Liver Hepatocytes Research Articles

Nutritional Significance, Antimicrobial, Antioxidants, Anticancer, and Antiviral Activities of Lemongrass Leaves Extract and Its Application as Hepatoprotective Agent against CCl4-Induced Hepatic Injury in Rats.

This work investigated the antioxidant and hepatoprotective activities of lemongrass extract and its effects on rat hepatotoxicity. The lemongrass extract (LGE) contains bioactive components such as phenolic acids, flavonoid components, vitamin C, fibers, and tannins. The LGE had high phenolic content (397 mg/100g) and flavonoids (164 mg/100g), influencing its antioxidant activity of 91.25%. Additionally, it inhibited 81% of breast cancer, also, inhibited the growth of pathogenic bacteria and Candida at a concentration of 20-40 µg/mL. Additionally, it inhibited SARS-Cov-2 by 75%; however, increasing the activity of Cas-3. Quercetin-3-rhamnoside was the main phenolic compound in the HPLC profile; the phenolic compounds may be attributable to the beneficial effects of LGE. In this study, the CCl4-challenged rats delivered two levels of LGE (100 and 300 mg/kg BW). LGE reduced ALT, AST, creatinine and urea by 50 and 37%, respectively. Generally, LGE mitigated the oxidative stress induced by CCl4, which is evident in the histology of liver and kidney tissues, where significant improvement, with no cytoplasmic degradation in undamaged liver hepatocytes, improved kidney performance and shape. It can be concluded that polyphenolic-rich LGE can mitigate the oxidative stress induced by CCl4 and other parameters while enhancing kidney and liver performance.

Influence of drop on the ultrastructure lipid composition of liver hepatocytes of pregnant rats

The influence of a toxic agent on the structure of the liver is inevitable. Of interest are the effects of pesticides on intrauterine formation and their teratogenic and embryotoxic properties. The influence of teratogens may manifest itself at different levels of organization of a living organism, and some aspects of cell metabolism change. Using advanced electron microscopy techniques combined with modern knowledge of membranology and toxicology, we conducted a detailed study of morphological changes in the subcellular architecture of the liver after repeated dropplet exposure. Ultrastructural studies were carried out on pregnant rats and their embryos on the 3rd, 13th, 19th days of pregnancy and embryonic development. Poisoning of animals with the pesticide dropp causes clear degenerative processes in the cellular and subcellular structures of the liver.

The effect of crude extract of Cyathula prostrata leaves on serum c-reactive proteins levels of normal albino Wistar rats after an administration period of 7 days

Food fortification is considered a sustainable public health strategy because it can reach wider populations through existing food delivery systems without requiring major changes in existing consumption patterns (Das et al., 2013.). C-reactive Protein (CRP) is a major acute phase reactant synthesized primarily in the liver hepatocytes. CRP shows the strongest association with cardiovascular events. It is detectable on the surface of about 4% of normal peripheral blood lymphocytes. This research was designed to study the effects of the administration of graded doses (100mg/kg and 200mg/kg) of crude extracts of Cyathulaprotrata leaves on albino Wistar rats after a period of 7 days. The animals were sacrificed after the experimental period and blood collected and prepared for analysis to access the level of blood C-reactive protein. After the study, it was observed that the blood C-reactive protein levels for groups II and III animals were elevated significantly, indicating that there was tissue damage due to toxicity of xenobiotics. This research has led to the conclusion that the consumption of Cyathulaprostrata leaves extract was safe though it generated oxidative stress. Results show that experimental animals of group II, and III indicated elevated C-reactive protein levels (3.21±0.19 and 3.14±1.11 mg/dL respectively) significantly at p<0.5 when compared to experimental animals of group 1 (control, 2.38±1.04 mg/dL) significantly at p<0.05.

The protective effects of Nigella sativa, thymoquinone and bentonite in experimental aflatoxicosis model in broilers

The aim of this study was to determine the preventive efficacy of black seed (Nigella sativa L.), thymoquinone and Na bentonite on biomarkers of tissue degeneration in the liver and other organs induced by aflatoxin (AF) in broilers. One hundred broiler chicks were divided into 10 equal groups and fed for 28 days. The animals received feed with 2 mg/kg total aflatoxin, Nigella sativa (NS; 5%), thymoquinone (TMQ; 300 mg/kg) and Na bentonite (BNT; 10 g/kg) both individually and in combination. At the end of the experiment, blood and liver tissue samples were collected. AF treatment significantly increased serum and liver 8-hydroxy-2’-deoxyguanosine(8-OHdG), serum transforming growth factor β (TGF-β), and liver total antioxidant capacity (TAC) levels, whereas it significantly decreased liver TGF-β and serum TAC levels compared to the control. However, there were no changes in the serum and liver hepatocyte growth factor (HGF) levels from AF. Both individual and combined addition of NS, BNT and TMQ to AF-contaminated feed significantly improved liver 8-OHdG, liver TAC and serum TGF-β levels. Serum 8-OHdG levels were ameliorated only in the AF+BNT+TMQ group. In addition, the AF+BNT+NS and AF+BNT+TMQ groups showed significant improvement in liver 8-OHdG, liver TGF-β and liver TAC levels. We conclude that NS and TMQ supplementation to AF-contaminated feed may be beneficial against aflatoxicosis in broilers.

Investigating SHP and PCSK9 Interactions in Cholesterol-Mediated Cardiovascular Diseases: A Research Protocol

Introduction: Improper cholesterol metabolism results in accumulation of low-density lipoproteins (LDL). High levels of LDL cholesterol deposits in blood vessels, forming plaques and contributing to various cardiovascular diseases (CVD). The nuclear farnesoid X receptor (FXR) regulates the transcription of genes involved in cholesterol metabolism and is a therapeutic target for cholesterol dysregulation. Studies conducted on immortalized human hepatocytes demonstrate FXR signaling-induced downregulation of proprotein convertase subtilisin/kexin type 9 (PCSK9) expression. PCSK9 is an LDL receptor-degrading enzyme whose upregulation is implicated in cholesterol-mediated diseases. Specifically, the FXR target gene SHP (small heterodimer partner) is a transcriptional regulator that has been implicated in an inverse relationship with PCSK9 expression. The biomolecular mechanism mediating this relationship has not been explored, meriting investigation into a potential novel axis of cholesterol metabolism. We hypothesize that SHP is a direct repressor of PCSK9 transcription. Methods: To investigate, we will knock out SHP expression in the liver hepatocyte cell line AML12 using small interfering RNAs (siRNAs). To confirm SHP knockout on transcriptomic and proteomic levels, reverse transcription quantitative PCR (RT-qPCR) and Western blotting will be performed. To assess SHP binding to the promoter region of PCSK9, an electrophoretic mobility supershift (EMSA) assay will be performed on unstimulated or chenodeoxycolic acid (CDCA)-stimulated AML12 cells that have undergone SHP or control knockouts. Western blotting will quantitate PCSK9 protein expression following SHP knockout in CDCA-stimulated and unstimulated conditions. Results: Results from EMSA are expected to demonstrate SHP binding to the promoter region of PCSK9 in a transcription factor complex to repress transcription. SHP knockout models are expected to show upregulated PCSK9 expression at transcriptomic and proteomic levels. Discussion: If successful, our study presents a novel perspective on cholesterol metabolism by characterizing the inhibitory effect of SHP on PCSK9 expression. This underlines the critical role of FXR signaling in PCSK9 regulation, and knockout models and assay techniques provide valuable evidence of this regulatory role. Conclusion: This study will establish an enhanced understanding of the SHP/PCSK9 pathway within broader pathways of cholesterol metabolism. Further research may explore therapies targeting the SHP/PCSK9 pathway to manage CVD downstream of cholesterol dysregulation.

Constitutive androstane receptor agonist initiates metabolic activity required for hepatocite proliferation

Constitutive androstane receptor (CAR, NR1I3) activation by chemical compounds evokes liver hyperplasia in rodent. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most commonly used to study chemically induced liver hyperplasia and hepatocyte proliferation in vivo. TCPOBOP is potent murine liver chemical mitogen, which induces rapid direct liver hyperplasia independent of liver injury. In recent years, a lot of data has been accumulated on the transcription program that characterizes TCPOBOP-induced hepatocyte proliferation. However, there are scarce data about metabolic requirements of hepatocytes dividing upon treatment with xenobiotics. In present study, we employed liquid chromatography - mass spectrometry technology combined with statistical analysis to develop a metabolite profile of small biomolecules, to identify key metabolic changes in male mouse liver tissue after TCPOBOP administration. Analysis of biochemical pathways of the differentially affected metabolites in mouse livers demonstrated significant TCPOBOP-mediated enrichment of several processes including those relevant to nucleotide metabolism, amino acid metabolism, and energy substrate metabolism. Our findings provide evidence to support the conclusion that CAR agonist, TCPOBOP, initiates an intracellular program that promotes the global coordinated metabolic activities required for hepatocyte proliferation. Our metabolic data may provide novel insight into the biological mechanisms that occur during TCPOBOP-induced hepatocyte proliferation in mice.

Discovery and characterization of cross-reactive intrahepatic antibodies in severe alcoholic hepatitis.

The pathogenesis of antibodies in severe alcoholic hepatitis (SAH) remains unknown. We analyzed immunoglobulins (Ig) in explanted livers from SAH patients (n=45) undergoing liver transplantation and tissues from corresponding healthy donors (HD, n=10) and found massive deposition of IgG and IgA isotype antibodies associated with complement fragment C3d and C4d staining in ballooned hepatocytes in SAH livers. Ig extracted from SAH livers, but not patient serum exhibited hepatocyte killing efficacy. Employing human and Escherichia coli K12 proteome arrays, we profiled the antibodies extracted from explanted SAH, livers with other diseases, and HD livers. Compared with their counterparts extracted from livers with other diseases and HD, antibodies of IgG and IgA isotypes were highly accumulated in SAH and recognized a unique set of human proteins and E. coli antigens. Further, both Ig- and E. coli-captured Ig from SAH livers recognized common autoantigens enriched in several cellular components including cytosol and cytoplasm (IgG and IgA), nucleus, mitochondrion, and focal adhesion (IgG). Except IgM from primary biliary cholangitis livers, no common autoantigen was recognized by Ig- and E. coli-captured Ig from livers with other diseases. These findings demonstrate the presence of cross-reacting anti-bacterial IgG and IgA autoantibodies in SAH livers.

Effects of long-term dehydration on stress markers, blood parameters, and tissue morphology in the dromedary camel (Camelus dromedarius).

Dromedary camels robustly withstand dehydration, and the rough desert environment but the adaptation mechanisms are not well understood. One of these mechanisms is that the dromedary camel increases its body temperature to reduce the process of evaporative cooling during the hot weather. Stress in general, has deleterious effects in the body. In this study, we sought to determine the effects of dehydration and rehydration on stress parameters in the dromedary camels and how it pacifies these effects. Nineteen male camels were randomly divided into control, dehydrated and rehydrated groups, and fed alfalfa hay ad-libitum. The dehydrated and rehydrated groups were water-restricted for 20 days after which the rehydrated camels were provided with water for 72 h. The control and dehydrated camels were slaughtered at day 20 from the start of experiment whereas the rehydrated group was killed 72 h later. Many biochemical, hematological histopathological parameters and gene analysis were performed in relevant tissues collected including blood, plasma, and tissues. It was observed that severely dehydrated camels lost body weight, passed very hard feces, few drops of concentrated urine, and were slightly stressed as reflected behaviorally by loss of appetite. Physiologically, the stress of dehydration elicited modulation of plasma stress hormones for water preservation and energy supply. Our results showed significant increase in cortisol, norepinephrine and dopamine, and significant decrease in epinephrine and serotonin. The significant increase in malondialdehyde was accompanied with significant increase in antioxidants (glutathione, retinol, thiamin, tocopherol) to provide tissue protection from oxidative stress. The physiological blood changes observed during dehydration serve different purposes and were quickly restored to normality by rehydration. The dehydrated/rehydrated camels showed reduced hump size and serous atrophy of perirenal and epicardial fat. The latter changes were accompanied by significantly increased expression of genes encoding proteins for energy production (ANGPTL4, ACSBG1) from fat and significantly decreased expression of genes (THRSP; FADS 1&2) encoding proteins enhancing energy expenditure. This process is vital for camel survival in the desert. Dehydration induced no major effects in the vital organs. Only minor degenerative changes were observed in hepatic and renal cells, physiological cardiomyocyte hypertrophy in heart and follicular hyperplasia in splenic but lipidosis was not depicted in liver hepatocytes. Ketone bodies were not smelled in urine, sweat and breathing of dehydrated animals supporting the previous finding that the ß hydroxybutyrate dehydrogenase, a key enzyme in ketone body formation, is low in the camel liver and rumen. Rehydration restored most of blood and tissues to normal or near normal. In conclusion, camels are adapted to combat dehydration stress and anorexia by increasing anti-stressors and modulating genes involved in fat metabolism.

C–C chemokine receptor 5 is essential for conventional NK cell trafficking and liver injury in a murine hepatitis virus-induced fulminant hepatic failure model

BackgroundPrevious studies have demonstrated that natural killer (NK) cells migrated into the liver from peripheral organs and exerted cytotoxic effects on hepatocytes in virus-induced liver failure.AimThis study aimed to investigate the potential therapeutic role of chemokine receptors in the migration of NK cells in a murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatic failure (MHV-3-FHF) model and its mechanism.ResultsBy gene array analysis, chemokine (C–C motif) receptor 5 (CCR5) was found to have remarkably elevated expression levels in hepatic NK cells after MHV-3 infection. The number of hepatic CCR5+ conventional NK (cNK) cells increased and peaked at 48 h after MHV-3 infection, while the number of hepatic resident NK (rNK) cells steadily declined. Moreover, the expression of CCR5-related chemokines, including macrophage inflammatory protein (MIP)-1α, MIP-1β and regulated on activation, normal T-cell expressed and secreted (RANTES) was significantly upregulated in MHV-3-infected hepatocytes. In an in vitro Transwell migration assay, CCR5-blocked splenic cNK cells showed decreased migration towards MHV-3-infected hepatocytes, and inhibition of MIP-1β or RANTES but not MIP-1α decreased cNK cell migration. Moreover, CCR5 knockout (KO) mice displayed reduced infiltration of hepatic cNK cells after MHV-3 infection, accompanied by attenuated liver injury and improved mouse survival time. Adoptive transfer of cNK cells from wild-type mice into CCR5 KO mice resulted in the abundant accumulation of hepatic cNK cells and aggravated liver injury. Moreover, pharmacological inhibition of CCR5 by maraviroc reduced cNK cell infiltration in the liver and liver injury in the MHV-3-FHF model.ConclusionThe CCR5-MIP-1β/RANTES axis played a critical role in the recruitment of cNK cells to the liver during MHV-3-induced liver injury. Targeted inhibition of CCR5 provides a therapeutic approach to ameliorate liver damage during virus-induced acute liver injury.

Selective Inhibition of Repulsive Guidance Molecule C/Hemojuvelin (RGMc/HJV) with SRK-256 to Mobilize Iron in Functional Iron Deficiency

Functional iron deficiency is a serious condition marked by serum iron restriction in the presence of adequate iron stores that can lead to anemia, worsening quality of life, and leading to poor patient outcomes. Functional iron deficiency is characterized by abnormally high levels of hepcidin given serum iron status; it is frequently observed in diseases with chronic inflammation, such as anemia of chronic disease, chronic kidney disease, and myelofibrosis. Current treatments to address this patient population involve non-specifically targeting hepcidin and come with risks of off-target side effects. The secretion of hepcidin from the liver hepatocytes is linked to circulating serum iron levels and is controlled by the Bone Morphogenic Protein (BMP) 2/6 signaling pathway. Repulsive Guidance Molecule C or Hemojuvelin (RGMc/HJV) is a BMP 2/6 co-receptor in liver hepatocytes. Human genetic mutations in this gene result in juvenile hemochromatosis and genetic mouse studies have demonstrated that RGMc/HJV has a specific role in maintaining systemic iron homeostasis through modulation of hepcidin expression. We have generated a highly selective, fully human antibody (SRK-256) against RGMc/HJV which binds to the target with 39 pM affinity. SRK-256 has no detectable binding to RGMa or RGMb, thus avoiding potential side effects of targeting these gene family members with key physiological roles outside of iron metabolism. A high-resolution crystal structure (2.2 Å) elucidates not only the mechanism of action of SRK-256 directly competing for BMP binding to RGMc via contacts within the BMP binding domain, but also identifies binding to regions within this domain highly unique to RGMc over RGMa and RGMb, thus supporting the selectivity of the molecule. This mechanism of action is corroborated by in vitro assay data demonstrating that SRK-256 blocks signaling by BMP 2, 4, and 6. Pharmacodynamic and pharmacokinetic data supportive of a monthly subcutaneous dosing regimen in human patients has been generated in cynomolgus macaques, where SRK-256 demonstrates a dose-proportional duration of effect on suppression of hepcidin expression and elevation of transferrin saturation and achieves ~95% bioavailability via subcutaneous dosing. Furthermore, in the rat peptidoglycan-polysaccharide (PGPS) model of anemia of chronic disease, we have shown that in contrast to treatment with erythropoiesis stimulating agent alone (EPO, serum iron 47.7 ± 9.8 µg/dL), SRK-256 can restore serum iron to normal levels (Healthy control, serum iron 231.9 ± 12.0 µg/dL; SRK-256, serum iron 248.7 ± 60.9 µg/dL; and SRK-256+EPO, serum iron 277.6 ± 92.9 µg/dL) in the context of inflammation-driven, iron-restricted anemia (Figure 1). SRK-256 represents a potent and selective potential therapy for the treatment of patients with iron-restricted anemias across diseases of chronic inflammation.

Notch1 Regulates Hepatic Thrombopoietin Production

Notch signaling is highly conserved and regulates cell-fate decisions in several developmental processes and cell functions. However, a role for Notch in hepatic thrombopoietin (TPO) production has not been described. We noted that mice with a deficiency in hepatic Notch1 had thrombocytopenia, and so investigated TPO production and other features of platelets in these mice. We found that the liver ultrastructure and hepatocyte function were comparable between control mice and Notch1-deficient mice. However, the Notch1-deficient mice had significantly lower plasma TPO and hepatic TPO mRNA levels, concomitant with lower numbers of circulating platelets and impaired megakaryocyte differentiation and maturation. Addition of exogenous TPO rescued megakaryocyte maturation and circulating platelet numbers. Additionally, JAK2/STAT3 phosphorylation was significantly inhibited in Notch1-deficient hepatocytes. RNA-seq analysis showed significantly reduced JAK-STAT signaling. JAK2/STAT3 phosphorylation and TPO production was also impaired in purified cultured Notch1-deficient hepatocytes after treatment with desialylated platelets. Interestingly, Notch1 deficiency downregulated the expression of HES5 but not HES1. Moreover, desialylated platelets promoted the binding of HES5 to JAK2/STAT3, leading to JAK2/STAT3 phosphorylation and pathway activation. Furthermore, blockage of Dll4 on desialylated platelets inhibited hepatocyte Notch1 activation and HES5 expression, JAK2/STAT3 phosphorylation and subsequent TPO production. In conclusion, our study identifies a novel regulatory role of Notch1 in hepatic TPO production, indicating that it might be a target for modulating TPO level.

Object detection: A novel AI technology for the diagnosis of hepatocyte ballooning.

Metabolic dysfunction-associated fatty liver disease (MAFLD) has reached epidemic proportions worldwide and is the most frequent cause of chronic liver disease in developed countries. Within the spectrum of liver disease in MAFLD, steatohepatitis is a progressive form of liver disease and hepatocyte ballooning (HB) is a cardinal pathological feature of steatohepatitis. The accurate and reproducible diagnosis of HB is therefore critical for the early detection and treatment of steatohepatitis. Currently, a diagnosis of HB relies on pathological examination by expert pathologists, which may be a time-consuming and subjective process. Hence, there has been interest in developing automated methods for diagnosing HB. This narrative review briefly discusses the development of artificial intelligence (AI) technology for diagnosing fatty liver disease pathology over the last 30 years and provides an overview of the current research status of AI algorithms for the identification of HB, including published articles on traditional machine learning algorithms and deep learning algorithms. This narrative review also provides a summary of object detection algorithms, including the principles, historical developments, and applications in the medical image analysis. The potential benefits of object detection algorithms for HB diagnosis (specifically those combined with a transformer architecture) are discussed, along with the future directions of object detection algorithms in HB diagnosis and the potential applications of generative AI on transformer architecture in this field. In conclusion, object detection algorithms have huge potential for the identification of HB and could make the diagnosis of MAFLD more accurate and efficient in the near future.

"Small Hepatocytes" in the Liver.

Mature hepatocytes (MHs) in an adult rodent liver are categorized into the following three subpopulations based on their proliferative capability: type I cells (MH-I), which are committed progenitor cells that possess a high growth capability and basal hepatocytic functions; type II cells (MH-II), which possess a limited proliferative capability; and type III cells (MH-III), which lose the ability to divide (replicative senescence) and reach the final differentiated state. These subpopulations may explain the liver's development and growth after birth. Generally, small-sized hepatocytes emerge in mammal livers. The cells are characterized by being morphologically identical to hepatocytes except for their size, which is substantially smaller than that of ordinary MHs. We initially discovered small hepatocytes (SHs) in the primary culture of rat hepatocytes. We believe that SHs are derived from MH-I and play a role as hepatocytic progenitors to supply MHs. The population of MH-I (SHs) is distributed in the whole lobules, a part of which possesses a self-renewal capability, and decreases with age. Conversely, injured livers of experimental models and clinical cases showed the emergence of SHs. Studies demonstrate the involvement of SHs in liver regeneration. SHs that appeared in the injured livers are not a pure population but a mixture of two distinct origins, MH-derived and hepatic-stem-cell-derived cells. The predominant cell-derived SHs depend on the proliferative capability of the remaining MHs after the injury. This review will focus on the SHs that appeared in the liver and discuss the significance of SHs in liver regeneration.

Hepatoprotective Effects of Biochanin A on Thioacetamide-Induced Liver Cirrhosis in Experimental Rats.

The protective effect of biochanin A (BCA) on the histopathology, immunohistochemistry, and biochemistry of thioacetamide (TAA)-induced liver cirrhosis in vivo was investigated. There was a significant reduction in liver weight and hepatocyte propagation, with much lower cell injury in rat groups treated with BCA (25 mg/kg and 50 mg/kg) following a TAA induction. These groups had significantly lower levels of proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA). The liver homogenates showed increased antioxidant enzyme activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as decreased malondialdehyde (MDA) levels. The serum biomarkers associated with liver function, namely alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma glutamyl transaminase (GGT), returned to normal levels, comparable to those observed in both the normal control group and the reference control group. Taken together, the normal microanatomy of hepatocytes, the inhibition of PCNA and α-SMA, improved antioxidant enzymes (SOD, CAT, and GPx), and condensed MDA with repairs of liver biomarkers validated BCA's hepatoprotective effect.

Qijia rougan formula ameliorates ECM deposition in hepatic fibrosis by regulating the JAK1/STAT6-microRNA-23a feedback loop in macrophage M2 polarization

Hepatic fibrosis is the critical pathological stage in the progression of chronic liver disease to cirrhosis and hepatocellular carcinoma (HCC). However, no approved anti-hepatic fibrosis drugs are available currently. Qijia Rougan Formula (QRF) is a traditional Chinese medicine (TCM) with significant clinical efficacy on hepatic fibrosis. It was derived from Sanjiasan, a famous decoction documented in the Book of Treatise on the Pestilence in the Ming Dynasty of China. However, the underlying regulatory mechanisms remain elusive. This study further confirmed the therapeutic effects of QRF on hepatic fibrosis and dissected its underlying molecular mechanisms from the perspective of macrophage M2 polarization, one of the critical events in hepatic fibrosis. Experimentally, QRF significantly improved extracellular matrix (ECM) deposition and fibrosis in the liver of model rats. QRF diminished the proportion of M2 macrophages, decreased the levels of TGF-β, PDGFB and IL-10, and regulated the expression of p-JAK1, p-STAT6, JAK1 and microRNA-23a both in vitro and in vivo. Collectively, it was confirmed that QRF effectively improves liver function and hepatocyte damage, and reduces ECM deposition. QRF ameliorates hepatic fibrosis by regulating JAK1/STAT6-microRNA-23a negative feedback loop to inhibit macrophage M2 polarization and thus reduce ECM deposition. Our study illustrates the potential of QRF for hepatic fibrosis therapy, suggesting that QRF is a promising anti-hepatic fibrosis drug candidate.

Extracellular matrix modulates the spatial hepatic features in hepatocyte-like cells derived from human embryonic stem cells

BackgroundHuman pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) can provide a valuable in vitro model for disease modelling and drug development. However, generating HLCs with characteristics comparable to hepatocytes in vivo is challenging. Extracellular matrix (ECM) plays an important role in supporting liver development and hepatocyte functions, but their impact on hepatocyte differentiation and maturation during hPSC differentiation remains unclear. Here, we investigate the effects of two ECM components—Matrigel and type I collagen on hepatic differentiation of human embryonic stem cells (hESCs).MethodshESC-derived HLCs were generated through multistage differentiation in two-dimensional (2D) and three-dimensional (3D) cultures, incorporating either type I collagen or Matrigel during hepatic specification and maturation. The resulting HLCs was characterized for their gene expression and functionality using various molecular and cellular techniques.ResultsOur results showed that HLCs cultured with collagen exhibited a significant increase in albumin and alpha-1 anti-trypsin expression with reduced AFP compared to HLCs cultured with Matrigel. They also secreted more urea than Matrigel cultures. However, these HLCs exhibited lower CYP3A4 activity and glycogen storage than those cultured with Matrigel. These functional differences in HLCs between collagen and Matrigel cultures closely resembled the hepatocytes of periportal and pericentral zones, respectively.ConclusionOur study demonstrates that Matrigel and collagen have differential effects on the differentiation and functionality of HLCs, which resemble, to an extent, hepatic zonation in the liver lobules. Our finding has an important impact on the generation of hPSC-HLCs for biomedical and medical applications.

Metabolic Stability of Eight Airborne OrganoPhosphate Flame Retardants (OPFRs) in Human Liver, Skin Microsomes and Human Hepatocytes

The waste of electrical and electronic equipment (WEEE) is generally considered a secondary raw material for the recovery of valuable components. However, emerging issues regarding the impact of suspended particles arising from WEEE recycling operations are a concern. It was recently demonstrated that samples from three different WEEE plants were rich in organophosphate flame retardants (OPFRs). Since exposure to a xenobiotic can lead to its biotransformation through human metabolism routes, in the present study, the metabolism of eight OPFRs of interest in our sampling campaign (triphenyl phosphate (TPhP), tri-m-tolyl phosphate (TMTP), ethylhexyl diphenyl phosphate (EHDPhP), tributoxyethyl phosphate (TBOEP), diphenyl phosphate (DPhP), trichloroethyl phosphate (TCEP), tris(1,3-dichloropropan-2-yl) phosphate (TDClPP) and bisphenol A bis(diphenyl phosphate) (BDP)) was investigated. Their metabolism was studied at different time points in three matrices: human liver microsomes, human hepatocytes and human skin microsomes. This study, which was run using a common experimental setting, allowed easy comparison of results for each OPFR of interest, and a comparison with other data in the literature was performed. In particular, a number of metabolites not previously described were detected, and for the first time, it was shown that TPhP could be metabolized in human skin microsomes.

Generation of Fibrotic Liver Organoids Using Hepatocytes, Primary Liver Sinusoidal Endothelial Cells, Hepatic Stellate Cells, and Macrophages.

Liver organoids generated with single or multiple cell types have been used to investigate liver fibrosis development, toxicity, pathogenesis, and drug screening. However, organoid generation is limited by the availability of cells isolated from primary tissues or differentiated from various stem cells. To ensure cell availability for organoid formation, we investigated whether liver organoids could be generated with cell-line-based Huh-7 hepatocellular carcinoma cells, macrophages differentiated from THP-1 monocytes, and LX-2 hepatic stellate cells (HSCs) and primary liver sinusoidal endothelial cells (LSECs). In liver organoids, hepatocyte-, LSEC-, macrophage-, and HSC-related gene expression increased relative to that in two-dimensional (2D)-cultured Huh-7/LSEC/THP-1/LX-2 cells without Matrigel. Thioacetamide (TAA) increased α-smooth muscle actin expression in liver organoids but not in 2D-cultured cells, whereas in TAA-treated organoids, the expression of hepatic and LSEC markers decreased and that of macrophage and HSC markers increased. TAA-induced fibrosis was suppressed by treatment with N-acetyl-L-cysteine or tumor-necrosis-factor-stimulated gene 6 protein. The results showed that liver toxicants could induce fibrotic and inflammatory responses in liver organoids comprising Huh-7/LSEC/macrophages/LX-2 cells, resulting in fibrotic liver organoids. We propose that cell-line-based organoids can be used for disease modeling and drug screening to improve liver fibrosis treatment.

Caspase 6 promotes innate immune activation by functional crosstalk between RIPK1-IκBα axis in liver inflammation

BackgroundCaspase 6 is an essential regulator in innate immunity, inflammasome activation and host defense. We aimed to characterize the causal mechanism of Caspase 6 in liver sterile inflammatory injury.MethodsHuman liver tissues were harvested from patients undergoing ischemia-related hepatectomy to evaluate Caspase 6 expression. Subsequently, we created Caspase 6-knockout (Caspase 6KO) mice to analyze roles and molecular mechanisms of macrophage Caspase 6 in murine models of liver ischemia/reperfusion (IR) injury.ResultsIn human liver biopsies, Caspase 6 expression was positively correlated with more severe histopathological injury and higher serum ALT/AST level at one day postoperatively. Moreover, Caspase 6 was mainly elevated in macrophages but not hepatocytes in ischemic livers. Unlike in controls, the Caspase 6-deficient livers were protected against IR injury, as evidenced by inhibition of inflammation, oxidative stress and iron overload. Disruption of macrophage NF-κB essential modulator (NEMO) in Caspase 6-deficient livers deteriorated liver inflammation and ferroptosis. Mechanistically, Caspase 6 deficiency spurred NEMO-mediated IκBα phosphorylation in macrophage. Then phosphorylated-inhibitor of NF-κBα (p-IκBα) co-localized with receptor-interacting serine/ threonine-protein kinase 1 (RIPK1) in the cytoplasm to degradate RIPK1 under inflammatory conditions. The disruption of RIPK1-IκBα interaction preserved RIPK1 degradation, triggering downstream apoptosis signal-regulating kinase 1 (ASK1) phosphorylation and inciting NIMA-related kinase 7/NOD-like receptor family pyrin domain containing 3 (NEK7/NLRP3) activation in macrophages. Moreover, ablation of macrophage RIPK1 or ASK1 diminished NEK7/NLRP3-driven inflammatory response and dampened hepatocyte ferroptosis by reducing HMGB1 release from macrophages.ConclusionsOur findings underscore a novel mechanism of Caspase 6 mediated RIPK1-IκBα interaction in regulating macrophage NEK7/NLRP3 function and hepatocytes ferroptosis, which provides therapeutic targets for clinical liver IR injury.Graphical DRFRodggEVQV_MBi1PPYnNVideo

Quantitative Translation of Substrate Intrinsic Clearance from Recombinant CYP1A1 to Humans.

CYP1A1 is a cytochrome P450 family 1 enzyme that is mostly expressed in the extrahepatic tissues. To understand the CYP1A1 contribution to drug clearance in humans, we examined the in vitro-in vivo extrapolation (IVIVE) of intrinsic clearance (CLint) for a set of drugs that are in vitro CYP1A1 substrates. Despite being strong in vitro CYP1A1 substrates, 82% of drugs gave good IVIVE with predicted CLint within 2-3-fold of the observed values using human liver microsomes and hepatocytes, suggesting they were not in vivo CYP1A1 substrates due to the lack of extrahepatic contribution to CLint. Only three drugs (riluzole, melatonin and ramelteon) that are CYP1A2 substrates yielded significant underprediction of in vivo CLint up to 11-fold. The fold of CLint underprediction was linearly proportional to human recombinant CYP1A1 (rCYP1A1) CLint, indicating they were likely to be in vivo CYP1A1 substrates. Using these three substrates, a calibration curve can be developed to enable direct translation from in vitro rCYP1A1 CLint to in vivo extrahepatic contributions in humans. In vivo CYP1A1 substrates are planar and small, which is consistent with the structure of the active site. This is in contrast to the in vitro substrates, which include large and nonplanar molecules, suggesting rCYP1A1 is more accessible than what is in vivo. The impact of CYP1A1 on first-pass intestinal metabolism was also evaluated and shown to be minimal. This is the first study providing new insights on in vivo translation of CYP1A1 contributions to human clearance using in vitro rCYP1A1 data.