characterized in human Huh7 cells or primary non-human primate hepatocytes using Correlative Light-Electron Microscopy (CLEM). Specifically, CLEM approaches that rely on GFP-expressing parasites (GFP-CLEM) or on an immunofluorescence assay (IFA-CLEM) were used for imaging liver stages. The results from P. berghei showed that host and parasite organelles can be identified and imaged at high resolution using both CLEM approaches. While IFA-CLEM was associated with more pronounced extraction of cellular content, samples features were generally well preserved. Using IFA-CLEM, a collection of micrographs was acquired for P. cynomolgi liver stage schizonts and hypnozoites, demonstrating the potential of this approach for characterizing the liver stages of Plasmodium species causing relapsing malaria. CONCLUSIONS: A CLEM approach that does not rely on parasites expressing genetically encoded tags was developed, therefore suitable for imaging the liver stages of Plasmodium species that lack established protocols to perform genetic engineering. This study also provides a dataset that characterizes the ultrastructural features of liver stage schizonts and hypnozoites from the simian parasite species P. cynomolgi.

Interactions between adipose tissue, liver and immune system are at the center of metabolic dysfunction-associated steatotic liver disease and type 2 diabetes. To address the need for an accurate in vitro model, we establish an interconnected microphysiological system (MPS) containing white adipocytes, hepatocytes and proinflammatory macrophages derived from isogenic human induced pluripotent stem cells. Using this MPS, we find that increasing the adipocyte-to-hepatocyte ratio moderately affects hepatocyte function, whereas macrophage-induced adipocyte inflammation causes lipid accumulation in hepatocytes and MPS-wide insulin resistance, corresponding to initiation of metabolic dysfunction-associated steatotic liver disease. We also use our MPS to identify and characterize pharmacological intervention strategies for hepatic steatosis and systemic insulin resistance and find that the glucagon-like peptide-1 receptor agonist semaglutide improves hepatocyte function by acting specifically on adipocytes. These results establish our MPS modeling the adipose tissue-liver axis as an alternative to animal models for mechanistic studies or drug discovery in metabolic diseases.

Background & aimsPathologists quantify liver steatosis as the fraction of lipid droplet-containing hepatocytes out of all hepatocytes, whereas the magnetic resonance-determined proton density fat fraction (PDFF) reflects the tissue triacylglycerol concentration. We investigated the linearity, agreement, and correspondence thresholds between histological steatosis and PDFF across the full clinical spectrum of liver fat content associated with non-alcoholic fatty liver disease.MethodsUsing individual patient-level measurements, we conducted a systematic review and meta-analysis of studies comparing histological steatosis with PDFF determined by magnetic resonance spectroscopy or imaging in adults with suspected non-alcoholic fatty liver disease. Linearity was assessed by meta-analysis of correlation coefficients and by linear mixed modelling of pooled data, agreement by Bland-Altman analysis, and thresholds by receiver operating characteristic analysis. To explain observed differences between the methods, we used RNA-seq to determine the fraction of hepatocytes in human liver biopsies.ResultsEligible studies numbered 9 (N = 597). The relationship between PDFF and histology was predominantly linear (r = 0.85 [95% CI, 0.80-0.89]), and their values approximately coincided at 5% steatosis. Above 5% and towards higher levels of steatosis, absolute values of the methods diverged markedly, with histology exceeding PDFF by up to 3.4-fold. On average, 100% histological steatosis corresponded to a PDFF of 33.0% (29.5-36.7%). Targeting at a specificity of 90%, optimal PDFF thresholds to predict histological steatosis grades were ≥5.75% for ≥S1, ≥15.50% for ≥S2, and ≥21.35% for S3. Hepatocytes comprised 58 ± 5% of liver cells, which may partly explain the lower values of PDFF vs. histology.ConclusionsHistological steatosis and PDFF have non-perfect linearity and fundamentally different scales of measurement. Liver fat values obtained using these methods may be rendered comparable by conversion equations or threshold values.Impact and implicationsMagnetic resonance-proton density fat fraction (PDFF) is increasingly being used to measure liver fat in place of the invasive liver biopsy. Understanding the relationship between PDFF and histological steatosis fraction is important for preventing misjudgement of clinical status or treatment effects in patient care. Our analysis revealed that histological steatosis fraction is often significantly higher than PDFF, and their association varies across the spectrum of fatty liver severity. These findings are particularly important for physicians and clinical researchers, who may use these data to interpret PDFF measurements in the context of histologically evaluated liver fat content.

Liver fibrosis of different etiologies is a serious health problem worldwide. There is no effective therapy available for liver fibrosis except the removal of the underlying cause of injury or liver transplantation. Development of liver fibrosis is caused by fibrogenic myofibroblasts that are not present in the normal liver, but rather activate from liver resident mesenchymal cells in response to chronic toxic or cholestatic injury. Many studies indicate that liver fibrosis is reversible when the causative agent is removed. Regression of liver fibrosis is associated with the disappearance of activated myofibroblasts and resorption of the fibrous scar. In this review, we discuss the results of genetic tracing and cell fate mapping of hepatic stellate cells and portal fibroblasts, their specific characteristics, and potential phenotypes. We summarize research progress in the understanding of the molecular mechanisms underlying the development and reversibility of liver fibrosis, including activation, apoptosis, and inactivation of myofibroblasts.

Chordin (CHRD) is a secreted protein important in early development, yet a role for CHRD in human disease has not been identified. In this study we investigated CHRD in cancer and normal adult tissues using the wealth of genome-wide data available in public databases. We found that Chordin is amplified in the DNA of specific cancers such as lung squamous cell and others, although copy number variation did not strictly correlate with higher mRNA expression. In some cancers, such as renal and stomach carcinomas, increased CHRD expression significantly correlated with poor survival. In normal adult human tissues, CHRD mRNA was highest in hepatocytes. Crossveinless-2/BMPER, a component of the Chordin morphogenetic pathway expressed at the opposite side in embryos, was expressed in liver stellate cells. This raises the intriguing possibility that a BMP gradient might be established in the extracellular matrix of the space of Disse that surrounds portal sinusoid capillaries.

ImportanceThe hepatitis C virus (HCV) causes liver disease, affecting millions. Even though we have effective antivirals that cure HCV, they cannot stop terminal liver disease. We used an adult stem cell-derived liver organoid system to understand how HCV infection leads to the progression of terminal liver disease. Here, we show that HCV maintains low-grade infections in liver organoids for the first time. HCV infection in liver organoids leads to transcriptional reprogramming causing cancer cell development and altered immune response. Our finding shows how HCV infection in liver organoids mimics HCV infection and patient pathogenesis. These results reveal that HCV infection in liver organoids contributes to liver disease progression.

The Hedgehog (Hh) signaling pathway is essential for maintaining homeostasis during embryogenesis and in adult tissues. In the liver, dysregulation of this pathway often leads to liver cancer development. Recent studies also suggest that disturbances in the Hh pathway can affect liver metabolism in healthy livers through interactions with other signaling pathways, such as the Wnt/β-catenin pathway. As a result, the Hh pathway has emerged as a promising target for therapeutic intervention. However, little is known about the effects of Hh modulators on healthy hepatocytes. In our study, we investigated the effects of the Hh agonists SAG (300 nM) and triamcinolone acetonide (40 µM), as well as the antagonists RU-SKI 43 (100 nM), cyclopamine (5 µM), budesonide (25 µM), GANT61 (0.5 µM), and vismodegib (1 µM) on healthy mouse and human primary hepatocytes in vitro. We employed toxicological, transcriptomic, proteomic, and functional assays, including proliferation and Seahorse assays. Our results show that these compounds significantly impact metabolic pathways such as lipid and glucose metabolism at both transcriptional and protein levels. Mechanistically, our data suggest the involvement of both canonical and non-canonical Hedgehog pathways, a phenomenon not previously described in hepatocytes. These findings highlight the diverse effects of these compounds on signaling and key metabolic functions in the liver, which emphasizes the need to investigate the hepatic Hh cascade and its metabolic control in depth. As the compounds regulate different aspects of metabolism, they need to be carefully studied in appropriate model systems for specific therapeutic use. [ABSTRACT FROM AUTHOR]

Abstract Background Xenobiotic exposures can extensively influence the expression and alternative splicing of drug-metabolizing enzymes, including cytochromes P450 (CYPs), though their transcriptome-wide impact on splicing remains underexplored. This study used a well-characterized splicing event in the Cyp2b2 gene to validate a sandwich-cultured primary rat hepatocyte model for studying global splicing in vitro. Using endpoint PCR, RNA sequencing, and bioinformatics tools (rSeqDiff, rMATs, IGV), we analyzed differential gene expression and splicing in CYP and nuclear receptor genes, as well as the entire transcriptome, to understand how xenobiotic exposures shape alternative splicing and activate xenosensors. Methods Primary rat hepatocytes in sandwich culture were exposed to two methylenedioxybenzene (MDB) congeners and carbamazepine, with gene expression and splicing assessed. A 3D-clustergram integrating KEGG pathway analysis with differential gene expression provided distinct splicing landscapes for each xenobiotic, showing that splicing diversity does not always align with gene expression changes. Results Endpoint PCR revealed a Cyp2b2v to wild-type Cyp2b2 splicing ratio near 1:1 (100%) under most conditions, while RNA-seq showed a stable baseline closer to 40%. C6-MDB reduced this ratio to ~ 50% by PCR and ~ 39% by RNA-seq, showing slight method-dependent variations yet consistent trends. In contrast, exon 6 skipping in Cyp1a1 occurred only with MDB exposure, implicating AHR activation. Xenobiotic treatments also induced alternative splicing in defensome and stress-responsive genes, including the phase II enzyme Gstm3, Albumin, Orm1, and Fxyd1, highlighting their roles in xenobiotic response modulation. Significant splicing changes in factors such as SRSF1, SRSF7, and METTL3 suggest a coordinated feedback loop involving epitranscriptomic modulation and cross-talk within SR protein networks, refining splice site selection, transcript stability, and cellular fate. Conclusions This study demonstrates how xenobiotic structural features influence gene expression and splicing, revealing splicing patterns that expand our understanding of transcriptome diversity and function. By identifying regulatory mechanisms, including AHR activation, epitranscriptomic modulation, and crosstalk within SR protein networks, that shape adaptive responses to xenobiotic stress, this work offers insights into the splicing and transcriptional networks that maintain cellular homeostasis. These findings provide predictive biomarkers for toxic exposures and highlight the potential of splicing profiles as diagnostic tools for assessing the health impacts of chemical exposure.

The present experimental investigation was executed to evaluate the combined effect of probiotic bacterium Bacillus coagulans along with herb Mentha piperita on histological architecture; and extent of DNA damage after challenge trial with pathogenic bacterium Aeromonas hydrophila in Catla catla. In this experiment, six dietary treatments in triplicate were formulated; C0, M3 and M6 in which M. piperita was supplemented at 0, 3, 6 g kg−1 of feed, while in dietary treatments CP, MP3 and MP6; supplementation of probiotic bacterium B. coagulans at the rate of 3000 CFU g−1 was done along with different inclusion level of M. piperita (0, 3, 6 g kg−1 of feed). Fishes were fed for 60 days on different dietary treatments. Growth performance, histological study and extent of DNA damage using comet assay was examined. Significantly (p

The liver has a big potential for self-healing, but the activity of regeneration decreases with age. Changes are occurring, including in the functional activity of various liver cell populations, the study of the characteristics of which can become the basis for the development of new therapeutic approaches to the liver diseases treatment at older people. The aim of this research was to study the level of stem and progenitor cells and the functional activity of the healthy liver from age- different rats. Material and methods. Experiments were carried out on Wistar rats aged 6 and 12 months. Ultrasound and histological examination of the liver from rats was used to assess morphological changes. The lipid profile of blood serum was evaluated by biochemical methods. Cytometric methods were used to study the surface and intracellular antigens of stem and progenitor cells isolated from the bone marrow, arterial blood and liver of rats. Results and discussion. In 12-month-old male Wistar rats, compared with 6-month-old rats, excessive formation of extracellular matrix components, disruption of tissue architecture, development of portal hypertension, as well as an increase in the concentration of cholesterol, triglycerides, high- and low-density lipoproteins were revealed. We identified age- related differences in the content of hematopoietic and mesenchymal stem cells, epithelial cells (CD45–CD326+) in the bone marrow, blood and liver of rats. In the liver parenchyma, the populations of hepatocyte precursors (CD45– CD326+CD133+), oval cells (CD45–CD326+CD133+CD90+). At the same time, the level of all cell populations in the liver parenchyma of rats expressing the intracellular marker Sox9 was higher in one-year-old animals compared to younger ones, regardless of the cell phenotype. Conclusions. In the liver of 12-month-old rats, compared to 6-month-old rats, the number of cells expressing Sox9, lymphocytes with an inflammatory phenotype increases, the number of stem cells and various populations of epithelial and endothelial cells decreases, which leads to a decrease in the regenerative capacity of the liver, disruption of the tissue architecture of the organ and changes in lipid metabolism. These changes largely determine the increased susceptibility with age to the development of chronic liver diseases.

Abstract Background Cardiovascular function and metabolic homeostasis are closely linked, but the underlying mechanisms are not fully understood. Corin is a protease that activates atrial natriuretic peptide (ANP), an essential hormone for normal blood pressure and cardiac function. The goal of this study is to investigate a potential corin and ANP function in regulating liver glycogen metabolism and glucose homeostasis. Methods Liver glycogen and blood glucose levels were analyzed in Corin or Nppa (encoding ANP) knockout (KO) mice. ANP signaling was examined in livers from Corin and Nppa KO mice and in cultured human and mouse hepatocytes by western blotting. Results We found that Corin and Nppa KO mice had reduced liver glycogen contents and increased blood glucose levels. By analyzing conditional KO mice lacking either cardiac or renal Corin, we showed that cardiac corin and ANP act in an endocrine manner to enhance cGMP-protein kinase G (PKG)-AKT-GSK3 signaling in hepatocytes. In cultured hepatocytes, ANP treatment stimulated PKG signaling, glucose uptake, and glycogen production, which could be blocked by small molecule PKG and AKT inhibitors. Conclusions Our results indicate that corin and ANP are important regulators in liver glycogen metabolism and glucose homeostasis, suggesting that defects in the corin and ANP pathway may contribute to both cardiovascular and metabolic diseases.

Abstract Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.