OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with HO, gastrodin, gastrodin+HO, cyclosporin A (CsA), or CsA+gas+HO group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in HO-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in HO-exposed cells. CONCLUSIONS Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.
Adenosine Triphosphate ; analysis ; Apoptosis ; drug effects ; Benzyl Alcohols ; antagonists & inhibitors ; pharmacology ; Caspase 3 ; analysis ; Cell Line ; Cell Survival ; drug effects ; Cyclosporine ; pharmacology ; Cytochromes c ; analysis ; Glucosides ; antagonists & inhibitors ; pharmacology ; Humans ; Hydrogen Peroxide ; antagonists & inhibitors ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondrial Membrane Transport Proteins ; physiology ; Myocytes, Cardiac ; drug effects ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

OBJECTIVEScorpion (Hemiscorpius lepturus) stings are a public health concern in Iran, particularly in south and southwestern regions of Iran. The gold standard for the treatment of a scorpion sting is anti-venom therapy. However, immunotherapy can have serious side effects, such as anaphylactic shock (which can sometimes even lead to death). The aim of the current study was to demonstrate the protective effect of ozone against toxicity induced by Hemiscorpius lepturus (H. lepturus) venom in mice.

METHODSEight hours after the injection of ozone to the experimental design groups, the male mice were decapitated and mitochondria were isolated from five different tissues (liver, kidney, heart, brain, and spinal cord) using differential ultracentrifugation. Then, assessment of mitochondrial parameters including mitochondrial reactive oxidative species (ROS) production, mitochondrial membrane potential (MMP), ATP level, and the release of cytochrome c from the mitochondria was performed.

RESULTSOur results showed that H. lepturus venom-induced oxidative stress is related to ROS production and MMP collapse, which is correlated with cytochrome c release and ATP depletion, indicating the predisposition to the cell death signaling.

CONCLUSIONIn general, ozone therapy in moderate dose can be considered as clinically effective for the treatment of H. lepturus sting as a protective and antioxidant agent.

Animals ; Brain ; drug effects ; metabolism ; Cytochromes c ; metabolism ; Heart ; drug effects ; Kidney ; drug effects ; metabolism ; Liver ; drug effects ; metabolism ; Male ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred BALB C ; Muscle, Skeletal ; drug effects ; metabolism ; Myocardium ; metabolism ; Ozone ; pharmacology ; Scorpion Venoms ; toxicity ; Scorpions ; physiology ; Spinal Cord ; drug effects ; metabolism

OBJECTIVETo explore the molecular mechanism of realgar-induced apoptosis of cervical cancer cells.

METHODSThe cervical cancer cell line Siha was used to determine the cell viability and apoptosis after treatment with realgar using MTT assay and flow cytometry. The activities of caspase-3, -8, and -9 were detected by fluorescence resonance energy transfer technology and colorimetric assay, while the levels of Bcl-2, cytochrome c, and Bax were detected by Western blot method.

RESULTSInduction of apoptosis by realgar was detected in Siha cell line in a dose-dependent manner. The apoptosis was accompanied by a significant increase in cytochrome c release and activation of caspase-3 and caspase-9 but not caspase-8. Further, the realgar-induced apoptosis was inhibited by a broad-spectrum caspase inhibitor, a caspase-3 inhibitor, and a caspase-9 inhibitor but not by a caspase-8 inhibitor. Bcl-2 and Bax protein expressions were not changed by realgar.

CONCLUSIONThe induction of apoptosis by realgar is mediated through a cytochrome c-dependent pathway, which sequentially activates caspase-9 and caspase-3.

Apoptosis ; drug effects ; physiology ; Arsenicals ; pharmacology ; Caspase 3 ; metabolism ; Caspase 8 ; metabolism ; Caspase 9 ; metabolism ; Caspase Inhibitors ; Cell Line, Tumor ; Cell Survival ; drug effects ; physiology ; Cytochromes c ; metabolism ; Dose-Response Relationship, Drug ; Enzyme Activation ; drug effects ; Enzyme Inhibitors ; pharmacology ; Female ; Fluorescence Resonance Energy Transfer ; Humans ; Sulfides ; pharmacology ; Uterine Cervical Neoplasms ; drug therapy ; metabolism ; pathology

BACKGROUNDNeuroblastoma (NB) is one of the most common malignant solid tumors of childhood. It is still not clear whether the apoptosis of tumor cells or the non-tumor cells contributes to the increase of concentration of cytochrome c (Cyt c) in the serum of the cancer patients. The aim of this research was to identify the source of the Cyt c in the serum when the tumor grows up by subcutaneous inoculation of human NB cells into nude mice.

METHODSWe subcutaneously inoculated human NB cells (KP-N-NS) into nude mice and collected the sera of tumor-bearing mice (n = 14) and control mice (n = 25) 4 weeks later in order to screen for and identify differentially expressed proteins in the serum. Differentially expressed proteins in the serum were screened by surface-enhanced laser desorption/ionization-time-of-flight (SELDI-TOF) mass spectrometry.

RESULTSThe relative intensity of a protein having a mass-to-charge ratio (m/z) of 11 609 was 3338.37 ± 3410.85 in the tumor group and 59.84 ± 40.74 in the control group, indicating that the expression level of this protein in the tumor group was 55.8 times higher than that in the control group. Serum proteins were separated and purified by high-performance liquid chromatography (HPLC). Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was performed to produce peptide mass fingerprints (PMFs). Spectrum analysis and a database search revealed that the highly expressed protein (m/z = 11 605.4) from the serum of tumor-bearing mice was the mouse Cyt c.

CONCLUSIONSIncreased concentration of Cyt c in the serum of tumor-bearing nude mice might be partially attributed to the secretion of this protein by non-tumor cells.

Animals ; Apoptosis ; physiology ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; Cytochromes c ; blood ; Female ; Humans ; Mice ; Mice, Nude ; Neuroblastoma ; blood ; Tandem Mass Spectrometry ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate cell apoptosis, mitochondrial membrane potential, cytochrome C and mechanisms of mitochondria in lymphocyte apoptosis of sepsis.

METHODSIn the research, female C57BL/6 mice whose body weight ranged from 17 to 25 grams were utilized and assigned randomly to two groups: sham operated group (Control), cecal ligation and puncture group (CLP). The present study was undertaken by using the mice splenic lymphocyte to investigate cell apoptosis, mitochondrial membrane potential, cytochrome C. The apoptosis alteration was evaluated by Annexin V-FITC/PI double staining with flow cytometry. The alteration of mitochondrial membrane potential was investigated by Rhodamine-123 staining of cells. Cytochrome C of mitochondria and cytosol was investigated by Western blot methods. Statistical analysis was performed using SPSS 11.5 for Windows software. Experiment data was indicated with mean ± standard.

RESULTSThe splenic lymphocyte apoptosis was significantly accelerated in the CLP group when compared with that in control group (17.3% ± 2.2% vs. 3.5% ± 0.5%, P < 0.05). The Rhodamine-123 fluorescent intensity in splenic lymphocyte apoptosis was reduced in CLP group (76.2% ± 1.6%). Comparison between sham group (99.6% ± 0.4%) and CLP group had statistical significance (P < 0.05). Apoptosis could induce mitochondrial cytochrome C release into cytoplasm. In the CLP group, elevation of cytochrome C in cytosol was concurrently in accordance with decline in mitochondrial cytochrome C content.

CONCLUSIONThese data suggest that mitochondria and mitochondria signal pathway play an important role in lymphocyte apoptosis of sepsis.

Animals ; Apoptosis ; Cells, Cultured ; Cytochromes c ; metabolism ; Disease Models, Animal ; Female ; Lymphocytes ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Mitochondria ; metabolism ; physiology ; Sepsis ; metabolism ; pathology

The present study aimed to investigate the change of cytochrome c in postconditioning-attenuated ischemia-reperfusion (I/R)-induced mucosal apoptosis in rat intestine compared with ischemic preconditioning (IPC). Using rat model of intestine I/R injury, male Sprague-Dawley rats weighing 220-250 g were divided into 4 groups which were Sham operation group, I/R group, IPC group and ischemic postconditioning (IPOST) group. In these groups, I/R procedure was performed by the occlusion of the superior mesenteric artery (SMA) for 45 min followed by reperfusion for 1 h. In Sham group, there was no intervention. In IPC group, SMA was occluded for 5 min and reperfused for 5 min, for two cycles, before the prolonged occlusion. In IPOST group, three cycles of 30-s reperfusion and 30-s reocclusion were preceded at the start of reperfusion. After the reperfusion, the small intestines were sampled for experimental detection. Intestinal mucosal mitochondrial membrane potential was detected by confocal laser scanning microscopy. Expressions of cytochrome c and caspase-3 proteins were detected using Western-blot method. The apoptosis of intestinal mucosal cells was determined with agarose gel electrophoresis and deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling (TUNEL) technique. Compared with I/R group, the mitochondrial membrane potentials and the expressions of cytochrome c protein were significantly increased, while the expressions of caspase-3 and the apoptotic rates were decreased in IPOST and IPC groups (P<0.05). There were no significant differences between IPOST and IPC groups (P>0.05). These data provide substantial evidence that IPOST attenuates I/R-induced mucosal apoptosis by reducing the release of cytochrome c from mitochondria in the rat small intestine.
Animals ; Apoptosis ; physiology ; Cytochromes c ; metabolism ; Intestinal Mucosa ; metabolism ; pathology ; Intestines ; blood supply ; Ischemic Postconditioning ; methods ; Male ; Membrane Potential, Mitochondrial ; Mitochondria ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control

Activated protein C (APC) is known to be beneficial on ischemia reperfusion injury in myocardium. However, the protection mechanism of APC is not fully understood. The purpose of this study was to investigate the effects and possible mechanisms of APC on myocardial ischemic damage. Artificially ventilated anaesthetized Sprague-Dawley rats were subjected to a 30 min of left anterior descending coronary artery occlusion followed by 2 hr of reperfusion. Rats were randomly divided into four groups; Sham, I/R, APC preconditioning and postconditioning group. Myocardial infarct size, apoptosis index, the phosphorylation of ERK1/2, Bcl-2, Bax and cytochrome c genes and proteins were assessed. In APC-administrated rat hearts, regardless of the timing of administration, infarct size was consistently reduced compared to ischemia/reperfusion (I/R) rats. APC improved the expression of ERK1/2 and anti-apoptotic protein Bcl-2 which were significantly reduced in the I/R rats. APC reduced the expression of pro-apoptotic genes, Bax and cytochrome c. These findings suggest that APC produces cardioprotective effect by preserving the expression of proteins and genes involved in anti-apoptotic pathways, regardless of the timing of administration.
Animals ; Apoptosis ; Cytochromes c/genetics/metabolism ; Hemodynamics/physiology ; Male ; Mitogen-Activated Protein Kinase 1/metabolism ; Mitogen-Activated Protein Kinase 3/metabolism ; Myocardial Reperfusion Injury/metabolism/pathology/*prevention & control ; Myocardium/*metabolism/pathology ; Phosphorylation ; Protein C/*therapeutic use ; Proto-Oncogene Proteins c-bcl-2/metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; bcl-2-Associated X Protein/metabolism

Apoptotic regulation is critical to organismal homeostasis and protection against many human disease processes such as cancer. Significant research efforts over the past several decades have illuminated many signaling molecules and effecter proteins responsible for this form of programmed cell death. Recent evidence suggests that transfer RNA (tRNA) regulates apoptotic sensitivity at the level of cytochrome c-mediated apoptosome formation. This finding unexpectedly places tRNA at the nexus of cellular biosynthesis and survival. Here we review the current understanding of both the apoptotic machinery and tRNA biology. We describe the evidence linking tRNA and cytochrome c in depth, and speculate on the implications of this link in cell biology.
Animals ; Apoptosis ; genetics ; physiology ; Caspase 9 ; physiology ; Cytochromes c ; physiology ; Humans ; Models, Biological ; Nucleic Acid Conformation ; RNA, Transfer ; chemistry ; genetics ; physiology

OBJECTIVETo investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase (PARP), and survivin of HT-29 cells.

METHODSHT-29 cells were treated with curcumin (0 approximately 80 micromol/L) for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSCurcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10 approximately 80 micromol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cytochrome c, the activation of caspase-3, and the cleavage of PARP in a dose-dependent manner.

CONCLUSIONThese data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitochondria-mediated pathway.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Cell Nucleus ; drug effects ; Cell Survival ; drug effects ; Curcumin ; pharmacology ; Cytochromes c ; secretion ; HT29 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Membrane Potential, Mitochondrial ; drug effects ; Microtubule-Associated Proteins ; genetics ; Mitochondria ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; RNA, Messenger ; analysis ; bcl-2-Associated X Protein ; genetics ; bcl-X Protein ; genetics

AIMTo explore the effect of propofol preconditioning on cardiomyocyte apoptosis and cytochrome C release from mitochondria during mild hypothermic ischemia/reperfusion in isolated rat hearts.

METHODS50 isolated SD rat hearts perfused on Langendorff apparatus were randomly divided into 5 groups (n=10): control group (C), DMSO group (D), 3 different concentrations of propofol groups of 25 micromol x L(-1) (P1), 50 micromol x L(-1) (P2), 100 micromol x L(-1) (P3) propofol respectively. All of the isolated rat hearts were subjected to 31 degrees C mild hypothermic ischemia for 55 min followed by 60 min reperfusion. The D, P1, P2, P3 groups were preconditioned by perfusing with K-H solution containing 20 micromol x L(-1) DMSO and 25, 50, 100 micromol x L(-1) propofol respectively for 10 min and then followed by 5 min K-H solution washing out before ischemia. The preconditioning procedure was repeated twice. Hemodynamics of the hearts was recorded after equilibration(baseline values) immediately before ischemia, 30 min and 60 min after reperfusion respectively. Cardiomyocyte apoptosis rate and contents of cytosolic and mitochondrial cytochrome C were measured at the end of reperfusion.

RESULTSAfter 30 min and 60 min reperfusion, LVEDP was significantly lower and LVDP was significantly higher in P3 group than those in C group ( P < 0.05, P < 0.01). Compared with C group, cardiomyocyte apoptosis rate of the hearts decreased significantly in P2,P3 groups at the end of reperfusion (P < 0.05, P < 0.01). Cytochrome C level increased significantly in mitochondria but decreased significantly in cytosol in P2, P3 groups as compared with C group (P < 0.05, P < 0.01).

CONCLUSIONPropofol preconditioning decreased cardiomyocyte apoptosis, protected the heart against 31 degrees C mild hypothermic ischemia/reperfusion injury by attenuation of the release of cytochrome C from mitochondria to cytosol.

Animals ; Apoptosis ; drug effects ; physiology ; Cytochromes c ; metabolism ; Hypothermia, Induced ; In Vitro Techniques ; Ischemic Preconditioning ; methods ; Male ; Mitochondria, Heart ; metabolism ; Myocardium ; metabolism ; pathology ; Propofol ; pharmacology ; therapeutic use ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; prevention & control