Liver Explant Cultures Research Articles

Mincle-GSDMD-mediated release of IL-1β small extracellular vesicles from hepatic macrophages in ethanol-induced liver injury.

Macrophage-inducible C-type lectin (Mincle) is expressed on hepatic macrophages and senses ethanol (EtOH)-induced danger signals released from dying hepatocytes and promotes IL-1β production. However, it remains unclear what and how EtOH-induced Mincle ligands activate downstream signaling events to mediate IL-1β release and contribute to alcohol-associated liver disease (ALD). In this study, we investigated the association of circulating β-glucosylceramide (β-GluCer), an endogenous Mincle ligand, with severity of ALD and examined the mechanism by which β-GluCer engages Mincle on hepatic macrophages to release IL-1β in the absence of cell death and exacerbates ALD. Concentrations of β-GluCer were increased in serum of patients with severe AH and correlated with disease severity. Challenge of hepatic macrophages with lipopolysaccharide and β-GluCer induced formation of a Mincle and Gsdmd-dependent secretory complex containing chaperoned full-length gasdermin D (Hsp90-CDC37-NEDD4) with polyubiquitinated pro-IL-1β and components of the Caspase 8-NLRP3 inflammasome loaded as cargo in small extracellular vesicles (sEVs). Gao-binge EtOH exposure to wild-type, but not Mincle-/- and Gsdmd-/-, mice increased release of IL-1β-containing sEVs from liver explant cultures. Myeloid-specific deletion of Gsdmd similarly decreased the formation of sEVs by liver explant cultures and protected mice from EtOH-induced liver injury. sEVs collected from EtOH-fed wild-type, but not Gsdmd-/-, mice promoted injury of cultured hepatocytes and, when injected into wild-type mice, aggravated Gao-binge EtOH-induced liver injury. β-GluCer functions as a danger-associated molecular pattern activating Mincle-dependent gasdermin D-mediated formation and release of IL-1β-containing sEVs, which in turn exacerbate hepatocyte cell death and contribute to the pathogenesis of ALD.

Evaluation of Rasna panchaka (indigenous drug) as oxidative stress down-regulator using serum-free explant culture system.

CONTEXT:The importance of phytochemicals/natural products as potential therapeutic agents in the present context is gaining a lot of importance. India with a rich heritage of such preparations needs evaluation as potent drugs. Explant culture system is a method, which is sensitive, reliable, reproducible and is capable of mimicking the in situ conditions maintaining the tissue in sufficiently high level of integration.AIM:The current study aimed to test the antioxidant activity of test compounds, namely, traditional aqueous (4212) and aqueous-methanolic (4308) extracts of Rasna panchaka using liver explant cultures.MATERIALS AND METHODS:Dose-response optima of extracts (0.2–10 μg/mL) were determined using mouse liver explant culture system up to 48 h. The antioxidant property of extracts was assessed by primary oxidative defense parameters, namely, superoxide-dismutase (SOD), catalase, reduced glutathione (GSH), and malondialdehyde (MDA).RESULTS:The results indicated that the cellular architecture of the cultured tissue was well conserved in the first 6 h with a gradual display of specific changes in the next 24 h. There was a significant increase in MDA levels in experimental groups indicating the oxidative stress induction in explants. A dose of 2.0 μg/mL extracts have shown statistically significant (P < 0.05) protection against oxidative stress. MDA levels, a measure of lipid peroxidation, were significantly (P < 0.01) reduced by 50% in extract treated explants compared to control. This effect was accompanied by the increase in the first defense enzymes SOD (50%) and catalase (18%) with no change in reduced GSH levels.CONCLUSION:The study enforces the importance of “explant culture system,” as it not only reduces the use of nonclinical/animal model but also is rapid and sensitive. Further, results of the current study also suggest that aqueous-methanolic extract of Rasna panchaka is having superior antioxidant activity compared to traditional water extract.

In-Vitro Effect of Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and MDA in Liver Explant Culture

In-Vitro Effect of Picroliv on Plasmodium Berghei Induced Alterations in the Activity of Adenosine Triphosphatase, Aryl Hyrocarbon Hydroxylase Enzymes and MDA in Liver Explant Culture

Assessment of hepatoprotective potential of N. indicum leaf on haloalkane xenobiotic induced hepatic injury in Swiss albino mice

CCl4 is a potent environmental toxin which cause liver damage through free radical mediated inflammatory processes. In this study, hepatoprotective capacity of Nerium indicum leaf extract (NILE) was evaluated on CCl4 induced acute hepatotoxicity in murine model. Animals were divided into 5 groups and treated as following: control group (received only normal saline), CCl4 group (received only CCl4), silymarin group (received CCl4 and 100mg/kg silymarin), NILE low group (received CCl4 and 50mg/kg NILE) and NILE high group (received CCl4 and 200mg/kg NILE). After 10 consecutive days of treatment, the levels of hepatic biochemical markers, malondialdehyde (MDA) content, peroxidase and catalase activities were measured as well as histopathological study was performed. Furthermore, liver explant cultures were set up as following: control (no treatment), CCl4 group (contained 25μl/ml CCl4), silymarin group (contained 25μl/ml CCl4 and 100μg/ml silymarin), NILE low group (contained 25μl/ml CCl4 and 25μg/ml NILE) and NILE high group (contained 25μl/ml CCl4 and 100μg/ml NILE). Hepatic transaminases and phosphatases, tumor necrosis factor-α (TNF-α) expression, nitric oxide (NO) release and cell viability were studied on the explant cultures. Phytochemical fingerprinting of NILE was performed by gas chromatography–mass spectrometry (GC–MS). The results showed that the biochemical parameters were overexpressed due to CCl4 administration, which were significantly normalized by NILE treatment. The findings were further supported by histopathological evidences showing less hepatocellular necrosis, inflammation and fibrosis in NILE and silymarin treated groups, compared to CCl4 group. GC–MS analysis revealed presence of different bioactive phytochemicals with hepatoprotective and antioxidant properties. Therefore, the present study indicate that NILE possesses potent hepatoprotective capacity to ameliorate haloalkane xenobiotic induced injured liver in murine model.

Bioassay Guided Fractionation and Hepatoprotective Activity of Oleanolic Acid Acetate Isolated from Vitex negundo Linn.

Hepatoprotective activity of dichloromethane-methanol extracts of Vitex negundo (Linn.) roots was tested against CCl -induced hepatotoxicity in rats in vivo by bioassay guided fractionation. The extract was fractionated by column chromatography and the resulting fractions were analyzed for their hepatoprotective action in rat liver explant cultures in-vitro. The active fraction was further purified by chromatographic method. The 1H & 13C NMR, IR and mass spectral studies of the isolated compound suggested its pentacyclic triterpenic nature, characterized as oleanolic acid acetate, was evaluated for its in vitro hepatoprotective activity. Dichloromethane-methanol extract of Vitex negundo (Linn) root showed a dose dependent in vivo hepatoprotective activity. Treatment of CCl -intoxcicated liver explant culture sets (in vitro) with oleanolic acid acetate at a concentration of 3.3 mg/ml attenuated the marker enzymes (GOT: 27.8±0.73 IU/l; GPT: 17.5±0.09 IU/l and ALP: 16.1±0.66 IU/l) level in culture supernatants significantly (P<0.001) as compared to the toxic. The study revealed significant anti-hepatoxic potential of Vitex negundo (Linn) roots extract and isolated oleanolic acid acetate.

A new in vitro screening bioassay for the ecotoxicological evaluation of the estrogenic responses of environmental chemicals using roach (Rutilus rutilus) liver explant culture

There is growing evidence that many chemicals released in the environment are able to disturb the normal endocrinology of organisms affecting the structure and function of their reproductive system. This has prompted the scientific community to develop appropriate testing methods to identify active compounds and elucidate mechanisms of action. Of particular interest are in vitro screening methods that can document the effects of these endocrine disrupting compounds on fish. In this study, an in vitro bioassay was developed in the roach (Rutilus rutilus) for evaluating the estrogenicity or antiestrogenicity potency of environmental pollutants by measuring vitellogenin (VTG) induction in cultured liver explants. The cell viability was assessed by the measurement of nonspecific esterase activity using a fluorescein diacetate hydrolysis assay. Results showed that explants could be cultured for 72 h without any significant loss of activity. Dose-dependent responses have been measured with estrogenic model compounds such as 17-β-estradiol (E2) and 17-α-ethynylestradiol (EE2) or antiestrogenic compounds such as tamoxifen. Lowest observable effective concentrations were 1 nM for E2, 1 nM for EE2, and 100 nM for tamoxifen, showing a good sensitivity of the test system. Estrogenicity of butyl 4-hydroxybenzoate, 4-nonylphenol, and bisphenol A was tested. bisphenol A (100 μM) or butylparaben induced a twofold increase in VTG production when compared with 100 nM E2, whereas this production was only 20% with 100 μM 4-nonylphenol. Overall, this study shows that the bioassay could provide valuable information on endocrine disrupting chemicals including metabolites and mixtures of compounds.

Role of metalloproteinases at the onset of liver development

At the onset of liver development, the hepatic precursor cells, namely, the hepatoblasts, derive from the ventral foregut endoderm and form a bud surrounded by a basement membrane (BM). To initiate liver growth, the hepatoblasts migrate across the BM and invade the neighboring septum transversum mesenchyme. In the present study, carried out in the mouse embryo, we searched for effectors involved in this process and we examined the role of matrix metalloproteinases (MMPs). We found expression of a broad range of MMPs, among which MMP-2 was predominantly expressed in the septum transversum and MMP-14 in the hepatoblasts. Using a new liver explant culture system we showed that inhibition of MMP activity represses migration of the hepatoblasts. We conclude that MMPs are required to initiate expansion of the liver during development and that our culture system provides a new model to study hepatoblast migration.

Loss of cooperative function of transforming growth factor‐β signaling proteins, smad3 with embryonic liver fodrin, a β‐spectrin, in primary biliary cirrhosis

Modulation of fibrogenesis, epithelial, and mesenchymal cell fates are prominent effects of transforming growth factor-beta (TGF-beta) signaling by Smad proteins. We have previously shown that Smad2 and Smad3 insufficiency leads to a loss of bile ducts. In addition, Smad3/4 activity is mediated by embryonic liver fodrin (ELF), a beta-Spectrin. In mouse elf(-/-) mutants and in liver explant cultures, loss of ELF function results in T lymphocytic proliferation and absent intrahepatic bile ducts. A similar phenotype is seen in a number of cholestatic diseases with progressive loss of intrahepatic bile ducts and fibrosis. However, the expression patterns of Smads or role of ELF in cholestatic and fibrotic liver diseases are not yet known. We investigated the role of ELF in primary biliary cirrhosis (PBC), autoimmune hepatitis C, chronic viral hepatitis and in livers from mice deficient in Smad2/Smad3. We generated elf(+/-) mutant mice and analyzed for chronic liver disease and hepatocellular cancer (HCC) from 6 to 12 months. Perturbations in ELF expression were consistently seen only in PBC tissues. ELF expression was similarly aberrant in tissues from Smad2(+/-)/Smad3(+/-) mutant mice. Further studies indicated that ELF mislocalization is correlated with aberrant localization of Smad3 in some PBC tissues. Thirteen of 17 elf(+/-) mutant mice developed steatosis, fibrosis, hepatic dysplasia, with HCC in two mice. These results suggest that a compromised cytoarchitecture and polarized trafficking of TGF-beta signaling molecules, ELF and Smad3 are involved in the pathogenesis of PBC as well as HCC.

Itih-4, a serine protease inhibitor regulated in interleukin-6-dependent liver formation: role in liver development and regeneration.

Inter-alpha-trypsin inhibitor-4 (Itih-4) is a liver-restricted member of the serine protease inhibitor family with diverse functions as an anti-apoptotic and matrix stabilizing molecule that are important throughout development. We investigate the functional role of Itih-4 in liver formation, regeneration (LR) and examine its role in calcium and hyaluronic acid binding. Itih-4 expression is prominent in early liver development at E9 and later at E16, being restricted to hepatoblasts, immature hepatocytes, and differentiated hepatocytes. We note a marked and differential increase in Itih-4 labeling in proliferating hepatocytes, compared with bile duct cells in liver explant cultures treated with interleukin-6 (IL-6). After partial hepatectomy, maximal Itih-4 expression occurs in a bimodal manner at 30 min and at 12 hr, with a predominant centrizonal distribution. There is no detectable binding of glutathione transferase-fusion Itih-4 protein to calcium and hyaluronic acid, indicating a possible requirement for posttranslational modifications for these functions. These results suggest that in LR, Itih-4 expression corresponds to that of immediate early genes and may contribute to the entry of normally quiescent hepatocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggests a prominent role for Itih-4 at key points in liver formation.

The role of the RON receptor tyrosine kinase in a murine model of fulminant hepatic failure

as well as colorimetric immunoassays. Results: 1. In LR, maximal Itih4 expression was observed at 30 minutes and 12 hours, predominantly centrizonal in distribution. 2. Itih-4 expression was prominent in early liver development at day 9 and reached a second peak at day 16, being restricted to hepatoblasts, immature hepatncytes and differentiated hepatocytes. 3. A marked increase in Itih-4 labeling was noted in proliferating hepatocytes, but not bile duct cells in liver explant cultures treated with I L-6 (100 units/ml). Itih-4 expression was not altered in explants cultured with TNF alpha. 4. The fluorescence emission spectrum of metal free ITIH-4 showed a maximum at 332 nm with no change on addition of 5 mM Ca. The GST-fusion protein was recovered in the void fraction with no protein binding to the column. Similarly covalent binding of Itih-4 to hyaiuronic acid was not detected. Conclusions: In LR, Itih-4 expression corresponds to that of immediate early genes, such as c-myc, c-los, c-jun, IGFBP-2 and may contribute to the entry of normally quiescent hepetocytes into the early stages of the cell cycle. The markedly high expression of Itih-4 in early liver development and in explants treated with IL-6 suggest a prominent role for Itih-4 in the tissue remodeling that occurs during regenerative and developmental aspects of liver formation.

Expansion of Hepatic and Hematopoietic Stem Cells Utilizing Mouse Embryonic Liver Explants.

Ex vivo embryonic liver explant culture is a novel and attractive approach to obtain abundant hepatic and hematopoietic stem cells. Gene therapy of autologous hepatic and hematopoietic stem cells represents an alternative therapeutic approach to liver transplantation for genetic and metabolic disorders. In this study we characterize the growth and differentiation of hepatic stem cells utilizing embryonic liver cultures. Day 9.5 liver buds are microdissected and cultured under specific conditions. Modulation of growth conditions by addition of hepatocyte growth factor, Flt-3 ligand, and stem cell factor leads to enrichment of hepatic progenitor cells in embryonic liver explants. Under these conditions, we also demonstrate the role of a novel marker PRAJA-1 to identify hepatic stem cells and transitional hepatocytes. Utilization of dexamethasone enhanced pseudolobule formation with increased hepatocytic and biliary differentiation. Transforming growth factor-β leads to enrichment of biliary cells in the culture. Gut formation is enhanced in the presence of interleukin-3 and blood formation by increasing the mesodermal tissue in these cultures. We also show increased retroviral-mediated expression of the green fluorescent protein expression in the expanded hepatic and hematopoietic stem cells under different culture conditions. Thus, the embryonic liver explant culture is an attractive source for hepatic progenitors and is a possible step towards generating nontumorigenic immortalized hepatocytes with possible transplantation applications.

Praja1, a novel gene encoding a RING-H2 motif in mouse development.

As part of a cloning strategy to identify genes involved in early mouse liver development we have isolated Praja1, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl) which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja1 is a 2.1 kb gene encoding a putative 396 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, we have localized Praja1 on chromosome X at 36 cM, which may be a candidate gene for mouse sla (sex linked sideroblastic anemia), near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, p.c.). In vitro transcription/translation yielded a product with an Mr of 59 kD. Immunohistochemical staining of in vitro liver explant cultures using a heterologous antibody against praja1 demonstrated cytoplasmic staining of cuboidal cells that have hepatocyte morphology and organization. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja1 product is possibly involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of Praja1 to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

Coordinate Estrogen-Regulated Instability of Serum Protein-Coding Messenger RNAs inXenopus laevis

Estrogen causes the cytoplasmic destabilization of albumin and gamma-fibrinogen mRNA in Xenopus laevis liver. The purpose of the present study was to determine whether mRNA destabilization is a generalized phenomenon in response to estrogen, or whether this process is restricted to a particular class of mRNAs. To address this, we have expanded our bank of serum protein-coding cDNA clones to include transferrin, the second protein of inter-alpha-trypsin inhibitor and clone 12B, for which there is no mammalian homolog. Together with albumin and gamma-fibrinogen, these represent more than 85% of the mRNAs encoding liver secreted proteins. Estrogen administration to male Xenopus or to liver explant cultures causes the generalized disappearance of all of these mRNAs. In contrast, estrogen has no effect on actin, ferritin, or poly(A)-binding protein mRNA, all of which encode intracellular proteins. We have previously demonstrated that albumin mRNA is degraded in both messenger ribonucleoprotein and polysome fractions. Sucrose gradient analysis demonstrates the same pattern for degradation of all other serum protein-coding mRNAs. Estrogen has no effect on the amounts or gradient distribution of actin, ferritin, or poly(A)-binding protein mRNA. We conclude that regulated destabilization of mRNAs encoding secreted proteins is a generalized phenomenon in response to estrogen stimulation of Xenopus liver.

The estrogen-regulated destabilization of Xenopus albumin mRNA is independent of translation

Protein synthesis inhibitors have been shown to increase the stability of a number of labile mRNAs. In Xenopus laevis serum albumin mRNA is destabilized in the liver cell cytoplasm following estrogen administration. The present study examined the effect of translation inhibitors on this process. The initiation inhibitor 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide causes accumulation of albumin mRNA in 20–80S mRNP particles whereas the elongation inhibitor cycloheximide causes albumin mRNA to accumulate in polysomes. Neither inhibitor blocked the disappearance of albumin mRNA from liver cell cytoplasm when added with estradiol to the medium of liver explant cultures. We conclude that unlike a number of labile mRNAs the instability of Xenopus albumin mRNA following estradiol is independent of translation.

Induction of Porphyrin Biosynthesis in the Non-Animal Experimental Model of Tissue Explant Cultures: Prevention and Reversal by the Antimitotic Colchicine

The addition of different concentrations (5–20mg/dish) of allyl isopropylacetamide (AIA) and veronal (VER) to mouse liver, skin and heart tissue explant cultures increased the biosynthesis of porphyrin from endogenous precursors several-fold as compared to controls. Maximal effects were observed after a 12-hour incubation in liver culture for both AIA and VER (at 5mg drug/dish); after 2 and 6 hours in skin for AIA (10mg/dish) and VER (5mg/dish). In heart, with AIA (5mg/dish) and VER (20mg/dish), a peak was found after 2 hours, but a second increase was observed later after AIA induction. Colchicine (O.1mg/dish) did not by itself alter porphyrin biosynthesis by any tissue However, when added before or after porphyrin induction by AIA, it was able to both prevent and reverse the effect of the inducer in liver explants only, having no action whatever on other tissues or when VER was used. The usefulness of the non-animal experimental model of tissue explant cultures for investigating the porphyrinogenic properties and/or toxicity of drugs and their mechanism of action is emphasised.

62] Cultured avian liver explants for studies of lipogenic enzymes

Publisher Summary This chapter describes the cultured avian liver explants for the studies of lipogenic enzymes. Most primary liver cell culture systems for the study of lipid metabolism involve disassociated hepatocytes and utilize serum that contains many hormones, unknown growth factors, and lipids, and thus makes it difficult to evaluate the role of hormones and lipids in the regulation of lipogenic enzymes. The characterization of an embryonic avian liver explant culture system maintained in a chemically defined culture medium, not containing serum, has made it possible to probe into the mechanism of hormonal induction of lipogenic enzymes, such as fatty acid synthase, stearoyl-CoA desaturase, and malic enzyme. The explants cultured in control medium lacking hormones maintained their initial specific activities of [U- 14 C]glucose oxidation to CO 2 , [ 3 H]uridine incorporation into RNA, and [ 3 H]leucine incorporation into cellular proteins. These results indicate that the functional viability of the liver can be maintained in explant culture for at least 120 h. The liver explant culture system is well suited for the investigation of mechanism by which insulin induces lipogenic enzymes.

Neurotrophic effects on the electrical properties of cultured muscle produced by conditioned medium from spinal cord explants

The passive electrical properties of cultured chick skeletal muscle are significantly altered when the muscle is co-cultured with spinal cord explants. A reduced transverse membrane resistance appears to be responsible for the smaller values of input resistance, space constant, and time constant observed in co-cultures relative to those observed in pure muscle cultures. In this report, we show that neuromuscular junctions are not required in order to observe this neurotrophic effect because medium from spinal cord explant cultures is capable of producing the same reduction in transverse membrane resistance as the co-culturing of spinal cord explants with muscle. Control medium from liver explant cultures has no effect on muscle passive electrical properties. These results indicate that a trophic substance which is capable of regulating the electrical properties of excitable cells is released into the culture medium by spinal cord explants.

Mechanism of cellular damage by virus: A study of antihistamine drugs: I. Murine hepatitis virus and liver explant cultures

1. 1. Antihistamine drugs, notably diphenhydramine, afford considerable protection against liver injury caused by MHV3 virus (Gledhill virus). 2. 2. This protective effect is obtained both in vivo and in vitro. The evidence strongly supports the view that protection is not dependent upon a direct effect on the virus or on its multiplication. 3. 3. It is suggested that hepatitis virus has a twofold action on the liver: one a specific action, and the second a nonspecific component which is inhibited by antihistamine drugs, and which is common to many types of cellular injury. The nature of this common component is discussed. 4. 4. Under the protective influence of diphenhydramine, an artificial carrier state has been induced in the cell-virus system. Up to a point, this is reversible, and removal of the drug leads to cell destruction. After this point, the virus fails to destroy the cells. This final state has been studied to see if resistance to further infection has been conferred upon the cells.