OBJECTIVE: To investigate the effects of salvianolic acid A (SAA) on cardiomyocyte apoptosis and mitochondrial dysfunction in response to hypoxia/reoxygenation (H/R) injury and to determine whether the Akt signaling pathway might play a role. METHODS: An in vitro model of H/R injury was used to study outcomes on primary cultured neonatal rat cardiomyocytes. The cardiomyocytes were treated with 12.5, 25, 50 μg/mL SAA at the beginning of hypoxia and reoxygenation, respectively. Adenosine triphospate (ATP) and reactive oxygen species (ROS) levels were assayed. Cell apoptosis was evaluated by flow cytometry and the expression of cleaved-caspase 3, Bax and Bcl-2 were detected by Western blotting. The effects of SAA on mitochondrial dysfunction were examined by determining the mitochondrial membrane potential (△Ψm) and mitochondrial permeability transition pore (mPTP), followed by the phosphorylation of Akt (p-Akt) and GSK-3β (p-GSK-3β), which were measured by Western blotting. RESULTS: SAA significantly preserved ATP levels and reduced ROS production. Importantly, SAA markedly reduced the number of apoptotic cells and decreased cleaved-caspase 3 expression levels, while also reducing the ratio of Bax/Bcl-2. Furthermore, SAA prevented the loss of △Ψm and inhibited the activation of mPTP. Western blotting experiments further revealed that SAA significantly increased the expression of p-Akt and p-GSK-3β, and the increase in p-GSK-3β expression was attenuated after inhibition of the Akt signaling pathway with LY294002. CONCLUSION SAA has a protective effect on cardiomyocyte H/R injury; the underlying mechanism may be related to the preservation of mitochondrial function and the activation of the Akt/GSK-3β signaling pathway.
Adenosine Triphosphate ; analysis ; Animals ; Animals, Newborn ; Caffeic Acids ; pharmacology ; Cell Hypoxia ; Cells, Cultured ; Glycogen Synthase Kinase 3 beta ; physiology ; Lactates ; pharmacology ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Myocytes, Cardiac ; drug effects ; Proto-Oncogene Proteins c-akt ; physiology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; physiology

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

The opening of mitochondrial permeability transition pore (MPTP) plays a critical role in platelet activation. However, the potential trigger of the MPTP opening in platelet activation remains unknown. Inflammation is the crucial trigger of platelet activation. In this study, we aimed to explore whether and how the important inflammatory cytokine IL-17 is associated with MPTP opening in platelets activation by using MPTP inhibitor cyclosporine-A (CsA). The mitochondrial membrane potential (ΔΨm) was detected to reflect MPTP opening levels. And the platelet aggregation, activation, and the primary signaling pathway were also tested. The results showed that the MPTP opening levels were increased and Δψm reduced in platelets administrated with IL-17. Moreover, the levels of aggregation, CD62P, PAC-1, P53 and the phosphorylation of ERK2 were enhanced along with the MPTP opening in platelets pre-stimulated with IL-17. However, CsA attenuated these effects triggered by IL-17. It was suggested that IL-17 could induce MPTP opening through ERK2 and P53 signaling pathway in platelet activation and aggregation.
Blood Platelets ; cytology ; drug effects ; metabolism ; Cell Separation ; Cyclosporine ; pharmacology ; Dual Specificity Phosphatase 2 ; genetics ; metabolism ; Gene Expression Regulation ; Humans ; Interleukin-17 ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; agonists ; antagonists & inhibitors ; genetics ; metabolism ; Mitogen-Activated Protein Kinase 1 ; genetics ; metabolism ; P-Selectin ; genetics ; metabolism ; Phosphorylation ; drug effects ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Primary Cell Culture ; Signal Transduction ; Tumor Suppressor Protein p53 ; genetics ; metabolism

OBJECTIVETo explore the effects of Panax Quinquefolium Saponin (PQS) on phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway of neonatal rat myocardial cells subjected to hypoxia.

METHODSNeonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups: the normal group, the model group, the positive control group (Ciclosporin A, 2 µ mol/L), the low-dose PQS group (PQSL, 25mg/L), and the high-dose PQS group (PQSH, 50 mg/L). Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase (LDH) leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase (GSK)-3β and phospho-Akt as well as cytochrome C were determined by Western blot

RESULTSLDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group (P<0.05). Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group (P<0.05, P<0.01). Compared with the model group (16.41±1.74; 35.28±6.30), both the integrated optical density of mitochondrial permeability transition pore (MPTP) and the mitochondrial transmembrane potential significantly increased in the PQSH group (42.74±2.12; 71.36±6.54) and the PQSL group (39.58±1.49; 66.99±5.45; P<0.05, P<0.01). However, the protein of cytochrome C outside the mitochondrion decreased in the PQSH group (273.66±14.61) and the PQSL group (259.62±17.31) compared with the model group (502.41±17.76; P<0.05).

CONCLUSIONThrough activation of the PI3K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

Animals ; Animals, Newborn ; Cell Hypoxia ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; L-Lactate Dehydrogenase ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; enzymology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein-Serine-Threonine Kinases ; metabolism ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo observe the impairing effects of triptolide on liver mitochondria in isolated rat-liver mitochondria and human normal liver HL7702 cell line.

METHODSRat-liver mitochondria were isolated from adult female Sprague-Dawley (SD) rats. Liver mitochondria were incubated with 0, 1.25, 2.5, 5 and 10 μmol/L triptolide for detecting mitochondrial swelling and with 0, 2.5, 5 and 10 μmol/L triptolide for mitochondrial permeability transition pore (MPTP) activity. Mitochondrial swelling was estimated by measuring the apparent absorbance change during 600 s in the mitochondrial suspensions at 520 nm with a mitochondrial swelling examining kit. The effect of triptolide on MPTP was determined with a fluorescence detection kit by detecting the fluorescence intensity at an excitation wavelength of 488 nm emitted at 527 nm. Human normal liver HL7702 cells were treated without or with 0.02, 0.1 and 0.5 μmol/L triptolide for 24 h for analyzing mitochondrial transmembrane potential (Δψm) and reactive oxygen species (ROS). Δψm was measured using the fluorescent probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). ROS was measured using fluorescent probe 2',7'-dichlorofluorescin diacetate (DCFH-DA). The cells were harvested and dyed with JC-1 and DCFH-DA, and analyzed by flow cytometry, respectively.

RESULTSIncubation of isolated mitochondria with triptolide results in swollen mitochondria in a concentration-dependent manner. Moreover, triptolide significantly activated mitochondrial permeability transition at 5 and 10 μmol/L (P<0.05 and P<0.01). When HL7702 cells were exposed to a various concentration triptolide for 24 h, mitochondrial membrane depolarization and increase of ROS were caused by triptolide in a concentration-dependent manner. Triptolide significantly induced the mitochondrial membrane depolarization at 0.1 and 0.5 μmol/L (P<0.05 and P<0.01) and the increase of ROS at 0.1 and 0.5 μmol/L (P<0.05 and P<0.01).

CONCLUSIONTriptolide could induce mitochondrial impairment, which may be one of the mechanisms by which hepatotoxicity occurs.

Animals ; Cell Line ; Diterpenes ; chemistry ; pharmacology ; Epoxy Compounds ; chemistry ; pharmacology ; Female ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Swelling ; drug effects ; Phenanthrenes ; chemistry ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the intervention effect and the possibly mechanism of the glutamine (Gln) on the opening change of the permeability transition pore (PTP) in the myocardial mitochondrial membrane under the overtraining state.

METHODS30 SD rats were randomly divided into 3 groups (n = 10): control group (CG group), overtraining group (OG group) and supplementary (Gln) + overtraining group group). Spectrophotometry was used to test the openness of the permeability transition pore in the myocardial mitochondrial membrane. Electrochemistry was used to test the malondialdehyde (MDA) and the glutathione (GSH) content and the phospholipase A2 (PLA2) activity.

RESULTSOG group compared with the GOG group, the absorbance (A0) and the absorbance change (Delta A) were decreased significantly (P < 0.05). Rh123 fluorescence (F0) intensity was significantly increased (P < 0.05). Rhodamine123 (Rh123) fluorescence change (delta F) was significantly decreased (P < 0.05). Compared with the GOG, the mitochondrial GSH was significantly decreased (P < 0.05), the PLA2 activity and the content of MDA were significantly increased (P <0.05).

CONCLUSIONOvertraining could lead to opening increase of permeability transition pore in the myocardial mitochondrial membrane, after overtraining, the production of the reactive oxygen species (ROS) and PLA2 activity were increased, GSH content was decreased. But added exogenous Gln had a significant intervention effect for these changes.

Animals ; Glutamine ; pharmacology ; Glutathione ; metabolism ; Male ; Mitochondria, Heart ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; metabolism ; Mitochondrial Membranes ; drug effects ; physiology ; Myocardium ; metabolism ; Permeability ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism

OBJECTIVETo explore the effect of MnCl(2) on the mitochondrial function of human lung cells, and to study the changes of protein expression level of nuclear respiratory factor-1 (NRF-1) in mitochondrial dysfunction induced by MnCl(2).

METHODSThe effects of MnCl(2) on cell survival rate were assessed by the reductions of tetrazolium dye (MTT) in cultured cell lines 16HBE and A549 cells. All tested16HBE and A549 cells were incubated with different concentrations of MnCl(2). The permeability transition pore (PTP) of mitochondria, mitochondrial membrane potential and the inhibition rate of mitochondrial enzymes as indicators of mitochondrial damage were measured by fluorescent spectrometry and MTT assay, respectively. Apoptosis was determined by flow cytometry. Protein levels of NRF-1 and mtTFA were measured by Western blot assay.

RESULTSMnCl(2) decreased the survival rate of the two cell lines. The IC(50) of 16HBE and A549 cells were 1.91 mmol/L and 1.98 mmol/L, respectively. MnCl(2) caused a concentration-dependent decrease of mitochondrial enzymes and the inhibition rate of mitochondrial enzymes of the two cell lines induced by 1.00 mmol/L MnCl(2) were (52.8 ± 5.4)% and (50.6 ± 2.2)%, respectively. The PTP opening increased in MnCl(2)-treated cells in a dose- and time-dependent manner. Compared with the control group, mitochondrial membrane potential in the two cell lines was decreased by MnCl(2), by (7.9 ± 3.0)%, (26.2 ± 2.2)% and (27.8 ± 4.1)% in the 16HBE cells, and (4.7 ± 1.0)%, (14.9 ± 2.4)% and (27.5 ± 1.2)% in the A549 cells. Increased apoptosis rates of the two cell lines were induced by 1.00 mmol/L MnCl(2), (12.3 ± 1.9)% and (6.0 ± 0.4)%, respectively. The results of Western blot assay revealed that the protein levels of NRF-1 and mtTFA were decreased in manganese-treated cells in a dose-dependent manner, with a significant difference compared with that of the control cells (P < 0.05).

CONCLUSIONMnCl(2) induces mitochondrial dysfunction in 16HBE and A549 cells, and decreases the expression level of nuclear respiratory factor-1 (NRF-1), indicating that NRF-1 may play an important role in mitochondrial dysfunction.

Apoptosis ; drug effects ; Bronchi ; cytology ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Chlorides ; administration & dosage ; toxicity ; DNA-Binding Proteins ; metabolism ; Dose-Response Relationship, Drug ; Epithelial Cells ; cytology ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Manganese Compounds ; administration & dosage ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; physiology ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Proteins ; metabolism ; Nuclear Respiratory Factor 1 ; metabolism ; Transcription Factors ; metabolism

OBJECTIVETo study effects of three pentacyclic triterpenoids, oleanolic acid (OA), ursolic acid (UA) and asiatic acid (AA) on Ca(2+)-induced liver mitochondrial permeability transition (MPT).

METHODEffects of three compounds on liver MPT induced by Ca2+ were assessed by measuring the change in mitochondrial swelling, mitochondrial membrane potential and release of matrix Ca2+ in vitro.

RESULTObvious mitochondrial swelling, loss of mitochondrial membrane potential and release of matrix Ca2+ occurred after the addition of 50 micromol x L(-1) Ca2+. However, preincubation with 50 mg x L(-1) OA, UA or AA significantly blocked the above changes. In addition, it was also found that there are differences in the inhibitions of three compounds on liver MPT induced by Ca2+.

CONCLUSIONThree pentacyclic triterpenoids, OA, UA and AA, have significant mitochondrial protection through blocking on liver MPT and the inhibition on liver MPT of AA is stronger than that of UA and OA.

Animals ; Calcium ; metabolism ; pharmacology ; Membrane Potential, Mitochondrial ; drug effects ; Mice ; Mice, Inbred ICR ; Mitochondria, Liver ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; drug effects ; Mitochondrial Swelling ; drug effects ; Pentacyclic Triterpenes ; pharmacology

OBJECTIVETo determine whether the cardioprotection of puerarin (Pue) against hypoxia/reoxygenation injury is mediated by mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) and/or mitochondria calcium-activated potassium channel(mitoK(Ca)).

METHODSCardiomyocytes were isolated from male Sprague-Dawley rats and hypoxia/reoxygenation injury was induced by myocyte pelleting model. Cell viability was assessed by trypan blue exclusion and mitochondrial membrane potential was measured by loading with TMRE. The opening of mitochondrial permeability transition pore was determined spectrophotometrically.

RESULTSPretreatment with Pue at 0.24 mmol/L for 5 min increased the cell viability against hypoxia/reoxygenation injury, while mitochondrial ATP-sensitive potassium channel inhibitor 5-hydroxydecanoate (5-HD, 100 micromol/L, 20 min) or mitochondrial calcium-activated potassium channel blocker paxilline (Pax, 1 micromol/L, 5 min) attenuated the effect of puerarin. The pretreatment with Pue at 0.24 mmol/L for 5 min attenuated collapse of delta-psim induced by hypoxia/reoxygenation injury, 5-HD and Pax abrogated the effect of Pue. In mitochondria isolated from hearts pretreated with Pue, a significant inhibition of Ca(2+)-induced swelling was observed, and this inhibition was attenuated by 5-HD and Pax.

CONCLUSIONThese findings indicate that Pue protects cardiomyocytes against hypoxia/reoxygenation injury via activating mitoK(ATP) channel and mitoK(Ca) channel, and inhibiting mitochondrial permeability transition pore opening.

Androsterone ; pharmacology ; Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Indoles ; pharmacology ; Isoflavones ; pharmacology ; Male ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocardium ; cytology ; Myocytes, Cardiac ; drug effects ; metabolism ; Potassium Channels ; metabolism ; Potassium Channels, Calcium-Activated ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo determine whether the caidioprotection of acetylcholine (ACh) against ischeniia/reperftision (I/R) injury is re-kited to mitochondrial permeability transition pore (MEW) and mitochondrial AW-sensitive potassium channel (mitoK(ATP)).

METHODSMale Sprague-Dawley rats were used for Langendorif isolated bean perkision. The hearts were subjected to global ischemia for 30 mm followed by 120 rein of reperfusion and the left ventricular hemodynaniic parameters were measured. Formazan, a product of 2,3, 5-triphenyl-tetrazolium chloride (TTC), which is proportional to myocardial viability, was measured at 490 nm, and the level of lactate dehydrogenase (LDH) in the coronary effluent was measured to evaluate the cardiac injury.

RESULTSThe pretreatment with ACh (0.1 mol/L, 5 mm) before I/R markedly increased myocardial formazan content, reduced LDH release, improved the recovery of the left veritficular developed pressure, +/- dP/dtmax, and rate pressure product (left ventricular developed pressure multiplied by hean rate) and attenuated the decrease of coronary flow during reperfusion. The opener of MPTP, atiractyloside (20 mmoL/L) or the inhibitor of mitoK(ATP), 5-hydroxydecanoate (100 micromol/L) abolisbed the beneficial effect of ACh.

CONCLUSIONIn the isolated rat bean, ACh protects myocardium against ischemia/reperfusion injury via inhibiting the opening of MPTP and increasing the opening of mitoKATP in heart.

Acetylcholine ; pharmacology ; Animals ; Cardiotonic Agents ; pharmacology ; In Vitro Techniques ; Ischemic Preconditioning ; methods ; Male ; Mitochondrial Membrane Transport Proteins ; drug effects ; metabolism ; Myocardial Ischemia ; physiopathology ; Myocardial Reperfusion Injury ; prevention & control ; Potassium Channels ; metabolism ; Rats ; Rats, Sprague-Dawley