Molecular Mechanisms of the Cytotoxic Effect of Recombinant Selenoprotein SELENOM on Human Glioblastoma Cells.

Currently, selenobiology is an actively developing area, primarily due to the study of the role of the trace element selenium and its organic and inorganic compounds in the regulation of vital processes occurring in the cell. In particular, the study of the functions of selenium nanoparticles has gained great popularity in recent years. However, a weak point in this area of biology is the study of the functions of selenoproteins, of which 25 have been identified in mammals to date. First of all, this is due to the difficulties in obtaining native forms of selenoproteins in preparative quantities, due to the fact that the amino acid selenocysteine is encoded by one of the three stop codons of the TGA universal genetic code. A complex system for recognizing a given codon as a selenocysteine codon has a number of features in pro- and eukaryotes. The selenoprotein SELENOM is one of the least studied mammalian selenoproteins. In this work, for the first time, studies of the molecular mechanisms of regulation of the cytotoxic effect of this protein on human glioblastoma cells were carried out. The cytotoxicity of cancer cells in our experiments was already observed when cells were exposed to 50 µg of SELENOM and increased in proportion to the increase in protein concentration. Apoptosis of human glioblastoma cells was accompanied by an increase in mRNA expression of a number of pro-apoptotic genes, an increase in endoplasmic reticulum stress, and activation of the UPR IRE1α signaling pathway. The results obtained also demonstrate a dose-dependent depletion of the Ca2+ pool under the action of SELENOM, which proves the important role of this protein in the regulation of calcium homeostasis in the cell.

Ca2+-Dependent Effects of the Selenium-Sorafenib Nanocomplex on Glioblastoma Cells and Astrocytes of the Cerebral Cortex: Anticancer Agent and Cytoprotector.

Despite the fact that sorafenib is recommended for the treatment of oncological diseases of the liver, kidneys, and thyroid gland, and recently it has been used for combination therapy of brain cancer of various genesis, there are still significant problems for its widespread and effective use. Among these problems, the presence of the blood-brain barrier of the brain and the need to use high doses of sorafenib, the existence of mechanisms for the redistribution of sorafenib and its release in the brain tissue, as well as the high resistance of gliomas and glioblastomas to therapy should be considered the main ones. Therefore, there is a need to create new methods for delivering sorafenib to brain tumors, enhancing the therapeutic potential of sorafenib and reducing the cytotoxic effects of active compounds on the healthy environment of tumors, and ideally, increasing the survival of healthy cells during therapy. Using vitality tests, fluorescence microscopy, and molecular biology methods, we showed that the selenium-sorafenib (SeSo) nanocomplex, at relatively low concentrations, is able to bypass the mechanisms of glioblastoma cell chemoresistance and to induce apoptosis through Ca2+-dependent induction of endoplasmic reticulum stress, changes in the expression of selenoproteins and selenium-containing proteins, as well as key kinases-regulators of oncogenicity and cell death. Selenium nanoparticles (SeNPs) also have a high anticancer efficacy in glioblastomas, but are less selective, since SeSo in cortical astrocytes causes a more pronounced activation of the cytoprotective pathways.

Pilot Study of Cytoprotective Mechanisms of Selenium Nanorods (SeNrs) under Ischemia-like Conditions on Cortical Astrocytes.

The cytoprotective properties of the trace element selenium, its nanoparticles, and selenium nanocomplexes with active compounds are shown using a number of models. To date, some molecular mechanisms of the protective effect of spherical selenium nanoparticles under the action of ischemia/reoxygenation on brain cells have been studied. Among other things, the dependence of the effectiveness of the neuroprotective properties of nanoselenium on its diameter, pathways, and efficiency of penetration into astrocytes was established. In general, most research in the field of nanomedicine is focused on the preparation and study of spherical nanoparticles of various origins due to the ease of their preparation; in addition, spherical nanoparticles have a large specific surface area. However, obtaining and studying the mechanisms of action of nanoparticles of a new form are of great interest since nanorods, having all the positive properties of spherical nanoparticles, will also have a number of advantages. Using the laser ablation method, we managed to obtain and characterize selenium nanorods (SeNrs) with a length of 1 µm and a diameter of 100 nm. Using fluorescence microscopy and inhibitory analysis, we were able to show that selenium nanorods cause the generation of Ca2+ signals in cortical astrocytes in an acute experiment through the mobilization of Ca2+ ions from the thapsigargin-sensitive pool of the endoplasmic reticulum. Chronic use of SeNrs leads to a change in the expression pattern of genes encoding proteins that regulate cell fate and protect astrocytes from ischemia-like conditions and reoxygenation through the inhibition of a global increase in the concentration of cytosolic calcium ([Ca2+]i). An important component of the cytoprotective effect of SeNrs during ischemia/reoxygenation is the induction of reactive A2-type astrogliosis in astrocytes, leading to an increase in both baseline and ischemia/reoxygenation-induced phosphoinositide 3-kinase (PI3K) activity and suppression of necrosis and apoptosis. The key components of this cytoprotective action of SeNrs are the actin-dependent process of endocytosis of nanoparticles into cells and activation of the Ca2+ signaling system of astrocytes.

Cerium Oxide Nanoparticles Protect Cortical Astrocytes from Oxygen-Glucose Deprivation through Activation of the Ca2+ Signaling System.

Most of the works aimed at studying the cytoprotective properties of nanocerium are usually focused on the mechanisms of regulation of the redox status in cells while the complex effects of nanocerium on calcium homeostasis, the expression of pro-apoptotic and protective proteins are generally overlooked. There is a problem of a strong dependence of the effects of cerium oxide nanoparticles on their size, method of preparation and origin, which significantly limits their use in medicine. In this study, using the methods of molecular biology, immunocytochemistry, fluorescence microscopy and inhibitory analysis, the cytoprotective effect of cerium oxide nanoparticles obtained by laser ablation on cultured astrocytes of the cerebral cortex under oxygen-glucose deprivation (OGD) and reoxygenation (ischemia-like conditions) are shown. The concentration effects of cerium oxide nanoparticles on ROS production by astrocytes in an acute experiment and the effects of cell pre-incubation with nanocerium on ROS production under OGD conditions were studied. The dose dependence for nanocerium protection of cortical astrocytes from a global increase in calcium ions during oxygen-glucose deprivation and cell death were demonstrated. The concentration range of cerium oxide nanoparticles at which they have a pro-oxidant effect on cells has been identified. The effect of nanocerium concentrations on astrocyte preconditioning, accompanied by increased expression of protective proteins and limited ROS production induced by oxygen-glucose deprivation, has been investigated. In particular, a correlation was found between an increase in the concentration of cytosolic calcium under the action of nanocerium and the suppression of cell death. As a result, the positive and negative effects of nanocerium under oxygen-glucose deprivation and reoxygenation in astrocytes were revealed at the molecular level. Nanocerium was found to act as a "double-edged sword" and to have a strictly defined concentration therapeutic "window".

Socrates: A Novel N-Ethyl-N-nitrosourea-Induced Mouse Mutant with Audiogenic Epilepsy.

Epilepsy is one of the common neurological diseases that affects not only adults but also infants and children. Because epilepsy has been studied for a long time, there are several pharmacologically effective anticonvulsants, which, however, are not suitable as therapy for all patients. The genesis of epilepsy has been extensively investigated in terms of its occurrence after injury and as a concomitant disease with various brain diseases, such as tumors, ischemic events, etc. However, in the last decades, there are multiple reports that both genetic and epigenetic factors play an important role in epileptogenesis. Therefore, there is a need for further identification of genes and loci that can be associated with higher susceptibility to epileptic seizures. Use of mouse knockout models of epileptogenesis is very informative, but it has its limitations. One of them is due to the fact that complete deletion of a gene is not, in many cases, similar to human epilepsy-associated syndromes. Another approach to generating mouse models of epilepsy is N-Ethyl-N-nitrosourea (ENU)-directed mutagenesis. Recently, using this approach, we generated a novel mouse strain, soc (socrates, formerly s8-3), with epileptiform activity. Using molecular biology methods, calcium neuroimaging, and immunocytochemistry, we were able to characterize the strain. Neurons isolated from soc mutant brains retain the ability to differentiate in vitro and form a network. However, soc mutant neurons are characterized by increased spontaneous excitation activity. They also demonstrate a high degree of Ca2+ activity compared to WT neurons. Additionally, they show increased expression of NMDA receptors, decreased expression of the Ca2+-conducting GluA2 subunit of AMPA receptors, suppressed expression of phosphoinositol 3-kinase, and BK channels of the cytoplasmic membrane involved in protection against epileptogenesis. During embryonic and postnatal development, the expression of several genes encoding ion channels is downregulated in vivo, as well. Our data indicate that soc mutation causes a disruption of the excitation-inhibition balance in the brain, and it can serve as a mouse model of epilepsy.

A Comparative Analysis of Neuroprotective Properties of Taxifolin and Its Water-Soluble Form in Ischemia of Cerebral Cortical Cells of the Mouse.

Cerebral ischemia, and, as a result, insult, attacks up to 15 million people yearly in the world. In this connection, the development of effective preventive programs and methods of therapy has become one of the most urgent problems in modern angiology and pharmacology. The cytoprotective action of taxifolin (TAX) in ischemia is well known, but its limitations are also known due to its poor solubility and low capacity to pass through the hematoencephalic barrier. Molecular mechanisms underlying the protective effect of TAX in complex systems such as the brain remain poorly understood. It is known that the main cell types of the brain are neurons, astrocytes, and microglia, which regulate the activity of each other through neuroglial interactions. In this work, a comparative study of cytoprotective mechanisms of the effect of TAX and its new water-soluble form aqua taxifolin (aqTAX) was performed on cultured brain cells under ischemia-like conditions (oxygen-glucose deprivation (OGD)) followed by the reoxygenation of the culture medium. The concentration dependences of the protective effects of both taxifolin forms were determined using fluorescence microscopy, PCR analysis, and vitality tests. It was found that TAX began to effectively inhibit necrosis and the late stages of apoptosis in the concentration range of 30-100 µg/mL, with aqTAX in the range of 10-30 µg/mL. At the level of gene expression, aqTAX affected a larger number of genes than TAX; enhanced the basic and OGD/R-induced expression of genes encoding ROS-scavenging proteins with a higher efficiency, as well as anti-inflammatory and antiapoptotic proteins; and lowered the level of excitatory glutamate receptors. As a result, aqTAX significantly inhibited the OGD-induced increase in the Ca2+ levels in the cytosol ([Ca2+]i) in neurons and astrocytes under ischemic conditions. After a 40 min preincubation of cells with aqTAX under hypoxic conditions, these Ca2+ signals were completely inhibited, resulting in an almost complete suppression of necrotic death of cerebral cortical cells, which was not observed with the use of classical TAX.

Interleukin-10 restores glutamate receptor-mediated Ca2+-signaling in brain circuits under loss of Sip1 transcription factor.

OBJECTIVE: This study aimed to investigate the connection between the mutation of the Sip1 transcription factor and impaired Ca2+-signaling, which reflects changes in neurotransmission in the cerebral cortex in vitro. METHODS: We used mixed neuroglial cortical cell cultures derived from Sip1 mutant mice. The cells were loaded with a fluorescent ratiometric calcium-sensitive probe Fura-2 AM and epileptiform activity was modeled by excluding magnesium ions from the external media or adding a GABA(A) receptor antagonist, bicuculline. Intracellular calcium dynamics were recorded using fluorescence microscopy. To identify the level of gene expression, the Real-Time PCR method was used. RESULTS: It was found that cortical neurons isolated from homozygous (Sip1fl/fl) mice with the Sip1 mutation demonstrate suppressed Ca2+ signals in models of epileptiform activity in vitro. Wild-type cortical neurons are characterized by synchronous high-frequency and high-amplitude Ca2+ oscillations occurring in all neurons of the network in response to Mg2+-free medium and bicuculline. But cortical Sip1fl/fl neurons only single Ca2+ pulses or attenuated Ca2+ oscillations are recorded and only in single neurons, while most of the cell network does not respond to these stimuli. This signal deficiency of Sip1fl/fl neurons correlates with a suppressed expression level of the genes encoding the subunits of NMDA, AMPA, and KA receptors; protein kinases PKA, JNK, CaMKII; and also the transcription factor Hif1α. These negative effects were partially abolished when Sip1fl/fl neurons are grown in media with anti-inflammatory cytokine IL-10. IL-10 increases the expression of the above-mentioned genes but not to the level of expression in wild-type. At the same time, the amplitudes of Ca2+ signals increase in response to the selective agonists of NMDA, AMPA and KA receptors, and the proportion of neurons responding with Ca2+ oscillations to a Mg2+-free medium and bicuculline increases. CONCLUSION: IL-10 restores neurotransmission in neuronal networks with the Sip1 mutation by regulating the expression of genes encoding signaling proteins.

Size-Dependent Cytoprotective Effects of Selenium Nanoparticles during Oxygen-Glucose Deprivation in Brain Cortical Cells.

It is known that selenium nanoparticles (SeNPs) obtained on their basis have a pleiotropic effect, inducing the process of apoptosis in tumor cells, on the one hand, and protecting healthy tissue cells from death under stress, on the other hand. It has been established that SeNPs protect brain cells from ischemia/reoxygenation through activation of the Ca2+ signaling system of astrocytes and reactive astrogliosis. At the same time, for a number of particles, the limitations of their use, associated with their size, are shown. The use of nanoparticles with a diameter of less than 10 nm leads to their short life-time in the bloodstream and rapid removal by the liver. Nanoparticles larger than 200 nm activate the complement system and are also quickly removed from the blood. The effects of different-sized SeNPs on brain cells have hardly been studied. Using the laser ablation method, we obtained SeNPs of various diameters: 50 nm, 100 nm, and 400 nm. Using fluorescence microscopy, vitality tests, PCR analysis, and immunocytochemistry, it was shown that all three types of the different-sized SeNPs have a cytoprotective effect on brain cortex cells under conditions of oxygen-glucose deprivation (OGD) and reoxygenation (R), suppressing the processes of necrotic death and inhibiting different efficiency processes of apoptosis. All of the studied SeNPs activate the Ca2+ signaling system of astrocytes, while simultaneously inducing different types of Ca2+ signals. SeNPs sized at 50 nm- induce Ca2+ responses of astrocytes in the form of a gradual irreversible increase in the concentration of cytosolic Ca2+ ([Ca2+]i), 100 nm-sized SeNPs induce stable Ca2+ oscillations without increasing the base level of [Ca2+]i, and 400 nm-sized SeNPs cause mixed patterns of Ca2+ signals. Such differences in the level of astrocyte Ca2+ signaling can explain the different cytoprotective efficacy of SeNPs, which is expressed in the expression of protective proteins and the activation of reactive astrogliosis. In terms of the cytoprotective efficiency under OGD/R conditions, different-sized SeNPs can be arranged in descending order: 100 nm-sized > 400 nm-sized > 50 nm-sized.

Neuronal Calcium Sensor-1 Protects Cortical Neurons from Hyperexcitation and Ca2+ Overload during Ischemia by Protecting the Population of GABAergic Neurons.

A defection of blood circulation in the brain leads to ischemia, damage, and the death of nerve cells. It is known that individual populations of GABAergic neurons are the least resistant to the damaging factors of ischemia and therefore they die first of all, which leads to impaired inhibition in neuronal networks. To date, the neuroprotective properties of a number of calcium-binding proteins (calbindin, calretinin, and parvalbumin), which are markers of GABAergic neurons, are known. Neuronal calcium sensor-1 (NCS-1) is a signaling protein that is expressed in all types of neurons and is involved in the regulation of neurotransmission. The role of NCS-1 in the protection of neurons and especially their individual populations from ischemia and hyperexcitation has not been practically studied. In this work, using the methods of fluorescence microscopy, vitality tests, immunocytochemistry, and PCR analysis, the molecular mechanisms of the protective action of NCS-1 in ischemia/reoxygenation and hyperammonemia were established. Since NCS-1 is most expressed in GABAergic neurons, the knockdown of this protein with siRNA led to the most pronounced consequences in GABAergic neurons. The knockdown of NCS-1 (NCS-1-KD) suppressed the basic expression of protective proteins without significantly reducing cell viability. However, ischemia-like conditions (oxygen-glucose deprivation, OGD) and subsequent 24-h reoxygenation led to a more massive activation of apoptosis and necrosis in neurons with NCS-1-KD, compared to control cells. The mass death of NCS-1-KD cells during OGD and hyperammonemia has been associated with the induction of a more pronounced network hyperexcitation symptom, especially in the population of GABAergic neurons, leading to a global increase in cytosolic calcium ([Ca2+]i). The symptom of hyperexcitation of neurons with NCS-1-KD correlated with a decrease in the level of expression of the calcium-binding protein-parvalbumin. This was accompanied by an increase in the expression of excitatory ionotropic glutamate receptors, N-methyl-D-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (NMDAR and AMPAR) against the background of suppression of the expression of glutamate decarboxylase (synthesis of γ-aminobutyric acid).

Modulation of the Functional State of Mouse Neutrophils by Selenium Nanoparticles In Vivo.

This study aimed to discover the immunomodulatory effect of selenium nanoparticles (SeNPs) on the functional state of neutrophils in vivo. Intraperitoneal injections of SeNPs (size 100 nm) 2.5 mg/kg/daily to BALB/c mice for a duration of 7-28 days led to the development of an inflammatory reaction, which was registered by a significant increase in the number of neutrophils released from the peritoneal cavity, as well as their activated state, without additional effects. At the same time, subcutaneous injections of the same SeNPs preparations at concentrations of 0.1, 0.5, and 2.5 mg/kg, on the contrary, modulated the functional state of neutrophils depending on the concentration and duration of SeNPs administration. With the use of fluorescence spectroscopy, chemiluminescence, biochemical methods, and PCR analysis, it was found that subcutaneous administration of SeNPs (0.1, 0.5, and 2.5 mg/kg) to mice for a short period of time (7-14 days) leads to modification of important neutrophil functions (adhesion, the number of migrating cells into the peritoneal cell cavity, ROS production, and NET formation). The obtained results indicated the immunostimulatory and antioxidant effects of SeNPs in vivo during short-term administration, while the most pronounced immunomodulatory effects of SeNPs were observed with the introduction of a low concentration of SeNPs (0.1 mg/kg). Increase in the administration time of SeNPs (0.1 mg/kg or 2.5 mg/kg) up to 28 days led to a decrease in the adhesive abilities of neutrophils and suppression of the expression of mRNA of adhesive molecules, as well as proteins involved in the generation of ROS, with the exception of NOX2; there was a tendency to suppress gene expression pro-inflammatory factors, which indicates the possible manifestation of immunosuppressive and anti-inflammatory effects of SeNPs during their long-term administration. Changes in the expression of selenoproteins also had features depending on the concentration and duration of the administered SeNPs. Selenoprotein P, selenoprotein M, selenoprotein S, selenoprotein K, and selenoprotein T were the most sensitive to the introduction of SeNPs into the mouse organism, which indicates their participation in maintaining the functional status of neutrophils, and possibly mediated the immunomodulatory effect of SeNPs.

Immunomodulatory and Anti-Inflammatory Properties of Selenium-Containing Agents: Their Role in the Regulation of Defense Mechanisms against COVID-19.

The review presents the latest data on the role of selenium-containing agents in the regulation of diseases of the immune system. We mainly considered the contributions of selenium-containing compounds such as sodium selenite, methylseleninic acid, selenomethionine, and methylselenocysteine, as well as selenoproteins and selenium nanoparticles in the regulation of defense mechanisms against various viral infections, including coronavirus infection (COVID-19). A complete description of the available data for each of the above selenium compounds and the mechanisms underlying the regulation of immune processes with the active participation of these selenium agents, as well as their therapeutic and pharmacological potential, is presented. The main purpose of this review is to systematize the available information, supplemented by data obtained in our laboratory, on the important role of selenium compounds in all of these processes. In addition, the presented information makes it possible to understand the key differences in the mechanisms of action of these compounds, depending on their chemical and physical properties, which is important for obtaining a holistic picture and prospects for creating drugs based on them.

Comparative Analysis of the Cytotoxic Effect of a Complex of Selenium Nanoparticles Doped with Sorafenib, "Naked" Selenium Nanoparticles, and Sorafenib on Human Hepatocyte Carcinoma HepG2 Cells.

Despite the use of sorafenib as one of the most effective drugs for the treatment of liver cancer, its significant limitations remain-poor solubility, the need to use high doses with the ensuing complications on healthy tissues and organs, and the formation of cell resistance to the drug. At the same time, there is more and more convincing evidence of the anticancer effect of selenium-containing compounds and nanoparticles. The aim of this work was to develop a selenium-sorafenib nanocomplex and study the molecular mechanisms of its anticancer effect on human hepatocyte carcinoma cells, where nanoselenium is not only a sorafenib transporter, but also an active compound. We have created a selenium-sorafenib nanocomplex based on selenium nanoparticles with size 100 nm. Using vitality tests, fluorescence microscopy, and PCR analysis, it was possible to show that selenium nanoparticles, both by themselves and doped with sorafenib, have a pronounced pro-apoptotic effect on HepG2 cells with an efficiency many times greater than that of sorafenib (So). "Naked" selenium nanoparticles (SeNPs) and the selenium-sorafenib nanocomplex (SeSo), already after 24 h of exposure, lead to the induction of the early stages of apoptosis with the transition to the later stages with an increase in the incubation time up to 48 h. At the same time, sorafenib, at the studied concentrations, began to exert a proapoptotic effect only after 48 h. Under the action of SeNPs and SeSo, both classical pathways of apoptosis induction and ER-stress-dependent pathways involving Ca2+ ions are activated. Thus, sorafenib did not cause the generation of Ca2+ signals by HepG2 cells, while SeNPs and SeSo led to the activation of the Ca2+ signaling system of cells. At the same time, the selenium-sorafenib nanocomplex turned out to be more effective in activating the Ca2+ signaling system of cells, inducing apoptosis and ER stress by an average of 20-25% compared to "naked" selenium nanoparticles. Our data on the mechanisms of action and the created nanocomplex are promising as a platform for the creation of highly selective and effective drugs with targeted delivery to tumors.

Novel Hydroxypyridine Compound Protects Brain Cells against Ischemic Damage In Vitro and In Vivo.

A non-surgical pharmacological approach to control cellular vitality and functionality during ischemic and/or reperfusion-induced phases of strokes remains extremely important. The synthesis of 2-ethyl-6-methyl-3-hydroxypyridinium gammalactone-2,3-dehydro-L-gulonate (3-EA) was performed using a topochemical reaction. The cell-protective effects of 3-EA were studied on a model of glutamate excitotoxicity (GluTox) and glucose-oxygen deprivation (OGD) in a culture of NMRI mice cortical cells. Ca2+ dynamics was studied using fluorescent bioimaging and a Fura-2 probe, cell viability was assessed using cytochemical staining with propidium iodide, and gene expression was assessed by a real-time polymerase chain reaction. The compound anti-ischemic efficacy in vivo was evaluated on a model of irreversible middle cerebral artery (MCA) occlusion in Sprague-Dawley male rats. Brain morphological changes and antioxidant capacity were assessed one week after the pathology onset. The severity of neurological disorder was evaluated dynamically. 3-EA suppressed cortical cell death in a dose-dependent manner under the excitotoxic effect of glutamate and ischemia/reoxygenation. Pre-incubation of cerebral cortex cells with 10-100 µM 3-EA led to significant stagnation in Ca2+ concentration in a cytosol ([Ca2+]i) of neurons and astrocytes suffering GluTox and OGD. Decreasing intracellular Ca2+ and establishing a lower [Ca2+]i baseline inhibited necrotic cell death in an acute experiment. The mechanism of 3-EA cytoprotective action involved changes in the baseline and ischemia/reoxygenation-induced expression of genes encoding anti-apoptotic proteins and proteins of the oxidative status; this led to inhibition of the late irreversible stages of apoptosis. Incubation of brain cortex cells with 3-EA induced an overexpression of the anti-apoptotic genes BCL-2, STAT3, and SOCS3, whereas the expression of genes regulating necrosis and inflammation (TRAIL, MLKL, Cas-1, Cas-3, IL-1ß and TNFa) were suppressed. 3-EA 18.0 mg/kg intravenous daily administration for 7 days following MCA occlusion preserved rats' cortex neuron population, decreased the severity of neurological deficit, and spared antioxidant capacity of damaged tissues. 3-EA demonstrated proven short-term anti-ischemic activity in vivo and in vitro, which can be associated with antioxidant activity and the ability to target necrotic and apoptotic death. The compound may be considered a potential neuroprotective molecule for further pre-clinical investigation.

Astrocytes Modulate Baroreflex Sensitivity at the Level of the Nucleus of the Solitary Tract.

Maintenance of cardiorespiratory homeostasis depends on autonomic reflexes controlled by neuronal circuits of the brainstem. The neurophysiology and neuroanatomy of these reflex pathways are well understood, however, the mechanisms and functional significance of autonomic circuit modulation by glial cells remain largely unknown. In the experiments conducted in male laboratory rats we show that astrocytes of the nucleus of the solitary tract (NTS), the brain area that receives and integrates sensory information from the heart and blood vessels, respond to incoming afferent inputs with [Ca2+]i elevations. Astroglial [Ca2+]i responses are triggered by transmitters released by vagal afferents, glutamate acting at AMPA receptors and 5-HT acting at 5-HT2A receptors. In conscious freely behaving animals blockade of Ca2+-dependent vesicular release mechanisms in NTS astrocytes by virally driven expression of a dominant-negative SNARE protein (dnSNARE) increased baroreflex sensitivity by 70% (p < 0.001). This effect of compromised astroglial function was specific to the NTS as expression of dnSNARE in astrocytes of the ventrolateral brainstem had no effect. ATP is considered the principle gliotransmitter and is released by vesicular mechanisms blocked by dnSNARE expression. Consistent with this hypothesis, in anesthetized rats, pharmacological activation of P2Y1 purinoceptors in the NTS decreased baroreflex gain by 40% (p = 0.031), whereas blockade of P2Y1 receptors increased baroreflex gain by 57% (p = 0.018). These results suggest that glutamate and 5-HT, released by NTS afferent terminals, trigger Ca2+-dependent astroglial release of ATP to modulate baroreflex sensitivity via P2Y1 receptors. These data add to the growing body of evidence supporting an active role of astrocytes in brain information processing.SIGNIFICANCE STATEMENT Cardiorespiratory reflexes maintain autonomic balance and ensure cardiovascular health. Impaired baroreflex may contribute to the development of cardiovascular disease and serves as a robust predictor of cardiovascular and all-cause mortality. The data obtained in this study suggest that astrocytes are integral components of the brainstem mechanisms that process afferent information and modulate baroreflex sensitivity via the release of ATP. Any condition associated with higher levels of "ambient" ATP in the NTS would be expected to decrease baroreflex gain by the mechanism described here. As ATP is the primary signaling molecule of glial cells (astrocytes, microglia), responding to metabolic stress and inflammatory stimuli, our study suggests a plausible mechanism of how the central component of the baroreflex is affected in pathological conditions.

Mechanosensory Signaling in Astrocytes.

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca2+ responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y1 receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca2+ responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca2+ signals via P2Y1 receptors and Ca2+ recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENT There is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca2+ responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.

THE MAIN CYTOTOXIC EFFECTS OF METHYLSELENINIC ACID ON VARIOUS CANCER CELLS.

Studies of recent decades have repeatedly demonstrated the cytotoxic effect of selenium-containing compounds on cancer cells of various origins. Particular attention in these studies is paid to methylseleninic acid, a widespread selenium-containing compound of organic nature, for several reasons: it has a selective cytotoxic effect on cancer cells, it is cytotoxic in small doses, it is able to generate methylselenol, excluding the action of the enzyme ß-lyase. All these qualities make methylseleninic acid an attractive substrate for the production of anticancer drugs on its basis with a well-pronounced selective effect. However, the studies available to date indicate that there is no strictly specific molecular mechanism of its cytotoxic effect in relation to different cancer cell lines and cancer models. This review contains generalized information on the dose- and time-dependent regulation of the toxic effect of methylseleninic acid on the proliferative properties of a number of cancer cell lines. In addition, special attention in this review is paid to the influence of this selenium-containing compound on the regulation of endoplasmic reticulum stress and on the expression of seven selenoproteins, which are localized in the endoplasmic reticulum.

The Mechanisms Underlying the Protective Action of Selenium Nanoparticles against Ischemia/Reoxygenation Are Mediated by the Activation of the Ca2+ Signaling System of Astrocytes and Reactive Astrogliosis.

In recent years, much attention has been paid to the study of the therapeutic effect of the microelement selenium, its compounds, especially selenium nanoparticles, with a large number of works devoted to their anticancer effects. Studies proving the neuroprotective properties of selenium nanoparticles in various neurodegenerative diseases began to appear only in the last 5 years. Nevertheless, the mechanisms of the neuroprotective action of selenium nanoparticles under conditions of ischemia and reoxygenation remain unexplored, especially for intracellular Ca2+ signaling and neuroglial interactions. This work is devoted to the study of the cytoprotective mechanisms of selenium nanoparticles in the neuroglial networks of the cerebral cortex under conditions of ischemia/reoxygenation. It was shown for the first time that selenium nanoparticles dose-dependently induce the generation of Ca2+ signals selectively in astrocytes obtained from different parts of the brain. The generation of these Ca2+ signals by astrocytes occurs through the release of Ca2+ ions from the endoplasmic reticulum through the IP3 receptor upon activation of the phosphoinositide signaling pathway. An increase in the concentration of cytosolic Ca2+ in astrocytes leads to the opening of connexin Cx43 hemichannels and the release of ATP and lactate into the extracellular medium, which trigger paracrine activation of the astrocytic network through purinergic receptors. Incubation of cerebral cortex cells with selenium nanoparticles suppresses ischemia-induced increase in cytosolic Ca2+ and necrotic cell death. Activation of A2 reactive astrocytes exclusively after ischemia/reoxygenation, a decrease in the expression level of a number of proapoptotic and proinflammatory genes, an increase in lactate release by astrocytes, and suppression of the hyperexcitation of neuronal networks formed the basis of the cytoprotective effect of selenium nanoparticles in our studies.

The Protective Mechanism of Deuterated Linoleic Acid Involves the Activation of the Ca2+ Signaling System of Astrocytes in Ischemia In Vitro.

Ischemia-like (oxygen-glucose deprivation, OGD) conditions followed by reoxygenation (OGD/R) cause massive death of cerebral cortex cells in culture as a result of the induction of necrosis and apoptosis. Cell death occurs as a result of an OGD-induced increase in Ca2+ ions in the cytosol of neurons and astrocytes, an increase in the expression of genes encoding proapoptotic and inflammatory genes with suppression of protective genes. The deuterated form of linoleic polyunsaturated fatty acid (D4-Lnn) completely inhibits necrosis and greatly reduces apoptotic cell death with an increase in the concentration of fatty acid in the medium. It was shown for the first time that D4-Lnn, through the activation of the phosphoinositide calcium system of astrocytes, causes their reactivation, which correlates with the general cytoprotective effect on the cortical neurons and astrocytes in vitro. The mechanism of the cytoprotective action of D4-Lnn involves the inhibition of the OGD-induced calcium ions, increase in the cytosolic and reactive oxygen species (ROS) overproduction, the enhancement of the expression of protective genes, and the suppression of damaging proteins.

Deregulation of Ca2+-Signaling Systems in White Adipocytes, Manifested as the Loss of Rhythmic Activity, Underlies the Development of Multiple Hormonal Resistance at Obesity and Type 2 Diabetes.

Various types of cells demonstrate ubiquitous rhythmicity registered as simple and complex Ca2+-oscillations, spikes, waves, and triggering phenomena mediated by G-protein and tyrosine kinase coupled receptors. Phospholipase C/IP3-receptors (PLC/IP3R) and endothelial NO-synthase/Ryanodine receptors (NOS/RyR)-dependent Ca2+ signaling systems, organized as multivariate positive feedback generators (PLC-G and NOS-G), underlie this rhythmicity. Loss of rhythmicity at obesity may indicate deregulation of these signaling systems. To issue the impact of cell size, receptors' interplay, and obesity on the regulation of PLC-G and NOS-G, we applied fluorescent microscopy, immunochemical staining, and inhibitory analysis using cultured adipocytes of epididumal white adipose tissue of mice. Acetylcholine, norepinephrine, atrial natriuretic peptide, bradykinin, cholecystokinin, angiotensin II, and insulin evoked complex [Ca2+]i responses in adipocytes, implicating NOS-G or PLC-G. At low sub-threshold concentrations, acetylcholine and norepinephrine or acetylcholine and peptide hormones (in paired combinations) recruited NOS-G, based on G proteins subunits interplay and signaling amplification. Rhythmicity was cell size- dependent and disappeared in hypertrophied cells filled with lipids. Contrary to control cells, adipocytes of obese hyperglycemic and hypertensive mice, growing on glucose, did not accumulate lipids and demonstrated hormonal resistance being non responsive to any hormone applied. Preincubation of preadipocytes with palmitoyl-L-carnitine (100 nM) provided accumulation of lipids, increased expression and clustering of IP3R and RyR proteins, and partially restored hormonal sensitivity and rhythmicity (5-15% vs. 30-80% in control cells), while adipocytes of diabetic mice were not responsive at all. Here, we presented a detailed kinetic model of NOS-G and discussed its control. Collectively, we may suggest that universal mechanisms underlie loss of rhythmicity, Ca2+-signaling systems deregulation, and development of general hormonal resistance to obesity.

Mechanisms Underlying the Protective Effect of the Peroxiredoxin-6 Are Mediated via the Protection of Astrocytes during Ischemia/Reoxygenation.

Ischemia-like conditions reflect almost the entire spectrum of events that occur during cerebral ischemia, including the induction of oxidative stress, Ca2+ overload, glutamate excitotoxicity, and activation of necrosis and apoptosis in brain cells. Mechanisms for the protective effects of the antioxidant enzyme peroxiredoxin-6 (Prx-6) on hippocampal cells during oxygen-glucose deprivation/reoxygenation (OGD/R) were investigated. Using the methods of fluorescence microscopy, inhibitory analysis, vitality tests and PCR, it was shown that 24-h incubation of mixed hippocampal cell cultures with Prx-6 does not affect the generation of a reversible phase of a OGD-induced rise in Ca2+ ions in cytosol ([Ca2+]i), but inhibits a global increase in [Ca2+]i in astrocytes completely and in neurons by 70%. In addition, after 40 min of OGD, cell necrosis is suppressed, especially in the astrocyte population. This effect is associated with the complex action of Prx-6 on neuroglial networks. As an antioxidant, Prx-6 has a more pronounced and astrocyte-directed effect, compared to the exogenous antioxidant vitamin E (Vit E). Prx-6 inhibits ROS production in mitochondria by increasing the antioxidant capacity of cells and altering the expression of genes encoding redox status proteins. Due to the close bond between [Ca2+]i and intracellular ROS, this effect of Prx-6 is one of its protective mechanisms. Moreover, Prx-6 effectively suppresses not only necrosis, but also apoptosis during OGD and reoxygenation. Incubation with Prx-6 leads to activation of the basic expression of genes encoding protective kinases-PI3K, CaMKII, PKC, anti-apoptotic proteins-Stat3 and Bcl-2, while inhibiting the expression of signaling kinases and factors involved in apoptosis activation-Ikk, Src, NF-κb, Caspase-3, p53, Fas, etc. This effect on the basic expression of the genome leads to the cell preconditions, which is expressed in the inhibition of caspase-3 during OGD/reoxygenation. A significant effect of Prx-6 is directed on suppression of the level of pro-inflammatory cytokine IL-1ß and factor TNFα, as well as genes encoding NMDA- and kainate receptor subunits, which was established for the first time for this antioxidant enzyme. The protective effect of Prx-6 is due to its antioxidant properties, since mutant Prx-6 (mutPrx-6, Prx6-C47S) leads to polar opposite effects, contributing to oxidative stress, activation of apoptosis and cell death through receptor action on TLR4.