Serelaxin Protects H9c2 Cardiac Myoblasts against Hypoxia and Reoxygenation-Induced Damage through Activation of AMP Kinase/Sirtuin1: Further Insight into the Molecular Mechanisms of the Cardioprotection of This Hormone.

Serelaxin (RLX), namely the human recombinant Relaxin-2 hormone, protects the heart from ischemia/reperfusion (I/R)-induced damage due to its anti-inflammatory, anti-apoptotic and antioxidant properties. RLX acts by binding to its specific RXFP1 receptor whereby it regulates multiple transduction pathways. In this in vitro study, we offer the first evidence for the involvement of the AMP kinase/Sirtuin1 (AMPK/SIRT1) pathway in the protection by RLX against hypoxia/reoxygenation (H/R)-induced damage in H9c2 cells. The treatment of the H/R-exposed cells with RLX (17 nmol L-1) enhanced SIRT1 expression and activity. The inhibition of SIRT1 signaling with EX527 (10 µmol L-1) reduced the beneficial effect of the hormone on mitochondrial efficiency and cell apoptosis. Moreover, RLX upregulated the AMPK pathway, as shown by the increase in the expression of phospho-AMPK-activated protein. Finally, AMPK pathway inhibition by Compound C (10 and 20 µmol L-1) abrogated the increase in SIRT1 expression induced by RLX, thus suggesting the involvement of the AMPK pathway in this effect of RLX. These results strengthen the concept that RLX exerts its cardioprotective effects against H/R-induced injury through multiple pathways which also include AMPK/SIRT1. These new findings support the use of RLX or RLX-derived molecules as a promising therapeutic for those diseases in which I/R and oxidative stress play a pathogenic role.

Chronic lactate exposure promotes cardiomyocyte cytoskeleton remodelling.

We investigated the effect of growing on lactate instead of glucose in human cardiomyocyte assessing their viability, cell cycle activity, oxidative stress and metabolism by a proteomic and metabolomic approach. In previous studies performed on elite players, we found that adaptation to exercise is characterized by a chronic high plasma level of lactate. Lactate is considered not only an energy source but also a signalling molecule and is referred as "lactormone"; heart is one of the major recipients of exogenous lactate. With this in mind, we used a cardiac cell line AC16 to characterize the lactate metabolic profile and investigate the metabolic flexibility of the heart. Interestingly, our data indicated that cardiomyocytes grown on lactate (72 h) show change in several proteins and metabolites linked to cell hypertrophy and cytoskeleton remodelling. The obtained results could help to understand the effect of this metabolite on heart of high-performance athletes.

Mediterranean Diet and Oxidative Stress: A Relationship with Pain Perception in Endometriosis.

BACKGROUND: Endometriosis is a chronic and inflammatory disease associated with pelvic pain. Dietary changes may be therapeutic for chronic inflammatory processes, reducing visceral input. The aim was to evaluate the role of dietary changes according to the Mediterranean Diet (MD) on pain perception in endometriosis and their relationship with oxidative stress. METHODS: in this prospective study, we included 35 endometriosis women. At baseline (T0) and after 3 (T1) and 6 (T2) months from the start of the diet, we investigated pain intensity with VAS (Visual Analogue Scale, from 0 to 10), vitamin profile, and oxidative stress. RESULTS: we found a significant increase in the diet score (p < 0.001). At T1, patients reduced pain in terms of dyspareunia (p = 0.04), non-menstrual pelvic pain (p = 0.06), dysuria (p = 0.04), and dyschezia (p < 0.001). Dyspareunia (p = 0.002) and dyschezia (p < 0.001) were further significantly reduced also at T2. We observed a significant positive correlation between lipid peroxidation and VAS non-menstrual pelvic pain and dysuria and a significant negative correlation between Oxygen radical absorbance capacity and VAS non-menstrual pain and dyschezia. CONCLUSIONS: our findings show a clear tendency toward a relationship between pain relief in endometriosis and MD. This appears promising to treat endometriosis-related symptoms and could be considered a new effective strategy for chronic pain management in the long term.

ROS-driven structural and functional fibrinogen modifications are reverted by interleukin-6 inhibition in Giant Cell Arteritis.

BACKGROUND: Cranial and extra-cranial vascular events are among the major determinants of morbidity and mortality in Giant Cell Arteritis (GCA). Vascular events seem mostly of inflammatory nature, although the precise pathogenetic mechanisms are still unclear. We investigated the role of oxidation-induced structural and functional fibrinogen modifications in GCA. The effects of the anti-IL6R tocilizumab in counteracting these mechanisms were also assessed. MATERIALS AND METHODS: A cross-sectional study was conducted on 65 GCA patients and 65 matched controls. Leucocyte reactive oxygen species (ROS) production, redox state, and fibrinogen structural and functional features were compared between patients and controls. In 19 patients receiving tocilizumab, pre vs post treatment variations were assessed. RESULTS: GCA patients displayed enhanced blood lymphocyte, monocyte and neutrophil ROS production compared to controls, with an increased plasma lipid peroxidation and a reduced total antioxidant capacity. This oxidative impairment resulted in a sustained fibrinogen oxidation (i.e. dityrosine content 320 (204-410) vs 136 (120-176) Relative Fluorescence Units (RFU), p < 0.0001), with marked alterations in fibrinogen secondary and tertiary structure [intrinsic fluorescence: 134 (101-227) vs 400 (366-433) RFU, p < 0.001]. Structural alterations paralleled a remarkable fibrinogen functional impairment, with a reduced ability to polymerize into fibrin and a lower fibrin susceptibility to plasmin-induced lysis. In patients receiving tocilizumab, a significant improvement in redox status was observed, accompanied by a significant improvement in fibrinogen structural and functional features (p < 0.001). CONCLUSIONS: An impaired redox status accounts for structural and functional fibrinogen modifications in GCA, suggesting a potential role of tocilizumab for cardiovascular prevention in GCA.

Gastric Cancer Vascularization and the Contribution of Reactive Oxygen Species.

Blood vessels are the most important way for cancer cells to survive and diffuse in the body, metastasizing distant organs. During the process of tumor expansion, the neoplastic mass progressively induces modifications in the microenvironment due to its uncontrolled growth, generating a hypoxic and low pH milieu with high fluid pressure and low nutrients concentration. In such a particular condition, reactive oxygen species play a fundamental role, enhancing tumor proliferation and migration, inducing a glycolytic phenotype and promoting angiogenesis. Indeed, to reach new sources of oxygen and metabolites, highly aggressive cancer cells might produce a new abnormal network of vessels independently from endothelial cells, a process called vasculogenic mimicry. Even though many molecular markers and mechanisms, especially in gastric cancer, are still unclear, the formation of such intricate, leaky and abnormal vessel networks is closely associated with patients' poor prognosis, and therefore finding new pharmaceutical solutions to be applied along with canonical chemotherapies in order to control and normalize the formation of such networks is urgent.

Serum Interleukin-36 α as a Candidate Biomarker to Distinguish Behçet's Syndrome and Psoriatic Arthritis.

Behçet's syndrome (BS) is a rare systemic vasculitis characterized by different clinical manifestations. As no specific laboratory tests exist, the diagnosis relies on clinical criteria, and the differential diagnosis with other inflammatory diseases can be challenging. Indeed, in a relatively small proportion of patients, BS symptoms include only mucocutaneous, articular, gastrointestinal, and non-typical ocular manifestations, which are frequently found also in psoriatic arthritis (PsA). We investigate the ability of serum interleukin (IL)-36α-a pro-inflammatory cytokine involved in cutaneous and articular inflammatory diseases-to differentiate BS from PsA. A cross-sectional study was performed on 90 patients with BS, 80 with PsA and 80 healthy controls. Significantly lower IL-36α concentrations were found in patients with BS as compared to PsA, although in both groups IL-36α was significantly increased compared to healthy controls. An empirical cut-off of 420.6 pg/mL displayed a specificity of 0.93, with a sensitivity of 0.70 (AUC 0.82) in discriminating PsA from BS. This cut-off displayed a good diagnostic performance also in BS patients lacking highly specific BS manifestations. Our results indicate that IL-36α might be involved in the pathogenesis of both BS and PsA, and might be a candidate biomarker to support the differential diagnosis of BS.

Chemical Modification of Auranofin Yields a New Family of Anticancer Drug Candidates: The Gold(I) Phosphite Analogues.

A panel of four novel gold(I) complexes, inspired by the clinically established gold drug auranofin (1-Thio-ß-D-glucopyranosatotriethylphosphine gold-2,3,4,6-tetraacetate), was prepared and characterized. All these compounds feature the replacement of the triethylphosphine ligand of the parent compound auranofin with a trimethylphosphite ligand. The linear coordination around the gold(I) center is completed by Cl-, Br-, I- or by the thioglucose tetraacetate ligand (SAtg). The in-solution behavior of these gold compounds as well as their interactions with some representative model proteins were comparatively analyzed through 31PNMR and ESI-MS measurements. Notably, all panel compounds turned out to be stable in aqueous media, but significant differences with respect to auranofin were disclosed in their interactions with a few leading proteins. In addition, the cytotoxic effects produced by the panel compounds toward A2780, A2780R and SKOV-3 ovarian cancer cells were quantitated and found to be in the low micromolar range, since the IC50 of all compounds was found to be between 1 µM and 10 µM. Notably, these novel gold complexes showed large and similar inhibition capabilities towards the key enzyme thioredoxin reductase, again comparable to those of auranofin. The implications of these results for the discovery of new and effective gold-based anticancer agents are discussed.

Atherosclerosis and Endometriosis: The Role of Diet and Oxidative Stress in a Gender-Specific Disorder.

Background: Accelerated atherosclerosis in patients with endometriosis has been hypothesised, and lifestyle improvement might control cardiovascular risk. We explored cardiometabolic markers and oxidative stress and evaluated the effects of the Mediterranean Diet (MD) in modulating these markers. Methods: In this prospective study, we included 35 women with endometriosis. At baseline (T0) and after 3 (T1) and 6 (T2) months from the start of the diet, we investigated cardiometabolic parameters, lifestyle and oxidative stress. Results: After a 3-month intervention with MD, we observed a significant reduction in total cholesterol (p = 0.01) and LDL-c (p = 0.003). We observed at T1 an increase in B12 and E vitamins, folate and zinc. After 6 months, zinc (p = 0.04) and folate (p = 0.08) increased in comparison to T0. A reduction in homocysteine from T0 to T1 (p = 0.01) was found. After 3 months, an increase in Rapid Assessment of Physical Activity tool 1 (RAPA) (p < 0.001) and RAPA 2 was observed (p = 0.009). We observed high levels of oxidative stress markers at baseline. After 6 months of MD, a significant improvement in lymphocyte Reactive Oxygen Species (ROS) (p < 0.001) and total antioxidant capacity was observed (p = 0.02). Conclusions: The improvement of lifestyle, and in particular the Mediterranean dietary intervention, allowed the improvement of the metabolic and oxidative profile and overall health-related quality of life.

Blood Leukocyte ROS Production Reflects Seminal Fluid Oxidative Stress and Spermatozoa Dysfunction in Idiopathic Infertile Men.

A large proportion of infertile men do not receive a clear diagnosis, being considered as idiopathic or unexplained cases due to infertility diagnosis based on standard semen parameters. Particularly in unexplained cases, the search for new indicators seems mandatory to provide specific information. In the etiopathogenesis of male infertility oxidative stress displays important roles by negatively affecting sperm quality and function. In this study, performed in a population of 34 idiopathic infertile men and in 52 age-matched controls, redox parameters were assessed in blood, leukocytes, spermatozoa, and seminal fluid and related to semen parameters. The main findings indicate that blood oxidative stress markers reflect seminal oxidative stress. Interestingly, blood leukocyte ROS production was significantly correlated to sperm ROS production and to semen parameters. Overall, these results suggest the potential employ of blood redox markers as a relevant and adjunctive tool for sperm quality evaluation aimed to preconception care.

Effects of benzo[a]pyrene on the reproductive axis: Impairment of kisspeptin signaling in human gonadotropin-releasing hormone primary neurons.

The neuroendocrine control of reproduction is strictly coordinated at the central level by the pulsatile release of gonadotropin-releasing hormone (GnRH) by the hypothalamic GnRH neurons. Alterations of the GnRH-network, especially during development, lead to long-term reproductive and systemic consequences, also causing infertility. Recent evidence shows that benzo[a]pyrene (BaP), a diffuse pollutant that can play a role as an endocrine disruptor, affects gonadal function and gamete maturation, whereas data demonstrating its impact at hypothalamic level are very scarce. This study investigated the effects of BaP (10 µM) in a primary cell culture isolated from the human fetal hypothalamus (hfHypo) and exhibiting a clear GnRH neuron phenotype. BaP significantly decreased gene and protein expression of both GnRH and kisspeptin receptor (KISS1R), the master regulator of GnRH neuron function. Moreover, BaP exposure increased phospho-ERK1/2 signaling, a well-known mechanism associated with KISS1R activation. Interestingly, BaP altered the electrophysiological membrane properties leading to a significant depolarizing effect and it also significantly increased GnRH release, with both effects being not affected by kisspeptin addition. In conclusion, our findings demonstrate that BaP may alter GnRH neuron phenotype and function, mainly interfering with KISS1R signaling and GnRH secretion and therefore with crucial mechanisms implicated in the central neuroendocrine control of reproduction.

Oxidative Stress and Redox-Dependent Pathways in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a primary liver tumor that accounts for 2% of all cancer-related deaths worldwide yearly. It can arise from cholangiocytes of biliary tracts, peribiliary glands, and possibly from progenitor cells or even hepatocytes. CCA is characterized by high chemoresistance, aggressiveness, and poor prognosis. Potentially curative surgical therapy is restricted to a small number of patients with early-stage disease (up to 35%). Accumulating evidence indicates that CCA is an oxidative stress-driven carcinoma resulting from chronic inflammation. Oxidative stress, due to enhanced reactive oxygen species (ROS) production and/or decreased antioxidants, has been recently suggested as a key factor in cholangiocyte oncogenesis through gene expression alterations and molecular damage. However, due to different experimental models and conditions, contradictory results regarding oxidative stress in cholangiocarcinoma have been reported. The role of ROS and antioxidants in cancer is controversial due to their context-dependent ability to stimulate tumorigenesis and support cancer cell proliferation or promote cell death. On these bases, the present narrative review is focused on illustrating the role of oxidative stress in cholangiocarcinoma and the main ROS-driven intracellular pathways. Heterogeneous data about antioxidant effects on cancer development are also discussed.

SIRT1 and thrombosis.

Thrombosis is a major cause of morbidity and mortality worldwide, with a complex and multifactorial pathogenesis. Recent studies have shown that SIRT1, a member of the sirtuin family of NAD + -dependent deacetylases, plays a crucial role in regulating thrombosis, modulating key pathways including endothelial activation, platelet aggregation, and coagulation. Furthermore, SIRT1 displays anti-inflammatory activity both in vitro, in vivo and in clinical studies, particularly via the reduction of oxidative stress. On these bases, several studies have investigated the therapeutic potential of targeting SIRT1 for the prevention of thrombosis. This review provides a comprehensive and critical overview of the main preclinical and clinical studies and of the current understanding of the role of SIRT1 in thrombosis.

Erythrocyte oxidative stress and thrombosis.

Thrombosis is a common disorder with a relevant burden of morbidity and mortality worldwide, particularly among elderly patients. Growing evidence demonstrated a direct role of oxidative stress in thrombosis, with various cell types contributing to this process. Among them, erythrocytes produce high quantities of intracellular reactive oxygen species (ROS) by NADPH oxidase activation and haemoglobin autoxidation. Concomitantly, extracellular ROS released by other cells in the blood flow can be uptaken and accumulate within erythrocytes. This oxidative milieu can alter erythrocyte membrane structure, leading to an impaired erythrocyte function, and promoting erythrocytes lysis, binding to endothelial cells, activation of platelet and of coagulation factors, phosphatidylserine exposure and release of microvesicles. Moreover, these abnormal erythrocytes are able to adhere to the vessel wall, contributing to thrombin generation within the thrombus. This process results in accelerated haemolysis and in a hypercoagulable state, in which structurally impaired erythrocytes contribute to increase thrombus size, to reduce its permeability and susceptibility to lysis. However, the wide plethora of mechanisms by which oxidised erythrocytes contribute to thrombosis is not completely elucidated. This review discusses the main biochemical aspects linking erythrocytes, oxidative stress and thrombosis, addressing their potential implication for clinical and therapeutic management.

Doxorubicin-induced senescence in normal fibroblasts promotes in vitro tumour cell growth and invasiveness: The role of Quercetin in modulating these processes.

Ageing is a complex biological phenomenon representing the major risk factor for developing age-related diseases, such as cardiovascular pathologies, neurodegenerative diseases, and cancer. Geroscience, the new vision of gerontology, identifies cellular senescence as an interconnected biological process that characterises ageing and age-related diseases. Therefore, many strategies have been employed in the last years to reduce the harmful effects of senescence, and among these, the most intriguing ones use nutraceutical compounds. Here we show that a pre-treatment with Quercetin, a bioactive flavonoid present in many fruits and vegetables, increasing cellular antioxidant defence, can alleviate Doxorubicin (Doxo)-induced cellular senescence in human normal WI-38 fibroblasts. Furthermore, our work demonstrates that Quercetin pre-treatment, reducing the number of senescent cells and the production of the senescence-associated secretory phenotype (SASP) factors, can decrease the pro-tumour effects of conditioned medium from Doxo-induced senescent fibroblasts on osteosarcoma cells. Overall, our findings are consistent with the hypothesis that targeting senescent cells can be an emerging strategy for cancer treatment, especially in elderly patients, in which senescent cells are already abundant in several tissues and organs.

The Protection of Zinc against Acute Cadmium Exposure: A Morphological and Molecular Study on a BBB In Vitro Model.

Cadmium (Cd) is a well-known occupational and environmental pollutant worldwide, and its toxicity is widely recognised. Cd is reported to increase the permeability of the blood-brain barrier (BBB) and to penetrate and accumulate in the brain. Although many lines of evidence show that Cd toxicity is induced by different mechanisms, one of the best known is the Cd-dependent production of reactive oxygen species (ROS). Zinc is a trace element known as coenzyme and cofactor for many antioxidant proteins, such as metallothioneins and superoxide dismutase enzymes. To date, very little is known about the role of Zn in preventing Cd-induced blood-brain barrier (BBB) alterations. The goal of this study was to test the Zn antioxidant capacity against Cd-dependent alterations in a rat brain endothelial cell line (RBE4), as an in vitro model for BBB. In order to mimic acute Cd poisoning, RBE4 cells were treated with CdCl2 30 µM for 24 h. The protective role of ZnCl2 (50 µM) was revealed by evaluating the cell viability, reactive oxygen species (ROS) quantification, cytochrome C distribution, and the superoxide dismutase (SOD) protein activity. Additionally, the effectiveness of Zn in counteracting the Cd-induced damage was investigated by evaluating the expression levels of proteins already known to be involved in the Cd signalling pathway, such as GRP78 (an endoplasmic reticulum (ER) stress protein), caspase3 pro- and cleaved forms, and BAX. Finally, we evaluated if Zn was able to attenuate the alterations of zonula occludens-1 (ZO-1), one of the tight-junction (TJ) proteins involved in the formation of the BBB. Our data clearly demonstrate that Zn, by protecting from the SOD activity impairment induced by Cd, is able to prevent the triggering of the Cd-dependent signalling pathway that leads to ZO-1 dislocation and downregulation, and BBB damage.