Decreased Bax Protein Expression Research Articles

Abstract 17828: Preconditioning of Mice with a Prolyl Hydroxylase Inhibitor, Dimethyloxalylglycine, Prevents Skin Flap Necrosis

Background- Local skin flaps often present with flap necrosis caused by critical disturbance of blood supply. Because hypoxia-inducible factor 1 (HIF-1) has a pivotal role in ischemic vascular responses, we assessed the hypothesis that preoperative stabilization of HIFs using prolyl hydroxylases (PHD) inhibitor contributes to improvement of skin flap survival. Methods and Results- Mice with ischemic skin flap on the dorsum were treated with PHD inhibitor dimethyloxalylglycine (DMOG) 48hr prior to operation by intraperitoneal administration. Immunoblot assay revealed sustained up-regulation of HIF-1α protein at pre- and postoperative states in the skin. Survival area of flap and the number of CD31 positive vessels at proximal part of flaps in DMOG-treated mice were significantly increased at day seven (20.6±4.9% vs. 55.5±13.2%; P<0.001 and 11.3±1.7 vs. 17.3±3.3; P<0.05). Serum vascular endothelial growth factor (VEGF) protein at the time of operation and its receptor Flk-1 protein at proximal part of flap on day one were significantly increased in DMOG-treated mice. Furthermore, flow cytometric analysis revealed a marked induction of circulating endothelial progenitor cells (EPCs) in DMOG-treated mice on day one (0.75±0.01% vs. 1.49±0.11% of CD45 gated CD34/CD133 positive cells; P<0.005). Of interest, flow cytometric bromodeoxyuridine (BrdU)/DNA analysis showed a marked induction of proliferative progenitor cells in bone marrow 48hr after DMOG treatment. In addition, TUNEL staining at distal part of flaps revealed significantly decreasing apoptotic cells accompanied by increasing protein level of hexokinase II and decreasing Bax protein expression in DMOG-treated mice. HIF-1α heterozygous-null mice showed the reduction of flap survival areas, decreasing the number of circulating EPCs, and increasing the number of apoptotic cells, but these mutant mice with DMOG pretreatment demonstrated complete recovery of these parameters. Conclusion- These findings indicate that prevention of flap necrosis by pretreatment of DMOG is due to enhancement of both anti-apoptotic effects and angiogenesis at the flap region via HIF-1 functions, and that pretreatment of DMOG could be a potential therapeutic strategy for prevention of flap necrosis.

Effect of emulsified isoflurane preconditioning on focal cerebral ischemia-reperfusion injury in rats

Objective To investigate the effect of emulsified isoflurane preconditioning on focal cerebral ischemia-reperfusion (I/R) injury in rats and the mechanism. Methods Fifty-six healthy male adult SD rats weighing 250-280 g were randomly divided into 4 groups (n = 14): group Ⅰ sham operation (group S); group Ⅱfocal cerebral I/R; group Ⅲ emulsified isoflurane preconditioning and group Ⅳ fat emulsion preconditioning. Focal cerebral ischemia was induced by middle cerebral artery occlusion (MCAO) using a nylon thread with rounded tip inserted into internal carotid artery and advanced cranially until resistance was met. MCAO was maintained for 2 h followed by 24 h reperfusion. In group Ⅲ and Ⅳ 8% emulsified isoflurane 7.5 ml/kg and 3% fat emulsion 7.5 ml/kg were injected intraperitoneally at 24 h before MCAO respectively. Neurologic outcome was evaluated at 24 h of reperfusion and scored (0 = no deficit, 4 = unable to crawl, loss of consciousness). The animals were then killed and brains removed. The infarct size was assessed. The apoptotic cells were detected by TUNEL and calculated. The expression of Bax, Bcl-2, cytochrome C and caspase-3 protein was detected by immunohistochemical staining. Results The neurologic deficit scores were significantly lower in emulsified isoflurane preconditioning group than in I/R group. Preconditioning with emulsified isoflurane significantly decreased the infarct size and the number of apoptotic cells and increased the expression of Bcl-2 protein and inhibited the expression of Bax, cytochrome C and caspace-3 protein. Conclusion Emulsified iscflurane preconditioning can protect the brain from focal cerebral I/R by increasing Bcl-2 protein expression and decreasing Bax protein expression, and reducing cytochrome C release from mitochondria, caspase-3 activation and neuronal apoptosis. Key words: Isoflurane; Fat emulsions, intravenous; Ischemic preconditioning; Reperfusion injury; Brain

Apelin suppresses apoptosis of human vascular smooth muscle cells via APJ/PI3-K/Akt signaling pathways

Apoptosis of vascular smooth muscle cells (VSMCs) plays an important role in regulating vascular remodeling during cardiovascular diseases. Apelin is the endogenous ligand for the G-protein-coupled receptor APJ and plays an important role in the cardiovascular system. However, the mechanisms of apelin on apoptosis of VSMCs have not been elucidated. Using a culture of human VSMCs as a model for the study of apoptosis, the relationship between apelin and apoptosis of human VSMCs and the signal pathway involved were investigated. Using western blotting, we confirmed that VSMCs could express APJ. To evaluate the possible role of apelin in VSMC apoptosis, we assessed its effect on apoptosis of human VSMCs. The results showed that apelin inhibited human VSMCs apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Apelin increased Bcl-2 protein expression, but decreased Bax protein expression. An increase in activation of extracellular signal-regulated protein kinase (ERK) and Akt (a downstream effector of phosphatidylinositol 3-kinase) was shown after apelin stimulation. Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. LY294002 (a PI3-K inhibitor) blocked apelin-induced activation of Akt and abolished the apelin-induced antiapoptotic activity. Our study suggests that apelin suppresses serum deprivation-induced apoptosis of human VSMCs, and that the anti-apoptotic action is mediated through the APJ/PI3-K/Akt signaling pathways.

Effect of postconditioning with propofol and ischemia on hepatic ischemia-reperfusion injury in rats

Objective To investigate the effect of postconditioning with propofol and ischemia on the hepatic ischemia-reperfusion (I/R) injury in rats. Methods Thirty male SD rats weighing 200-250 g were randomly divided into 5 groups of 6 animals each: group I sham operation (group S); groupⅡ I/R; group Ⅲ ischemic postconditioning (group IPC); group Ⅳ propofol postconditioning (group PPC) and group V IPC + PPC. In group Ⅱ-Ⅳ the hepatic arteries and veins of middle and left lobes were occluded for 1 h followed by 4 h reperfusion. Ischemia of the liver was confirmed by the color of the liver turning from red to gray. In group Ⅲ and Ⅴ the livers were subjected to six episodes of 10 s ischemia at 10 s intervals at the end of 1 h ischemia before 4 h reperfusion. In group Ⅳ and V 0.5 % propofol 10 mg/kg was given iv at the end of ischemia followed by propofol infusion at 40 mg·g~(-1) ·h~(-1). Blood samples were taken at the end of 4 h reperfusion for determination of serum ALT activity. Mean-while liver specimens were taken for electron microscopic examination and determination of MDA content and SOD activity. Results I/R significantly increased serum ALT activity and MDA content in the liver and decreased liver SOD activity in group Ⅱ . The I/R-induced changes were significantly attenuated by propofol and/or ischemic postconditioning in group Ⅲ ,Ⅱ and Ⅴ . I/R significantly increased Bel-2 and Bax protein expression in the liver cells. Propofol and/or ischemic postconditioning increased Bel-2 protein expression further but decreased Bax protein expression in group Ⅲ , Ⅳ and Ⅴ as compared with group Ⅱ (group I/R).Electron microscopic examination showed that the pathologic changes induced by I/R were less severe in group Ⅲ, Ⅳ and Ⅴ than in group I/R. Conclusion Postconditioning with propofol and ischemia can reduce the hepatic I/R injury and the mechanism may be related to inhibition of lipid peroxidation and apoptosis, but the efficacy is the same as that of propofol postconditioning alone. Key words: Propofol; Reperfusion injury; Hepatic; Ischemic postconditioning

Cardioprotective effects of benazepril, an angiotensin-converting enzyme inhibitor, in an ischaemia-reperfusion model of myocardial infarction in rats

The present study evaluated the effects of benazepril, an angiotensin-converting enzyme inhibitor on haemodynamic, biochemical, and immunohistochemical (Bax and Bcl-2 protein) indices in ischaemia and reperfusion (IR) injury. Male Wistar albino rats were divided into three groups and were orally administered saline once daily (IR-sham and IR-control) or benazepril (30 mg/kg/day; IR-benazepril) for 14 days. On the 15(th) day, in the IR-control and IR-benazepril groups, rats were subjected to left anterior descending coronary artery occlusion for 45 minutes followed by a one-hour reperfusion. Haemodynamic parameters were recorded and rats were sacrificed; hearts were isolated for biochemical estimation and immunohistochemistry. In the IR-control group, significant ventricular dysfunctions (p<0.05 vs. IR-sham group) were observed along with enhanced expression of pro-apoptotic protein Bax. A decline in lactate dehydrogenase activity and increased content of thiobarbituric acid reactive substances, a marker of lipid peroxidation, were observed. Benazepril pretreatment significantly improved mean arterial pressure (p<0.01), reduced left ventricular end-diastolic pressure (p<0.05), and improved both inotropic and lusitropic function of the heart (+LVdP/dt and - LVdP/dt) (p<0.05; p<0.01) as compared to IR-control. Furthermore, benazepril treatment significantly decreased the level of thiobarbituric acid reactive substances and restored the activity of lactate dehydrogenase towards normal value (p<0.05 vs. IR-control). This study demonstrates that benazepril upregulated Bcl-2 protein and decreased Bax protein expression, thus exhibiting anti-apoptotic effects. These beneficial effects of benazepril will have an important implication in the therapeutic use of benazepril in ischaemic heart disease.

Apelin suppresses apoptosis of human osteoblasts

Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor APJ. Adipocytes can express and secrete apelin. Osteoblast can express apelin and APJ. The aim of this study was to investigate the action of apelin on apoptosis of human osteoblasts. Apelin inhibited human osteoblasts apoptosis induced by serum deprivation. Suppression of APJ with small-interfering RNA (siRNA) abolished the anti-apoptotic activity of apelin. Our study also showed an increased Bcl-2 protein expression and decreased Bax protein expression under the treatment of apelin. Apelin decreased cytochrome c release and caspase-3 activation in human osteoblasts. Apelin activated phosphatidylinositol-3 kinase (PI-3 kinase) and Akt. The apelin-induced activation of Akt was blocked by suppression of APJ with siRNA. LY294002 (a PI-3 kinase inhibitor) or 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO; an Akt inhibitor) abolished apelin induced activation of Akt, and, LY294002 or HIMO abolished the anti-apoptotic activity of apelin. Furthermore, apelin protects against apoptosis induced by the glucocorticoid dexamethasone. Apelin suppresses serum deprivation-induced apoptosis of human osteoblasts and the anti-apoptotic action is mediated via the APJ/PI-3 kinase/Akt signaling pathway.

Recombinant human granulocyte colony‐stimulating factor protects against MPTP‐induced dopaminergic cell death in mice by altering Bcl‐2/Bax expression levels

Granulocyte colony-stimulating factor (G-CSF) has been used for the treatment of neutropenia in hematologic disorders. The neuroprotective effects of G-CSF were reported in neurological disease models. In the present study, we examined whether G-CSF can protect dopaminergic neurons against MPTP-induced cell death in a mouse model of Parkinson's disease. Mice of one group were injected intraperitoneally with MPTP for five consecutive days, those of another group with MPTP and intraperitoneal G-CSF at 2 days and 1 day before the first MPTP injection, and 30 min before each MPTP injection, while control mice received saline injections. Immunohistochemistry, western blotting analysis, and HPLC were performed to evaluate damage of substantia nigra dopaminergic neurons and expression of Bcl-2 and Bax protein. MPTP induced dopaminergic cell death in the substantia nigra. G-CSF significantly prevented MPTP-induced loss of tyrosine hydroxylase-positive neurons (p < 0.05), increased Bcl-2 protein and decreased Bax protein expression. Our findings indicate that G-CSF provides neuroprotection against MPTP-induced cell death and this effect is mediated by increasing Bcl-2 expression levels and decreasing Bax expression levels in C57BL/6 mice.

Tissue Inhibitor of Matrix Metalloproteinase-1 Suppresses Apoptosis of Mouse Bone Marrow Stromal Cell Line MBA-1

We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

The effect of hypothermia on the expression of the apoptosis-regulating protein Bax after incomplete cerebral ischemia and reperfusion in rats.

This study investigated the effects of hypothermia on apoptosis-regulating proteins in a rat model of incomplete cerebral ischemia. Twenty-seven fasted male Sprague-Dawley rats (300-420 g) were anesthetized, intubated, and mechanically ventilated with 2.0% isoflurane and N(2)O/O(2) (FiO(2) = 0.33). Catheters were inserted and cerebral blood flow velocity was measured using bilateral laser Doppler flowmetry. At the end of preparation, the administration of isoflurane was replaced by fentanyl (25 microg. kg(-1). h(-1)). Animals were randomly assigned to one of the following groups: group 1 (n = 9, normothermia), normothermia (37.5 degrees C) during ischemia; group 2 (n = 9, hypothermia), 34 degrees C pericranial temperature during ischemia; and group 3 (n = 9, sham-operated animals), normothermia, no cerebral ischemia. Ischemia (30 minutes) was produced by unilateral common carotid artery occlusion plus hemorrhagic hypotension (mean arterial blood pressure 30-35 mm Hg). Arterial blood gas tensions and pH were maintained constant. Four hours after 30 minutes of incomplete cerebral ischemia, the brains were removed for determination of the expression of the apoptosis-regulating proteins Bax, Bcl-2, p53, and Mdm-2 using immunofluorescence and Western blot analysis. Four hours after cerebral ischemia there was a significant increase in the expression of the pro-apoptotic protein Bax in normothermic animals compared with hypothermic (85-260%) and sham-operated animals (60-190%). The proteins Bcl-2, p53, and Mdm-2 showed no statistically significant differences between the groups or between the hemispheres. In conclusion, hypothermia during ischemia decreased Bax protein expression that is associated with programed cell death. This suggests that neuroprotection seen with hypothermia may be related to a reduction of pro-apoptotic events.