Effects of therapeutic and toxic phenobarbital doses on brain tissue caspase 3-activity in the neonatal rat

2006 ◽  
Vol 37 (03) ◽  
Author(s):  
R Husain ◽  
HS Fink ◽  
K Lang ◽  
B Merkle ◽  
R Bauer ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Caspase 3 ◽  
Neonatal Rat

scholarly journals Protective Effects of Shenfuyixin Granule on H2O2-Induced Apoptosis in Neonatal Rat Cardiomyocytes

2021 ◽  
Vol 2021 ◽  
pp. 1-10
Author(s):  
Xinlu Wang ◽  
Xuanxuan Hao ◽  
Youping Wang ◽  
Bin Li ◽  
Lin Cui ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Rat Cardiomyocytes ◽  
Expression Levels ◽  
Apoptosis Rate ◽  
Neonatal Rat Cardiomyocytes ◽  
Mitochondrion Membrane Potential

Shenfuyixin granule (SFYXG, i.e., Xinshuaikang granule) is a prescription, commonly used in the clinical experience, which plays a significant role in the treatment of heart failure. The purpose of this present research was to investigate the protective effect of SFYXG, and the mechanism about anti-H2O2-induced oxidative stress and apoptosis in the neonatal rat cardiomyocytes. Myocardial cells, as is well known, were divided into 4 groups: normal, model, SFYXG, and coenzyme Q10 group, respectively. Cells viability was determined by MTT assay. Flow cytometry and AO/EB staining were implemented to test the apoptosis rate and intracellular reactive oxygen species (ROS) level. Mitochondrion membrane potential (MMP) was evaluated by JC-1 fluorescence probe method. The myocardial ultrastructure of mitochondrion was measured by electron microscope. The related mRNA expression levels of Bax, Bcl-2, Caspase-3, caspase-8, and caspase-9 were detected by real-time polymerase chain reaction (PCR). Also, the expression levels of Bax and Bcl-2 protein were detected by Western blot, and the expression levels of caspase-3, caspase-8, and caspase-9 protein were tested by caspase-Glo®3 Assay, caspase-Glo®8 Assay, and caspase-Glo®9 Assay, respectively. GAPDH was used as the internal reference gene/protein. The results revealed that SFYXG (0.5 mg/ml) raised the viability of myocardial cell, weakened the apoptosis rate and ROS level, corrected the mitochondrion membrane potential stability, and improved cell morphology and ultrastructure of myocardial mitochondrion. Furthermore, SFYXG upregulated the antiapoptosis gene of Bcl-2, but downregulated the proapoptosis genes of Bax, caspase-3, and caspase-9. In conclusion, SFYXG could appear to attenuate myocardial injury by its antioxidative and antiapoptosis effect.

Role of F-actin organization in p38 MAP kinase-mediated apoptosis and necrosis in neonatal rat cardiomyocytes subjected to simulated ischemia and reoxygenation

2005 ◽  
Vol 289 (6) ◽  
pp. H2310-H2318 ◽  
Author(s):  
Takayuki Okada ◽  
Hajime Otani ◽  
Yue Wu ◽  
Shiori Kyoi ◽  
Chiharu Enoki ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Map Kinase ◽  
Dna Fragmentation ◽  
P38 Mapk ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Cytochrome C Release ◽  
Actin Reorganization ◽  
Ldh Release ◽  
Sb 203580

Activation of p38 mitogen-activated protein (MAP) kinase (MAPK) has been implicated in the mechanism of cardiomyocyte (CMC) protection and injury. The p38 MAPK controversy may be related to differential effects of this kinase on apoptosis and necrosis. We have hypothesized that p38 MAPK-mediated F-actin reorganization promotes apoptotic cell death, whereas it protects from osmotic stress-induced necrotic cell death. Cultured neonatal rat CMCs were subjected to 2 h of simulated ischemia followed by reoxygenation. p38 MAPK activity measured by phosphorylation of MAP kinase-activated protein (MAPKAP) kinase 2 was increased during simulated ischemia and reoxygenation. This was associated with translocation of heat shock protein 27 (HSP27) from the cytosolic to the cytoskeletal fraction and F-actin reorganization. Cytochrome c release from mitochondria, caspase-3 activation, and DNA fragmentation were increased during reoxygenation. Robust lactate dehydrogenase (LDH) release was observed under hyposmotic (140 mosM) reoxygenation. The p38 MAPK inhibitor SB-203580 abrogated activation of p38 MAPK, translocation of HSP27, and F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation. Conversely, SB-203580 enhanced LDH release during hyposmotic reoxygenation. The F-actin disrupting agent cytochalasin D inhibited F-actin reorganization and prevented cytochrome c release, caspase-3 activation, and DNA fragmentation, whereas it enhanced LDH release during hyposmotic reoxygenation. When CMCs were incubated under the isosmotic condition for the first 15 min of reoxygenation, SB-203580 and cytochalasin D increased ATP content of CMCs and prevented LDH release after the conversion to the hyposmotic condition. These results suggest that F-actin reorganization mediated by activation of p38 MAPK plays a differential role in apoptosis and protection against osmotic stress-induced necrosis during reoxygenation in neonatal rat CMCs; however, the sarcolemmal fragility caused by p38 MAPK inhibition can be reversed during temporary blockade of physical stress during reoxygenation.

Abstract 19908: 5-oxoprolinase: a Novel Cardiac Mediator of the Oxidative Stress Response in the Failing Heart

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Atze van der Pol ◽  
Jasper Tromp ◽  
Martijn F Hoes ◽  
Ibrahim J Domian ◽  
Wiek H van Gilst ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Caspase 3 ◽  
Differentially Expressed Gene ◽  
Embryonic Stem ◽  
Pressure Overload ◽  
Neonatal Rat ◽  
Novel Genes ◽  
Human Organ ◽  
Cardiac Progenitors ◽  
Micro Array

Introduction: A hallmark of Heart Failure (HF) is the re-emergence of the fetal gene program. The aim of this study was to identify novel genes associated with the fetal gene program in HF, by utilizing a mouse embryonic stem (mES) cell based micro-array. Methods/Results: The micro-array analysis was performed on mES cells, early and late mES-derived cardiac progenitors, and mouse neonatal right ventricular tissue. As expected, our screen effectively identified established fetal genes, including alpha smooth muscle actin. We further uncovered several novel genes behaving like fetal genes. The top 5 hits were validated in an in vivo pressure overload HF model. 5-oxoprolinase (OPLAH) was found to be the most significantly differentially expressed gene in the HF model. In a human organ panel, OPLAH gene expression was highest in cardiac tissue, and found to be expressed mainly in the cytosol of cardiomyocytes. Additionally OPLAH protein levels were reduced by 50-70% in both pressure overload and ischemia induced HF (p=0.001 and p=0.009, respectively). OPLAH is an ATP-hydrolyzing enzyme involved in the γ-Glutamyl cycle, responsible for the conversion of the metabolite 5-oxoproline into glutamate. To study OPLAH function we used siRNA and/or over-expression by means of viral transfection of neonatal rat ventricular myocytes. In vitro silencing of OPLAH resulted in an increased susceptibility towards oxidative stress induced apoptosis (40% increase in cleaved caspase-3 positive cells, p=0.02). Furthermore, exogenous administration of 5-oxoproline increased apoptosis in cardiomyocytes (40% increase in cleaved caspase-3 positive cells, p=0.04). Conclusions: We identified OPLAH as a novel enzyme associated with HF, behaving like fetal genes, which is involved in the innate protection against oxidative stress, by scavenging excess 5-oxoproline.

scholarly journals Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
Xu Yan ◽  
Jinwen Tian ◽  
Hongjin Wu ◽  
Yuna Liu ◽  
Jianxun Ren ◽  
...  
Keyword(s):  
Caspase 3 ◽  
Neonatal Rat ◽  
Ginsenoside Rb1 ◽  
Caspase 9 ◽  
Apoptotic Pathway ◽  
Rat Cardiomyocytes ◽  
Mitochondrial Apoptotic Pathway ◽  
Neonatal Rat Cardiomyocytes ◽  
Hypoxia Ischemia

Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1) on hypoxia/ischemia (H/I) injury in cardiomyocytesin vitroand the mitochondrial apoptotic pathway mediated mechanism.Methods. Neonatal rat cardiomyocytes (NRCMs) for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP) were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit.Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS) burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm). Its administration also inhibited activities of caspase-9 and caspase-3.Conclusion. Administration of GS-Rb1 during H/Iin vitrois involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

scholarly journals Sorcin modulates mitochondrial Ca2+ handling and reduces apoptosis in neonatal rat cardiac myocytes

2013 ◽  
Vol 304 (3) ◽  
pp. C248-C256 ◽  
Author(s):  
Jorge Suarez ◽  
Patrick M. McDonough ◽  
Brian T. Scott ◽  
Angelica Suarez-Ramirez ◽  
Hong Wang ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cardiac Myocytes ◽  
Caspase 3 ◽  
Neonatal Rat ◽  
Caspase Activation ◽  
Cytochrome C Release ◽  
Permeabilized Cells ◽  
Neonatal Rat Cardiac Myocytes ◽  
Rat Cardiac Myocytes ◽  
Uptake And Release

Sorcin localizes in cellular membranes and has been demonstrated to modulate cytosolic Ca2+ handling in cardiac myocytes. Sorcin also localizes in mitochondria; however, the effect of sorcin on mitochondrial Ca2+ handling is unknown. Using mitochondrial pericam, we measured mitochondrial Ca2+ concentration and fluxes in intact neonatal cardiac myocytes overexpressing sorcin. Our results showed that sorcin increases basal and caffeine-stimulated mitochondrial Ca2+ concentration. This effect was associated with faster Ca2+ uptake and release. The effect of sorcin was specific for mitochondria, since similar results were obtained with digitonin-permeabilized cells, where cytosolic Ca2+ flux was disrupted. Furthermore, mitochondria of cardiac myocytes in which sorcin was overexpressed were more Ca2+-tolerant. Experiments analyzing apoptotic signaling demonstrated that sorcin prevented 2-deoxyglucose-induced cytochrome c release. Furthermore, sorcin prevented hyperglycemia-induced cytochrome c release and caspase activation. In contrast, antisense sorcin induced caspase-3 activation. Thus, sorcin antiapoptotic properties may be due to modulation of mitochondrial Ca2+ handling in cardiac myocytes.

scholarly journals NMDA receptor activation induces mitochondrial dysfunction, oxidative stress and apoptosis in cultured neonatal rat cardiomyocytes

2007 ◽  
pp. 559-569
Author(s):  
X Gao ◽  
X Xu ◽  
J Pang ◽  
C Zhang ◽  
JM Ding ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Caspase 3 ◽  
Receptor Activation ◽  
Nmda Receptor Antagonist ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Rat Cardiomyocytes ◽  
Peripheral Tissues ◽  
Excitatory Neurotransmitter ◽  
Neonatal Rat Cardiomyocytes

Glutamate is a well-characterized excitatory neurotransmitter in the central nervous system (CNS). Recently, glutamate receptors (GluRs) were also found in peripheral tissues, including the heart. However, the function of GluRs in peripheral organs remains poorly understood. In the present study, we found that N-methyl-D-aspartate (NMDA) could increase intracellular calcium ([Ca(2+)]i) level in a dose-dependent manner in cultured neonatal rat cardiomyocytes. NMDA at 10(-4) M increased the levels of reactive oxygen species (ROS), cytosolic cytochrome c (cyto c), and 17-kDa caspase-3, but depolarized mitochondrial membrane potential, leading to cardiomyocyte apoptosis. In addition, NMDA treatment induced an increase in bax mRNA but a decrease in bcl-2 mRNA expression in the cardiomyocytes. The above effects of NMDA were blocked by the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), and by ROS scavengers glutathione (GSH) and N-acetylcystein (NAC). These results suggest that stimulation of NMDA receptor in the cardiomyocyte may lead to apoptosis via a Ca(2+), ROS, and caspase-3 mediated pathway. These findings suggest that NMDA receptor may play an important role in myocardial pathogenesis.

scholarly journals Hypaconitine confers protection on ketamine-induced neuronal injury in neonatal rat brain via a mechanism involving PI3K/Akt/Bcl-2 pathway

2021 ◽  
Vol 18 (9) ◽  
pp. 1839-1844
Author(s):  
Junbao Liu ◽  
Yubo Hu ◽  
Linlin Li ◽  
Longyun Li
Keyword(s):  
Cell Viability ◽  
Caspase 3 ◽  
Neuroprotective Effect ◽  
Neuronal Cells ◽  
Specific Inhibitor ◽  
Neuronal Cell ◽  
Neuronal Injury ◽  
Neonatal Rat ◽  
Control Group ◽  
Neonatal Rats

Purpose: To investigate the neuroprotective effect of hypaconitine against ketamine-induced neuronal injury in the brains of neonatal rats, and the underlying mechanism of action. Methods: Seven day-old Sprague-Dawley pups weighing 15.0 to 20.0 g (mean weight = 17.5 ± 2.5 g), and aged 7 days were used for this study. The pups were sacrificed, and their forebrains isolated and used to prepare cell suspensions. The isolated cells were treated with ketamine (100 µM) or varied concentrations of hypaconitine (0.1 – 2 µM) or LY294002 (10 µM). The cells were trypsinized and cultured at 37 °C in 10 % fetal bovine serum (FBS) supplemented Dulbecco's modified Eagle's medium (DMEM) in a humidified incubator containing 5 % CO2. Cell viability was determined using MTT assay, while TUNEL assay was used to determine the extent of apoptosis in the cells. The expressions of pAkt, Bcl-2 and caspase-3 were determined using Western blotting. Results: There were only few viable cells in the ketamine-treated group, and cell viability was significantly and dose-dependently increased in hypaconitine-treated groups (p < 0.05). The extent of apoptosis was significantly higher in ketamine-treated cells than in control cells, but treatment with hypaconitine significantly reduced the number of apoptotic cells (p < 0.05). However, in the presence of LY294002 (a PI3K-specific inhibitor), the effect of hypaconitine on neuronal cell apoptosis was significantly reversed (p < 0.05). The expressions of p-Akt and Bcl-2 were significantly down-regulated while the expression of caspase-3 was significantly upregulated in ketamine-treated neuronal cells, when compared with control group (p < 0.05). However, in cells treated with hypaconitine, the expressions of p-Akt and Bcl-2 were significantly upregulated, while the expression of caspase-3 was significantly down-regulated (p < 0.05). Treatment of neuronal cells with hypaconitine in the presence of LY294002 significantly reversed the effect of hypaconitine on the expressions of p-Akt, Bcl-2 and caspase-3 (p < 0.05). Conclusion: These results suggest that hypaconitine ameliorates ketamine-induced neuronal injury in neonatal rats via a mechanism involving the PI3K/Akt/Bcl-2 pathway.

scholarly journals Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia In Vitro

2014 ◽  
Vol 120 (1) ◽  
pp. 50-61 ◽  
Author(s):  
Lisa V. Doan ◽  
Olga Eydlin ◽  
Boris Piskoun ◽  
Richard P. Kline ◽  
Esperanza Recio-Pinto ◽  
...  
Keyword(s):  
Dorsal Root Ganglia ◽  
Local Anesthetics ◽  
Dorsal Root ◽  
Caspase 3 ◽  
Cytosolic Calcium ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Drg Neurons ◽  
Induced Changes

Abstract Background: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. Methods: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. Results: The mean KCl-induced calcium transient was greater in P7 neurons (P &lt; 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P &lt; 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Conclusions: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis

2000 ◽  
Vol 279 (5) ◽  
pp. H2124-H2132 ◽  
Author(s):  
Genevieve C. Sparagna ◽  
Diane L. Hickson-Bick ◽  
L. Maximilian Buja ◽  
Jeanie B. McMillin
Keyword(s):  
Cardiac Myocytes ◽  
Caspase 3 ◽  
Cardiac Myocyte ◽  
Mitochondrial Permeability Transition ◽  
Permeability Transition ◽  
Carnitine Palmitoyltransferase ◽  
Complex Iii ◽  
Neonatal Rat ◽  
Metabolic Role ◽  
Carnitine Palmitoyltransferase I

After cardiac ischemia, long-chain fatty acids, such as palmitate, increase in plasma and heart. Palmitate has previously been shown to cause apoptosis in cardiac myocytes. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. Phosphatidylserine translocation and caspase 3-like activity confirmed the apoptotic action of palmitate. Cytosolic cytochrome cwas detected at 8 h and plateaued at 12 h. The mitochondrial membrane potential (ΔΨ) in tetramethylrhodamine ethyl ester-loaded cardiac myocytes decreased significantly in individual mitochondria by 8 h. This loss was heterogeneous, with a few energized mitochondria per myocyte remaining at 24 h. Total ATP levels remained high at 16 h. The ΔΨ loss was delayed by cyclosporin A, a mitochondrial permeability transition inhibitor. Mitochondrial swelling accompanied changes in ΔΨ. Carnitine palmitoyltransferase I activity fell at 16 h; this decline was accompanied by ceramide increases that paralleled decreased complex III activity. We conclude that carnitine palmitoyltransferase I inhibition, ceramide accumulation, and complex III inhibition are downstream events in cardiac apoptosis mediated by palmitate and occur independent of events leading to caspase 3-like activation.

scholarly journals Hydrogen-Rich Saline Attenuates Brain Injury Induced by Cardiopulmonary Bypass and Inhibits Microvascular Endothelial Cell Apoptosis Via the PI3K/Akt/GSK3β Signaling Pathway in Rats

2017 ◽  
Vol 43 (4) ◽  
pp. 1634-1647 ◽  
Author(s):  
Keyan Chen ◽  
Nan Wang ◽  
Yugang Diao ◽  
Wanwei Dong ◽  
YingJie Sun ◽  
...  
Keyword(s):  
Cardiopulmonary Bypass ◽  
Brain Injury ◽  
Signaling Pathway ◽  
Brain Tissue ◽  
Caspase 3 ◽  
Cell Structure ◽  
Regulatory Gene ◽  
Open Heart Surgery ◽  
Inflammatory Factors ◽  
Microvascular Endothelial

Background/Aims: Cardiopulmonary bypass (CPB) is prone to inducing brain injury during open heart surgery. A hydrogen-rich solution (HRS) can prevent oxidation and apoptosis, and inhibit inflammation. This study investigated effects of HRS on brain injury induced by CPB and regulatory mechanisms of the PI3K/Akt/GSK3β signaling pathway. Methods: A rat CPB model and an in vitro cell hypoxia model were established. After HRS treatment, Rat behavior was measured using neurological deficit score; Evans blue (EB) was used to assess permeability of the blood-brain barrier (BBB); HE staining was used to observe pathological changes; Inflammatory factors and brain injury markers were detected by ELISA; the PI3K/Akt/GSK3β pathway-related proteins and apoptosis were assessed by western blot, immunohistochemistry and qRT –PCR analyses of brain tissue and neurons. Results: After CPB, brain tissue anatomy was disordered, and cell structure was abnormal. Brain tissue EB content increased. There was an increase in the number of apoptotic cells, an increase in expression of Bax and caspase-3, a decrease in expression of Bcl2, and increases in levels of Akt, GSK3β, P-Akt, and P-GSK3β in brain tissue. HRS treatment attenuated the inflammatory reaction ,brain tissue EB content was significantly reduced and significantly decreased expression levels of Bax, caspase-3, Akt, GSK3β, P-Akt, and P-GSK3β in the brain. After adding the PI3K signaling pathway inhibitor, LY294002, to rat cerebral microvascular endothelial cells (CMECs), HRS could reduce activated Akt expression and downstream regulatory gene phosphorylation of GSK3β expression, and inhibit CMEC apoptosis. Conclusion: The PI3K/Akt/GSK3β signaling pathway plays an important role in the mechanism of CPB-induced brain injury. HRS can reduce CPB-induced brain injury and inhibit CMEC apoptosis through the PI3K/Akt/GSK3β signaling pathway.

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