Features of the cytoprotective effect of selenium nanoparticles on primary cortical neurons and astrocytes during oxygen-glucose deprivation and reoxygenation.

The study is aimed at elucidating the effect of selenium nanoparticles (SeNPs) on the death of cells in the primary culture of mouse cerebral cortex during oxygen and glucose deprivation (OGD). A primary cell culture of the cerebral cortex containing neurons and astrocytes was subjected to OGD and reoxygenation to simulate cerebral ischemia-like conditions in vitro. To evaluate the neuroprotective effect of SeNPs, cortical astrocytes and neurons were incubated for 24 h with SeNPs, and then subjected to 2-h OGD, followed by 24-h reoxygenation. Vitality tests, fluorescence microscopy, and real-time PCR have shown that incubation of primary cultured neurons and astrocytes with SeNPs at concentrations of 2.5-10 µg/ml under physiological conditions has its own characteristics depending on the type of cells (astrocytes or neurons) and leads to a dose-dependent increase in apoptosis. At low concentration SeNPs (0.5 µg/ml), on the contrary, almost completely suppressed the processes of basic necrosis and apoptosis. Both high (5 µg/ml) and low (0.5 µg/ml) concentrations of SeNPs, added for 24 h to the cells of cerebral cortex, led to an increase in the expression level of genes Bcl-2, Bcl-xL, Socs3, while the expression of Bax was suppressed. Incubation of the cells with 0.5 µg/ml SeNPs led to a decrease in the expression of SelK and SelT. On the contrary, 5 µg/ml SeNPs caused an increase in the expression of SelK, SelN, SelT, SelP. In the ischemic model, after OGD/R, there was a significant death of brain cells by the type of necrosis and apoptosis. OGD/R also led to an increase in mRNA expression of the Bax, SelK, SelN, and SelT genes and suppression of the Bcl-2, Bcl-xL, Socs3, SelP genes. Pre-incubation of cell cultures with 0.5 and 2.5 µg/ml SeNPs led to almost complete inhibition of OGD/R-induced necrosis and greatly reduced apoptosis. Simultaneously with these processes we observed suppression of caspase-3 activation. We hypothesize that the mechanisms of the protective action of SeNPs involve the activation of signaling cascades recruiting nuclear factors Nrf2 and SOCS3/STAT3, as well as the activation of adaptive pathways of ESR signaling of stress arising during OGD and involving selenoproteins SelK and SelT, proteins of the Bcl-2 family ultimately leading to inactivation of caspase-3 and inhibition of apoptosis. Thus, our results demonstrate that SeNPs can act as neuroprotective agents in the treatment of ischemic brain injuries.

[Effects of Sodium Selenite and Dithiothreitol on Expression of Endoplasmic Reticulum Selenoproteins and Apoptosis Markers in MSF7 Breast Adenocarcinoma Cells].

The endoplasmic reticulum (ER) stress inducers dithiothreitol (DTT) and sodium selenite (SS) were tested for effect on expression of ER selenoproteins and apoptosis markers in MCF7 breast adenocarcinoma cells. DTT used at 1 or 5 mM did not affect the survival of MCF7 cells. Based on the real-time PCR data and the protein expression levels of ER stress markers, ER stress was assumed to evolve along an adaptation pathway in MCF7 cells treated with 1 or 5 mM DTT, involving mainly the transcription factors IRE1 and ATF6 and the selenoproteins SELS, SELK, SELT, SELM, and SELN. Cell treatment with 0.01 µM SS decreases the mRNA levels of all genes examined. When the SS concentration was increased to 0.1 µM, an increase in expression was observed for key ER stress genes and apoptosis markers, including CHOP, GADD34, PUMA, BIM, ATF4, sXBP, uXBP, AKT1, BAX, and BAK. Higher SS concentrations were assumed to trigger the unfolded protein response (UPR) via a proapoptic signaling pathway involving PERK and an alternative IRE1 signaling pathway. Used at 1 µM, SS increased the mRNA levels of apoptosis markers, upregulated expression of a spliced form of XBP1, and substantially decreased the cell survival. SS (1 µM) was assumed to trigger apoptosis in MCF7 cells. The results indicate that both adaptive and proapoptic UPR signaling pathways are activated in cells, depending on the nature and concentration of the ER stress inducer.

Expression of ER-resident selenoproteins and activation of cancer cells apoptosis mechanisms under ER-stress conditions caused by methylseleninic acid.

The aim of this work was to study changes in gene expression levels of 7 ER-resident selenoproteins under ER-stress caused by the action of a selenium-containing compound of organic nature, methylselenic acid using three human cancer cell lines DU 145 (prostate carcinoma), MCF 7 (breast adenocarcinoma)and HT-1080 (fibrosarcoma). According to the obtained results, we can speak of a synchronous changes in the expression of SELT and SEP15 mRNA depending on the concentration of MSA for 24 h, while the pattern of SELM expression was completely opposite and was radically different from other selenoproteins. It should be noted that in HT-1080 cells, the expression pattern of SELM differed from the expression pattern in two other cancer cells, while the expression patterns of other ER-resident selenoproteins (SELT, SEP15, SELK, SELS, SELN and DIO2) differed slightly depending on the cell line. Also we investigated the molecular mechanisms of UPR caused by MSA-induced ER stress in three cancer cell lines. According to the obtained results, it can be assumed that in DU 145 cells, MSA promotes activation of the PERK signaling pathway of UPR. In fibrosarcoma cells MSA was promoted the activation of ATF-6 UPR signaling pathway. In MCF 7 cells, MSA promoted the activation of two pro-apoptotic UPR signaling pathways at once: IRE1 and ATF-6.The results of this work once again demonstrate that the mechanisms of ER-stress regulation caused by the same agent, in this case, MSA, lead to the activation of different UPR signaling pathways in different cancer cells, and about their relationship.

The selective BDNF overexpression in neurons protects neuroglial networks against OGD and glutamate-induced excitotoxicity.

Objective: Cerebral ischemia is accompanied by damage and death of a significant number of neurons due to glutamate excitotoxicity with subsequent a global increase of cytosolic Ca2+ concentration ([Ca2+]i). This study aimed to investigate the neuroprotective action of BDNF overexpression in hippocampal neurons against injury under ischemia-like conditions (oxygen and glucose deprivation) and glutamate-induced excitotoxicity (GluTox).Methods: The overexpression of BDNF was reached by the transduction of cell cultures with the adeno-associated (AAV)-Syn-BDNF-EGFP virus construct. Neuroprotective effects were mediated by Ca2+-dependent BDNF release followed by activation of the neuroprotective signaling cascades and changes of the gene expression. Thus, BDNF overexpression modulates Ca2+ homeostasis in cells, preventing Ca2+ overload and initiation of apoptotic and necrotic processes.Results:Antiapoptotic effect of BDNF overexpression is mediated via activation of phosphoinositide-3-kinase (PI3K) pathway and changing the expression of PI3K, HIF-1, Src and an anti-inflammatory cytokine IL-10. On the contrary, the decrease of expression of proapoptotic proteins such as Jun, Mapk8, caspase-3 and an inflammatory cytokine IL-1ß was observed. These changes of expression were accompanied by the decrease of quantity of IL-1ß receptors and the level of TNFα in cells in control, as well as 24 h after OGD. Besides, BDNF overexpression changes the expression of GABA(B) receptors. Also, the expression of NMDA and AMPA receptor subunits was altered towards a change in the conductivity of the receptors for Ca2+.Conclusion: Thus, our results demonstrate that neuronal BDNF overexpression reveals complex neuroprotective effects on the neurons and astrocytes under OGD and GluTox via inhibition of Ca2+ responses and regulation of gene expression.

Taxifolin protects neurons against ischemic injury in vitro via the activation of antioxidant systems and signal transduction pathways of GABAergic neurons.

Cerebral blood flow disturbances lead to the massive death of brain cells. The death of >80% of cells is observed in hippocampal cell cultures after 40 min of oxygen and glucose deprivation (ischemia-like conditions, OGD). However, there are some populations of GABAergic neurons which are characterized by increased vulnerability to oxygen-glucose deprivation conditions. Using fluorescent microscopy, immunocytochemical assay, vitality tests and PCR-analysis, we have shown that population of GABAergic neurons are characterized by a different (faster) Ca2+ dynamics in response to OGD and increased basal ROS production under OGD conditions. A plant flavonoid taxifolin inhibited an excessive ROS production and an irreversible cytosolic Ca2+ concentration increase in GABAergic neurons, preventing the death of these neurons and further excitation of a neuronal network; neuroprotective effect of taxifolin increased after incubation of 24 h and correlated with increased expression of antiapoptocic and antioxidant genes Stat3 Nrf-2 Bcl-2, Bcl-xL, Ikk2, and genes coding for AMPA and kainate receptor subunits; in addition, taxifolin decreased expression of prooxidant enzyme NOS and proinflammatory cytokine IL-1ß.

[The effect of infrared and X-ray radiation on the production of reactive oxygen species in blood and induction of cytogenetic damage in the bone marrow of mice in vivo].

The goal of the present work was to study the effect of infrared light (IRL) at a wavelength of 850 nm modulated by a frequency of 101 Hz with a mode of power 22 mW/cm2 and X-rays with a voltage of 200 kV at a dose rate of 1 Gy/min on the production of reactive oxygen species (ROS) in blood cells using luminol-dependent chemiluminescence, as well as on the induction of a cytogenetic damage in bone marrow cells of mice by the in vivo micronucleus test. The experiments performed have shown: 1) the level of the ROS production in blood of the mice exposed to IRL and X-rays at an adapting dose of 0.1 Gy reaches the peak value after 0.5 h and drops to the ROS level in untreated animals 5 h after either exposure; 2) irradiation of mice with IRL and X-rays at a dose of 0.1 Gy induces adaptive responses both in blood cells and bone marrow cells of mice. These adaptive responses were revealed only 5 h after both exposures, when the level of ROS production decreased to the ROS level in untreated animals; they are equal in magnitude and dynamics and persist up to 2 months.

Common regularities in changes of the neutrophil functional activity during in vivo growth of tumors of different immunogenic activity.

Studies on BALB/c mice with tumors of different immunogenic activity (nonimmunogenic J774, WEHI 164 and immunogenic NS0) have showed that the development of a tumor is associated with changes in the neutrophil morphology and functions: the counts and size of the cells migrating to the focus increase and their capacity to produce active oxygen species is changed.

[Ambiguity role of neutrophils in oncogenesis].

The review is focused on the participation of polymorphonuclear granulocytes (neutrophils) in development and spreading of a tumor. We consider both the well known functions of neutrophils (degranulation, production of reactive oxygen species (ROS)) and the recently shown one (presentation of an antigene). The special attention is focused on the ambiguity of the neutrophil role in oncogenesis. The dominant view is that neutrophils display exclusively antitumor properties. The update information testifies about protumoral activity of neutrophils: they migrate to a tumor and promote angiogenesis and metastasis at late stages of the tumor. It is interesting that certain components of neutrophil cytotoxic arsenal (ROS, cytokines, specific enzymes) participate both in antitumoral defenses of an organism and protumoral activity.

Evaluation of the Role of FMLF chemotaxic peptide receptors in umbilical cord blood granulocytes from newborns at risk of infectious inflammatory diseases.

We studied the role of receptors with high and low affinity for fMLF chemotaxic peptide in the generation of active oxygen species by umbilical cord blood granulocytes from newborns with normal neonatal period, born after normal or complicated gestation, in children with manifestations of bacterial infection born after complicated pregnancy, and in granulocytes of non-pregnant women with normal reproductive function. Granulocytes of children born after complicated pregnancy exhibited high reactivity in induction of respiratory burst in a wide range of fMLF concentrations. The presentation of receptors with high and low affinity on granulocytes during initiation of the respiratory burst differs in children born after complicated pregnancy and in healthy babies born after normal gestation. Presumably, the detected differences result from high expression of receptors with low affinity for fMLF and disorders or immaturity of mechanisms responsible for receptor inactivation.

[Effect of negative air ions on respiratory organs and blood].

The effect of negatively charged ions on respiratory organs and blood of rats has been studied. It was shown that the inhaling of negative air ions (NAI) for 60 min with a concentration of NAI at the place of location of animals 320-350 000 ions/cm2 activated the secretion of goblet cells without damaging the mucosa of the trachea and changed the spectrum of proteins of bronchopulmonary lavage. It was also found that the spontaneous production of reactive oxygen species (ROS) by cells of nonfractionated blood after the exposure to NAI increased in both males and females; the intensity of ROS generation induced by opsonized zymosan increased only in females. Different sensitivity of the antioxidant enzymes superoxide dismutase and glutathione reductase of blood to NAI in females and males was revealed. These results enable one to consider the effect of NAI as priming and a weak activation of the respiratory organs through the direct action on the mucosa of the primary target organs of the respiratory tract and then on the blood.

[Dynamic analysis of modification of peripheral neutrophils functional activity and its regulation during tumor growth in vivo].

Polymorphonuclear granulocytes (neutrophils) release the reactive oxygen species (ROS) for destruction of pathogens, providing quicker of an organism from infections and own defective of transformed cells. Reactive oxygen species are also potential carcinogens because they facilitate mutagenesis, tumor promotion and progression. Balance between these opposite influences is supported by coordinated interrelations in intracellular signaling systems. Tumor growth influence on the NADPH oxidase in peripheral innate immune cells is unclear. A solid cancer model was developed after an intramuscular injection of Ehrlich carcinoma cells into hind leg of NMRI strain mice. Intensity of the respiratory burst was estimated by luminol-dependent chemiluminescence technique. Transformation of inflammatory reaction was revealed during tumor growth: greater amounts of neutrophils were recruited into peritoneal cavity; sizes of the cells, their nuclei and granules were enlarged; the ratio of different cell types in peritoneal exudation was changed. The study revealed that tumor progression was accompanied by significant changes in functional activity of neutrophils. Dynamic increase in spontaneous level of ROS production and concentration-dependent change of intensity of the respiratory burst induced with chemotactic peptide N-formyl-Met-Leu-Phe (fMLF) was revealed in peripheral neutrophils under tumor growth conditions. It was found that effects of inhibitors of tyrosine protein kinases, protein kinase C, mitogen-activated protein kinase p38MAPK (p38MAPK) and phosphatidylinositol 3-kinase (PI3K) were altered in neutrophils from tumor-bearing mice in comparison with the cells of control mice. This indicates a change in the role of the enzymes in regulation of the neutrophil respiratory burst. Data obtained show that p38MAPK and PI3K entangle up- and down-regulation of NADPH oxidase in peripheral neutrophils during tumor growth.

Generation of reactive oxygen species by umbilical blood cells and immune status of newborns at risk of infectious inflammatory diseases.

We carried out a comparative clinical and immunological examination of newborns whose mothers were at risk of infectious inflammatory diseases. Umbilical blood cell phenotype was evaluated by flow cytofluorometry. ROS level was evaluated by chemiluminescence intensity. Spontaneous production of ROS and phagocytic activity of cells in the whole umbilical blood was reduced in newborns born after complicated pregnancy. Low immunoregulatory index indicating changed CD4+/CD8+ ratio and low percentage of natural killer cells were observed in children with manifestations of bacterial infection. ROS production by isolated granulocytes and the effects of PI3K and p38 MAPK (kinases involved in the regulation of activity of NADPH oxidase responsible for the production of ROS) in the risk group infants differed from the corresponding parameters in the control group. The results indicate shifts in the phagocytosis system, immune status, and the receptor-conjugated regulatory systems of ROS generation by granulocytes in newborns at risk of infectious inflammatory diseases.