Suppressor of cytokine signaling 1 protects mice against concanavalin A-induced hepatitis by inhibiting apoptosis

Iron overload exacerbates various liver diseases. In hepatocytes, a portion of non-heme iron is sequestered in lysosomes and endosomes. The precise mechanisms by which lysosomal iron participates in hepatocellular injury remain uncertain. Here, our aim was to determine the role of intracellular movement of chelatable iron in oxidative stress-induced killing to cultured hepatocytes from C3Heb mice and Sprague-Dawley rats. Mitochondrial polarization and chelatable iron were visualized by confocal microscopy of tetramethylrhodamine methylester (TMRM) and quenching of calcein, respectively. Cell viability and hydroperoxide formation (a measure of lipid peroxidation) were measured fluorometrically using propidium iodide and chloromethyl dihydrodichlorofluorescein, respectively. After collapse of lysosomal/endosomal acidic pH gradients with bafilomycin (50 nM), an inhibitor of the vacuolar proton-pumping adenosine triphosphatase, cytosolic calcein fluorescence became quenched. Deferoxamine mesylate and starch-deferoxamine (1 mM) prevented bafilomycin-induced calcein quenching, indicating that bafilomycin induced release of chelatable iron from lysosomes/endosomes. Bafilomycin also quenched calcein fluorescence in mitochondria, which was blocked by 20 microM Ru360, an inhibitor of the mitochondrial calcium uniporter, consistent with mitochondrial iron uptake by the uniporter. Bafilomycin alone was not sufficient to induce mitochondrial depolarization and cell killing, but in the presence of low-dose tert-butylhydroperoxide (25 microM), bafilomycin enhanced hydroperoxide generation, leading to mitochondrial depolarization and subsequent cell death. CONCLUSION: Taken together, the results are consistent with the conclusion that bafilomycin induces release of chelatable iron from lysosomes/endosomes, which is taken up by mitochondria. Oxidative stress and chelatable iron thus act as two "hits" synergistically promoting toxic radical formation, mitochondrial dysfunction, and cell death. This pathway of intracellular iron translocation is a potential therapeutic target against oxidative stress-mediated hepatotoxicity.     

Nitric oxide protects rat hepatocytes against reperfusion injury mediated by the mitochondrial permeability transition

We investigated the effects of nitric oxide (NO) on hepatocellular killing after simulated ischemia/reperfusion and characterized signaling factors triggering cytoprotection by NO. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 hours and reoxygenated at pH 7.4 for 2 hours. During reoxygenation, some hepatocytes were exposed to combinations of NO donors (S-nitroso-N-acetylpenicillamine [SNAP] and others), a cGMP analogue (8-bromoguanosine-3,5-cGMP [8-Br-cGMP]), and a cGMP-dependent protein kinase inhibitor (KT5823). Cell viability was determined by way of propidium iodide fluorometry. Inner membrane permeabilization and mitochondrial depolarization were monitored by confocal microscopy. SNAP, but not oxidized SNAP, increased cGMP during reperfusion and decreased cell killing. Other NO donors and 8-Br-cGMP also prevented cell killing. Both guanylyl cyclase and cGMP-dependent kinase inhibition blocked the cytoprotection of NO. However, 5-hydroxydecanoate and diazoxide- mitochondrial K(ATP) channel modulators-did not affect NO-dependent cytoprotection or reperfusion injury. During reoxygenation, confocal microscopy showed mitochondrial repolarization, followed by depolarization, inner membrane permeabilization, and cell death. In the presence of either SNAP or 8-Br-cGMP, mitochondrial repolarization was sustained after reperfusion preventing inner membrane permeabilization and cell death. In isolated rat liver mitochondria, a cGMP analogue in the presence of a cytosolic extract and adenosine triphosphate blocked the Ca(2+)-induced mitochondrial permeability transition (MPT), an effect that was reversed by KT5823. In conclusion, NO prevents MPT-dependent necrotic killing of ischemic hepatocytes after reperfusion through a guanylyl cyclase and cGMP-dependent kinase signaling pathway, events that may represent the target of NO cytoprotection in preconditioning.     

Melatonin induces mitochondrial-mediated apoptosis in human myeloid HL-60 cells

Abstract:  The role of melatonin in the mediation of apoptotic events has recently gained attention, especially after recent studies have reported that melatonin exerts antiapoptotic actions in normal cells but may activate proapoptotic pathways in some tumor cells. Here, we have evaluated the effect of melatonin on apoptosis in the human leukemia cell line HL-60. Melatonin treatment (1 m m ) induced a significant increase in caspase-3 and -9 activities. The effect of melatonin on the activation of caspases was time dependent, reaching a maximum after 12 hr of stimulation, and then decreasing to a minimum after 72 hr. Treatment with melatonin also evoked mitochondrial membrane depolarization and permeability transition pore induction, which caused loss of mitochondrial staining by calcein, and increased cell death by apoptosis/necrosis as demonstrated by propidium iodide positive-staining of cells after 72 hr of stimulation. In addition, the exposure of cells to melatonin resulted in an activation and association of the proapoptotic proteins Bax and Bid, as well as promoting detectable increases in the expression of both proteins. We conclude that melatonin has proapoptotic and/or oncostatic effects in the human myeloid cell line HL-60.     

Evaluation of annexin V and Calcein-AM as markers of mononuclear cell apoptosis during human immunodeficiency virus infection

Evaluation of apoptosis by flow cytometry is generally accomplished by methods that use annexin V-FITC as vital dye, which access phosphatidylserine exposed on the external membrane at the beginning of this process. In addition, the concomitant use of propidium iodide makes possible to verify the characteristic nuclear alterations in the late stages of apoptosis, as a consequence of the increase in membrane permeability. On the other hand, the use of calcein-AM in association with ethidium homodimer (EthD-1) allows the evaluation of cell apoptosis through detection of esterase activity and cellular membrane physical and chemical alterations. The aim of this study was to compare the sensibility of calcein-AM and EthD-1 with annexin V-FITC and propidium iodide for early apoptosis evaluation in peripheral blood mononuclear cell culture, obtained from HIV-infected patients. Apoptosis and cellular viability were detected and quantified by flow cytometry after 24 and 48 hours incubation times. Our results showed that calcein-AM/EthD-1 was more sensitive for apoptotic cell quantification in both incubation times than annexin V-FITC/propidium iodide (mean of 46.95% +/- 3.56, p < 0.0001, for 24 hours and mean of 37.67% +/- 2.47, p < 0.0014 for 48 hours), besides allowing to clearly define viable, apoptotic and dead cell populations.     

Cyclosporine A attenuates mitochondrial permeability transition and improves mitochondrial respiratory function in cardiomyocytes isolated from dogs with heart failure

We used isolated cardiomyocytes to investigate a possible role of mitochondrial permeability transition pore in mitochondrial abnormalities associated with heart failure. Cardiomyocytes were isolated from LV myocardium of normal control dogs and dogs with heart failure produced by intracoronary microembolizations. Mitochondrial permeability transition was measured in isolated cardiomyocytes with intact sarcolemma with and without 0.2 microM cyclosporin A using calcein AM and the fluorometer. State-3 mitochondrial respiration was also measured with the Clark electrode. Mitochondrial membrane potential was measured with JC-1 probe using the fluorometer. Propidium iodide was used to ensure sarcolemma integrity. 200 min after loading with calcein AM, mitochondria of failing cardiomyocytes showed only 50% of maximal level of calcein fluorescence while it remained unchanged in normal cells. The mitochondrial membrane potential in failing cardiomyocytes was significantly decreased by 38% compared to normal cardiomyocytes. Cyclosporine A significantly slowed the exit of calcein from mitochondria of failing cardiomyocytes and increased mitochondrial membrane potential by 29%. State-3 respiration was not affected with cyclosporine A in normal cardiomyocytes while it was significantly increased in failing cardiomyocytes by 20%. Exit of calcein (m.w. 1.0 kDa) from mitochondria of viable failing cardiomyocytes with intact sarcolemma suggests an existence of a reversible transitory permeability transition opening in high conductance mode. Attenuation of calcein exit, DeltaPsi(m) and improvement of state-3 respiration achieved with CsA (0.2 microM) show that permeability transition opening could be a cause of mitochondrial dysfunction described in the failing heart.     

Hypoxic conformance of metabolism in primary rat hepatocytes: a model of hepatic hibernation

Following prolonged hypoxia, mammalian cells invoke adaptive mechanisms to enhance oxygen delivery and promote energy conservation. We previously reported that hepatocytes subjected to prolonged moderate hypoxia (PO2 = 20-50 mmHg for > 3 hours) demonstrated a reversible inhibition of cellular respiration with maintenance of cell viability, associated with a decrease in mitochondrial adenosine triphosphate (ATP) synthesis; acute hypoxia (similar PO2 for < 30 minutes) did not induce a similar suppression of respiration and ATP synthesis. In the current study, using an in vitro model of primary rat hepatocytes, we measured the changes in metabolic demand for ATP during hypoxic conformance, and tested whether viability is maintained by preferentially suppressing nonessential processes while sustaining processes essential for maintaining cell homeostasis. In addition, the rate of recovery of oxygen consumption and ATP concentrations following reoxygenation after prolonged and acute hypoxia/anoxia was compared. Oxygen consumption and ATP concentrations decreased during prolonged hypoxia compared with acute hypoxia. However, ouabain-inhibitable respiration did not decrease during prolonged hypoxia, indicating that membrane Na+/K+ ATPase activity, an essential process for cell viability, was maintained. In contrast, ATP-dependent glucuronidation and sulfation of acetaminophen, deemed "non-essential" processes, were decreased significantly compared with normoxic cells. After reoxygenation, cells exposed to prolonged moderate hypoxia demonstrated a more rapid recovery of respiration compared to acute hypoxia/anoxia. Conclusion: This "hepatic hibernation" during prolonged moderate hypoxia may represent an anticipatory adaptation that seeks to maintain cell viability while delaying or preventing the onset of lethal hypoxia, and facilitates rapid recovery after the resumption of normoxia.     

Cytochrome P450-generated reactive metabolites cause mitochondrial permeability transition, caspase activation, and apoptosis in rat hepatocytes

Although cytochrome P-450 (CYP)-generated reactive metabolites can cause hepatocyte apoptosis, the mechanism of this effect is incompletely understood. In the present study, we assessed the hepatotoxicity of skullcap, a diterpenoid-containing herbal remedy. Male rat hepatocytes were incubated for 2 hours with skullcap diterpenoids (100 microg/mL). This treatment decreased cell glutathione and protein thiols and increased cell [Ca(2+)]. This activated Ca(2+)-dependent tissue transglutaminase, forming a cross-linked protein scaffold, and also opened the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, increased cytosolic cytochrome c, activation of procaspase 3, internucleosomal DNA fragmentation, and ultrastructural features of apoptosis. Cell death was increased by a CYP3A inducer (dexamethasone) or a sulfur amino acid-deficient diet increasing glutathione depletion. In contrast, cell death was prevented by decreasing CYP3A activity (with troleandomycin), preventing glutathione depletion (with cysteine or cystine), blocking Ca(2+)-modulated events (with calmidazolium), preventing mitochondrial permeability transition (with cyclosporin A), or inhibiting caspase 3 (with acetyl-Asp-G u-Va-Asp-a dehyde). Both calmidazolium and cyclosporin A also prevented the increase in cytosolic cytochrome c and procaspase 3 activation. In conclusion, CYP3A activates skullcap diterpenoids into reactive metabolites that deplete cellular thiols and increase cell [Ca(2+)]. This activates Ca(2+)-dependent transglutaminase and also opens the mitochondrial permeability transition pore, causing outer mitochondrial membrane rupture, cytochrome c release, and caspase activation. Preventing mitochondrial permeability transition pore opening and/or caspase activity blocks apoptosis, showing the fundamental role of these final events in metabolite-mediated hepatotoxicity.     

Early midzonal oxidative stress preceding cell death in hypoperfused rat liver.

Intralobular heterogeneity of oxidative stress and its topographic relationship with cell death during low-flow hypoxia were shown in perfused rat liver by digital microfluorography using dichlorofluorescin diacetate, a fluorochrome sensitive to intracellular hydroperoxide formation, and propidium iodide, which labels the nuclei of nonviable cells. The surface of the liver loaded with two precursors was microscopically visualized, and the fluorescence of dichlorofluorescein, a highly fluorescent molecule generated by hydroperoxide-mediated dichlorofluorescin oxidation, was digitally processed. Dichlorofluorescein fluorescence significantly increased in midzonal regions as early as 20 minutes after starting the 25% low-flow hypoxia. At 40 minutes the fluorograph showed multiple dotted patterns, and the intensity peaked at 60 minutes. The onset of cell death studied by propidium iodide was observed at 40 minutes, and its topographic distribution corresponded to the dichlorofluorescein-enhanced midzonal regions. Allopurinol diminished the early midzonal oxidative stress and retarded the onset of cell death. The current findings show that xanthine oxidase-dependent oxidative stress and the resultant cell death during low-flow hypoxia are spatially restricted in the intermediate zone between the periportal and pericentral regions.     

The effect of phenothiazines upon maintenance of membrane integrity in the cultured myocardial cell

The cultured myocardial cell provides a defined model for examining factors which are responsible for maintaining cellular viability and sarcolemmal integrity. Our data indicates that the spontaneous loss of myocyte membrane integrity is a calcium-dependent process and thus provides a method for examining the mechanism through which calcium exerts this effect. Antimyosin antibody staining and propidium iodide uptake were used to quantitate membrane integrity. The integrity of the cell membrane was inversely related to the calcium concentration in the culture medium. This loss of membrane integrity was calmodulin-dependent as demonstrated by the following: phenothiazines (trifluoperazine greater than chlorpromazine greater than promethazine) and structurally dissimilar calmodulin-inhibitors prevented the formation of sarcolemmal defects at concentrations similar to those known to inhibit calmodulin; phenothiazines and calcium demonstrated a competitive interaction with respect to this effect on membrane integrity. Electron microscopy confirmed the integrity of the sarcolemma of the cells exposed to high phenothiazine concentrations although metabolic alterations occurred in these cells as evidenced by an increased membrane permeability to the low molecular weight probe propidium iodide, degenerative changes in the fine structure of the mitochondria, the accumulation of autophagic vacuoles in the cytoplasm and the loss of contractile ability. These findings indicate that calmodulin inhibitory compounds are capable of preserving the membrane integrity of cardiac myocytes, interfering with a calcium-dependent process that is associated with the spontaneous attrition of these cells in culture. Significant intracellular alterations appear at high doses of these agents even while the sarcolemma is free of gross defects.     

Contribution of increased mitochondrial free Ca2+ to the mitochondrial permeability transition induced by tert-butylhydroperoxide in rat hepatocytes

Previously, we showed that the oxidant chemical, tert-butylhydroperoxide (t-BuOOH), induces a mitochondrial permeability transition (MPT) in intact hepatocytes, causing lethal cell injury. Here, we investigated the role of mitochondrial free Ca2+ in t-BuOOH cytotoxicity to 1-day-cultured rat hepatocytes using confocal microscopy of autofluorescence and parameter-indicating fluorophores. t-BuOOH (100 micromol/L) caused an early increase of mitochondrial free Ca2+, as assessed by confocal microscopy of Rhod-2 fluorescence. Increased mitochondrial Ca2+ was followed by onset of the MPT, as evidenced by permeation of cytosolic calcein into mitochondria and loss of the mitochondrial membrane potential-indicating dye, tetramethylrhodamine methylester. Preincubation with an intracellular Ca2+ chelator (BAPTA-AM and its derivatives) partially blocked the late phase of mitochondrial NAD(P)H oxidation after t-BuOOH, but failed to prevent the early oxidation of mitochondrial NAD(P)H. Ca2+ chelation also prevented the increase of mitochondrial Ca2+, generation of mitochondrial reactive oxygen species (ROS), onset of the MPT, and subsequent cell death. Confocal images showed that protection occurred when loading of the Ca2+ chelator was predominantly mitochondrial. The antioxidant, desferal, also diminished increased mitochondrial Ca2+ after t-BuOOH and prevented cell death. We conclude that oxidative stress induced by t-BuOOH enhances mitochondrial Ca2+ uptake, leading to increased matrix Ca2+, increased ROS formation, onset of the MPT, and cell death.     

Ventilator-induced cell wounding and repair in the intact lung

We tested the hypothesis that cells of ventilator-injured lungs are subject to reversible plasma membrane stress failure. Rat lungs were perfused with the membrane impermeable fluorescent marker propidium iodide and randomized to one of four ventilation strategies. Subpleural lung regions were imaged with confocal microscopy, and cell injury was quantified as the number of propidium iodide-positive cells per alveolus. The number of injured cells was significantly greater in lungs ventilated with large tidal volumes and zero end-expiratory pressure than in lungs ventilated with small tidal volumes and positive end-expiratory pressure (p < 0.01). Cell injury correlated with lung weight gain, change in dynamic compliance, and histologic injury scores. In a second set of experiments, lungs were mechanically ventilated for 30 minutes at high tidal volume settings, whereas propidium iodide was perfused either during or after injurious ventilation. Labeling after removal of injurious stress revealed significantly fewer injured cells (0.25 +/- 0.09 to 0.08 +/- 0.08, p < 0.01). We conclude that cells of ventilator-injured lungs are subject to reversible plasma membrane stress failure.     

Opening of the mitochondrial permeability transition pore causes matrix expansion and outer membrane rupture in Fas-mediated hepatic apoptosis in mice

Although Fas stimulation has been reported to cause outer mitochondrial membrane rupture in Jurkat cells, the mechanism of this effect is debated, and it is not known if outer membrane rupture also occurs in hepatocyte mitochondria. We studied the in vivo effects of Fas stimulation on ultrastructural lesions and mitochondrial function in mice. Four hours after administration of an agonistic anti-Fas antibody (8 microg/animal), caspase activity increased 5.4-fold. Nuclear DNA showed internucleosomal fragmentation, whereas supercoiled mitochondrial DNA was replaced by circular and linear forms. Mitochondrial cytochrome c was partly released into the cytosol. Ultrastructurally, mitochondrial lesions were observed in both apoptotic hepatocytes (with nuclear chromatin condensation/fragmentation) and nonapoptotic hepatocytes (without nuclear changes). In nonapoptotic cells, outer mitochondrial membrane rupture allowed herniation of the inner membrane and matrix through the outer membrane gap. In apoptotic hepatocytes, the matrix became electron-lucent and no longer protruded through the outer membrane gap. Mitochondria clustered around the nucleus, whereas rough endoplasmic reticulum cisternae became peripheral. In liver mitochondria isolated after Fas stimulation, the membrane potential decreased, whereas basal respiration increased. Pretreatment with either z-VAD-fmk (an inhibitor of caspases) or cyclosporin A (a permeability transition inhibitor) totally or mostly prevented mitochondrial outer membrane rupture, membrane potential decrease, cytochrome c release, and apoptosis. In conclusion, in vivo Fas stimulation causes caspase activation, mitochondrial permeability transition (decreasing the membrane potential and increasing basal respiration), mitochondrial matrix expansion (as shown by matrix herniation), outer mitochondrial membrane rupture, and cytochrome c release.     

Expanding hepatocytes in vitro before cell transplantation: donor age-dependent proliferative capacity of cultured human hepatocytes

BACKGROUND: For hepatocyte transplantation as well as experimental purposes, it would be advantageous to be able to expand human hepatocytes in vitro. However, under serum-free conditions, even with supplements of HGF (hepatic growth factor) and EGF (epidermal growth factor), proliferation of human hepatocytes is hampered. The aim of this study was to identify differences in the proliferative capacity of cultured primary human hepatocytes related to the age of the liver donors. METHODS: Proliferation was determined by BrdU-uptake, ploidy was measured using propidium iodide staining and flow cytometry, and the expression of cell cycle related proteins was determined by Western blotting. RESULTS: During the initial culture, juvenile hepatocytes proliferated better than adult hepatocytes. The proliferation rate declined to barely detectable levels after 8 days in culture in both juvenile and adult hepatocytes. The higher proliferative capacity of juvenile hepatocytes was associated with a larger fraction of diploid cells and a higher viability. The expression of regulatory cell cycle related proteins was higher in juvenile than in adult hepatocytes. CONCLUSIONS: The proliferation of human hepatocytes in vitro is critically related to a large fraction of diploid hepatocytes. The expression of regulatory cell cycle proteins reflects the proliferative capacity of cultured human hepatocytes. Juvenile as compared to adult human hepatocytes may be better suited for expansion in culture and could have a stronger repopulation capacity in vivo.     

Epimorphin protects hepatocytes from oxidative stress by inhibiting mitochondrial injury

Background and Aims: Many investigations have demonstrated that cell injuries caused by generation of reactive oxygen species (ROS) is a common mechanism of various hepatic disorders. Recently, we have demonstrated that epimorphin, originally cloned as a mesenchymal protein, protects cultured intestinal epithelial cells from ROS. We therefore examine whether epimorphin protects primary cultured hepatocytes from ROS‐induced cell injury. Methods: We explored the cell viability and the intracellular ROS levels of purified murine hepatocytes after exposure to 0.5 mM H₂O₂ with or without pretreatment of epimorphin. Then, we observed mitochondrial permeability transition (MPT) and depolarization using confocal microscopy to make clear the mechanism that epimorphin inhibited cell injuries after exposure to H₂O₂. In addition, to clarify the signaling pathways related to cell survival, we carried out Western blotting analysis with phosphorylated stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) polyclonal antibody to evaluate the inhibition of JNK by epimorphin. Finally, we evaluated the cell viability in hepatocytes administered JNK inhibitor. Results: Epimorphin protected primary cultured hepatocytes from H₂O₂‐induced cell injuries independent of intracellular ROS levels. Epimorphin also inhibited onset of MPT, depolarization of the mitochondrial membrane potential, and eventually cell killing. The cell protective function of epimorphin after exposure to H₂O₂ was not dependent on Akt signaling but on JNK signaling. Conclusion: Epimorphin can protect hepatocytes from MPT‐dependent cell injury induced by ROS. Since hepatic disorders could be caused by MPT‐dependent cell injuries with excessive ROS, epimorphin might open a new therapeutic avenue for hepatic disorders.     

Mechanistically distinct steps in the mitochondrial death pathway triggered by oxidative stress in cardiac myocytes

Oxidative stress plays an important role in the pathogenesis of cardiovascular diseases. In the present study, we characterize three distinct phases of the H2O2-induced response, which leads to loss of mitochondrial membrane potential (DeltaPsi(m)) and subsequent cell death in cultured cardiac myocytes. (1) Priming: After H2O2 exposure (100 micromol/L), cells maintain a constant DeltaPsi(m) for the cell-to-cell specific latency but at the same time undergo progressive changes in inner mitochondrial membrane structure (swelling and loss of cristae by electron microscopy). An increase of matrix calcium is required, but not sufficient, for this process. (2) Depolarization: Priming is followed by sudden depolarization of DeltaPsi(m), which is mediated by mitochondrial permeability transition pore opening, as evidenced by the concomitant release of calcein from mitochondria. This process is rapid (<4 minutes), complete, and irreversible. The duration of depolarization is constant and does not depend on the length of the priming process in any given cell. (3) Fragmentation: Along with massive mitochondrial swelling and release of cytochrome c into the cytoplasm, cells undergo surface membrane alterations, such as exposure of phosphatidylserine and eventual loss of membrane integrity and cellular fragmentation. Thus, oxidant stress elicits reproducible and stereotyped responses in cardiac cells. The priming phase, during which mitochondria undergo major ultrastructural alterations but remain functional, represents a particularly attractive target for intervention in the prevention of cell death.     

HepaRG cells: a human model to study mechanisms of acetaminophen hepatotoxicity

Acetaminophen (APAP) overdose is the leading cause of acute liver failure in Western countries. In the last four decades much progress has been made in our understanding of APAP-induced liver injury through rodent studies. However, some differences exist in the time course of injury between rodents and humans. To study the mechanism of APAP hepatotoxicity in humans, a human-relevant in vitro system is needed. Here we present evidence that the cell line HepaRG is a useful human model for the study of APAP-induced liver injury. Exposure of HepaRG cells to APAP at several concentrations resulted in glutathione depletion, APAP-protein adduct formation, mitochondrial oxidant stress and peroxynitrite formation, mitochondrial dysfunction (assessed by JC-1 fluorescence), and lactate dehydrogenase (LDH) release. Importantly, the time course of LDH release resembled the increase in plasma aminotransferase activity seen in humans following APAP overdose. Based on propidium iodide uptake and cell morphology, the majority of the injury occurred within clusters of hepatocyte-like cells. The progression of injury in these cells involved mitochondrial reactive oxygen and reactive nitrogen formation. APAP did not increase caspase activity above untreated control values and a pancaspase inhibitor did not protect against APAP-induced cell injury. CONCLUSION: These data suggest that key mechanistic features of APAP-induced cell death are the same in human HepaRG cells, rodent in vivo models, and primary cultured mouse hepatocytes. Thus, HepaRG cells are a useful model to study mechanisms of APAP hepatotoxicity in humans.     

Uncoupling protein-2 overexpression inhibits mitochondrial death pathway in cardiomyocytes

Uncoupling proteins (UCPs) are located in the mitochondrial inner membrane and partially dissipate the transmembrane proton electrochemical gradient. UCP2 is expressed in various human and rodent tissues, including the heart, where its functional role is unknown. In the present study, we tested the hypothesis that UCP2 overexpression could protect cardiomyocytes from oxidative stress-induced cell death by reducing reactive oxygen species (ROS) production in mitochondria. Using an adenoviral vector containing human UCP2, we investigated the effects of UCP2 overexpression on the mitochondrial death pathway induced by oxidative stress (100 micromol/L H2O2) in cultured neonatal cardiomyocytes. UCP2 overexpression significantly suppressed markers of cell death, including TUNEL positivity, phosphatidylserine exposure, propidium iodide uptake, and caspase-3 cleavage. Furthermore, UCP2 remarkably prevented the catastrophic loss of mitochondrial inner membrane potential induced by H2O2, which is a critical early event in cell death. Ca2+ overload and the production of ROS in mitochondria, both of which contribute to mitochondrial inner membrane potential loss, were dramatically attenuated by UCP2 overexpression. Thus, overexpression of UCP2 attenuates ROS generation and prevents mitochondrial Ca2+ overload, revealing a novel mechanism of cardioprotection.     

Protective effect of nitric oxide on isolated rat hepatocytes submitted to an oxidative stress

We have previously suggested that the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway protects both hepatocytes and endothelial cells against liver ischemia-reperfusion injury in rat. We study here the ability of NO to protect isolated hepatocytes against an in vitro oxidative stress induced with hypochlorite solution (ClO(-)). The severity of ClO(-)-induced stress was quantified by the measurement of total glutathione and membrane lipid peroxidation. Cell damage was assessed by morphologic (cell viability and bleb formation) and biologic (transaminase release) criteria. A 30-minute incubation of hepatocytes with 100 micromol/L ClO(-) maximally decreased cell viability (-40%) and increased bleb formation (+300%) and release of transaminases activities (aspartate transaminase [AST] = +60% and alanine transaminase [ALT] = +300%). A good correlation was observed between morphologic and biologic criteria. A preincubation of cells with 50 micromol/L 8-Br-cGMP, did not affect the adverse ClO(-) effects on the morphologic criteria. In the presence of 20 micromol/L spermineNONOate, an NO donor, ClO(-) did not decrease cell viability, whereas its deleterious effects on bleb formation was unchanged. A preincubation with a specific inhibitor of the soluble guanylate cyclase, the 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 micromol/L), did not affect the beneficial effect of NO on the cell viability. Our results suggest that NO protects hepatocytes against oxidative stress by a mechanism, which is cGMP-independent. However, taking into account the cytoprotective effects of cGMP in the liver, it is likely that the rapid effect of NO observed in vitro is relayed in vivo by a more long-lasting mechanism, which would be inhibited by ODQ and mimicked by 8-Br-cGMP.     

Induction of apoptosis and cell cycle arrest in human HCC MHCC97H cells with Chrysanthemum indicum extract

AIM： To investigate the effects of Chrysanthemum indicum extract （CIE） on inhibition of proliferation and on apoptosis, and the underlying mechanisms, in a human hepatocellular carcinoma （HCC） MHCC97H cell line. METHODS： Viable rat hepatocytes and human endothelial ECV304 cells were examined by trypan blue exclusion and MTT assay, respectively, as normal controls. The proliferation of MHCC97H cells was determined by MTT assay. The cellular morphology of MHCC97H cells was observed by phase contrast microscopy. Flow cytometry was performed to analyze cell apoptosis with annexin V/propidium iodide （PI）, mitochondrial membrane potential with rhodamine 123 and cell cycle with PI in MHCC97H cells. Apoptotic proteins such as cytochrome C, caspase-9, caspase-3 and cell cycle proteins, including P21 and CDK4, were measured by Western blotting. RESULTS： CIE inhibited proliferation of MHCC97H cells in a timeand dose-dependent manner without cytotoxicity in rat hepatocytes and human endothelial ceils. CIE induced apoptosis of MHCC97H cells in a concentration-dependent manner, as determined by flow cytometry. The apoptosis was accompanied by a decrease in mitochondrial membrane potential, release of cytochrome C and activation of caspase-9 and caspase-3. CIE arrested the cell cycle in the S phase by increasing P21 and decreasing CDK4 protein expression. CONCLUSION： CIE exerted a significant apoptotic effect through a mitochondrial pathway and arrested the cell cycle by regulation of cell cycle-related proteins in MHCC97H cells without an effect on normal cells. The cancer-specific selectivity shown in this study suggests that the plant extract could be a promising novel treatment for human cancer.     

线粒体膜电位对缺氧人肺动脉平滑肌细胞内氧自由基的变化及细胞增殖的作用

目的研究线粒体膜上ATP敏感钾通道(MitoKATP)的开放剂二氮嗪和线粒体膜电位在缺氧引起的人肺动脉平滑肌细胞(HPASMC)内氧自由基的变化及细胞增殖/凋亡失衡中的作用。方法培养HPASMC并将所培养的细胞分为6组正常对照组(A组);MitoKATP阻断剂5-羟基癸酸盐(5-HD)组(B组);MitoKATP开放剂二氮嗪组(C组);慢性缺氧组(D组);慢性缺氧+二氮嗪组(E组);慢性缺氧+5-HD组(F组),每组样本数均为6。利用激光共焦显微镜成像检测线粒体膜电位,荧光染色检测细胞内氧自由基含量,免疫组化法检测增殖细胞核抗原(PCNA)、c-fos及c-jun的蛋白表达和四甲基偶氮唑盐(MTT)法检测细胞增殖情况。结果C、D、E组细胞线粒体膜电位(以R123的荧光强度表示)分别为105±4、95±13、126±8,较A组(75±7)明显去极化(q值分别为5.474、3.659、9.213,P均<0.05);C、D、E组细胞内氧自由基含量分别为3045±126、3116±34、3236±31,与A组(2772±49)比较差异有统计学意义(q值分别为6.882、7.448、16.289,P均<0.05);C、D、E组细胞增殖活性[以MTT法检测出的A值表示]分别为0.305±0.022、0.328±0.078、0.440±0.023,与A组(0.237±0.013)比较差异有统计学意义(q值分别为2.993、4.017、8.919,P均<0.05),且E组线粒体膜电位、细胞内氧自由基含量、细胞增殖活性与D组比较差异有统计学意义(q值分别为5.554、8.841、4.902,P均<0.05)。F组线粒体膜电位、细胞内氧自由基含量、细胞增殖活性分别为71±4、2863±132、0.264±0.045,与D组(95±13、3116±34、0.328±0.078)比较差异有统计学意义(q值分别为4.367、5.907、2.832,P均<0.05)。结论二氮嗪或缺氧能够通过开放HPASMC线粒体膜上ATP敏感的钾通道,引起线粒体膜电位去极化,增加细胞内氧自由基的含量,最终导致HPASMC的增殖/凋亡的失衡,从而促进了缺氧性肺动脉重塑过程。