anisomycin and Hypoxia

Stress granules (SGs) could be formed under different stimulation to inhibit cell injury.. To investigate whether SGs could protect hepatocytes from hypoxia-induced damage during acute liver failure (ALF) by reducing endoplasmic reticulum stress (ERS) mediated apoptosis.. The agonist of SGs, arsenite (Ars) was used to intervene hypoxia-induced hepatocyte injury cellular model and ALF mice models. Further, the siRNA of activating transcription factor 4 (ATF4) and SGs inhibitor anisomycin was then used to intervene in cell models.. With the increase of hypoxia time from 4 h to 12 h, the levels of HIF-1α, ERS and apoptosis gradually increased, and the expression of SGs marker G3BP1 and TIA-1 was increased and then decreased. Compared with the hypoxia cell model group and ALF mice model, the levels of HIF-1α, apoptosis and ERS were increased in the Ars intervention group. After siRNA-ATF4 intervention, the level of SGs in cells increased, and the levels of HIF-1α, ERS and apoptosis decreased. Compared with the siRNA-ATF4 group, the levels of G3BP1 in the siRNA-ATF4+anisomycin group were decreased, and the levels of HIF-1α, ERS and apoptosis were increased. Moreover, compared with the ALF group, the degree of liver injury and liver function, the levels of HIF-1α, ERS and apoptosis in the Ars intervention group were decreased, the level of SGs was increased.. SGs could protect hepatocytes from hypoxia-induced damage during ALF by reducing ERS-mediated apoptosis.

Topics: Animals; Anisomycin; Apoptosis; DNA Helicases; Endoplasmic Reticulum Stress; Hypoxia; Liver Failure, Acute; Mice; Poly-ADP-Ribose Binding Proteins; RNA Helicases; RNA Recognition Motif Proteins; RNA, Small Interfering; Stress Granules

Morroniside can prevent myocardial injury caused by ischemia and hypoxia, which can be used to treat acute myocardial infarction (AMI). Hypoxia can cause apoptosis and autophagic death of cardiomyocytes. Morroniside has the ability to inhibit apoptosis and autophagy. However, the relationship between Morroniside-protected cardiomyocytes and two forms of death is unclear. The effects of Morroniside on the proliferation, apoptosis level, and autophagic activity of rat cardiomyocyte line H9c2 under hypoxia were first observed. Next, the roles of Morroniside in the phosphorylation of JNK and BCL2, BCL2-Beclin1, and BCL2-Bax complexes as well as mitochondrial membrane potential in H9c2 cells were evaluated upon hypoxia. Finally, the significance of BCL2 or JNK in Morroniside-regulated autophagy, apoptosis, and proliferation in H9c2 cells was assessed by combining Morroniside and BCL2 competitive inhibitor (ABT-737) or JNK activator (Anisomycin). Our results showed that hypoxia promoted autophagy and apoptosis of H9c2 cells, and inhibited their proliferation. However, Morroniside could block the effect of hypoxia on H9c2 cells. In addition, Morroniside could inhibit JNK phosphorylation, BCL2 phosphorylation at the Ser70 and Ser87 sites, and the dissociation of BCL2-Beclin1 and BCL2-Bax complexes in H9c2 cells upon hypoxia. Moreover, the reduction of mitochondrial membrane potential in H9c2 cells caused by hypoxia was improved by Morroniside administration. Importantly, the inhibited autophagy, apoptosis, and promoted proliferation in H9c2 cells by Morroniside were reversed by the application of ABT-737 or Anisomycin. Overall, Morroniside inhibits Beclin1-dependent autophagic death and Bax-dependent apoptosis via JNK-mediated BCL2 phosphorylation, thereby improving the survival of cardiomyocytes under hypoxia.

Topics: Animals; Anisomycin; Apoptosis; Autophagy; bcl-2-Associated X Protein; Beclin-1; Hypoxia; Myocytes, Cardiac; Phosphorylation; Rats

Metformin is a first-line drug for the management of type 2 diabetes. Recent studies suggested cardioprotective effects of metformin against ischemia/reperfusion injury. However, it remains elusive whether metformin provides direct protection against hypoxia/reoxygenation (H/R) injury in cardiomyocytes under normal or hyperglycemic conditions. This study in H9C2 rat cardiomyoblasts was designed to determine cell viability under H/R and high-glucose (HG, 33 mM) conditions and the effects of cotreatment with various concentrations of metformin (0, 1, 5, and 10 mM). We further elucidated molecular mechanisms underlying metformin-induced cytoprotection, especially the possible involvement of AMP-activated protein kinase (AMPK) and Jun NH(2)-terminal kinase (JNK). Results indicated that 5 mM metformin improved cell viability, mitochondrial integrity, and respiratory chain activity under HG and/or H/R (P < 0.05). The beneficial effects were associated with reduced levels of reactive oxygen species generation and proinflammatory cytokines (TNF-α, IL-1α, and IL-6) (P < 0.05). Metformin enhanced phosphorylation level of AMPK and suppressed HG + H/R induced JNK activation. Inhibitor of AMPK (compound C) or activator of JNK (anisomycin) abolished the cytoprotective effects of metformin. In conclusion, our study demonstrated for the first time that metformin possessed direct cytoprotective effects against HG and H/R injury in cardiac cells via signaling mechanisms involving activation of AMPK and concomitant inhibition of JNK.

Topics: AMP-Activated Protein Kinases; Animals; Anisomycin; Cell Survival; Cytokines; Electron Transport; Glucose; Hyperglycemia; Hypoglycemic Agents; Hypoxia; In Vitro Techniques; JNK Mitogen-Activated Protein Kinases; Metformin; Mitochondria; Myocardial Reperfusion Injury; Myocytes, Cardiac; Rats; Reactive Oxygen Species

Many neurological insults are accompanied by a marked acute inflammatory reaction, involving the activation of microglia. Using a model of exogenous application of fluorescence-labeled BV2 microglia in pathophysiologically relevant concentrations onto organotypic hippocampal slice cultures, we investigated the specific effects of microglia on neuronal damage after ischemic injury. Neuronal cell death after oxygen-glucose deprivation (OGD) was determined by propidium iodide incorporation and Nissl staining. Migration and interaction with neurons were analyzed by time resolved 3-D two-photon microscopy. We show that microglia protect against OGD-induced neuronal damage and engage in close physical cell-cell contact with neurons in the damaged brain area. Neuroprotection and migration of microglia were not seen with integrin regulator CD11a-deficient microglia or HL-60 granulocytes. The induction of migration and neuron-microglia interaction deep inside the slice was markedly increased under OGD conditions. Lipopolysaccharide-prestimulated microglia failed to provide neuroprotection after OGD. Pharmacological interference with microglia function resulted in a reduced neuroprotection. Microglia proved to be neuroprotective even when applied up to 4 h after OGD, thus defining a "protective time window." In acute injury such as trauma or stroke, appropriately activated microglia may primarily have a neuroprotective role. Anti-inflammatory treatment within the protective time window of microglia would therefore be counterintuitive.

Topics: Animals; Anisomycin; Anti-Bacterial Agents; Brain Ischemia; CD11a Antigen; Cell Death; Cell Line; Glucose; Granulocytes; Hippocampus; HL-60 Cells; Humans; Hypoxia; Mice; Mice, Transgenic; Microglia; Minocycline; Neurons; Rats; Rats, Wistar

Hypoxic pulmonary vasoconstriction (HPV) and pulmonary hypertension present common and formidable clinical problems for thoracic, transplant, and trauma surgeons. We hypothesized that acute hypoxia causes pulmonary artery (PA) contraction and that p38 mitogen-activated protein (MAP) kinase is a key mediator in that process. To test this hypothesis, we measured isometric force displacement in isolated rat pulmonary artery rings during hypoxia in the presence and absence of the selective p38 MAP kinase inhibitor SB-20358, and stimulator anisomycin. In separate experiments, we measured the functional effects in isolated rat pulmonary artery rings of inhibiting p38 MAP kinase during normoxic conditions. p38 MAP kinase inhibition significantly attenuated the delayed, but not early, contractile phase of HPV. Additionally, stimulation of p38 MAP kinase significantly decreased the phase I vasodilation of HPV. Under normoxia conditions, there was no statistically significant difference in isometric force displacement between control and p38 MAPK inhibitor-treated pulmonary artery rings. We conclude that p38 MAP kinase may be a key mediator in the pathogenesis of HPV and that further understanding may lead to new therapies for HPV associated with acute lung injury.

Topics: Animals; Anisomycin; Enzyme Activation; Enzyme Inhibitors; Hypoxia; Imidazoles; Isometric Contraction; Lung Diseases; Male; p38 Mitogen-Activated Protein Kinases; Phenylephrine; Pulmonary Artery; Pyridines; Rats; Rats, Sprague-Dawley; Vasoconstriction; Vasodilation

WW domain-containing oxidoreductase WOX1, also named WWOX or FOR, undergoes Tyr33 phosphorylation at its first N-terminal WW domain and subsequent nuclear translocation in response to sex steroid hormones and stress stimuli. The activated WOX1 binds tumor suppressor p53, and both proteins may induce apoptosis synergistically. Functional suppression of WOX1 by antisense mRNA or a dominant negative abolishes p53-mediated apoptosis. Here, we determined that UV light, anisomycin, etoposide, and hypoxic stress rapidly induced phosphorylation of p53 at Ser46 and WOX1 at Tyr33 (phospho-WOX1) and their binding interactions in several tested cancer cells. Mapping by yeast two-hybrid analysis and co-immunoprecipitation showed that phospho-WOX1 physically interacted with Ser46-phosphorylated p53. Knockdown of WOX1 protein expression by small interfering RNA resulted in L929 fibroblast resistance to apoptosis by tumor necrosis factor, staurosporine, UV light, and ectopic p53, indicating an essential role of WOX1 in stress stimuli-induced apoptosis. Notably, UV light could not induce p53 protein expression in these WOX1 knockdown cells, although p53 mRNA levels were not reduced. Suppression of WOX1 by dominant negative WOX1 (to block Tyr33 phosphorylation) also abolished UV light-induced p53 protein expression. Time course analysis showed that the stability of ectopic wild type p53, tagged with DsRed, was decreased in WOX1 knockdown cells. Inhibition of MDM2 by nutlin-3 increased the binding of p53 and WOX1 and stability of p53. Together, our data show that WOX1 plays a critical role in conferring cellular sensitivity to apoptotic stress and that Tyr33 phosphorylation in WOX1 is essential for binding and stabilizing Ser46-phosphorylated p53.

Topics: Active Transport, Cell Nucleus; Animals; Anisomycin; Cell Line, Tumor; Cell Nucleus; Cytoplasm; Cytosol; DNA, Complementary; Dose-Response Relationship, Drug; Etoposide; Fibroblasts; Genes, Dominant; Humans; Hypoxia; Imidazoles; Immunoprecipitation; Luminescent Proteins; Mice; Microscopy, Fluorescence; Models, Biological; Oxidoreductases; Phosphorylation; Piperazines; Proteasome Endopeptidase Complex; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serine; Staurosporine; Time Factors; Tumor Necrosis Factor-alpha; Tumor Suppressor Protein p53; Tumor Suppressor Proteins; Two-Hybrid System Techniques; Tyrosine; U937 Cells; Ultraviolet Rays; WW Domain-Containing Oxidoreductase

Severe, intermittent hypoxia (hypoxic conditioning, HC) increases survival time during subsequent lethal hypoxia in mice. This protective effect was blocked by naloxone, suggesting an opioid-dependent mechanism. We proposed and evaluated three potential mechanisms of this acute adaptation: 1) increased hematocrit (Hct), 2) protein synthesis, and 3) decreased set point for temperature regulation (set point). Increased hematocrit is a well-studied adaptation to chronic hypoxia and could be acutely initiated by sympathetically mediated splenic contraction. Survival during stress can be prolonged by synthesis of stress proteins. We tested this hypothesis using two protein synthesis inhibitors, anisomycin and cycloheximide. Our third hypothesis is that set point is decreased after HC. A regulated decrease in body temperature would lower oxygen demand during hypoxia. Our studies indicate that hematocrit and protein synthesis are not dominant mechanisms of acute adaptation to hypoxia. However, we have observed a naloxone blockable decrease in set point after HC, supporting a mechanism in which acute adaptation involves an endogenous opioid-dependent decrease in set point. These studies also demonstrate that set point could be a more dominant contributor than body temperature to hypoxic tolerance.

Topics: Acclimatization; Analysis of Variance; Animals; Anisomycin; Body Temperature; Body Temperature Regulation; Cycloheximide; Heat-Shock Proteins; Hematocrit; Hypoxia; Male; Mice; Naloxone; Reference Values