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

The aim of this study was to determine the effects of extremely low frequency electromagnetic field (ELF-EMF) on energy metabolism and oxidative stress in Caenorhabditis elegans (C. elegans). Worms in three adult stages (young adult stage, egg-laying stage and peak egg-laying stage) were investigated under 50 Hz, 3 mT ELF-EMF exposure. ATP levels, ATP synthase activity in vivo, reactive oxygen species (ROS) content, and changes of total antioxidant capacity (TAC) were detected, and worms' oxidative stress responses were also evaluated under ELF-EMF exposure. The results showed that ATP levels were significantly increased under this ELF-EMF exposure, and mitochondrial ATP synthase activity was upregulated simultaneously. In young adult stage, worms' ROS level was significantly elevated, together with upregulated TAC but with a decreased ROS-TAC score indicated by principal component analysis. ROS level and TAC of worms had no significant changes in egg-laying and peak egg-laying stages. Based on these results, we concluded that ELF-EMF can enhance worm energy metabolism and elicit oxidative stress, mainly manifesting as ATP and ROS level elevation together with ATP synthase upregulation and ROS-TAC score decrease in young adult C. elegans.
Adenosine Triphosphate ; metabolism ; Animals ; Caenorhabditis elegans ; radiation effects ; Electromagnetic Radiation ; Energy Metabolism ; Mitochondrial Proton-Translocating ATPases ; metabolism ; Oxidative Stress ; Reactive Oxygen Species ; analysis

ObjectiveTo investigate the correlation of the levels of reproductive hormones and oxidative stress in the seminal plasma with semen parameters in obese males.

METHODSBased on the body mass index (BMI), we divided 138 infertile men into three groups: normal (BMI <24 kg/m2, n = 48), overweight (24 kg/m2≤BMI<28 kg/m2, n = 47), and obesity (BMI ≥28 kg/m2, n = 43). We determined the concentrations of follicle-stimulating hormone (FSH), luteotropic hormone (LH), prolactin (PRL), testosterone (T) and estradiol (E2) in the serum by electrochemiluminescence and measured the levels of superoxide dismutase (SOD), glutathione-S-transferases (GSTs), reactive oxygen species (ROS) and malondialdehyde (MDA) in the seminal plasma by ELISA, compared the above indexes among the three groups, and analyzed their correlation with the semen volume, sperm concentration, total sperm count, and percentage of progressively motile sperm (PMS).

RESULTSThe semen volume was significantly lower in the obesity than in the normal group (［2.63 ± 0.74］ vs ［3.37 ± 1.00］ ml, P < 0.05), and so was the percentage of PMS in the overweight and even lower in the obesity than in the normal group (［47.91 ± 12.89］ and ［41.27 ± 15.77］ vs ［54.04 ± 13.29］%, P < 0.05). Compared with the normal group, both the overweight and obesity groups showed markedly decreased levels of serum T (［4.83 ± 1.42］ vs ［3.71 ± 1.22］ and ［3.49 ± 1.12］ ng/ml, P<0.05), T/LH ratio (1.53 ± 0.57 vs 1.19 ± 0.54 and 0.97 ± 0.51, P<0.05), SOD (［112.05 ± 10.54］ vs ［105.85 ± 6.93］ and ［99.33 ± 8.39］ U/ml, P<0.05), and GSTs (［31.75±6.03］ vs ［29.54±5.78］ and ［29.02±4.52］ U/L, P<0.05), but remarkably increased seminal plasma ROS (［549.93±82.41］ vs ［620.61±96.13］ and ［701.47±110.60］ IU/ml, P<0.05) and MDA (［7.46 ± 2.13］ vs ［8.72 ± 1.89］ and ［10.47 ± 2.10］ nmol/L, P<0.05). BMI was correlated positively with ROS and MDA, but negatively with the semen volume, PMS, T, T/LH, SOD and GSTs (P<0.05); LH negatively with sperm concentration, total sperm count and GSTs (P<0.05); PRL negatively GSTs (P<0.05); E2 positively with SOD (P<0.05); T positively with SOD (P<0.05) but negatively with MDA (P<0.05); T/LH positively with PMS and SOD (P<0.05) but negatively with ROS and MDA (P<0.05); SOD positively with semen volume, PMS and GSTs (P<0.05) but negatively with ROS and MDA (P<0.05); GSTs negatively with sperm concentration; total sperm count and MDA (P<0.05); ROS positively with MDA (P<0.01) but negatively with PMS (P<0.05); and MDA negatively with semen volume (P<0.05). Multivariate logistic regression analysis showed that the independent factors influencing the semen volume were BMI and GSTs, those influencing the total sperm count were BMI and T, and those influencing PMS were BMI and MDA.

CONCLUSIONSIncreased BMI induces changes in the levels of male reproductive hormones and seminal plasma oxidative stress and affects semen quality, which may be associated with male infertility.

Body Mass Index ; Estradiol ; blood ; Follicle Stimulating Hormone ; blood ; Humans ; Infertility, Male ; blood ; classification ; metabolism ; Luteinizing Hormone ; blood ; Male ; Malondialdehyde ; analysis ; Obesity ; blood ; metabolism ; Oxidative Stress ; Prolactin ; blood ; Reactive Oxygen Species ; analysis ; Reproduction ; Semen ; metabolism ; Semen Analysis ; Sperm Count ; Testosterone ; blood

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

Neurodegenerative diseases are the consequences of imbalance between the production of oxidative stress and its nullification by cellular defense mechanisms. Hydrogen peroxide (HO), a precursor of deleterious reactive oxygen species, elicits oxidative stress, resulting in severe brain injuries. Bacopa monnieri is well known for its nerve relaxing and memory enhancing properties. The present study was designed to evaluate the protective effects of extracts from Bacopa monnieri against HO induced oxidative stress using a cellular model, neuroblastoma IMR32 cell line. The protective potential of methanolic, ethanolic, and water extracts of B. monnieri (BM-MEx, BM-EEx, and BM-WEx) was evaluated using MTT assay. Although, all the B. monnieri extracts were found to protect cells against HO-mediated stress but BM-MEx showed significantly greater protection. UPLC analysis of BM-MEx revealed various polyphenols, including quercetin, catechin, umbelliferone, and caffeic acid predominance. Further, BM-MEx was found to possess considerable greater neuroprotective potential in comparison to the standard polyphenols such as quercetin, catechin, umbelliferone, and caffeic acid. The levels of antioxidant enzymes were significantly elevated after the pretreatment of BM-MEx and quercetin. The expression levels of oxidative stress markers, such as NF200, HSP70, and mortalin, were significantly alleviated after the pretreatment of BM-MEx as shown by immunofluorescence and RT-PCR. In conclusion, the present study demonstrated the protective effects of BM-MEx, suggesting that it could be a candidate for the development of neuropathological therapeutics.
Antioxidants ; metabolism ; pharmacology ; Bacopa ; chemistry ; Cell Line ; Humans ; Hydrogen Peroxide ; Neuroblastoma ; Neurodegenerative Diseases ; metabolism ; Neuroprotective Agents ; pharmacology ; Oxidative Stress ; drug effects ; Plant Extracts ; pharmacology ; Polyphenols ; analysis ; pharmacology ; Reactive Oxygen Species ; metabolism

Oxidative stress, a predominant cause of apoptosis cascades triggered in neurodegenerative disorders, has been regarded as a critical inducement in the pathogenesis of Alzheimer's disease (AD). Gou Teng-San (GTS) is a traditional Chinese herbs preparation commonly utilized to alleviate cognitive dysfunction and psychological symptoms of patients with dementia. The present study aimed to investigate the protective effects of GTS40, an active fraction of GTS, on HO-induced oxidative damage and identify the potential active ingredients. Our results revealed that GTS40 exhibited radical scavenging activity, elevated cell viability, decreased the levels of intracellular reactive oxygen species (ROS), and stabilized mitochondrial transmembrane potential (MMP) in HO-treated PC12 cells. In addition, GTS40 blocked the apoptotic cascade by reversing the imbalance of Bcl-2/Bax and inhibiting the activity of caspase-3. Furthermore, an HPLC-QTOFMS method was developed to characterize major chemical constituents in GTS40. Our results revealed twenty-seven identified or tentatively characterized compounds through comparing their retention time (t) and MS spectra with reference standards. These results suggested that GTS40 was a promising active fraction that may be beneficial in the prevention and treatment of oxidative stress-mediated neurodegenerative disorders.
Animals ; Antioxidants ; analysis ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; genetics ; metabolism ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Hydrogen Peroxide ; toxicity ; Neurons ; cytology ; drug effects ; metabolism ; Neuroprotective Agents ; analysis ; pharmacology ; Oxidative Stress ; drug effects ; PC12 Cells ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; Rats ; Reactive Oxygen Species ; metabolism

BACKGROUND: To the best of our knowledge, the association between pediatric AML and mitochondrial aberrations has not been studied. We investigated various mitochondrial aberrations in pediatric AML and evaluated their impact on clinical outcomes. METHODS: Sequencing, mitochondrial DNA (mtDNA) copy number determination, mtDNA 4,977-bp large deletion assessments, and gene scan analyses were performed on the bone marrow mononuclear cells of 55 pediatric AML patients and on the peripheral blood mononuclear cells of 55 normal controls. Changes in the mitochondrial mass, mitochondrial membrane potential, and intracellular reactive oxygen species (ROS) levels were also examined. RESULTS: mtDNA copy numbers were about two-fold higher in pediatric AML cells than in controls (P<0.0001). Furthermore, a close relationship was found between mtDNA copy number tertiles and the risk of pediatric AML. Intracellular ROS levels, mitochondrial mass, and mitochondrial membrane potentials were all elevated in pediatric AML. The frequency of the mtDNA 4,977-bp large deletion was significantly higher (P< 0.01) in pediatric AML cells, and pediatric AML patients harboring high amount of mtDNA 4,977-bp deletions showed shorter overall survival and event-free survival rates, albeit without statistical significance. CONCLUSIONS: The present findings demonstrate an association between mitochondrial genome alterations and the risk of pediatric AML.
Bone Marrow Cells/metabolism ; Case-Control Studies ; Child ; Cohort Studies ; DNA, Mitochondrial/chemistry/genetics/metabolism ; Female ; Flow Cytometry ; Gene Deletion ; Gene Dosage ; *Genome, Mitochondrial ; Humans ; Leukemia, Myeloid, Acute/genetics/mortality/*pathology ; Male ; Membrane Potential, Mitochondrial ; Minisatellite Repeats/genetics ; Odds Ratio ; Reactive Oxygen Species/metabolism ; Sequence Analysis, DNA ; Survival Rate

This study was undertaken to investigate the antioxidant/oxidant status in recurrent miscarriage patients. Antioxidants including glutathione peroxidase (GPx), catalase (CAT), glutathione reductase (GR), reduced glutathione (GSH) and selenium (Se), as well as the oxidants hydrogen peroxide (H2O2), oxidised glutathione (GSSG) and lipid peroxidation were assayed in plasma, whole blood and placental tissue of non-pregnant women (NP), healthy pregnant women (HP), and recurrent miscarriage (RM) patients. Results indicated that all antioxidant activities and levels in plasma and whole blood of HP women were consistently moderately lower, and much more significantly lower in RM patients when both were compared to those seen in NP women (P<0.05 and P<0.001, respectively). Furthermore, whereas plasma antioxidant activities and levels were significantly lower in RM patients, those of whole blood and placental tissue were much more significantly lower when compared with HP women (P<0.001). Concurrent with these findings there were consistent increases of equal statistical significance and magnitude in the levels of all investigated oxidants assayed in all samples when compared in between subjects of the study as indicated above. Data thus illustrated a distinct shift in favor of oxidative reactions and reactive oxygen species (ROS) generation, and very significant decreases in the GSH/GSSG ratios in whole blood and placental tissue of RM patients when compared to HP and NP women (P<0.001). The above noted oxidative stress could have been a major causative factor of recurrent miscarriage.
Abortion, Habitual/*blood/*epidemiology ; Adult ; Antioxidants/analysis ; Biomarkers/*blood ; Catalase/blood ; Female ; Glutathione/blood ; Glutathione Peroxidase/blood ; Glutathione Reductase/blood ; Humans ; Hydrogen Peroxide/analysis ; Lipid Peroxidation ; *Oxidative Stress ; Placenta/metabolism ; Pregnancy ; Reactive Oxygen Species/metabolism ; Saudi Arabia/epidemiology ; Selenium/blood

PURPOSE: Reactive oxygen species (ROS) are generated as an indirect product of radiation therapy (RT). Genetic variation in genes related to ROS metabolism may influence the level of RT-induced adverse effects. We evaluated the potential association of single nucleotide polymorphism (SNP)-related response to radiotherapy injury in breast cancer patients undergoing RT. MATERIALS AND METHODS: Eighty patients receiving conventional RT were included. Acute effects were evaluated according to the Radiation Therapy Oncology Group (RTOG) scores. DNA was extracted from blood and buccal swab samples. SNPs were genotyped for GSTP1, GSTA1, SOD2, and NOS3 genes by polymerase chain reaction-based restriction fragment length polymorphism. Univariate analysis (odds ratios [ORs] and 95% confidence interval [CI]) and principal component analysis were used for correlation of SNPs and factors related to risk of developing ≥ grade 2 acute effects. RESULTS: Sixty-five patients (81.2%) showed side effects, 32 (40%) presented moderate to severe acute skin toxicity, and 33 (41.2%) manifested minimal acute skin reactions by the end of treatment. In both univariate and multivariate analyses, nominally significant associations were found among body mass index (OR, 3.14; 95% CI, 8.5338 to 1.1274; p=0.022), breast size (OR, 5.11; 95% CI, 17.04 to 1.54; p=0.004), and grade ≥ 2 acute radiation skin toxicity. A significant association was also observed between NOS3 G894T polymorphism (OR, 9.8; 95% CI, 211.6 to 0.45; p=0.041) and grade ≥ 2 acute radiation skin toxicity in patients with neo-adjuvant chemotherapy treatment. CONCLUSION: The analysis of the factors involved in individual radiosensitivity contributed to the understanding of the mechanisms underlying this trait.
Body Mass Index ; Breast Neoplasms* ; Breast* ; DNA ; Drug Therapy ; Genetic Variation ; Humans ; Metabolism ; Multivariate Analysis ; Oxidative Stress* ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single Nucleotide ; Principal Component Analysis ; Radiation Tolerance ; Radiotherapy* ; Reactive Oxygen Species ; Skin

OBJECTIVETo provide in vitro evidence of Psorinum treatment against cancer cells in a controlled study.

METHODSEffects of homeopathic Psorinum 6× on cell viability were initially determined in several cancer cell lines, including A549, HepG2 and MCF-7, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and an ethanol 6× control. The cell line that exhibited highest inhibition was selected and used in the following experiments. A range of Psorinum 6× doses was used to explore treatment effects on cell cycle arrest, cell death (apoptosis), generation of reactive oxygen species (ROS) and change in mitochondrial membrane potential (MMP) using flow cytometry and fluorescence microscopy, respectively. Expression of several signal proteins related to apoptosis and cell survival were quantified with Western blotting and confocal microscopy. Further, circular dichroism (CD) spectroscopy was used to determine possible drug-DNA interactions, as well as the induction of conformational changes.

RESULTSTreatment of cancer cell lines with Psorinum showed greater anticancer effects in A549 cells than in others. In A549 cells Psorinum treatment inhibited cell proliferation at 24 h after treatment, and arrested cell cycle at sub-G1 stage. It also induced ROS generation, MMP depolarization, morphological changes and DNA damage, as well as externalization of phosphatidyl serine. Further, increases in p53 expression, Bax expression, cytochrome c release, along with reduction of Bcl-2 level and caspase-3 activation were observed after Psorinum 6× treatment, which eventually drove A549 cells towards the mitochondria-mediated caspase-3-dependent pathway. CD spectroscopy revealed direct interaction of Psorinum with DNA, using calf thymus-DNA as target.

CONCLUSIONPsorinum 6× triggered apoptosis in A549 cells via both up- and down-regulations of relevant signal proteins, including p53, caspase-3, Bax and Bcl-2.

Caspase 3 ; metabolism ; Cell Cycle Checkpoints ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Homeopathy ; Humans ; Lung Neoplasms ; drug therapy ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Reactive Oxygen Species ; metabolism ; bcl-2-Associated X Protein ; analysis