cytochrome-c-t and cobaltous-chloride

Ischemia reperfusion injury (IRI) is a leading cause of acute kidney injury (AKI) in both native and transplanted kidneys. The objective of the present study was to evaluate whether low-molecular-weight fucoidan (LMWF) could attenuate renal IRI in an animal model and in vitro cell models and study the mechanisms in which LMWF protected from IRI.. Male mice were subjected to right renal ischemia for 30 min and reperfusion for 24 h, or to a sham operation with left kidney removed. Kidneys undergone IR showed characteristic morphological changes, such as tubular dilatation, and brush border loss. However, LMWF significantly corrected the renal dysfunction and the abnormal levels of MPO, MDA and SOD induced by IR. LMWF also inhibited the activation of MAPK pathways, which consequently resulted in a significant decrease in the release of cytochrome c from mitochondria, ratios of Bax/Bcl-2 and cleaved caspase-3/caspase-3, and phosphorylation of p53. LMWF alleviated hypoxia-reoxygenation or CoCl(2) induced cell viability loss and ΔΨm dissipation in HK2 renal tubular epithelial cells, which indicates LMWF may result in an inhibition of the apoptosis pathway through reducing activity of MAPK pathways in a dose-dependent manner.. Our in vivo and in vitro studies show that LMWF ameliorates acute renal IRI via inhibiting MAPK signaling pathways. The data provide evidence that LMWF may serve as a potential therapeutic agent for acute renal IRI.

Topics: Acute Kidney Injury; Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Cell Line; Cell Survival; Cobalt; Cytochromes c; Humans; Male; Malondialdehyde; MAP Kinase Signaling System; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Molecular Weight; Peroxidase; Phosphorylation; Polysaccharides; Proto-Oncogene Proteins c-bcl-2; Reperfusion Injury; Superoxide Dismutase; Tumor Suppressor Protein p53

Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl2-induced hypoxic/ischemic conditions. CoCl2 enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl2-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G2/M phase of the cell cycle. CoCl2 augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨm) and upregulated the voltage-dependent anion channel. In addition, CoCl2-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl2 led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl2 induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.

Topics: Animals; Apoptosis; Caspases; Cell Hypoxia; Cobalt; Cytochromes c; Embryonic Stem Cells; Fas Ligand Protein; fas Receptor; Hypoxia-Inducible Factor 1, alpha Subunit; Membrane Potential, Mitochondrial; Mice; Mitochondria; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Receptors, Death Domain; Signal Transduction; Transcriptional Activation; Tumor Suppressor Protein p53

To investigate whether mitochondria permeability transition pore (mPTP) opening was involved in ginsenoside Rb1 (Gs-Rb1) induced anti-hypoxia effects in neonatal rat cardiomyocytes ex vivo.. Cardiomyocytes were randomly divided into 7 groups: control group, hypoxia group (500 micromol/L CoCl(2)), Gs-Rb1 200 micromol/L group (CoCl(2) intervention+Gs-Rb1), wortmannin (PI3K inhibitor) 0.5 micromol/L group, wortmannin+Gs-Rb1 group, adenine 9-beta-D-arabinofuranoside (Ara A, AMPK inhibitor) 500 micromol/L group, and Ara A and Gs-Rb1 group. Apoptosis rate was determined by using flow cytometry. The opening of the transient mPTP was assessed by using co-loading with calcein AM and CoCl(2) in high conductance mode. Expression of GSK-3beta, cytochrome c, caspase-3 and poly (ADP-ribose) polymerase (PARP) was measured by using Western blotting. DeltaGSK-3beta was defined as the ratio of p-Ser9-GSK-3beta to total GSK-3beta.. CoCl(2) significantly stimulated mPTP opening and up-regulated the level of DeltaGSK-3beta. There was a statistically significant positive correlation between apoptosis rate and mPTP opening, between apoptosis rate and DeltaGSK-3beta, and between mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly inhibited mPTP opening induced by hypoxia (41.3%+/-2.0%, P<0.001) . Gs-Rb1 caused a 77.3%+/-3.2% reduction in the expression of GSK-3beta protein (P<0.001) and a significant increase of 1.182+/-0.007-fold (P=0.0001) in p-Ser9-GSK-3beta compared with control group. Wortmannin and Ara A significantly inhibited the effect of Gs-Rb1 on mPTP opening and DeltaGSK-3beta. Gs-Rb1 significantly decreased expression of cytochrome c (66.1%+/-1.7%, P=0.001), caspase-3 (56.5%+/-2.7%, P=0.001) and cleaved poly ADP-ribose polymerase (PARP) (57.9%+/-1.4%, P=0.001).. Gs-Rb1 exerted anti-hypoxia effect on neonatal rat cardiomyocytes by inhibiting GSK-3beta-mediated mPTP opening.

Topics: Androstadienes; Animals; Animals, Newborn; Apoptosis; Blotting, Western; Cardiotonic Agents; Caspase 3; Cell Hypoxia; Cells, Cultured; Cobalt; Cytochromes c; Flow Cytometry; Ginsenosides; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunohistochemistry; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Protein Kinase Inhibitors; Random Allocation; Rats; Wortmannin

20-Hydroxyecdysone, which is found in the rhizomes, roots and the stems of many plants, is an ecdysteroid hormone that regulates molting in insects. We have previously shown that 20-Hydroxyecdysone could alleviate neurological deficits induced by subarachnoid hemorrhage in rabbits. Thus, we hypothesized that 20-Hydroxyecdysone might protect neurons against hypoxic-ischemic injury. In present study, the effects of 20-Hydroxyecdysone on cobalt chloride (CoCl(2))-induced cellular injury in PC12 cells was investigated. The incubation of PC12 cells with CoCl(2) reduced the cell viability, increased the rate of apoptosis. However, when cells were treated with 20-Hydroxyecdysone before or after CoCl(2) exposure, the CoCl(2)-induced cellular injuries were significantly ameliorated. In addition, 20-Hydroxyecdysone dramatically reduced the CoCl(2)-induced production of reactive oxygen species (ROS), decreased the depolarization of the mitochondrial membrane, inhibited the release of cytochrome c into the cytosol and increased the Bax/Bcl-2 ratio. Furthermore, 20-Hydroxyecdysone eliminated the CoCl(2)-induced activation of caspase-3. Taken together, these results indicate that 20-Hydroxyecdysone may protect PC12 cells against CoCl(2)-induced cell injury by inhibiting ROS production and modulating components of the mitochondrial apoptosis pathway. Based on our results, 20-Hydroxyecdysone may be a potential candidate for intervention in hypoxic-ischemic brain injuries such as stroke.

Topics: Animals; Apoptosis; Blotting, Western; Caspase 3; Cell Survival; Cobalt; Cytochromes c; Ecdysterone; Enzyme Activation; Flow Cytometry; In Situ Nick-End Labeling; Membrane Potential, Mitochondrial; PC12 Cells; Rats; Reactive Oxygen Species

In hypoxic/ischemic conditions, neuronal apoptotic events are occurred, resulting in neuronal diseases. Estradiol is a female sex hormone with steroid structure known to provide neuroprotection through multiple mechanisms in the central nervous system. This study was aimed to investigate the signal transduction pathway leading to the inhibitory effects of estradiol against cobalt chloride (CoCl(2))-mediated hypoxic death in PC12 cells. Estradiol inhibits CoCl(2)-induced cell death with genomic DNA fragmentation and morphologic changes such as cell shrinkage and condensed nuclei. Pre-incubation of estradiol prior to CoCl(2) treatment attenuated CoCl(2)-mediated the reactive oxygen species (ROS) production and limited the activities of the caspase cascades, such as caspase-8, -9 and -3. Furthermore, estradiol downregulated the Bax:Bcl-2 ratio and decreased the release of cytochrome c from the mitochondria into the cytosol in CoCl(2)-treated cells, indicating that estradiol affect on mitochondrial pathway. Estradiol attenuated also CoCl(2)-induced upregulation of Fas-ligand (Fas-L) and truncated of Bid in sequence of death receptor-mediated pathway. In addition, estradiol increased the phosphorylation of Akt in CoCl(2)-treated cells, demonstrating that estradiol has no affect on upstream signaling through the PI3K/Akt in inhibition of CoCl(2)-induced apoptosis in PC12 cells. Taken together, estradiol was found to have a neuroprotective effect against CoCl(2)-induced apoptosis of PC12 cells by the attenuating ROS production and the modulating apoptotic signal pathway through Bcl-2 family, cytochrome c, Fas/Fas-L as well as PI3K/Akt pathway.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Cobalt; Cytochromes c; DNA Fragmentation; Electrophoresis, Agar Gel; Enzyme Activation; Estradiol; Estrogens; Fas Ligand Protein; fas Receptor; Mitochondria; PC12 Cells; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Rats; Reactive Oxygen Species; Reverse Transcriptase Polymerase Chain Reaction

A new, simple and sensitive method for the quantitative analysis of cytochrome C (Cyt C) based on the reduction wave of guanidine modified Co(II)-Cyt C complex at about -1.74 V (vs. SCE) by single sweep polarography in the solution containing 8 x 10(-6) mol L(-1) CoCl2, 0.04 mol L(-1) guanidine hydrochloride, 0.2 mol L(-1) NaOH and 0.5% Na2SO3. The peak height is linearly proportional to the concentration of Cyt C in the range of 0.005 approximately 1.500 mg L(-1) (correlation coefficient 0.999). Common amino acids, saccharide, organic acid and metal ions of appropriate concentrations have no interference on the Cyt C determination. The released Cyt C in the process of mitochondrial permeability transition of Hong-Lian cytoplasmic male sterile line of rice has been measured by the method, and the result is satisfactory.

Topics: Amino Acids; Cobalt; Cytochromes c; Guanidine; Mitochondria; Monosaccharides; Oryza; Polarography; Reproducibility of Results; Sodium Hydroxide; Sulfites

HL-1 cardiomyocytes were subjected to simulated hypoxia, in the presence of cobalt chloride, which resulted in reduction of cell viability and induction of DNA laddering, indicating the activation of the apoptotic cascade. In the presence of trolox, ascorbic acid, melatonin and the hybrid compound of trolox and lipoic acid (LaT 3a), cell viability was increased, with LaT 3a exhibiting the best effect. Antioxidant treatment restored ATP levels, abolished laddering of DNA, abrogated MPTP opening, Bax translocation to the mitochondria and cytochrome c release to the cytoplasm. Moreover, severe hypoxia, was found to destabilize hypoxia inducible factor-1alpha (Hif-1alpha) mRNA. Reduction of oxidative stress attenuated this effect, implying a possible anti-apoptotic action of the master regulator of hypoxia response. Our data suggest that antioxidants can maintain cell function and survival by inhibiting the mitochondrial apoptotic pathway and stabilizing Hif-1alpha.

Topics: Adenosine Triphosphate; Antioxidants; Apoptosis; bcl-2-Associated X Protein; bcl-X Protein; Cell Hypoxia; Cell Line; Cobalt; Cytochromes c; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Ion Channels; Mitochondria, Heart; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocytes, Cardiac; Oxidative Stress; Protein Transport; Reactive Oxygen Species; RNA, Messenger