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ABSTRACT: 1. After a severe burn, a marked decrease in myocardial blood flow results in ischaemic and hypoxic injury, which subsequently leads to apoptosis or necrosis. Phosphatidylinositol 3-kinase (PI3-K)/Akt is an important intracellular signal transduction molecule that regulates cell proliferation, differentiation, glucose metabolism and migration. However, the function and mechanisms of the PI3-K-Akt pathway in cardiomyocyte apoptosis after a burn remain unclear. 2. In the present study, an in vivo rat model of burn injury and an in vitro hypoxic model using rat cardiomyocytes were established. In burned rats, the expression of PI3-K and phosphorylated (p-) Akt expression increased, as did myocardial apoptosis. Inhibition of the PI3-K-Akt pathway with 1.4 mg/kg LY294002 caused a significant increase in the myocardial apoptotic index compared with hypoxia alone in the in vivo model. 3. Cardiomyocytes cultured under hypoxic conditions exhibited increased apoptosis, decreased cell viability, enhanced caspase 3 activity, a decreased mitochondrial membrane potential, increased cytoplasmic calcium transients and increased p53 and Bax mRNA expression. Pretreatment with 50 mumol/L LY294002 significantly enhanced all these negative indicators compared with hypoxia alone. In contrast, pretreatment of cells with 200 ng/mL insulin-like growth factor-1, an activator of PI3-K-Akt, significantly ameliorated the effects of hypoxia, although control levels were not reached. 4. These findings indicate that activation of the PI3-K-Akt pathway induced by ischaemia and hypoxia after a severe burn can protect cardiomyocytes from apoptosis. This anti-apoptotic effect is most likely mediated via the mitochondria and changes in p53 and Bax gene expression, intracellular [Ca(2+)] and caspase 3 activity.
Clinical and Experimental Pharmacology and Physiology 05/2010; 37(5-6):598-604. · 1.85 Impact Factor
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ABSTRACT: The aim of this study was to test the hypothesis that circulating factors released after a severe burn cause endothelial barrier dysfunction by triggering endothelial cell (EC) contraction through a p38 mitogen-activated protein (MAP) kinase-dependent mechanism. Human umbilical vein ECs (ECV304 cell line) were cultured to create a monolayer of cells that were then cultured with 20% human normal or burn serum. Monolayer permeability was measured by the influx of labeled albumin across the cells. Endothelial cells contraction was determined by alterations of cell surface area and formation of intracellular gaps. P38 MAP kinase activation, F-actin arrangement, and L-caldesmon phosphorylation were assessed by Western blots or immunofluorescence staining. These studies showed that exposure to burn serum resulted in a significant increase in endothelial permeability in a time-dependent manner, which was paralleled by a rapid and persistent activation of p38 MAP kinases. Morphologically, increased intercellular gaps, reduced cell surface area, and a unique rearrangement of F-actin cytoskeleton were observed in burn serum-treated ECs. Inhibition of p38 MAP kinase suppressed the rearrangement of F-actin cytoskeleton, reduced the occurrence of burn serum-induced formation of intercellular gaps, and ameliorated endothelial hyperpermeability. Further study showed that phosphorylation of L-caldesmon was enhanced in burn serum-treated cells via p38 MAP kinase; overexpression of L-caldesmon by adenovirus transfection, however, attenuated the increase in endothelial permeability by burn serum challenge. Collectively, these results have demonstrated for the first time that p38 MAP kinase is an important participant in mediating burn serum-induced endothelial barrier dysfunction through rearrangement of the F-actin cytoskeleton and phosphorylation of L-caldesmon. Inhibition of p38 MAP kinase in vivo, thus, would be a promising therapeutic strategy in ameliorating burn shock development.
Shock (Augusta, Ga.) 02/2010; 34(3):222-8. · 2.87 Impact Factor
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Clinical and Experimental Pharmacology and Physiology - CLIN EXP PHARMACOL PHYSIOL. 01/2010; 37(3):343-349.
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ABSTRACT: In both cardiomyocytes and HeLa cells, hypoxia (1% O(2)) quickly leads to microtubule disruption, but little is known about how microtubule dynamics change during the early stages of hypoxia. We demonstrate that microtubule associated protein 4 (MAP4) phosphorylation increases while oncoprotein 18/stathmin (Op18) phosphorylation decreases after hypoxia, but their protein levels do not change. p38/MAPK activity increases quickly after hypoxia concomitant with MAP4 phosphorylation, and the activated p38/MAPK signaling leads to MAP4 phosphorylation and to Op18 dephosphorylation, both of which induce microtubule disruption. We confirmed the interaction between phospho-p38 and MAP4 using immunoprecipitation and found that SB203580, a p38/MAPK inhibitor, increases and MKK6(Glu) overexpression decreases hypoxic cell viability. Our results demonstrate that hypoxia induces microtubule depolymerization and decreased cell viability via the activation of the p38/MAPK signaling pathway and changes the phosphorylation levels of its downstream effectors, MAP4 and Op18.
Cellular and Molecular Life Sciences CMLS 11/2009; 67(2):321-33. · 6.57 Impact Factor
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ABSTRACT: 1. Astragaloside IV (AST-IV) is purified from a natural plant product. Previous studies have shown that AST-IV has anti-oxidant activity. In the present study, we investigated the effect and mechanism of action AST-IV on rat cardiomyocytes subjected to hypoxic conditions (up to 12 h). 2. Cardiomyocytes were prepared from neonatal rats and cultured under normoxic or hypoxic conditions in the absence or presence of AST-IV (12.5, 25 or 50 microg/mL). Cell viability, malondialdehyde (MDA) levels, activity and expression of superoxide dismutase (SOD)-1 (mRNA and protein levels determined by reverse transcription-polymerase chain reaction and western blotting, respectively) and reactive oxygen species (ROS; determined by 2',7'-dichlorodihydrofluorescein diacetate) were investigated under these culture conditions. Intracellular localization of AST-IV was tested using fluorescein isothiocyanate-labelled AST-IV. 3. Hypoxic culture reduced the viability of cardiomyocytes, which was improved following treatment with 25 or 50 microg/mL AST-IV. Under hypoxic conditions, MDA levels were double those under control conditions. Astragaloside IV (25 and 50 microg/mL) dose-dependently reduced the increase in MDA seen in hypoxic cardiomyocytes. 4. Fluorescein isothiocyanate-labelled AST-IV entered cardiomyocytes and was localized mainly within the cytoplasm. 5. Under hypoxic conditions, SOD-1 activity was decreased, but mRNA and protein expression increased, compared with normoxia. Following treatment with 25 microg/mL AST-IV, SOD-1 activity and expression were increased under both normoxic and hypoxic conditions. The ROS scavenging effect of AST-IV was abolished in the presence of the SOD inhibitor sodium diethyl dithiocarbamate (25 micromol/L). 6. These in vitro results show that AST-IV protects cardiomyocytes from oxidative stress-mediated injury under hypoxic conditions. A major part of this action is achieved by upregulation of SOD-1 content and activity within the cell cytoplasm.
Clinical and Experimental Pharmacology and Physiology 11/2008; 36(4):351-7. · 1.85 Impact Factor
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ABSTRACT: To investigate the influence of insulin growth factor-I (IGF-I) on apoptosis of cardiomyocytes subjected to ischemia and hypoxia and its possible mechanism.
Cardiomyocytes were cultured in vitro, and randomized into hypoxia group, treatment group (T, the cells were treated with IGF-1 before subjected to hypoxia and ischemia) and control group (C, normal cardiomyocytes as controls). Changes in the OD value of cell apoptosis, mitochondrial membrane potential and relative amount of phospho-Akt protein were observed at different time-points by ELISA, laser scanning with TMRE staining and Western blot, respectively.
The OD value of cell apoptosis in control group was 0.18 +/- 0.03, while that in hypoxia group was gradually increased to 0.33 +/- 0.05, 0.61 +/- 0.06, 1.17 +/- 0.08, 2.25 +/- 0.11, respectively at 1, 3, 6, 12 post-hypoxia hours (PHH), showing an increasing tendency (P < 0.01). The OD values of cell apoptosis in T group were 0.26 +/- 0.04, 0.49 +/- 0.05, 0.84 +/- 0.06, 1.63 +/- 0.09, respectively, which were obviously lower than those in hypoxia group (P < 0.05 or P < 0.01). The mitochondrial membrane potential (Dymt) values in hypoxia group at 6 and 12 PHH were 18.7 +/- 5.1 and 6.3 +/- 1.9, respectively, which were obviously lower than that in control group (40.2 +/- 10.1, P < 0.01). The DYmt in T group at 6 and 12 PHH were 28.8 +/- 6.2, 12.5 +/- 3.1, respectively, which were obviously higher compared with those in hypoxia group (P < 0.05). The amount of phospho-Akt protein was increased by IGF-I administration.
IGF-I exhibits an anti-apoptotic effect on cardiomyocytes subjected to ischemia and hypoxia, and this may be related to the activation of PI3K/Akt signal pathway and stabilization of mitochondrial membrane potential.
Zhonghua shao shang za zhi = Zhonghua shaoshang zazhi = Chinese journal of burns 01/2008; 23(6):436-9.
