Apoptosis In Primary Hepatocytes Research Articles

Elevated cAMP Protects against Diclofenac-Induced Toxicity in Primary Rat Hepatocytes: A Protective Effect Mediated by the Exchange Protein Directly Activated by cAMP/cAMP-Regulated Guanine Nucleotide Exchange Factors.

Chronic consumption of the nonsteroidal anti-inflammatory drug diclofenac may induce drug-induced liver injury (DILI). The mechanism of diclofenac-induced liver injury is partially elucidated and involves mitochondrial damage. Elevated cAMP protects hepatocytes against bile acid-induced injury. However, it is unknown whether cAMP protects against DILI and, if so, which downstream targets of cAMP are implicated in the protective mechanism, including the classic protein kinase A (PKA) pathway or alternative pathways like the exchange protein directly activated by cAMP (EPAC). The aim of this study was to investigate whether cAMP and/or its downstream targets protect against diclofenac-induced injury in hepatocytes. Rat hepatocytes were exposed to 400 µmol/l diclofenac. Apoptosis and necrosis were measured by caspase-3 activity assay and Sytox green staining, respectively. Mitochondrial membrane potential (MMP) was measured by JC-10 staining. mRNA and protein expression were assessed by quantitative polymerase chain reaction (qPCR) and Western blot, respectively. The cAMP-elevating agent 7β-acetoxy-8,13-epoxy-1α,6β,9α-trihydroxylabd-14-en-11-one (forskolin), the pan-phosphodiesterase inhibitor IBMX, and EPAC inhibitors 5,7-dibromo-6-fluoro-3,4-dihydro-2-methyl-1(2H)-quinoline carboxaldehyde (CE3F4) and ESI-O5 were used to assess the role of cAMP and its effectors, PKA or EPAC. Diclofenac exposure induced apoptotic cell death and loss of MMP in hepatocytes. Both forskolin and IBMX prevented diclofenac-induced apoptosis. EPAC inhibition but not PKA inhibition abolished the protective effect of forskolin and IBMX. Forskolin and IBMX preserved the MMP, whereas both EPAC inhibitors diminished this effect. Both EPAC1 and EPAC2 were expressed in hepatocytes and localized in mitochondria. cAMP elevation protects hepatocytes against diclofenac-induced cell death, a process primarily involving EPACs. The cAMP/EPAC pathway may be a novel target for treatment of DILI. SIGNIFICANCE STATEMENT: This study shows two main highlights. First, elevated cAMP levels protect against diclofenac-induced apoptosis in primary hepatocytes via maintenance of mitochondrial integrity. In addition, this study proposes the existence of mitochondrial cAMP-EPAC microdomains in rat hepatocytes, opening new avenues for targeted therapy in drug-induced liver injury (DILI). Both EPAC1 and EPAC2, but not protein kinase A, are responsible for this protective effect. Our findings present cAMP-EPAC as a potential target for the treatment of DILI and liver injury involving mitochondrial dysfunction.

Co-exposure to multi-walled carbon nanotube and lead ions aggravates hepatotoxicity of nonalcoholic fatty liver via inhibiting AMPK/PPARγ pathway.

Multi-walled carbon nanotubes (MWCNTs) have been widely used in sewage disposal, water purification, and disinfection. Co-exposure to MWCNTs and heavy metal ions is common during water disposal. However, the hepatotoxicity of co-exposure to MWCNTs and lead ions for nonalcoholic fatty liver disease (NAFLD) subjects has not been investigated. NAFLD mice were fed intragastrically with MWCNTs and lead acetate (PbAc). Combined administration of MWCNTs and PbAc significantly damaged the liver function, and aggravated the nonalcoholic steatohepatitis phenotype as well as the hepatic fibrosis and steatosis in NAFLD mice. Furthermore, MWCNTs and PbAc significantly induced apoptosis in primary hepatocytes isolated from NAFLD mice. Combined administration of MWCNTs and PbAc also resulted in hepatic lipid peroxidation by inducing antioxidant defense system dysfunction, and significantly enhanced the expression levels of inflammatory cytokines in NAFLD mice livers. Meanwhile, combined administration of MWCNTs and PbAc may exert its hepatotoxicity in the NAFLD via inhibiting the adenosine 5‘-monophosphate activated protein kinase (AMPK)/peroxisome proliferator-activated receptors γ (PPARγ) pathway. Taken together, we conclude that co-exposure to MWCNTs and PbAc can remarkably aggravate the hepatotoxicity in NAFLD mice via inhibiting the AMPK/PPARγ pathway. This study may provide a biosafety evaluation for the application of nanomaterials in wastewater treatment.

Synergistic interaction of fatty acids and oxysterols impairs mitochondrial function and limits liver adaptation during nafld progression

The complete mechanism accounting for the progression from simple steatosis to steatohepatitis in nonalcoholic fatty liver disease (NAFLD) has not been elucidated. Lipotoxicity refers to cellular injury caused by hepatic free fatty acids (FFAs) and cholesterol accumulation. Excess cholesterol autoxidizes to oxysterols during oxidative stress conditions. We hypothesize that interaction of FAs and cholesterol derivatives may primarily impair mitochondrial function and affect biogenesis adaptation during NAFLD progression. We demonstrated that the accumulation of specific non-enzymatic oxysterols in the liver of animals fed high-fat+high-cholesterol diet induces mitochondrial damage and depletion of proteins of the respiratory chain complexes. When tested in vitro, 5α-cholestane-3β,5,6β-triol (triol) combined to FFAs was able to reduce respiration in isolated liver mitochondria, induced apoptosis in primary hepatocytes, and down-regulated transcription factors involved in mitochondrial biogenesis. Finally, a lower protein content in the mitochondrial respiratory chain complexes was observed in human non-alcoholic steatohepatitis. In conclusion, hepatic accumulation of FFAs and non-enzymatic oxysterols synergistically facilitates development and progression of NAFLD by impairing mitochondrial function, energy balance and biogenesis adaptation to chronic injury.

Inhibition of Drp1 protects against senecionine-induced mitochondria-mediated apoptosis in primary hepatocytes and in mice

Pyrrolizidine alkaloids (PAs) are a group of compounds found in various plants and some of them are widely consumed in the world as herbal medicines and food supplements. PAs are potent hepatotoxins that cause irreversible liver injury in animals and humans. However, the mechanisms by which PAs induce liver injury are not clear. In the present study, we determined the hepatotoxicity and molecular mechanisms of senecionine, one of the most common toxic PAs, in primary cultured mouse and human hepatocytes as well as in mice. We found that senecionine administration increased serum alanine aminotransferase levels in mice. H&E and TUNEL staining of liver tissues revealed increased hemorrhage and hepatocyte apoptosis in liver zone 2 areas. Mechanistically, senecionine induced loss of mitochondrial membrane potential, release of mitochondrial cytochrome c as well as mitochondrial JNK translocation and activation prior to the increased DNA fragmentation and caspase-3 activation in primary cultured mouse and human hepatocytes. SP600125, a specific JNK inhibitor, and ZVAD-fmk, a general caspase inhibitor, alleviated senecionine-induced apoptosis in primary hepatocytes. Interestingly, senecionine also caused marked mitochondria fragmentation in hepatocytes. Pharmacological inhibition of dynamin-related protein1 (Drp1), a protein that is critical to regulate mitochondrial fission, blocked senecionine-induced mitochondrial fragmentation and mitochondrial release of cytochrome c and apoptosis. More importantly, hepatocyte-specific Drp1 knockout mice were resistant to senecionine-induced liver injury due to decreased mitochondrial damage and apoptosis. In conclusion, our results uncovered a novel mechanism of Drp1-mediated mitochondrial fragmentation in senecionine-induced liver injury. Targeting Drp1-mediated mitochondrial fragmentation and apoptosis may be a potential avenue to prevent and treat hepatotoxicity induced by PAs.

Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway.

BackgroundNumerous pathological processes that affect liver function in patients with liver failure have been identified. Among them, hyperammonia is one of the most common phenomena.The purpose of this study was to determine whether hyperammonia could induced specific liver injury.MethodsHyperammonemic cells were established using NH4Cl. The cells were assessed by MTT, ELISA, and flow cytometric analyses. The expression levels of selected genes and proteins were confirmed by quantitative RT-PCR and western blot analyses.ResultsThe effects of 20 mM NH4Cl pretreatment on the cell proliferation and apoptosis of primary hepatocytes and other cells were performed by MTT assays and flow cytometric analyses. Significant increasing in cytotoxicity and apoptosis were only observed in hepatocytes. The cell damage was reduced after adding BAPTA-AM but unchanged after adding EGTA. The expression levels of caspase-3, cytochrome C, calmodulin, and inducible nitric oxide synthase were increased and that of bcl-2 was reduced. The Na+-K+-ATPase activities in hyperammonia liver cells was no signiaficant difference compaired with the control group, but was decreased in astrocytes. NH4Cl pretreatment of primary hepatocytes promoted the activation of mitochondrial permeability transition pores and the mitochondria swelled irregularly.ConclusionsHyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway.

OptPBN: An Optimisation Toolbox for Probabilistic Boolean Networks

BackgroundThere exist several computational tools which allow for the optimisation and inference of biological networks using a Boolean formalism. Nevertheless, the results from such tools yield only limited quantitative insights into the complexity of biological systems because of the inherited qualitative nature of Boolean networks.ResultsWe introduce optPBN, a Matlab-based toolbox for the optimisation of probabilistic Boolean networks (PBN) which operates under the framework of the BN/PBN toolbox. optPBN offers an easy generation of probabilistic Boolean networks from rule-based Boolean model specification and it allows for flexible measurement data integration from multiple experiments. Subsequently, optPBN generates integrated optimisation problems which can be solved by various optimisers.In term of functionalities, optPBN allows for the construction of a probabilistic Boolean network from a given set of potential constitutive Boolean networks by optimising the selection probabilities for these networks so that the resulting PBN fits experimental data. Furthermore, the optPBN pipeline can also be operated on large-scale computational platforms to solve complex optimisation problems. Apart from exemplary case studies which we correctly inferred the original network, we also successfully applied optPBN to study a large-scale Boolean model of apoptosis where it allows identifying the inverse correlation between UVB irradiation, NFκB and Caspase 3 activations, and apoptosis in primary hepatocytes quantitatively. Also, the results from optPBN help elucidating the relevancy of crosstalk interactions in the apoptotic network.SummaryThe optPBN toolbox provides a simple yet comprehensive pipeline for integrated optimisation problem generation in the PBN formalism that can readily be solved by various optimisers on local or grid-based computational platforms. optPBN can be further applied to various biological studies such as the inference of gene regulatory networks or the identification of the interaction's relevancy in signal transduction networks.

Alcohol potentiates HIV protease inhibitor‐induced ER stress and hepatic lipotoxicity (1001.3)

Alcohol consumption, which is highly prevalent in HIV‐infected individuals, has been associated with the amplification of liver toxicities induced by HIV protease inhibitors (PIs). We have demonstrated that HIV PI‐induced endoplasmic reticulum (ER) stress is linked to the dysregulation of hepatic lipid metabolism. However, little is known about the mechanistic pathways by which alcohol promotes HIV PI‐induced hepatic lipotoxicity. The aim of this study was to examine the cellular mechanisms by which alcohol promotes HIV PI‐induced hepatic lipotoxicity. Methods: Primary hepatocytes isolated from rat, wild type mouse or CHOP‐/‐ mouse were treated with individual HIV PIs with or without alcohol. Activation of the ER stress was determined by real time RT‐PCR and Western Blot. Apoptosis was detected by Annexin V‐FITC/propidium iodide staining. Intracellular lipid accumulation was determined by nile red staining. Results: Alcohol and HIV PIs synergistically induced ER stress, lipid accumulation and apoptosis in primary hepatocytes. Both alcohol and HIV PI‐induced lipid accumulation and apoptosis were significantly reduced in CHOP‐/‐ hepatocytes. Conclusions: Activation of the ER stress response is a key cellular mechanism underlying alcohol and HIV PI‐induced hepatotoxicity. Inhibition of ER stress activation may represent a new strategy to prevent alcohol and HIV PI‐associated hepatic lipotoxicity.Grant Funding Source: VA Merit Award

Ursodeoxycholyl Lysophosphatidylethanolamide Inhibits Lipoapoptosis by Shifting Fatty Acid Pools toward Monosaturated and Polyunsaturated Fatty Acids in Mouse Hepatocytes

Ursodeoxycholyl lysophosphatidylethanolamide (UDCA-LPE) is a hepatoprotectant in inhibiting apoptosis, inflammation, and hyperlipidemia in mouse models of nonalcoholic steatohepatitis (NASH). We studied the ability of UDCA-LPE to inhibit palmitate (Pal)-induced apoptosis in primary hepatocytes and delineate cytoprotective mechanisms. We showed that lipoprotection by UDCA-LPE was mediated by cAMP and was associated with increases in triglycerides (TGs) and phospholipids (PLs). An inhibitor of cAMP-effector protein kinase A partially reversed the protective effects of UDCA-LPE. Lipidomic analyses of fatty acids and PL composition revealed a shift of lipid metabolism from saturated Pal to monounsaturated and polyunsaturated fatty acids, mainly, oleate, docosapentaenoate, and docosahexaenoate. The latter two ω-3 fatty acids were particularly found in phosphatidylcholine and phosphatidylserine pools. The catalysis of Pal by stearoyl-CoA desaturase-1 (SCD-1) is a known mechanism for the channeling of Pal away from apoptosis. SCD-1 protein was upregulated during UDCA-LPE lipoprotection. SCD-1 knockdown of Pal-treated cells showed further increased apoptosis, and the extent of UDCA-LPE protection was reduced. Thus, the major mechanism of UDCA-LPE lipoprotection involved a metabolic shift from toxic saturated toward cytoprotective unsaturated fatty acids in part via SCD-1. UDCA-LPE may thus be a therapeutic agent for treatment of NASH by altering distinct pools of fatty acids for storage into TGs and PLs, and the latter may protect lipotoxicity at the membrane levels.

2,4-Dichlorophenol induces apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella) through mitochondrial pathway

2,4-Dichlorophenol (2,4-DCP), a major type of chlorophenols, has been widely used to produce some herbicides and pharmaceuticals, yet due to its incomplete degradation and bioaccumulation characteristics, it is toxic to aquatic organisms. Apoptosis is one of the most severe outcomes of cell poisoning and injury. So far, the potential molecular mechanism of 2,4-DCP-induced apoptosis has not been reported. This study showed that 2,4-DCP significantly induced apoptosis in primary hepatocytes of grass carp (Ctenopharyngodon idella). 2,4-DCP exposure upregulated mRNA of caspase-3, reduced the mitochondrial membrane potential (Δψm), increased intracellular reactive oxygen species (ROS) and the Bax/Bcl-2 ratio, while protection of mitochondria with acetyl-l-carnitine hydrochloride (ALC) rescued 2,4-DCP-induced apoptosis, restored the Δψm and reduced the Bax/Bcl-2 ratio. Taken together, this is the first study that has identified that 2,4-DCP exposure induced apoptosis through the mitochondria-dependent pathway in primary hepatocytes of grass carp.

Hepatoprotective effects of reynosin against thioacetamide-induced apoptosis in primary hepatocytes and mouse liver

The aim of this study was to identify the hepatoprotective effects of reynosin, sesquiterpenes from the leaves of Laurus nobilis, against thioacetamide (TAA)-induced apoptosis in primary hepatocyte cultures and an in vivo mouse model. Rat hepatocytes were isolated and pretreated with 0.13, 0.64, or 3.22 μM reynosin and then exposed to 100 mM TAA. Reynosin treatment significantly inhibited TAA-induced apoptosis and hepatocellular DNA damage in primary rat hepatocytes. We observed an increase in levels of antiapoptotic Bcl-2, Bcl-XL mRNA and a decrease in levels of proapoptotic Bax mRNA following reynosin treatment of hepatocytes. Apoptosis in BALB/c mice was induced with intra-peritoneal injection of 200 mg/kg TAA for 2 weeks every other day. Then reynosin (5 mg/kg) and TAA were intragastrically given for 3 weeks every other day. Aspartate aminotransferase and alanine aminotransferase levels in the blood of mice were decreased in the reynosin administration group. Bcl-2 and Bcl-XL mRNA levels were increased, and the Bax mRNA level was decreased in reynosin-treated mice. Thus, reynosin inhibited TAA-induced apoptosis in primary hepatocytes and an in vivo mouse model.

Effects of Cadmium on the Expression of Antioxidant Enzymes, Oxidative Stress and Apoptosis in Primary Hepatocytes of Carassius Auratus

The primary hepatocytes of Carassius auratus were incubated with 0, 0.01, 0.1, 1, 10 and 100 mg/l Cadmium(Cd2+) at 25°C for 8h in vitro. The results showed that the hepatocytes survival rate in the 1, 10 and 100 mg/l Cd2+ treated groups were significantly lower than that in the control, the percentage of apoptotic hepatocytes significantly increased in 0.1, 1 and 10 mg/l of Cd2+ treated groups, intracellular reactive oxygen species(ROS) and malondialdehyde (MDA) content significantly increased in 0.1, 1, 10 and 100 mg/l Cd2+ treated groups, typical DNA ladder was observed in 0.1, 1 and 10 mg/l Cd2+ treated groups, and ruleless DNA fragment occurred in 100 mg/l Cd2+ treated group. The activities of the antioxidant enzymes and the expression of their isozymes activities have similar change tendency, compared with the control group, the activities of superoxide dismutase (SOD) and their isozymes were activated by low concentration of Cd2+ (0.1 and 1 mg/l), however, their activities were inhibited by high concentration of Cd2+ (10 and 100 mg/l), especially, the special isozyme (SOD1) was found in the patterns of SOD isozymes in 0.1 and 1 mg/l Cd2+ treated groups, the activities of peroxidase(POD) and its isozymes significantly reduced with a concentration-dependent manner in all Cd Cd2+ treated groups, the activities of catalase(CAT) and its isozymes significantly decreased in higher concentration Cd2+ treated groups (1, 10 and 100mg/l). The results suggested that Cd2+ could damage the activities of the antioxidant enzymes and cause oxidative stress, lipid peroxidation, DNA fragmentation, apoptosis and necrosis in primary hepatocytes, contributing to the liver toxicity induced by Cd2+.

Modulation of Bax/Bcl-2 and caspases by probiotics during acetaminophen induced apoptosis in primary hepatocytes

Oxidative stress is an important factor in drug induced hepatotoxicity and antioxidants from natural sources have potential to ameliorate it. The present study was aimed to investigate cyto-protective potential of probiotic Enterococcus lactis IITRHR1 (El SN) and Lactobacillus acidophilus MTCC447 (La SN) lysate against acetaminophen (APAP) induced hepatotoxicity. Cultured rat hepatocytes pretreated with El SN/La SN showed higher cell viability under APAP stress. Pre-treatment with El SN, restored glutathione level and reduced ROS generation significantly which are major biomarkers of oxidative stress. It also reduced NO level, MDA formation and enhanced SOD activity. Pre-treatment with probiotic lysates significantly inhibited the translocation of pro-apoptotic protein (Bax), enhanced anti-apoptotic (Bcl-2) protein levels and prevented release of cyt c to cytosol; suggesting involvement of mitochondrial proteins in protection against APAP induced oxidative cellular damage. Loss in mitochondrial membrane potential due to APAP treatment was prevented in the presence of probiotic lysates. Protective action of El SN/La SN pretreatment was further supported by prevention of procaspase-3 activation, DNA fragmentation and chromatin condensation, in turn inhibiting APAP induced apoptotic cell death. The results indicate that probiotic preparations modulate crucial end points of oxidative stress induced apoptosis and may be used for management of drug induced liver injury.

Dual antitumour effect of 5-azacytidine by inducing a breakdown of resistance-mediating factors and epigenetic modulation

BackgroundThe cytokine tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) has shown promising anticancer activity in early clinical settings by selectively inducing apoptosis in different tumour types. However, some tumour entities such...

Tumor necrosis factor α sensitizes primary murine hepatocytes to Fas/CD95-induced apoptosis in a Bim- and Bid-dependent manner

Fas/CD95 is a critical mediator of cell death in many chronic and acute liver diseases and induces apoptosis in primary hepatocytes in vitro. In contrast, the proinflammatory cytokine tumor necrosis factor α (TNFα) fails to provoke cell death in isolated hepatocytes but has been implicated in hepatocyte apoptosis during liver diseases associated with chronic inflammation. Here we report that TNFα sensitizes primary murine hepatocytes cultured on collagen to Fas ligand (FasL)-induced apoptosis. This synergism is time-dependent and is specifically mediated by TNFα. Fas itself is essential for the sensitization, but neither Fas up-regulation nor endogenous FasL is responsible for this effect. Although FasL is shown to induce Bid-independent apoptosis in hepatocytes cultured on collagen, the sensitizing effect of TNFα is clearly dependent on Bid. Moreover, both c-Jun N-terminal kinase activation and Bim, another B cell lymphoma 2 homology domain 3 (BH3)-only protein, are crucial mediators of TNFα-induced apoptosis sensitization. Bim and Bid activate the mitochondrial amplification loop and induce cytochrome c release, a hallmark of type II apoptosis. The mechanism of TNFα-induced sensitization is supported by a mathematical model that correctly reproduces the biological findings. Finally, our results are physiologically relevant because TNFα also induces sensitivity to agonistic anti-Fas-induced liver damage. Our data suggest that TNFα can cooperate with FasL to induce hepatocyte apoptosis by activating the BH3-only proteins Bim and Bid.

Lipopolysaccharide triggered TNF-α-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model

Endogenous gut-derived bacterial endotoxins have been implicated as an important cofactor in the pathogenesis of liver injury, although their contribution to the progression of non-alcoholic steatohepatitis (NASH) remains unclear. Male C57BL/6 mice were fed a methionine-choline-deficient (MCD) diet or a standard diet for 17 days, following which they were injected with lipopolysaccharide (LPS) intraperitoneally and sacrificed after 6h. In an in vitro experiment, RAW264.7 cells, a mouse macrophage cell line, and primary mouse hepatocytes were co-treated with hydrogen peroxide (H(2)O(2)) and LPS or tumour necrosis factor (TNF)-alpha. Compared to the control mice, LPS treatment significantly increased hepatic TNF-alpha production in MCD mice. LPS also significantly increased TUNEL-positive cells, which were especially observed in the perivenular area. The apoptotic change was inhibited by co-treatment with a neutralizing anti-mouse TNF receptor antibody or pentoxifylline. In an in vitro experiment, treatment with H(2)O(2) synergistically enhanced LPS-induced TNF-alpha production in RAW264.7 cells, accompanied by an up-regulation of CD14 mRNA. Moreover, co-treatment with TNF-alpha- and H(2)O(2)-induced apoptosis in primary hepatocytes, although neither TNF-alpha nor H(2)O(2) could do so independently. LPS up-regulated TNF-alpha production, which induced hepatocyte apoptosis in a murine NASH model. LPS may play a key role in the pathogenesis of NASH.

Ferritin and FasL (CD95L) mediate density dependent apoptosis in primary rat hepatocytes

Based on a recent description of an apoptosis stimulating property for hepatocyte derived isoferritins, this investigation demonstrates that ferritin, released in vitro from hepatocytes substantially contributes to density dependent apoptosis in primary hepatocytes and is significantly (P < or = 0.05) inhibited by anti-H-ferritin antibody rH02. Furthermore, total protein release and albumin secretion rapidly decline in a time and density dependent mode under serum-free conditions, whereas ferritin secretion, which is upregulated at initial stages of primary culture is not affected by cell density. Supplementation with dexamethasone (DEX) or proliferative stimulation by epidermal growth factor (EGF) and insulin strongly suppresses density dependent apoptosis. Both regimens have previously been shown to inhibit isoferritin mediated apoptosis in hepatocytes, most likely by interrupting proapotitc mitochondrial signalling. Finally, FasL/Fas also participates in density dependent apoptosis, since apoptosis is significantly (P < or = 0.005) reduced in high density cultures supplemented with an anti-FasL antibody. This antibody has also been shown to neutralise ferritin mediated apoptosis in primary hepatocytes, suggesting a linkage of ferritin and Fas in density dependent apoptosis. In conclusion, ferritin contributes to apoptosis in primary hepatocytes in an autocrine, density dependent mode, involving Fas stimulation and proapoptotic mitochondrial signalling. With respect to liver physiology, these findings may indicate that ferritin plays a yet unrecognised role as an acute phase signalling molecule in early stages of tissue repair and liver regeneration, and may also be responsible for the limited ability to propagate human hepatocytes in culture and the limited expansion of donor cells in the recipient liver upon cell transplantation.

Regulating effect of apoptotic rate of hepatocyte on course of hepatic regeneration

Objective To investigate the role of different apoptotic rate induced by TGF-β1 in different stages of hepatic regeneration after partial hepatectomy(PH).Methods Rat model of 70% PH was established.Primary hepatocytes were isolated by in situ perfusion with collagenase solution from normal rats and those 1 d,3 d,5 d,7 d after PH.Flow cytometry was used to detect apoptotic rate in each group.The apoptosis of primary hepatocytes induced by TGF-β1(5 μg/L)was determined by staining with fluorescent dyes of DNA-binding Hoechst33258 and observed under fluorescent microscope.The effect of TGF-β1 on Bcl-2 expression in hepatoeytes was detected with immunocytochemistry.Results The viability of primary hepatocytes was 92%.The apoptotic rate of normal hepatocyte was 18.89%±3.14%,the apoptotic rate in 1 d was 11.85%±2.51%.After that it began to decrease,it was the lowest on the 3rd d.Then it began to increase gradually,it was 28.82%±5.53% on day 7 after PH.The apoptotic rate of normal hepatocytes induced by TGF-β1 was 58.13%±6.63%,which was higher than that in control groups(17.81%±3.19%,P＜0.05).However,the apoptotic rate of isolated primary hepatocytes had no difference between the groups induced and not induced by TGF-β1 1,3,5 d after PH.Bcl-2 expression in hepatocytes isolated in the early stage after PH was gradually increased and higher than that of normal group.The Bcl-2 expression was decreased significantly after being treated with TGF-β1.Conclusion TGF-β1 induced apoptotic rate has a marked difference in different stages of hepatic regeneration and it possesses an important regulating role for initiation and termination of hepatic regeneration. Key words: Liver regeneration; Apoptosis; TGF-β1

An inducible autocrine cascade regulates rat hepatocyte proliferation and apoptosis responses to tumor necrosis factor-α

Tumor necrosis factor-alpha (TNF) is an inflammatory cytokine that induces context-dependent proliferation, survival, and apoptosis responses in hepatocytes. TNF stimulates and enhances growth factor-mediated hepatocyte proliferation and survival following partial hepatectomy, but also acts in concert with other inflammatory cytokines of the innate immune response during viral infection to induce apoptosis in hepatocytes. In other epithelial cell types, TNF has recently been shown to stimulate autocrine release of transforming growth factor-alpha (TGF-alpha) and interleukin-1 (IL-1) family ligands. Here, we examine the role of these autocrine ligands in modulating TNF-induced proliferation and apoptosis in primary hepatocytes. We show that TNF-induced hepatocyte proliferation is regulated by an inducible, coupled, and self-antagonizing autocrine cascade involving the pro-proliferative TGF-alpha and IL-1 receptor antagonist (IL-1ra) ligands and antiproliferative IL-1alpha/beta ligands. Moreover, cooperative stimulation of hepatocyte proliferation by combined TNF and TGF-alpha treatment is self-limited through antiproliferative autocrine IL-1alpha/beta feedback. We show that TNF potently induces apoptosis of adenovirus-infected hepatocytes in a manner similarly determined through the integrated activity of a coupled TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade. Exogenous TGF-alpha can either enhance or diminish apoptosis in adenoviral vector-treated and TNF-treated hepatocytes, in a biphasic relationship also mediated by autocrine IL-1alpha/beta feedback. We demonstrate that TNF-induced hepatocyte proliferation and apoptosis are both governed by a self-antagonizing TGF-alpha-IL-1alpha/beta-IL-1ra autocrine cascade in vitro, and thus identify multiple molecular targets for control of TNF-regulated hepatocyte phenotypic responses related to liver regeneration and adenoviral gene therapy.

Interferon gamma promotes chemically induced apoptosis in primary hepatocytes by inducing p53 accumulation

Interferon gamma promotes chemically induced apoptosis in primary hepatocytes by inducing p53 accumulation

NF-κB protects rat ARL-6 hepatocellular carcinoma cells against hydrogen peroxide-induced apoptosis

Background/Aims: Refractoriness of some tumours to apoptosis has been related to over-expression of NF-κB, while NF-κB inhibition can promote apoptosis in several cell types. We compared NF-κB activation profiles between normal rat hepatocytes and ARL-6 rat hepatocellular carcinoma (HCC) cells exposed to hydrogen peroxide (H2O2), and examined whether NF-κB activation could explain the observed resistance to apoptosis of ARL-6 cells. We then infected ARL-6 cells with recombinant adenovirus containing mutant (non-degradable) I?B? (Adv-mI?B?), and examined whether this rendered ARL-6 cells more sensitive to oxidative stress-induced apoptosis. Methods: Cultured primary rat hepatocytes and ARL-6 cells were treated with graded doses of H2O2. To block NF-κB, ARL-6 cells were incubated with Adv-mIκBα for 24 h. Cytotoxicity, NF-κB activation, cell proliferation, and apoptosis were determined. Results: H2O2 induced more apoptosis in primary hepatocytes than ARL-6 cells, and the relative resistance of ARL-6 cells to H2O2-induced apoptosis was associated with more pronounced NF-κB activity. In ARL-6 cells, nuclear translocation of NF-κB took place within 2 h of administering H2O2 and remained prominent at 36 h. Adv-mI?B? sensitized ARL-6 cells to H2O2-induced apoptosis, but cell proliferation was minimally suppressed. Conclusions: Compared with normal hepatocytes, ARL-6 cells are refractory to apoptosis after exposure to H2O2, and this is associated with NF-κB activation. Conversely, NF-κB inhibition sensitises ARL-6 cells to H2O2-induced apoptosis. Sustained NF-?B activation in these HCC cells may protect them against apoptosis produced by oxidative stress.