The C-terminus of the prototypical M2 muscarinic receptor localizes to the mitochondria and regulates cell respiration under stress conditions.

Muscarinic acetylcholine receptors are prototypical G protein-coupled receptors (GPCRs), members of a large family of 7 transmembrane receptors mediating a wide variety of extracellular signals. We show here, in cultured cells and in a murine model, that the carboxyl terminal fragment of the muscarinic M2 receptor, comprising the transmembrane regions 6 and 7 (M2tail), is expressed by virtue of an internal ribosome entry site localized in the third intracellular loop. Single-cell imaging and import in isolated yeast mitochondria reveals that M2tail, whose expression is up-regulated in cells undergoing integrated stress response, does not follow the normal route to the plasma membrane, but is almost exclusively sorted to the mitochondria inner membrane: here, it controls oxygen consumption, cell proliferation, and the formation of reactive oxygen species (ROS) by reducing oxidative phosphorylation. Crispr/Cas9 editing of the key methionine where cap-independent translation begins in human-induced pluripotent stem cells (hiPSCs), reveals the physiological role of this process in influencing cell proliferation and oxygen consumption at the endogenous level. The expression of the C-terminal domain of a GPCR, capable of regulating mitochondrial function, constitutes a hitherto unknown mechanism notably unrelated to its canonical signaling function as a GPCR at the plasma membrane. This work thus highlights a potential novel mechanism that cells may use for controlling their metabolism under variable environmental conditions, notably as a negative regulator of cell respiration.

Endothelial cells from umbilical cord of women affected by gestational diabetes: A suitable in vitro model to study mechanisms of early vascular senescence in diabetes.

Human umbilical cord endothelial cells (HUVECs) obtained from women affected by gestational diabetes (GD-HUVECs) display durable pro-atherogenic modifications and might be considered a valid in vitro model for studying chronic hyperglycemia effects on early endothelial senescence. Here, we demonstrated that GD- compared to C-HUVECs (controls) exhibited oxidative stress, altered both mitochondrial membrane potential and antioxidant response, significant increase of senescent cells characterized by a reduced NAD-dependent deacetylase sirtuin-1 (SIRT1) activity together with an increase in cyclin-dependent kinase inhibitor-2A (P16), cyclin-dependent kinase inhibitor-1 (P21), and tumor protein p53 (P53) acetylation. This was associated with the p300 activation, and its silencing significantly reduced the GD-HUVECs increased protein levels of P300 and Ac-P53 thus indicating a persistent endothelial senescence via SIRT1/P300/P53/P21 pathway. Overall, our data suggest that GD-HUVECs can represent an "endothelial hyperglycemic memory" model to investigate in vitro the early endothelium senescence in cells chronically exposed to hyperglycemia in vivo.

Phytochemistry and Biological Activity of Medicinal Plants in Wound Healing: An Overview of Current Research.

Wound healing is a complicated process, and the effective management of wounds is a major challenge. Natural herbal remedies have now become fundamental for the management of skin disorders and the treatment of skin infections due to the side effects of modern medicine and lower price for herbal products. The aim of the present study is to summarize the most recent in vitro, in vivo, and clinical studies on major herbal preparations, their phytochemical constituents, and new formulations for wound management. Research reveals that several herbal medicaments have marked activity in the management of wounds and that this activity is ascribed to flavonoids, alkaloids, saponins, and phenolic compounds. These phytochemicals can act at different stages of the process by means of various mechanisms, including anti-inflammatory, antimicrobial, antioxidant, collagen synthesis stimulating, cell proliferation, and angiogenic effects. The application of natural compounds using nanotechnology systems may provide significant improvement in the efficacy of wound treatments. Increasing the clinical use of these therapies would require safety assessment in clinical trials.

VSL#3 probiotic differently influences IEC-6 intestinal epithelial cell status and function.

The data here reported introduce the wound-healing assay as a tool for testing probiotics aimed at protecting gastrointestinal mucosal surfaces and to verify the consistency of their manufacturing. At the scope, we compared the in vitro effects of two multi-strain high concentration formulations both commercialized under the same brand VSL#3 but sourced from different production sites (USA and Italy) on a non-transformed small-intestinal epithelial cell line, IEC-6. The effects on cellular morphology, viability, migration, and H2 O2 -induced damage, were assessed before and after the treatment with both VSL#3 formulations. While the USA-sourced product ("USA-made") VSL#3 did not affect monolayer morphology and cellular density, the addition of bacteria from the Italy-derived product ("Italy-made") VSL#3 caused clear morphological cell damage and strongly reduced cellularity. The treatment with "USA-made" lysate led to a higher rate of wounded monolayer healing, while the addition of "Italy-made" bacterial lysate did not influence the closure rate as compared to untreated cells. While lysates from "USA-made" VSL#3 clearly enhanced the formation of elongated and aligned stress fibers, "Italy-made" lysates had not similar effect. "USA-made" lysate was able to cause a total inhibition of H2 O2 -induced cytotoxic effect whereas "Italy-made" VSL#3 lysate was unable to protect IEC-6 cells from H2 O2 -induced damage. ROS generation was also differently influenced, thus supporting the hypotesis of a protective action of "USA-made" VSL#3 lysates, as well as the idea that "Italy-made" formulation was unable to prevent significantly the H2 O2 -induced oxidative stress.

Improved Mitochondrial and Methylglyoxal-Related Metabolisms Support Hyperproliferation Induced by 50 Hz Magnetic Field in Neuroblastoma Cells.

Extremely low frequency magnetic fields (ELF-MF) are common environmental agents that are suspected to promote later stages of tumorigenesis, especially in brain-derived malignancies. Even though ELF magnetic fields have been previously linked to increased proliferation in neuroblastoma cells, no previous work has studied whether ELF-MF exposure may change key biomolecular features, such as anti-glycative defence and energy re-programming, both of which are currently considered as crucial factors involved in the phenotype and progression of many malignancies. Our study investigated whether the hyperproliferation that is induced in SH-SY5Y human neuroblastoma cells by a 50 Hz, 1 mT ELF magnetic field is supported by an improved defense towards methylglyoxal (MG), which is an endogenous cancer-static and glycating α-oxoaldehyde, and by rewiring of energy metabolism. Our findings show that not only the ELF magnetic field interfered with the biology of neuron-derived malignant cells, by de-differentiating further the cellular phenotype and by increasing the proliferative activity, but also triggered cytoprotective mechanisms through the enhancement of the defense against MG, along with a more efficient management of metabolic energy, presumably to support the rapid cell outgrowth. Intriguingly, we also revealed that the MF-induced bioeffects took place after an initial imbalance of the cellular homeostasis, which most likely created a transient unstable milieu. The biochemical pathways and molecular targets revealed in this research could be exploited for future approaches aimed at limiting or suppressing the deleterious effects of ELF magnetic fields. J. Cell. Physiol. 231: 2014-2025, 2016. © 2016 Wiley Periodicals, Inc.

Pre-exposure of neuroblastoma cell line to pulsed electromagnetic field prevents H2 O2 -induced ROS production by increasing MnSOD activity.

Electromagnetic fields (EMFs) have been linked to increased risk of cancers and neurodegenerative diseases; however, EMFs can also elicit positive effects on biological systems, and redox status seems crucially involved in EMF biological effects. This study aimed to assess whether a short and repeated pulsed EMF (PEMF) could trigger adaptive responses against an oxidative insult in a neuronal cellular model. We found that a 40 min overall (four times a week, 10 min each) pre-exposure to PEMF did not affect major physiological parameters and led to a significant increase of Mn-dependent superoxide dismutase activity in the human neuroblastoma SH-SY5Y cell line. In addition, we found PEMF-pre-exposed cells exhibited decreased reactive oxygen species production following a 30 min H2 O2 challenge, with respect to non pre-exposed cells. Our findings might provide new insights on the role played by short and repeated PEMF stimulations in the enhancement of cellular defenses against oxidative insults. Although studies in normal neuronal cells would be useful to further confirm our hypothesis, we suggest that specific PEMF treatments may have potential biological repercussions in diseases where oxidative stress is implicated.

Dicarbonyl stress and glyoxalases in ovarian function.

The ovary is the main regulator of female fertility. Changes in maternal health and physiology can disrupt intraovarian homoeostasis thereby compromising oocyte competence and fertility. Research has only recently devoted attention to the involvement of dicarbonyl stress in ovarian function. On this basis, the present review focuses on clinical and experimental research supporting the role of dicarbonyl overload and AGEs (advanced glycation end-products) as key contributors to perturbations of the ovarian microenvironment leading to lower fertility. Particular emphasis has been given to oocyte susceptibility to methylglyoxal, a powerful glycating agent, whose levels are known to increase during aging and metabolic disorders. According to the literature, the ovary and the oocyte itself can rely on the glyoxalase system to counteract the possible dicarbonyl overload such as that which may occur in reproductive-age women and patients with PCOS (polycystic ovarian syndrome) or diabetes. Overall, although biochemical methods for proper evaluation of dicarbonyl stress in oocytes and the ovarian microenvironment need to be established, AGEs can be proposed as predictive markers and/or therapeutic targets in new strategies for improving reproductive counselling and infertility therapies.

SIRT1 signalling protects mouse oocytes against oxidative stress and is deregulated during aging.

STUDY QUESTION: Is SIRT1 involved in the oxidative stress (OS) response in mouse oocytes? SUMMARY ANSWER: SIRT1 plays a pivotal role in the adaptive response of mouse germinal vesicle (GV) oocytes to OS and promotes a signalling cascade leading to up-regulation of the MnSod gene. WHAT IS KNOWN ALREADY: OS is known to continuously threaten acquisition and maintenance of oocyte developmental potential during in vivo processes and in vitro manipulations. Previous studies in somatic cells have provided strong evidence for the role of SIRT1 as a sensor of the cell redox state and a protector against OS and aging. STUDY DESIGN, SIZE, DURATION: GV oocytes obtained from young (4-8 weeks) and reproductively old (48-52 weeks) CD1 mice were blocked in the prophase stage by 0.5 µM cilostamide. Groups of 30 oocytes were exposed to 25 µM H2O2 and processed following different times for the analysis of intracellular localization of SIRT1 and FOXO3A, and evaluation of Sirt1, miRNA-132, FoxO3a and MnSod gene expression. Another set of oocytes was cultured in the presence or absence of the SIRT1-specific inhibitor Ex527, and exposed to H2O2 in order to assess the involvement of SIRT1 in the activation of a FoxO3a-MnSod axis and ROS detoxification. In the last part of this study, GV oocytes were maturated in vitro in the presence of different Ex527 concentrations (0, 2.5, 5, 10, 20 µM) and assessed for maturation rates following 16 h. Effects of Ex527 on spindle morphology and ROS levels were also evaluated. PARTICIPANTS/MATERIALS, SETTING, METHODS: SIRT1 and FOXO3A intracellular distribution in response to OS was investigated by immunocytochemistry. Real-time RT-PCR was employed to analyse Sirt1, miR-132, FoxO3a and MnSod gene expression. Reactive oxygen species (ROS) production was evaluated by in vivo measurement of carboxy-H2DCF diacetate labelling. Spindle and chromosomal distribution in in vitro matured oocytes were analysed by immunocytochemistry and DNA fluorescent labelling, respectively. MAIN RESULTS AND THE ROLE OF CHANCE: Specific changes in the intracellular localization of SIRT1 and up-regulation of Sirt1 gene were detected in mouse oocytes in response to OS. Moreover, increased intracellular ROS were observed when SIRT1 activity was inhibited by Ex527. In aged oocytes Sirt1 was expressed more than in young oocytes but SIRT1 protein was undetectable. Upon OS, significant changes in miR-132 micro-RNA, a validated Sirt1 modulator, were observed. A negative correlation between Sirt1 mRNA and miR-132 levels was observed when young oocytes exposed to OS were compared with young control oocytes, and when aged oocytes were compared with young control oocytes. FoxO3a and MnSod transcripts were increased upon OS with the same kinetics as Sirt1 transcripts, and up-regulation of MnSod gene was prevented by oocyte treatment with Ex527, indicating that SIRT1 acts upstream to the FoxO3a-MnSod axis. Finally, the results of the in vitro maturation assay suggested that SIRT1 might be involved in oocyte maturation by regulating the redox state and ensuring normal spindle assembly. LIMITATIONS, REASONS FOR CAUTION: The main limitation of this study was the absence of direct quantification of SIRT1 enzymatic activity due to the lack of an appropriately sensitive method. WIDER IMPLICATIONS OF THE FINDINGS: The present findings may provide a valuable background for studying the regulation of SIRT1 during oogenesis and its relevance as a sensor of oocyte redox state and energy status. The antioxidant response orchestrated by SIRT1 in oocytes seems to decrease with aging. This suggests that SIRT1 could be an excellent pharmacological target for improving oocyte quality and IVF outcome in aging or aging-like diseases. STUDY FUNDING/COMPETING INTERESTS: The work was supported by the Ministero dell'Università e della Ricerca Scientifica (MIUR) to C.T., F.A., C.D., A.M.D. The authors declare no conflict of interest.

The constitutive activation of TLR4-IRAK1- NFκB axis is involved in the early NLRP3 inflammasome response in peripheral blood mononuclear cells of Rett syndrome patients.

Rett syndrome (RTT), a devastating neurodevelopmental disorder, is caused in 95% of the cases by mutations in the X-chromosome-localized MECP2 gene. To date, RTT is considered a broad-spectrum disease, due to multisystem disturbances affecting patients, associated with mitochondrial dysfunctions, subclinical inflammation and an overall OxInflammatory status. Inflammasomes are multi-protein complexes crucially involved in innate immune responses against pathogens and oxidative stress mediators. The assembly of NLRP3:ASC inflammasome lead to pro-caspase 1 activation, maturation of interleukins (IL)-1ß and 18 and proteolytic cleavage of Gasdermin D leading eventually to pyroptosis and systemic inflammation. The possible de-regulation of this system, in parallel with upstream nuclear factor (NF)-κB p65 pathway, were analyzed in peripheral blood mononuclear cells (PBMCs) and plasma isolated from RTT patients and matching controls. RTT PBMCs showed a constitutive activation of the axis TLR4 (Toll-like receptor 4)-IRAK1 (interleukin-1 receptor associated kinase 1)-NF-κB p65, together with augmented ROS generation and enhanced IL-18 mRNA levels and NLRP3:ASC co-localization. The deregulation of inflammasome components was even found in THP-1 cells silenced for MECP2 and importantly, in plasma compartment of RTT subjects, from the earliest stages of the pathology or in correlation with the severity of MeCP2 mutations. Taken together, these data provide new insights into the mechanisms involved in RTT sub-clinical inflammatory status present in RTT patients, thus helping to reveal new targets for future therapeutic approaches.

Molecular basis underlying the biological effects elicited by extremely low-frequency magnetic field (ELF-MF) on neuroblastoma cells.

Extremely low-frequency magnetic fields (ELF-MFs) may affect human health because of the possible associations with leukemia but also with cancer, cardiovascular, and neurological disorders. In the present work, human SH-SY5Y neuroblastoma cells were exposed to a 50 Hz, 1 mT sinusoidal ELF-MF at three different times, that is, 5 days (T5), 10 days (T10), and 15 days (T15) and then the effects of ELF-MF on proteome expression and biological behavior were investigated. Through comparative analysis between treated and control samples, we analyzed the proteome changes induced by ELF-MF exposure. Nine new proteins resolved in sample after a 15-day treatment were involved in a cellular defense mechanism and/or in cellular organization and proliferation such as peroxiredoxin isoenzymes (2, 3, and 6), 3-mercaptopyruvate sulfurtransferase, actin cytoplasmatic 2, t-complex protein subunit beta, ropporin-1A, and profilin-2 and spindlin-1. Our results indicated that ELF-MFs exposure altered the proliferative status and other important cell biology-related parameters, such as cell growth pattern, and cytoskeletal organization. These findings support our hypothesis that ELF radiation could trigger a shift toward a more invasive phenotype.

Evidence that carbonyl stress by methylglyoxal exposure induces DNA damage and spindle aberrations, affects mitochondrial integrity in mammalian oocytes and contributes to oocyte ageing.

BACKGROUND: Highly reactive carbonyl compounds formed during glycolysis, such as methylglyoxal (MG), can lead to the formation of 'advanced glycation end products' (AGE) and carbonyl stress. Toxic AGEs are suspected to accumulate and play a role in reducing quality and developmental potential of mammalian oocytes of aged females and in PCOS and diabetic patients. Whether and how MG and AGE affect young and aged oocytes at the cellular level is unknown. METHODS: The study consists of three parts. In Part A expression of MG-detoxifying enzymes glyoxalases 1 and 2 was analysed by RT-PCR at different stages of maturation in denuded oocytes (DO), cumulus-enclosed oocytes (CEO) and metaphase (M)II oocytes of the CD-1 mouse to obtain information on stage-specific susceptibility to carbonyl stress. DO and CEO from young and aged females and from stimulated cycles were exposed to MG during maturation in vitro to assess also age-related changes in sensitivity to carbonyl stress induced by MG. Induction of apoptosis by MG on in vitro maturing DO was assessed by terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling test. In Part B of the study, DO from large antral follicles of ovaries of adult, young MF-1 mice in late diestrous were exposed to MG to assess direct influences of MG and AGEs formed during continuous exposure to MG on rate and kinetics of maturation to MII, on DNA integrity (by γ-H2AX staining) in the germinal vesicle (GV) stage, and on spindle formation and chromosome alignment (by tubulin and pericentrin immunofluorescence and polarization microscopy), and chromosome segregation (by C-banding) during in vitro maturation. Since MG and AGEs can affect functionality of mitochondria in Part C, mitochondrial distribution and membrane potential was studied using JC-1 probe. Expression of a redox-sensitive mito-Grx1-roGFP2 protein in mitochondria of maturing oocytes by confocal laser scanning microscopy was employed to determine the inner mitochondrial glutathion (GSH)/glutathion disulfide (GSSG)-dependent redox potential. RESULTS: Part A revealed that mRNA for glyoxalases decreases during meiotic maturation. Importantly, cumulus from aged mice in CEO obtained from stimulated cycles does not protect oocytes efficiently from MG-induced meiotic arrest during in vitro maturation. Part B showed that the MG-induced meiotic delay or arrest is associated with significant rises in spindle aberrations, chromosome congression failure and aberrant telophase I in oocytes. MG exposure of meiotically arrested GV-stage oocytes significantly increases the numbers of γ-H2AX spots in the nucleus suggesting increased DNA damage, while MG exposure during maturation affects chromatin condensation and induces chromosome lagging at anaphase I. Moreover, Part C revealed that carbonyl stress by chronic exposure to MG is associated with delays in changes in mitochondrial distribution and altered inner-mitochondrial GSH/GSSG redox potential, which might be particularly relevant for cytoskeletal dynamics as well as processes after fertilization. Sensitivity to a meiotic block by MG appears dependent on the genetic background. CONCLUSIONS: The sensitivity to carbonyl stress by MG appears to increase with maternal age. Since MG-exposure induces DNA damage, meiotic delay, spindle aberrations, anaphase I lagging and epimutation, aged oocytes are particularly at risk for such disturbances in the absence of efficient protection by cumulus. Furthermore, disturbances in mitochondrial distribution and redox regulation may be especially critical for fertilization and developmental competence of oocytes exposed to MG and carbonyl stress before or during maturation, for instance, in aged females, or in PCOS or diabetic patients, in agreement with recent suggestions of correlations between poor follicular and embryonic development, lower pregnancy rate and presence of toxic AGEs in serum, irrespective of age.

AGEs-related dysfunctions in PCOS: evidence from animal and clinical research.

Polycystic ovary syndrome (PCOS) is the most common female endocrine disorder in women in their reproductive age. In recent years, the role of advanced glycation end products (AGEs) in PCOS has gained great attention. AGEs are highly reactive molecules that can be assumed by diet or endogenously synthesized as by-products of metabolic processes. AGE deposition increases with aging, hyperglycemia, insulin resistance, and glycotoxin-rich diet. Therefore, it has become imperative to understand the underlying mechanism of AGEs actions and its downstream effects in PCOS pathophysiology. By integrating evidence from human studies and experimental models, the present review points out that altered AGE deposition is a common feature in all PCOS phenotypes. Searching for possible mechanisms involved in the adaptive response against glycation injury in oocytes and ovaries, the role of SIRT1, the main member of the mammalian sirtuin family, has also recently emerged. Therefore, further studies based on anti-AGE interventions could be helpful in creating innovative strategies for counteracting PCOS and its effects on fertility.

Mitochondrial Sirtuins in Reproduction.

Mitochondria act as hubs of numerous metabolic pathways. Mitochondrial dysfunctions contribute to altering the redox balance and predispose to aging and metabolic alterations. The sirtuin family is composed of seven members and three of them, SIRT3-5, are housed in mitochondria. They catalyze NAD+-dependent deacylation and the ADP-ribosylation of mitochondrial proteins, thereby modulating gene expression and activities of enzymes involved in oxidative metabolism and stress responses. In this context, mitochondrial sirtuins (mtSIRTs) act in synergistic or antagonistic manners to protect from aging and aging-related metabolic abnormalities. In this review, we focus on the role of mtSIRTs in the biological competence of reproductive cells, organs, and embryos. Most studies are focused on SIRT3 in female reproduction, providing evidence that SIRT3 improves the competence of oocytes in humans and animal models. Moreover, SIRT3 protects oocytes, early embryos, and ovaries against stress conditions. The relationship between derangement of SIRT3 signaling and the imbalance of ROS and antioxidant defenses in testes has also been demonstrated. Very little is known about SIRT4 and SIRT5 functions in the reproductive system. The final goal of this work is to understand whether sirtuin-based signaling may be taken into account as potential targets for therapeutic applications in female and male infertility.

Role of p53 in the Regulation of Cellular Senescence.

The p53 transcription factor plays a critical role in cellular responses to stress. Its activation in response to DNA damage leads to cell growth arrest, allowing for DNA repair, or directs cellular senescence or apoptosis, thereby maintaining genome integrity. Senescence is a permanent cell-cycle arrest that has a crucial role in aging, and it also represents a robust physiological antitumor response, which counteracts oncogenic insults. In addition, senescent cells can also negatively impact the surrounding tissue microenvironment and the neighboring cells by secreting pro-inflammatory cytokines, ultimately triggering tissue dysfunction and/or unfavorable outcomes. This review focuses on the characteristics of senescence and on the recent advances in the contribution of p53 to cellular senescence. Moreover, we also discuss the p53-mediated regulation of several pathophysiological microenvironments that could be associated with senescence and its development.

Corrigendum to "Role of Mitochondria in the Oxidative Stress Induced by Electromagnetic Fields: Focus on Reproductive Systems".

[This corrects the article DOI: 10.1155/2018/5076271.].

Altered inflammasome machinery as a key player in the perpetuation of Rett syndrome oxinflammation.

Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the X-linked MECP2 gene. RTT patients show multisystem disturbances associated with an oxinflammatory status. Inflammasomes are multi-protein complexes, responsible for host immune responses against pathogen infections and redox-related cellular stress. Assembly of NLRP3/ASC inflammasome triggers pro-caspase-1 activation, thus, resulting in IL-1ß and IL-18 maturation. However, an aberrant activation of inflammasome system has been implicated in several human diseases. Our aim was to investigate the possible role of inflammasome in the chronic subclinical inflammatory condition typical of RTT, by analyzing this complex in basal and lipopolysaccharide (LPS)+ATP-stimulated primary fibroblasts, as well as in serum from RTT patients and healthy volunteers. RTT cells showed increased levels of nuclear p65 and ASC proteins, pro-IL-1ß mRNA, and NLRP3/ASC interaction in basal condition, without any further response upon the LPS + ATP stimuli. Moreover, augmented levels of circulating ASC and IL-18 proteins were found in serum of RTT patients, which are likely able to amplify the inflammatory response. Taken together, our findings suggest that RTT patients exhibited a challenged inflammasome machinery at cellular and systemic level, which may contribute to the subclinical inflammatory state feedback observed in this pathology.

Methylglyoxal-Dependent Glycative Stress and Deregulation of SIRT1 Functional Network in the Ovary of PCOS Mice.

Advanced glycation end-products (AGEs) are involved in the pathogenesis and consequences of polycystic ovary syndrome (PCOS), a complex metabolic disorder associated with female infertility. The most powerful AGE precursor is methylglyoxal (MG), a byproduct of glycolysis, that is detoxified by the glyoxalase system. By using a PCOS mouse model induced by administration of dehydroepiandrosterone (DHEA), we investigated whether MG-dependent glycative stress contributes to ovarian PCOS phenotype and explored changes in the Sirtuin 1 (SIRT1) functional network regulating mitochondrial functions and cell survival. In addition to anovulation and reduced oocyte quality, DHEA ovaries revealed altered collagen deposition, increased vascularization, lipid droplets accumulation and altered steroidogenesis. Here we observed increased intraovarian MG-AGE levels in association with enhanced expression of receptor for AGEs (RAGEs) and deregulation of the glyoxalase system, hallmarks of glycative stress. Moreover, DHEA mice exhibited enhanced ovarian expression of SIRT1 along with increased protein levels of SIRT3 and superoxide dismutase 2 (SOD2), and decreased peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC1α), mitochondrial transcriptional factor A (mtTFA) and translocase of outer mitochondrial membrane 20 (TOMM20). Finally, the presence of autophagy protein markers and increased AMP-activated protein kinase (AMPK) suggested the involvement of SIRT1/AMPK axis in autophagy activation. Overall, present findings demonstrate that MG-dependent glycative stress is involved in ovarian dysfunctions associated to PCOS and support the hypothesis of a SIRT1-dependent adaptive response.

Fifty hertz extremely low-frequency magnetic field exposure elicits redox and trophic response in rat-cortical neurons.

Large research activity has raised around the mechanisms of interaction between extremely low-frequency magnetic fields (ELF-MFs) and biological systems. ELF-MFs may interfere with chemical reactions involving reactive oxygen species (ROS), thus facilitating oxidative damages in living cells. Cortical neurons are particularly susceptible to oxidative stressors and are also highly dependent on the specific factors and proteins governing neuronal development, activity and survival. The aim of the present work was to investigate the effects of exposures to two different 50 Hz sinusoidal ELF-MFs intensities (0.1 and 1 mT) in maturing rat cortical neurons' major anti-oxidative enzymatic and non-enzymatic cellular protection systems, membrane peroxidative damage, as well as growth factor, and cytokine expression pattern. Briefly, our results showed that ELF-MFs affected positively the cell viability and concomitantly reduced the levels of apoptotic death in rat neuronal primary cultures, with no significant effects on the main anti-oxidative defences. Interestingly, linear regression analysis suggested a positive correlation between reduced glutathione (GSH) and ROS levels in 1 mT MF-exposed cells. On this basis, our hypothesis is that GSH could play an important role in the antioxidant defence towards the ELF-MF-induced redox challenge. Moreover, the GSH-based cellular response was achieved together with a brain-derived neurotrophic factor over-expression as well as with the interleukin 1beta-dependent regulation of pro-survival signaling pathways after ELF-MF exposure.

13-HODE, 9-HODE and ALOX15 as potential players in Rett syndrome OxInflammation.

Mutations in the MECP2 gene are the main cause of Rett syndrome (RTT), a pervasive neurodevelopmental disorder, that shows also multisystem disturbances associated with a metabolic component. The aim of this study was to investigate whether an increased production of oxidized linoleic acid metabolites, specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), can contribute to the altered the redox and immune homeostasis, suggested to be involved in RTT. Serum levels of 9- and 13-HODEs were elevated in RTT and associated with the expression of arachidonate 15-Lipoxygenase (ALOX15) in peripheral blood mononuclear cells (PBMCs). Omega-3 polyunsaturated fatty acids supplementation has shown to lower HODEs levels in RTT. Statistically significant correlation was demonstrated between the increased plasma HODEs levels and the lipoprotein-associated phospholipase A2 (Lp-PLA2) activity. Collectively, these findings reinforce the concept of the key role played by lipid peroxidation in RTT, and the possible ability of omega-3 polyunsaturated fatty acids supplementation in improving the oxinflammation status in RTT.

SIRT1-Dependent Upregulation of Antiglycative Defense in HUVECs Is Essential for Resveratrol Protection against High Glucose Stress.

Uncontrolled accumulation of methylglyoxal (MG) and reactive oxygen species (ROS) occurs in hyperglycemia-induced endothelial dysfunction associated with diabetes. Resveratrol (RSV) protects the endothelium upon high glucose (HG); however, the mechanisms underlying such protective effects are still debated. Here we identified key molecular players involved in the glycative/oxidative perturbations occurring in endothelial cells exposed to HG. In addition, we determined whether RSV essentially required SIRT1 to trigger adaptive responses in HG-challenged endothelial cells. We used primary human umbilical vein endothelial cells (HUVECs) undergoing a 24-h treatment with HG, with or without RSV and EX527 (i.e., SIRT1 inhibitor). We found that HG-induced glycative stress (GS) and oxidative stress (OS), by reducing SIRT1 activity, as well as by diminishing the efficiency of MG- and ROS-targeting protection. RSV totally abolished the HG-dependent cytotoxicity, and this was associated with SIRT1 upregulation, together with increased expression of GLO1, improved ROS-scavenging efficiency, and total suppression of HG-related GS and OS. Interestingly, RSV failed to induce effective response to HG cytotoxicity when EX527 was present, thus suggesting that the upregulation of SIRT1 is essential for RSV to activate the major antiglycative and antioxidative defense and avoid MG- and ROS-dependent molecular damages in HG environment.