Liver Parenchymal Cells Research Articles

Autophagy in liver diseases: A review

The liver is a highly dynamic metabolic organ that plays critical roles in plasma protein synthesis, gluconeogenesis and glycogen storage, cholesterol metabolism and bile acid synthesis as well as drug/xenobiotic metabolism and detoxification. Research from the past decades indicate that autophagy, the cellular catabolic process mediated by lysosomes, plays an important role in maintaining cellular and metabolic homeostasis in the liver. Hepatic autophagy fluctuates with hormonal cues and the availability of nutrients that respond to fed and fasting states as well as circadian activities. Dysfunction of autophagy in liver parenchymal and non-parenchymal cells can lead to various liver diseases including non-alcoholic fatty liver diseases, alcohol associated liver disease, drug-induced liver injury, cholestasis, viral hepatitis and hepatocellular carcinoma. Therefore, targeting autophagy may be a potential strategy for treating these various liver diseases. In this review, we will discuss the current progress on the understanding of autophagy in liver physiology. We will also discuss several forms of selective autophagy in the liver and the molecular signaling pathways in regulating autophagy of different cell types and their implications in various liver diseases.

Sub-Lethal Toxicity of Dichlorvos on Gills and Liver of Juvenile Heterobranchus longifilis

The study assessed the histological changes in the structure of gills and liver of juvenile Heterobranchus longifilis after exposure to varying sub-lethal concentrations of Dichlorvos. The fish, 120specimenswith mean weight of 47.33±2.31g and total length 10.67±0.58cm were selected and used for the experiment. Test fish were treated with concentrations of 0.00, 0.01, 0.015 and 0.02mg/l of Dichlorvos representing treatments T1, T2, T3 and T4 respectively. The study was carried out in a static renewal bioassay system. Fish samples were stocked in plastic bowls of 75litre capacity and the exposure period was7 days. All analyses were done using standard methods. The results showed prominent changes in the gills which included: partial epithelia lifting, hyperplasia, heavy degeneration changes, oedema, congestions and vacuolization. Changes in the liver included: congestion with sub-massive necrosis, liver parenchymal cell prominent with fatty/glycogen degeneration, hepatocytes and enlargement of portal tract. Histopathological changes on the two tissues increased with concentration of dichlorvos with severe damages observed at higher concentrations. This study showed that the use of dichlorvos as pesticides, which often find their way into the aquatic system, may have negative effects on fish. Thus, the use of organic pesticides should be encouraged.
 Keywords: Histopathology, sub-lethal, Heterobranchus longifilis, dichlorvos, gills and liver

Virus-Derived Peptides for Hepatic Enzyme Delivery.

Recently, a lipopeptide derived from the hepatitis B virus (HBV) large surface protein has been developed as an HBV entry inhibitor. This lipopeptide, called MyrcludexB (MyrB), selectively binds to the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral membrane of hepatocytes. Here, the feasibility of coupling therapeutic enzymes to MyrB was investigated for the development of enzyme delivery strategies. Hepatotropic targeting shall enable enzyme prodrug therapies and detoxification procedures. Here, horseradish peroxidase (HRP) was conjugated to MyrB via maleimide chemistry, and coupling was validated by SDS-PAGE and reversed-phase HPLC. The specificity of the target recognition of HRP-MyrB could be shown in an NTCP-overexpressing liver parenchymal cell line, as demonstrated by competitive inhibition with an excess of free MyrB and displayed a strong linear dependency on the applied HRP-MyrB concentration. In vivo studies in zebrafish embryos revealed a dominating interaction of HRP-MyrB with scavenger endothelial cells vs xenografted NTCP expressing mammalian cells. In mice, radiolabeled 125I-HRP-MyrBy, as well as the non-NTCP targeted control HRP-peptide-construct (125I-HRP-alaMyrBy) demonstrated a strong liver accumulation confirming the nonspecific interaction with scavenger cells. Still, MyrB conjugation to HRP resulted in an increased and NTCP-mediated hepatotropism, as revealed by competitive inhibition. In conclusion, the model enzyme HRP was successfully conjugated to MyrB to achieve NTCP-specific targeting in vitro with the potential for ex vivo diagnostic applications. In vivo, target specificity was reduced by non-NTCP-mediated interactions. Nonetheless, tissue distribution experiments in zebrafish embryos provide mechanistic insight into underlying scavenging processes indicating partial involvement of stabilin receptors.

Single-cell RNA sequencing of human liver reveals hepatic stellate cell heterogeneity

Background & AimsThe multiple vital functions of the human liver are performed by highly specialised parenchymal and non-parenchymal cells organised in complex collaborative sinusoidal units. Although crucial for homeostasis, the cellular make-up of the human liver remains to be fully elucidated. Here, single-cell RNA-sequencing was used to unravel the heterogeneity of human liver cells, in particular of hepatocytes (HEPs) and hepatic stellate cells (HSCs).MethodThe transcriptome of ~25,000 freshly isolated human liver cells was profiled using droplet-based RNA-sequencing. Recently published data sets and RNA in situ hybridisation were integrated to validate and locate newly identified cell populations.ResultsIn total, 22 cell populations were annotated that reflected the heterogeneity of human parenchymal and non-parenchymal liver cells. More than 20,000 HEPs were ordered along the portocentral axis to confirm known, and reveal previously undescribed, zonated liver functions. The existence of 2 subpopulations of human HSCs with unique gene expression signatures and distinct intralobular localisation was revealed (i.e. portal and central vein-concentrated GPC3+ HSCs and perisinusoidally located DBH+ HSCs). In particular, these data suggest that, although both subpopulations collaborate in the production and organisation of extracellular matrix, GPC3+ HSCs specifically express genes involved in the metabolism of glycosaminoglycans, whereas DBH+ HSCs display a gene signature that is reminiscent of antigen-presenting cells.ConclusionsThis study highlights metabolic zonation as a key determinant of HEP transcriptomic heterogeneity and, for the first time, outlines the existence of heterogeneous HSC subpopulations in the human liver. These findings call for further research on the functional implications of liver cell heterogeneity in health and disease.Lay summaryThis study resolves the cellular landscape of the human liver in an unbiased manner and at high resolution to provide new insights into human liver cell biology. The results highlight the physiological heterogeneity of human hepatic stellate cells.

OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation

In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.

Effect of amino acid imbalance in the nutrition on the morphology of animal liver

The article considers the influence of various forms of amino acid imbalance in diets on morphological changes in the liver of laboratory animals. An imbalance of amino acids caused a number of similar changes in the liver of animals. Liver sections revealed that hepatocytes had smoothed borders and areas with signs of granular dystrophy. The sinusoids and inter-protein spaces were overflowed with blood moulds. Small groups of lymphohistiocytic cells were presented in the parenchyma. Violation of the liver microcirculation contributed to the violation of metabolic processes in hepatocytes. Most of the hepatocytes were filled with yellow-orange fat droplets and had atrophied cytoplasm with nuclei shifted to the periphery. There were single cells with glycogen granules as well. The content of nucleic acids in tissues is an important indicator of the intensity of protein synthesis. Thus, in liver sections of groups 1, 2, 3 and 4, the cytoplasm of hepatocytes was less colored than in the control group, which indirectly indicates an increase in the intensity of protein synthesis in liver cells. Hemodynamic disorders in the form of dilated blood vessels and the appearance of stasis in groups 1, 2, 3 and 4 lead to difficulties in the penetration of amino acids to the sites of protein synthesis and, as a result, to a violation of metabolic processes in parenchymal liver cells.

Liver injury in Northern American patients with COVID-19

Presenter: Aleksandr Kalabin MD ;Columbia University at Harlem Hospital Background: Direct viral damage of organs/systems with subsequent inflammatory cascade is considered to be a major contributor to the pathogenesis of SARS-CoV-2 infection. Although lung is contemplated to be the primary organ targeted by the virus, recent reports predominantly from China and Europe provided evidence of affinity of COVID-19 to Angiotensin-converting enzyme 2(ACE 2) receptors expressed on gastrointestinal tract and liver parenchymal cells. The goal of our study was to investigate if SARS-CoV-2 virus affects liver function of Northern American population and if liver injury correlates with the disease severity in COVID-19 patients. Methods: Retrospective analysis of patients admitted with SARS-CoV-2 infection between March – April 2020 and liver function tests done within first 24h of admission. The primary outcome of interest was to determine if liver function is affected by COVOD-19 and assess the correlation with the disease severity. Patients were grouped into Non-intubated(NI-T) vs Intubated(I-T) patients and Survived(S) vs Deceased(D). Patients defined as survived were alive at the time of data accrual. Unpaired Student’s t-test was used for continues variables, Pearson Chi-square(χ2) test for categorical. P value < 0.05 was considered to represent statistically significant difference. Results: 183 patients were included, 73 Females(39.89%) and 110 males(60.11%). 114(62.30%) patients were African-Americans, 58(31.69%) Hispanics and 11 Asians(6.01%). Mean age was 64.77(SD 14.09), mean BMI 29.03(SD 7.36), mean body temperature 100.89(SD 1.68). 93 patients(50.82%) had GI complaints on presentation: most common symptoms were anorexia(54 patients, 29.51%), diarrhea(more than 3 loose stools per day, 41 patients, 22.40%), nausea/vomiting(33 patients, 18.03%) and abdominal pain(18 patients, 9.84%). Evaluation of LFTs on admission revealed mean aspartate aminotransferase(AST) of 63.83(SD 115.87), mean alanine transaminase(ALT) – 47.08(SD 85.64). Total bilirubin for the cohort was 0.64(SD 0.51), mean albumin 3.60(SD 0.52), mean Prothrombin time(PT) 14.38(SD 3.46), mean platelet count 220.94(83.59). Intubated patients tended to have significantly higher levels of AST(109.17 vs 53.97, P = 0.018), ALT(79.53 vs 40.03, P = 0.02) and total bilirubin(0.82 vs 0.60, P = 0.03) compared to non-intubated patents. This correlated with body temperature that was higher(101.38) for Intubated vs Non-intubated patients(100.70) (p = 0.0006), and acute phase reactants CRP(24.06 v 15.96, P = 0.019) and lactate(3.28 vs 2.13, P = 0.009). No difference between groups was observed for PT, platelets and albumin. Mean AST for Survived patients was 66.40 vs 50.41 for Deceased group(P = 0.51), mean ALT 48.89 vs 37.63(P = 0.53) and mean bilirubin 0.65 vs 0.60(P = 0.65) respectively. No difference between groups was observed for PT, platelets and albumin. CRP and lactate were higher in Deceased group compared to the Survived group:(27.09 vs 16.39, P = 0.008) and (3.33 vs 2.10 P = 0.005) respectively. Conclusion: Our study suggests direct viral injury to the liver parenchymal and biliary cells in Northern American patients with COVID-19 infection. Moreover, patients with elevated liver enzymes tended to have severe disease course, but no correlation with mortality was observed. Interestingly enough liver synthetic function of patients with SARS-CoV-2 infection was not significantly affected. While exact mechanisms of direct damage to the liver currently remain unclear and other explanations of elevated liver enzymes have been offered, more studies are needed to prove unambiguous interrelation between liver dysfunction and SARS-CoV-2 virus. [Formula presented]

Research advances in Kupffer cells participating in the regulation of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a common type of primary liver cancer with high mortality worldwide. Among the common malignant tumors in China, HCC ranks fourth in terms of incidence rate and ranks second in terms of mortality, which seriously threatens the health and life safety of the Chinese people. This article mainly introduces the dual role of Kupffer cells (KCs) in HCC and briefly describes its interaction with liver parenchymal cells and nonparenchymal cells and related targeted treatment methods. The analysis shows that in-depth research on KCs in the regulation of HCC helps to provide new ideas for further treatment of HCC.

Effects of genetically modified human skin fibroblasts, stably overexpressing hepatocyte growth factor, on hepatic functions of cocultured C3A cells.

Diseases leading to terminal hepatic failure are among the most common causes of death worldwide. Transplant of the whole organ is the only effective method to cure liver failure. Unfortunately, this treatment option is not available universally due to the serious shortage of donors. Thus, alternative methods have been developed that are aimed at prolonging the life of patients, including hepatic cells transplantation and bridging therapy based on hybrid bioartificial liver devices. Parenchymal liver cells are highly differentiated and perform many complex functions, such as detoxification and protein synthesis. Unfortunately, isolated hepatocytes display a rapid decline in viability and liver-specific functions. A number of methods have been developed to maintain hepatocytes in their highly differentiated state in vitro, amongst them the most promising being 3D growth scaffolds and decellularized tissues or coculture with other cell types required for the heterotypic cell-cell interactions. Here we present a novel approach to the hepatic cells culture based on the feeder layer cells genetically modified using lentiviral vector to stably produce additional amounts of hepatocyte growth factor and show the positive influence of these coculture conditions on the preservation of the hepatic functions of the liver parenchymal cells' model-C3A cells.

Single-Cell Transcriptomics Reveals Early Emergence of Liver Parenchymal and Non-parenchymal Cell Lineages

The cellular complexity and scale of the early liver have constrained analyses examining its emergence during organogenesis. To circumvent these issues, we analyzed 45,334 single-cell transcriptomes from embryonic day (E)7.5, when endoderm progenitors are specified, to E10.5 liver, when liver parenchymal and non-parenchymal cell lineages emerge. Our data detail divergence of vascular and sinusoidal endothelia, including a distinct transcriptional profile for sinusoidal endothelial specification by E8.75. We characterize two distinct mesothelial cell types as well as early hepatic stellate cells and reveal distinct spatiotemporal distributions for these populations. We capture transcriptional profiles for hepatoblast specification and migration, including the emergence of a hepatomesenchymal cell type and evidence for hepatoblast collective cell migration. Further, we identify cell-cell interactions during the organization of the primitive sinusoid. This study provides a comprehensive atlas of liver lineage establishment from the endoderm and mesoderm through to the organization of the primitive sinusoid at single-cell resolution.

Hepatic stellate cells contribute to liver regeneration through galectins in hepatic stem cell niche

BackgroundAs a critical cellular component in the hepatic stem cell niche, hepatic stellate cells (HSCs) play critical roles in regulating the expansion of hepatic stem cells, liver regeneration, and fibrogenesis. However, the signaling of HSCs, particularly that involved in promoting hepatic stem cell expansion, remains unclear. While the overexpression of galectins has been identified in regenerating liver tissues, their involvement in cell-cell interactions between HSCs and hepatic stem cells remains to be elucidated.MethodsTo generate a liver regeneration rat model and establish a hepatic oval cell microenvironment as a stem cell niche, 2-acetylaminofluorene treatment plus partial hepatectomy was performed. Immunofluorescence staining was conducted to detect the emergence of hepatic stem cells and their niche. Liver parenchymal cells, non-parenchymal cells, and HSCs were isolated for gene and protein expression analysis by qPCR or western blotting. To evaluate the effect of galectins on the colony-forming efficiency of hepatic stem cells, c-Kit−CD29+CD49f+/lowCD45−Ter-119− cells were cultured with recombinant galectin protein, galectin antibody, galectin-producing HSCs, and galectin-knockdown HSCs.ResultsFollowing liver injury, the cytokeratin 19+ ductal cells were robustly induced together with the emergence of OV6+CD44+CD133+EpCAM+ hepatic stem cells. The activated desmin+ HSCs were recruited around the periportal area and markedly enriched in the galectin-positive domain compared to the other non-parenchymal cells. Notably, the HSC fraction isolated from regenerating liver was accompanied by dramatically elevated gene and protein expression of galectins. Hepatic stem cells co-cultured with HSCs significantly enhanced colony-forming efficiency. Conversely, single or double knockdown of galectin-1 and galectin-3 led into a significant function loss, impaired the co-cultured hepatic stem cells to attenuated colony size, inhibited colony frequency, and reduced total cell numbers in colonies. On the other hand, the promotive function of galectins was further confirmed by recombinant galectin protein supplementation and galectins blocking antibodies.ConclusionsOur findings, for the first time, demonstrated that galectins from activated HSCs contribute to hepatic stem cell expansion during liver regeneration, suggesting that galectins serve as important stem cell niche components.

Covering the Cover

Covering the Cover

Development of highly functional bioengineered human liver with perfusable vasculature

Liver tissue engineering offers a promising strategy for liver failure patients. Since transplantation rejection resulting in vessel thrombosis is regarded as a major hurdle, vascular reconstruction is one of indispensable requirements of whole organ engineering. Here we demonstrated a novel strategy for reconstruction of a vascularized bioengineered human liver (VBHL) using decellularized liver scaffolds in an efficient manner. First we achieved fully functional endothelial coverage of scaffolds by adopting the anti-CD31 aptamer as a potent coating agent for re-endothelialization. Through an ex vivo human blood perfusion that recapitulates the blood coagulation response in humans, we demonstrated significantly reduced platelet aggregation in anti-CD31 aptamer coated scaffolds. We then produced VBHL constructs using liver parenchymal cells and nonparenchymal cells, properly organized into liver-like structures with an aligned vasculature. Interestingly, VBHL constructs displayed prominently enhanced long-term liver-specific functions that are affected by vascular functionality. The VBHL constructs formed perfusable vessel networks in vivo as evidenced by the direct vascular connection between the VBHL constructs and the renal circulation. Furthermore, heterotopic transplantation of VBHL constructs supported liver functions in a rat model of liver fibrosis. Overall, we proposed a new strategy to generate transplantable bioengineered livers characterized by highly functional vascular reconstruction.

Ultrastructure of Hepatocytes from Laboratory Mice Fed a Standard Dry Laboratory Animal Diet.

The significant destructive changes in ultrastructure of hepatocytes from laboratory mice kept in different vivariums in Moscow and fed with dry laboratory animal diets acquired from different domestic manufacturers that were not standardized for initial products were demonstrated using electron microscopy. Furthermore, disruption in the ultrastructure of liver parenchymal cells occurred regardless of the animal status (SPF or conventional), conditions of various vivariums, as well as the feed manufacturer. At the same time, studies on ultrastructure of liver hepatocytes from mice kept in the Charles River Laboratory facilities in Germany and fed with the Altromin Spezialfutter laboratory animal diet (GmbH & Co., Germany) that was produced using quality control of ingredients did not reveal destructive changes in the internal ultrastructure of hepatocytes. However, if these mice were later fed with the food produced in local manufactures, changes in the structure of liver cells developed after 2 months. Thus, feeding with dry diet from the domestic producers of an unspecified composition causes significant changes in the ultrastructure of hepatocytes in control animals, reflecting the development of some pathological processes in the body.

Hemostasis and Liver Regeneration.

The liver is unique in its remarkable regenerative capacity, which enables the use of liver resection as a treatment for specific liver diseases, including removal of neoplastic liver disease. After resection, the remaining liver tissue (i.e, liver remnant) regenerates to maintain normal hepatic function. In experimental settings as well as patients, removal of up to two-thirds of the liver mass stimulates a rapid and highly coordinated process resulting in the regeneration of the remaining liver. Mechanisms controlling the initiation and termination of regeneration continue to be discovered, and many of the fundamental signaling pathways controlling the proliferation of liver parenchymal cells (i.e., hepatocytes) have been uncovered. Interestingly, while hemostatic complications (i.e., bleeding and thrombosis) are primarily thought of as a complication of surgery itself, strong evidence suggests that components of the hemostatic system are, in fact, powerful drivers of liver regeneration. This review focuses on the clinical and translational evidence supporting a link between the hemostatic system and liver regeneration, and the mechanisms whereby the hemostatic system directs liver regeneration discovered using experimental settings.

Nanoparticular bisphosphonate induces an anti-fibrotic response by modulation of fibrosis associated genes and pathways in (non-) parenchymal liver cells in CCL4 fibrotic mice

Nanoparticular bisphosphonate induces an anti-fibrotic response by modulation of fibrosis associated genes and pathways in (non-) parenchymal liver cells in CCL4 fibrotic mice

THU178 - Deletion of XBP1 in liver parenchymal cells ameliorates acetaminophen (APAP)-induced hepatotoxicity via activation of IRE1alpha-JNK1-ATG5-dependent autophagy

THU178 - Deletion of XBP1 in liver parenchymal cells ameliorates acetaminophen (APAP)-induced hepatotoxicity via activation of IRE1alpha-JNK1-ATG5-dependent autophagy

Human Pluripotent Stem Cell-Derived Organoids as Models of Liver Disease

There are few invitro models for studying the 3-dimensional interactions among different liver cell types during organogenesis or disease development. We aimed to generate hepatic organoids that comprise different parenchymal liver cell types and have structural features of the liver, using human pluripotent stem cells. We cultured H1 human embryonic stem cells (WA-01, passage 27-40) and induced pluripotent stem cells (GM23338) with a series of chemically defined and serum-free media to induce formation of posterior foregut cells, which were differentiated in 3 dimensions into hepatic endoderm spheroids and stepwise into hepatoblast spheroids. Hepatoblast spheroids were reseeded in a high-throughput format and induced to form hepatic organoids; development of functional bile canaliculi was imaged live. Levels of albumin and apolipoprotein B were measured in cell culture supernatants using an enzyme-linked immunosorbent assay. Levels of gamma glutamyl transferase and alkaline phosphatase were measured in cholangiocytes. Organoids were incubated with troglitazone for varying periods and bile transport and accumulation were visualized by live-imaging microscopy. Organoids were incubated with oleic and palmitic acid, and formation of lipid droplets was visualized by staining. We compared gene expression profiles of organoids incubated with free fatty acids or without. We also compared gene expression profiles between liver tissue samples from patients with nonalcoholic steatohepatitis (NASH) versus without. We quantified hepatocyte and cholangiocyte populations in organoids using immunostaining and flow cytometry; cholangiocyte proliferation of cholangiocytes was measured. We compared the bile canaliculi network in the organoids incubated with versus without free fatty acids by live imaging. Cells in organoids differentiated into hepatocytes and cholangiocytes, based on the expression of albumin and cytokeratin 7. Hepatocytes were functional, based on secretion of albumin and apolipoprotein B and cytochrome P450 activity; cholangiocytes were functional, based on gamma glutamyl transferase and alkaline phosphatase activity and proliferative responses to secretin. The organoids organized a functional bile canaliculi system, which was disrupted by cholestasis-inducing drugs such as troglitazone. Organoids incubated with free fatty acids had gene expression signatures similar to those of liver tissues from patients with NASH. Incubation of organoids with free fatty acid-enriched media resulted in structural changes associated with nonalcoholic fatty liver disease, such as decay of bile canaliculi network and ductular reactions. We developed a hepatic organoid platform with human cells that can be used to model complex liver diseases, including NASH.

Total reflection X-ray fluorescence spectrometry for trace determination of iron and some additional elements in biological samples

Trace elements are essential for life and their concentration in cells and tissues must be tightly maintained and controlled to avoid pathological conditions. Established methods to measure the concentration of trace elements in biological matrices often provide only single element information, are time-consuming, and require special sample preparation. Therefore, the development of straightforward and rapid analytical methods for enhanced, multi-trace element determination in biological samples is an important and raising field of trace element analysis. Herein, we report on the development and validation of a reliable method based on total reflection X-ray fluorescence (TXRF) analysis to precisely quantify iron and other trace metals in a variety of biological samples, such as the liver, parenchymal and non-parenchymal liver cells, and bone marrow–derived macrophages. We show that TXRF allows fast and simple one-point calibration by addition of an internal standard and has the potential of multi-element analysis in minute sample amounts. The method was validated for iron by recovery experiments in homogenates in a wide concentration range from 1 to 1600 μg/L applying well-established graphite furnace atomic absorption spectrometry (GFAAS) as a reference method. The recovery rate of 99.93 ± 0.14% reveals the absence of systematic errors. Furthermore, the standard reference material “bovine liver” (SRM 1577c, NIST) was investigated in order to validate the method for further biometals. Quantitative recoveries (92–106%) of copper, iron, zinc, and manganese prove the suitability of the developed method. The limits of detection for the minute sample amounts are in the low picogram range.Graphical abstract

Loss of c-Jun N-terminal Kinase 1 and 2 Function in Liver Epithelial Cells Triggers Biliary Hyperproliferation Resembling Cholangiocarcinoma.

Targeted inhibition of the c‐Jun N‐terminal kinases (JNKs) has shown therapeutic potential in intrahepatic cholangiocarcinoma (CCA)‐related tumorigenesis. However, the cell‐type‐specific role and mechanisms triggered by JNK in liver parenchymal cells during CCA remain largely unknown. Here, we aimed to investigate the relevance of JNK1 and JNK2 function in hepatocytes in two different models of experimental carcinogenesis, the dethylnitrosamine (DEN) model and in nuclear factor kappa B essential modulator (NEMO)hepatocyte‐specific knockout (Δhepa) mice, focusing on liver damage, cell death, compensatory proliferation, fibrogenesis, and tumor development. Moreover, regulation of essential genes was assessed by reverse transcription polymerase chain reaction, immunoblottings, and immunostainings. Additionally, specific Jnk2 inhibition in hepatocytes of NEMOΔhepa/JNK1Δhepa mice was performed using small interfering (si) RNA (siJnk2) nanodelivery. Finally, active signaling pathways were blocked using specific inhibitors. Compound deletion of Jnk1 and Jnk2 in hepatocytes diminished hepatocellular carcinoma (HCC) in both the DEN model and in NEMOΔhepa mice but in contrast caused massive proliferation of the biliary ducts. Indeed, Jnk1/2 deficiency in hepatocytes of NEMOΔhepa (NEMOΔhepa/JNKΔhepa) animals caused elevated fibrosis, increased apoptosis, increased compensatory proliferation, and elevated inflammatory cytokines expression but reduced HCC. Furthermore, siJnk2 treatment in NEMOΔhepa/JNK1Δhepa mice recapitulated the phenotype of NEMOΔhepa/JNKΔhepa mice. Next, we sought to investigate the impact of molecular pathways in response to compound JNK deficiency in NEMOΔhepa mice. We found that NEMOΔhepa/JNKΔhepa livers exhibited overexpression of the interleukin‐6/signal transducer and activator of transcription 3 pathway in addition to epidermal growth factor receptor (EGFR)‐rapidly accelerated fibrosarcoma (Raf)‐mitogen‐activated protein kinase kinase (MEK)‐extracellular signal‐regulated kinase (ERK) cascade. The functional relevance was tested by administering lapatinib, which is a dual tyrosine kinase inhibitor of erythroblastic oncogene B‐2 (ErbB2) and EGFR signaling, to NEMOΔhepa/JNKΔhepa mice. Lapatinib effectively inhibited cystogenesis, improved transaminases, and effectively blocked EGFR‐Raf‐MEK‐ERK signaling. Conclusion: We define a novel function of JNK1/2 in cholangiocyte hyperproliferation. This opens new therapeutic avenues devised to inhibit pathways of cholangiocarcinogenesis.