Retention of vitamin A after goat milk processing into cheese: a nutritional strategy.

The deficiency in micronutrients is a public health problem, principally in lower-middle-income countries. Vitamin A (VA) is considered a micronutrient fundamental to the maintenance and development of different tissues in the organism. Therefore, it is an essential micronutrient in the human diet. In these terms, goat milk is the leading food consumed to provide nutritional support in innumerous lower-middle-income countries. Here our work aimed to produce goat cheese studying strategies to promote the retention of VA. Our experiment design also explores the use of the salting process to evaluate the levels of VA retention. The level of VA in goat cheese was determined using LC-MS/MS analysis. Additionally, the redox status of the goat cheese in terms of lipid peroxidation and protein oxidation was determined. The texture analysis was also evaluated to verify if the redox status and salting process influence the texture profile. The results showed that the salting process during goat cheese production improves the retention of VA in goat cheese. Moreover, the salting process also is related to alterations in the status redox of the goat cheese and texture parameters. Therefore, our results show that goat cheese production can be an alternative to produced dairy derivates with recognized concentrations of VA for human nutrition.

Anti-RAGE antibody selectively blocks acute systemic inflammatory responses to LPS in serum, liver, CSF and striatum.

Systemic inflammation induces transient or permanent dysfunction in the brain by exposing it to soluble inflammatory mediators. The receptor for advanced glycation endproducts (RAGE) binds to distinct ligands mediating and increasing inflammatory processes. In this study we used an LPS-induced systemic inflammation model in rats to investigate the effect of blocking RAGE in serum, liver, cerebrospinal fluid (CSF) and brain (striatum, prefrontal cortex, ventral tegmental area and substantia nigra). Intraperitoneal injection of RAGE antibody (50µg/kg) was followed after 1h by a single LPS (5mg/kg) intraperitoneal injection. Twenty-four hours later, tissues were isolated for analysis. RAGE antibody reduced LPS-induced inflammatory effects in both serum and liver; the levels of proinflammatory cytokines (TNF-α, IL-1ß) were decreased and the phosphorylation/activation of RAGE downstream targets (ERK1/2, IκB and p65) in liver were significantly attenuated. RAGE antibody prevented LPS-induced effects on TNF-α and IL-1ß in CSF. In striatum, RAGE antibody inhibited increases in IL-1ß, Iba-1, GFAP, phospho-ERK1/2 and phospho-tau (ser202), as well as the decrease in synaptophysin levels. These effects were caused by systemic RAGE inhibition, as RAGE antibody did not cross the blood-brain barrier. RAGE antibody also prevented striatal lipoperoxidation and activation of mitochondrial complex II. In conclusion, blockade of RAGE is able to inhibit inflammatory responses induced by LPS in serum, liver, CSF and brain.

Retinol (Vitamin A) Increases α-Synuclein, ß-Amyloid Peptide, Tau Phosphorylation and RAGE Content in Human SH-SY5Y Neuronal Cell Line.

Retinoids (vitamin A and derivatives) are recognized as essential factors for central nervous system (CNS) development. Retinol (vitamin A) also was postulated to be a major antioxidant component of diet as it modulates reactive species (RS) production and oxidative stress in biological systems. Oxidative stress plays a major role either in pathogenesis or development of neurodegenerative diseases, or even in both. Here we investigate the role of retinol supplementation to human neuron-derived SH-SY5Y cells over RS production and biochemical markers associated to neurodegenerative diseases expressed at neuronal level in Parkinson's disease and Alzheimer's disease: α-synuclein, ß-amyloid peptide, tau phosphorylation and RAGE. Retinol treatment (24 h) impaired cell viability and increased intracellular RS production at the highest concentrations (7 up to 20 µM). Antioxidant co-treatment (Trolox 100 µM) rescued cell viability and inhibited RS production. Furthermore, retinol (10 µM) increased the levels of α-synuclein, tau phosphorylation at Ser396, ß-amyloid peptide and RAGE. Co-treatment with antioxidant Trolox inhibited the increased in RAGE, but not the effect of retinol on α-synuclein, tau phosphorylation and ß-amyloid peptide accumulation. These data indicate that increased availability of retinol to neurons at levels above the cellular physiological concentrations may induce deleterious effects through diverse mechanisms, which include oxidative stress but also include RS-independent modulation of proteins associated to progression of neuronal cell death during the course of neurodegenerative diseases.

Effect of N-salicyloyltryptamine (STP), a novel tryptamine analogue, on parameters of cell viability, oxidative stress, and immunomodulation in RAW 264.7 macrophages.

Immunomodulatory actions exerted by some classes of tryptamines, such as benzoyltryptamine analogues, suggest these molecules as promising candidates to develop new therapies to treat conditions associated to acute and chronic pain and inflammation. N-salicyloyltryptamine (STP) was observed to act as an anticonvulsive agent and exert antinociceptive effects in mouse. In the present work, we performed a screening of cytotoxic, cytoprotective, immunomodulatory, and redox properties of STP in RAW 264.7 macrophages challenged with hydrogen peroxide and LPS. Our results show that STP presents no cytotoxicity in the range of 0.001 to 1 µg/mL, but doses of 50 and 100 µg/mL caused loss of cell viability (IC(50) = 22.75 µg/mL). Similarly, STP at 0.001 to 1 µg/mL did not cause oxidative stress to RAW 264.7 cells, although it did not prevent cell death induced by H(2)O(2) 0.5 mM. At 1 µg/mL, STP reversed some redox and inflammatory parameters induced by LPS. These include thiol (sulfhydryl) oxidation, superoxide dismutase activation, and morphological changes associated to macrophage activation. Besides, STP significantly inhibited LPS-induced TNF-α and IL-1ß release, as well as CD40 and TNF-α protein upregulation. Signaling events induced by LPS, such as phosphorylation of ERK 1/2 and IκBα and p65 nuclear translocation (NF-kB activation) were also inhibited by STP. These data indicate that STP is able to modulate inflammatory parameters at doses that do not interfere in cell viability.

L-NAME co-treatment prevent oxidative damage in the lung of adult Wistar rats treated with vitamin A supplementation.

Based on the fact that vitamin A in clinical doses is a potent pro-oxidant agent to the lungs, we investigated here the role of nitric oxide (NO•) in the disturbances affecting the lung redox environment in vitamin A-treated rats (retinol palmitate, doses of 1000-9000 IU•kg(-1)•day(-1)) for 28 days. Lung mitochondrial function and redox parameters, such as lipid peroxidation, protein carbonylation and the level of 3-nytrotyrosine, were quantified. We observed, for the first time, that vitamin A supplementation increases the levels of 3-nytrotyrosine in rat lung mitochondria. To determine whether nitric oxide (NO •) or its derivatives such as peroxynitrite (ONOO-) was involved in this damage, animals were co-treated with the nitric oxide synthase inhibitor L-NAME (30 mg•kg(-1), four times a week), and we analysed if this treatment prevented (or minimized) the biochemical disturbances resulting from vitamin A supplementation. We observed that L-NAME inhibited some effects caused by vitamin A supplementation. Nonetheless, L-NAME was not able to reverse completely the negative effects triggered by vitamin A supplementation, indicating that other factors rather than only NO• or ONOO- exert a prominent role in mediating the redox effects in the lung of rats that received vitamin A supplementation.

In vitro optimization of retinoic acid-induced neuritogenesis and TH endogenous expression in human SH-SY5Y neuroblastoma cells by the antioxidant Trolox.

Though, it is quite well-known how retinoic acid (RA) is able to induce neuritogenesis in different in vitro models, the putative role exerted by reactive oxygen species (ROS) during this process still need to be further studied. For such purpose, we used a neuronal-like cell line (SH-SY5Y cells) in order to investigate whether the antioxidant Trolox (a hydrophilic analog of alpha-tocopherol) could have any effect on the number of RA-induced neurites, and how significant changes in cellular redox homeostasis may affect the cellular endogenous expression of tyrosine hydroxylase (TH). Our results show a significant enhancement of RA (10 µM)-induced neuritogenesis and TH endogenous expression, when cells were co-treated with Trolox (100 µM) for 7 days. Moreover, this effect was associated with an improvement in cellular viability. The mechanism seems to mainly involve PI3 K/Akt rather than MEK signaling pathway. Therefore, our data demonstrate that concomitant decreases in basal reactive oxygen species (ROS) production could exert a positive effect on the neuritogenic process of RA-treated SH-SY5Y cells.

Influences of the polymorphisms of the Sod2 gene (rs4880) on the motility and vigor of X- and Y-bearing sperm at different pH values.

Superoxide dismutase 2 (SOD2) is an antioxidant enzyme that appears phylogenetically conserved. However, functional Sod2 polymorphisms have been studied, and the specific polymorphisms are related to activity alterations of the SOD2 enzyme. An example of a polymorphism of SOD2 is Val16Ala (rs4880), which has been identified in exon 2 of the human Sod2 gene. This polymorphism is recognized as a single nucleotide polymorphism (SNP) and alters the conformation of SOD2. Additionally, recent studies have shown that the Ala16 Val polymorphism in Sod2 can be related to different pathological diseases. In these terms, the objective of the present study was to evaluate whether the polymorphism of SOD2 in Val16Ala (rs4880) influences the motility and vigor of X- and Y-bearing sperm at different pH values promoting sperm selection. We found that polymorphism rs4880 at normal pH conditions can result in alterations in the activity of superoxide dismutase in the sperm through different assay analyses. Moreover, compelling modulation evidence indicates that this effect could also mediate seminal plasma redox alterations and consequently can play an important role in sperm physiology, fertilization, and postfertilization.

Anti-inflammatory and antixidant properties of blend formulated with compounds of Malpighia emarginata D.C (acerola) and Camellia sinensis L. (green tea) in lipopolysaccharide-stimulated RAW 264.7 macrophages.

The antioxidant and anti-inflammatory properties of Malpighia emarginata D.C (acerola) and Camellia sinensis L. (green tea) have been studied, particularly as an alternative in medicinal approach for different physio pathological conditions. Here we develop an powder blend formulated with both Malpighia emarginata D.C and Camellia sinensis L. which have in the composition higher content of ascorbic acid and epigallatocathechin-3-gallate respectively. Using different conditions for microencapsulation of biocompounds, we performed the powder production through spray-drying process. After, we evaluate the antioxidant and anti-inflammatory properties of blends formulated with Malpighia emarginata D.C and Camellia sinensis L. in an in vitro model of inflammation, using LPS-stimulated RAW-264.7 macrophage cell line. We observed that co-treatment with blends was able to modulate the redox parameters in cells during the in vitro inflammatory response. Moreover, the co-treatment with blends were able to modulate inflammatory response by altering the secretion of cytokines IL-1ß, IL-6, IL-10, and TNF-α. Taken together, our results demonstrate for the first time the synergistic effects antioxidant and anti-inflammatory of Malpighia emarginata D.C and Camellia sinensis L. These results warrant further use of the blend powder for use in the products to heath beneficial, principally in terms of prevention of chronic diseases.

Increased receptor for advanced glycation endproducts immunocontent in the cerebral cortex of vitamin A-treated rats.

Vitamin A, beyond its biological role, is an alternative choice in treating some life threatening pathologies, for instance leukemia and immunodeficiency. On the other hand, vitamin A therapy at moderate to high doses has caused concern among public health researchers due to the toxicological aspect resulting from such habit. It has been described hepatotoxicity, cognitive disturbances and increased mortality rates among subjects ingesting increased levels of vitamin A daily. Then, based on the previously reported data, we investigated here receptor for advanced glycation endproducts (RAGE) immunocontent and oxidative damage levels in cerebral cortex of vitamin A-treated rats at clinical doses (1,000-9,000 IU/kg day(-1)). RAGE immunocontent, as well as oxidative damage levels, were observed increased in cerebral cortex of vitamin A-treated rats. Whether increased RAGE levels exert negative effects during vitamin A supplementation it remains to be investigated, but it is very likely that deleterious consequences may arise from such alteration.

Retinol increases catalase activity and protein content by a reactive species-dependent mechanism in Sertoli cells.

Vitamin A (retinol) is widely used as an antioxidant in therapeutic interventions and dietary supplementations. However, the redox properties of retinoids have been the subject of intense debate in the last few years, as recent works observed deleterious effects caused by retinol supplementation in clinical trials. In the present work, we show that retinol treatment (7 microM, 24 h) led to catalase (EC 1.11.1.6; CAT) activation in cultured Sertoli cells by increasing its protein content in a reactive species-dependent manner. Retinol treatment also increased cell lipoperoxidation, assessed by determination of thiobarbituric acid-reactive substances (TBARS), and intracellular reactive species production, determined by the real-time dihydrochlorofluorescein (DCFH-DA) assay. However, no alterations on CAT mRNA expression (assessed by RT-PCR) were observed, indicating an effect independent of CAT gene-transcription regulation. Importantly, all the effects induced by retinol were inhibited by the antioxidant Trolox, a hydrophilic analogue of alpha-tocopherol. These results show for the first time that retinol increases CAT activity by a redox-dependent modulation of its protein content in a cell culture model. CAT activity or expression are widely used as indexes of oxidative stress in biological systems; since no changes in CAT mRNA expression were detected in these conditions, the use of CAT gene-transcription activation when assessing oxidative stress should be re-evaluated.

Retinol up-regulates the receptor for advanced glycation endproducts (RAGE) by increasing intracellular reactive species.

Retinol (vitamin A) and other retinoids have been suggested to exert an important antioxidant function in biological systems, besides their more established role as regulators of cell growth and differentiation. On the other hand, many authors have recently observed pro-oxidant activities of vitamin A and other retinoids in vitro and in vivo, resulting in cell death and/or transformation associated to increased oxidative damage. However, the mechanisms by which retinol causes oxidative stress are still not fully understood. Receptors for advanced glycation endproducts (RAGE) have been recently implied as promoters and/or amplifiers of oxidant-mediated cell death induced by diverse agents, and increased RAGE expression is observed in conditions related to unbalanced production of reactive species, such as in atherosclerosis and neurodegeneration. In the present work, we observed that retinol supplementation increases RAGE protein expression in cultured Sertoli cells, and antioxidant co-treatment reversed this effect. Retinol-increased RAGE expression was observed only at concentrations that induce intracellular reactive species production, as assessed by the DCFH assay. These results indicate that retinol is able to increase RAGE expression by an oxidant-dependent mechanism, and suggest that RAGE signaling may be involved in some of the deleterious effects observed in some retinol-supplementation therapies.

Vitamin A supplementation induces a prooxidative state in the striatum and impairs locomotory and exploratory activity of adult rats.

Although vitamin A has been reported to be essential to brain homeostasis, some central nervous system (CNS)-associated deleterious effects may be induced by vitamin A or by its metabolites. In this work, we investigated the effects of acute and chronic vitamin A supplementation at therapeutic (1,000 or 2,500 IU/kg/day) or excessive (4,500 or 9,000 IU/kg/day) doses on the redox state of the rat striatum. We found a 1.8- to 2.7-fold increase of lipid peroxidation in the striatum after acute or chronic supplementation (TBARS method). Therapeutic doses induced a 1.6- to 2.2-fold increase of protein carbonylation (dinitrophenylhydrazine (DNPH) derivatization). Vitamin A supplementation induced a 1.2- to 1.4-fold decrease of protein thiol content acutely and chronically. Superoxide dismutase (SOD) activity, assessed through the inhibition of epinephrine's autoxidation, was increased in a dose-dependent manner chronically. Acutely, both therapeutic and excessive vitamin A doses induced a 1.8- to 2.2-fold decrease of catalase (CAT) activity, as determined through the rate of decrease of hydrogen peroxide (H(2)O(2)). Glutathione peroxidase (GPx) activity did not change in this experimental model. Some vitamin A doses decreased the non-protein thiol content only chronically. Vitamin A supplementation decreased the striatal non-enzymatic antioxidant defenses (TRAP assay). Furthermore, our results show that vitamin A supplementation impaired the SOD/CAT ratio. Moreover, we observed a 1.6- to 2.0-fold decrease of locomotion and exploration in an open field after vitamin A supplementation. Therefore, our results suggest that vitamin A supplementation induces oxidative stress in the rat striatum and that it may be related to a metabolic impairment in such brain area.

Changes in Cell Cycle and Up-Regulation of Neuronal Markers During SH-SY5Y Neurodifferentiation by Retinoic Acid are Mediated by Reactive Species Production and Oxidative Stress.

Human neuroblastoma SH-SY5Y cells have been used as an in vitro model for neurodegenerative disorders such as Parkinson's disease and can be induced to a mature neuronal phenotype through retinoic acid (RA) differentiation. However, mechanisms of RA-induced differentiation remain unclear. Here, we investigate the role of reactive species (RS) on SH-SY5Y neuroblastoma cells under RA differentiation, using the antioxidant Trolox® as co-treatment. We found that RA treatment for 7 days reduced the cell number and proliferative capacity and induced the expression of adult catecholaminergic/neuronal markers such as tyrosine hydroxylase (TH), ß-III tubulin, and enolase-2. Evaluation of intracellular RS production by DCFH oxidation assay and quantification of cell non-enzymatic antioxidant activity by TRAP demonstrated that RA increases RS production. Furthermore, mitochondrial NADH oxidation showed to be inhibited under differentiation with RA. Cells subjected to co-treatment with antioxidant Trolox® demonstrated a remaining proliferative capacity and a decrease in the pro-oxidant state and RS production. Besides, antioxidant treatment restores the mitochondrial NADH oxidation. Importantly, Trolox® co-treatment inhibited the appearance of morphological characteristics such as neurite extension and branching, and decreased the expression of TH, ß-III tubulin, and enolase-2 after a seven-day differentiation with RA, indicating that RS production is a necessary step in this process. Trolox® also inhibited the phosphorylation of Akt and ERK1/2, which are involved in differentiation and survival, respectively, of these cells. Altogether, these data indicate the presence of a redox-dependent mechanism in SH-SY5Y RA-differentiation process and can be a useful insight to improve understanding of neuronal differentiation signaling.

NRF2 Mediates Neuroblastoma Proliferation and Resistance to Retinoic Acid Cytotoxicity in a Model of In Vitro Neuronal Differentiation.

Retinoic acid (RA) morphogenetic properties have been used in different kinds of therapies, from neurodegenerative disorders to some types of cancer such as promyelocytic leukemia and neuroblastoma. However, most of the pathways responsible for RA effects remain unknown. To investigate such pathways, we used a RA-induced differentiation model in the human neuroblastoma cells, SH-SY5Y. Our data showed that n-acetyl-cysteine (NAC) reduced cells' proliferation rate and increased cells' sensitivity to RA toxicity. Simultaneously, NAC pre-incubation attenuated nuclear factor erythroid 2-like factor 2 (NRF2) activation by RA. None of these effects were obtained with Trolox® as antioxidant, suggesting a cysteine signalization by RA. NRF2 knockdown increased cell sensibility to RA after 96 h of treatment and diminished neuroblastoma proliferation rate. Conversely, NRF2 overexpression limited RA anti-proliferative effects and increased cell proliferation. In addition, a rapid and non-genomic activation of the ERK 1/2 and PI3K/AKT pathways revealed to be equally required to promote NRF2 activation and necessary for RA-induced differentiation. Together, we provide data correlating NRF2 activity with neuroblastoma proliferation and resistance to RA treatments; thus, this pathway could be a potential target to optimize neuroblastoma chemotherapeutic response as well as in vitro neuronal differentiation protocols.

Gene Expression Profile of NF-κB, Nrf2, Glycolytic, and p53 Pathways During the SH-SY5Y Neuronal Differentiation Mediated by Retinoic Acid.

SH-SY5Y cells, a neuroblastoma cell line that is a well-established model system to study the initial phases of neuronal differentiation, have been used in studies to elucidate the mechanisms of neuronal differentiation. In the present study, we investigated alterations of gene expression in SH-SY5Y cells during neuronal differentiation mediated by retinoic acid (RA) treatment. We evaluated important pathways involving nuclear factor kappa B (NF-κB), nuclear E2-related factor 2 (Nrf2), glycolytic, and p53 during neuronal differentiation. We also investigated the involvement of reactive oxygen species (ROS) in modulating the gene expression profile of those pathways by antioxidant co-treatment with Trolox®, a hydrophilic analogue of α-tocopherol. We found that RA treatment increases levels of gene expression of NF-κB, glycolytic, and antioxidant pathway genes during neuronal differentiation of SH-SY5Y cells. We also found that ROS production induced by RA treatment in SH-SY5Y cells is involved in gene expression profile alterations, chiefly in NF-κB, and glycolytic pathways. Antioxidant co-treatment with Trolox® reversed the effects mediated by RA NF-κB, and glycolytic pathways gene expression. Interestingly, co-treatment with Trolox® did not reverse the effects in antioxidant gene expression mediated by RA in SH-SY5Y. To confirm neuronal differentiation, we quantified endogenous levels of tyrosine hydroxylase, a recognized marker of neuronal differentiation. Our data suggest that during neuronal differentiation mediated by RA, changes in profile gene expression of important pathways occur. These alterations are in part mediated by ROS production. Therefore, our results reinforce the importance in understanding the mechanism by which RA induces neuronal differentiation in SH-SY5Y cells, principally due this model being commonly used as a neuronal cell model in studies of neuronal pathologies.

Turnera subulata Anti-Inflammatory Properties in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

In South America, particularly in the Northeastern regions of Brazil, Turnera subulata leaf extract is used as an alternative traditional medicine approach for several types of chronic diseases, such as diabetes, hypertension, chronic pain, and general inflammation. Despite its widespread use, little is known about the medicinal properties of the plants of this genus. In this study, we evaluate the antioxidant and anti-inflammatory of T. subulata leaf extract in an in vitro model of inflammation, using lipopolysaccharide-stimulated RAW-264.7 macrophage cell line. We observed that cotreatment with T. subulata leaf extract was able to reduce the oxidative stress in cells due to inflammatory response. More importantly, we observed that the leaf extract was able to directly modulate inflammatory response by altering activity of members of the mitogen-activated protein kinase pathways. Our results demonstrate for the first time that T. subulata have antioxidant and anti-inflammatory properties, which warrant further investigation of the medicinal potential of this species.

Hecogenin acetate inhibits reactive oxygen species production and induces cell cycle arrest and senescence in the A549 human lung cancer cell line.

Cellular and molecular mechanisms related to lung cancer have been extensively studied in recent years, but the availability of effective treatments is still scarce. Hecogenin acetate, a natural saponin presenting a wide spectrum of reported pharmacological activities, has been previously evaluated for its anticancer/antiproliferative activity in some in vivo and in vitro models. Here, we investigated the effects of hecogenin acetate in a human lung cancer cell line. A549 non-small lung cancer cells were exposed to different concentrations of hecogenin acetate and reactive species production, ERK1/2 activation, matrix metalloproteinase expression, cell cycle arrest and cell senescence parameters were evaluated. Hecogenin acetate significantly inhibited increase in intracellular reactive species production induced by H2O2. In addition, hecogenin acetate blocked ERK1/2 phosphorylation and inhibited the increase in MMP-2 caused by H2O2. Treatment with hecogenin acetate induced G0/G1-phase arrest at two concentrations (75 and 100 µM, 74% and 84.3% respectively), and increased the staining of senescence-associated ß -galactosidase positive cells. These data indicate that hecogenin acetate is able to exert anti-cancer effects by modulating reactive species production, inducing cell cycle arrest and senescence and also modulating ERK1/2 phosphorylation and MMP-2 production.

Vitamin A (retinol) downregulates the receptor for advanced glycation endproducts (RAGE) by oxidant-dependent activation of p38 MAPK and NF-kB in human lung cancer A549 cells.

As an essential component of the diet, retinol supplementation is often considered harmless and its application is poorly controlled. However, recent works demonstrated that retinol may induce a wide array of deleterious effects, especially when doses used are elevated. Controlled clinical trials have demonstrated that retinol supplementation increased the incidence of lung cancer and mortality in smokers. Experimental works in cell cultures and animal models showed that retinol may induce free radical production, oxidative stress and extensive biomolecular damage. Here, we evaluated the effect of retinol on the regulation of the receptor for advanced glycation end-products (RAGE) in the human lung cancer cell line A549. RAGE is constitutively expressed in lungs and was observed to be down-regulated in lung cancer patients. A549 cells were treated with retinol doses reported as physiologic (2 µM) or therapeutic (5, 10 or 20 µM). Retinol at 10 and 20 µM increased free radical production, oxidative damage and antioxidant enzyme activity in A549 cells. These doses also downregulated RAGE expression. Antioxidant co-treatment with Trolox®, a hydrophilic analog of α-tocopherol, reversed the effects of retinol on oxidative parameters and RAGE downregulation. The effect of retinol on RAGE was mediated by p38 MAPK activation, as blockade of p38 with PD169316 (10 µM), SB203580 (10 µM) or siRNA to either p38α (MAPK14) or p38ß (MAPK11) reversed the effect of retinol on RAGE. Trolox also inhibited p38 phosphorylation, indicating that retinol induced a redox-dependent activation of this MAPK. Besides, we observed that NF-kB acted as a downstream effector of p38 in RAGE downregulation by retinol, as NF-kB inhibition by SN50 (100 µg/mL) and siRNA to p65 blocked the effect of retinol on RAGE, and p38 inhibitors reversed NF-kB activation. Taken together, our results indicate a pro-oxidant effect of retinol on A549 cells, and suggest that modulation of RAGE expression by retinol is mediated by the redox-dependent activation of p38/NF-kB signaling pathway.

Vitamin A (retinol) up-regulates the receptor for advanced glycation endproducts (RAGE) through p38 and Akt oxidant-dependent activation.

Retinol (vitamin A) is believed to exert preventive/protective effects against malignant, neurodegenerative and cardiovascular diseases by acting as an antioxidant. However, later clinical and experimental data show a pro-oxidant action of retinol and other retinoids at specific conditions. The receptor for advanced glycation endproducts (RAGE) is a pattern recognition receptor, being activated by different ligands such as S100 proteins, HMGB1 (amphoterin), ß-amyloid peptide and advanced glycation endproducts (AGE). RAGE activation influences a wide range of pathological conditions such as diabetes, pro-inflammatory states and neurodegenerative processes. Here, we investigated the involvement of different mitogen-activated protein kinases (MAPK: ERK1/2, p38 and JNK), PKC, PKA and Akt in the up-regulation of RAGE by retinol. As previously reported, we observed that the increase in RAGE immunocontent by retinol is reversed by antioxidant co-treatment, indicating the involvement of oxidative stress in this process. Furthermore, the p38 inhibitor SB203580 and the Akt inhibitor LY294002 also decreased the effect of retinol on RAGE levels, suggesting the involvement of these protein kinases in such effect. Both p38 and Akt phosphorylation were increased by treatment with pro-oxidant concentrations of retinol, and the antioxidant co-treatment blocked this effect, indicating that activation of p38 and Akt during retinol treatment is dependent on reactive species production. The 2',7'-dichlorohydrofluorescein diacetate (DCFH) assay also indicated that retinol treatment enhances cellular reactive species production. Altogether, these data indicate that RAGE up-regulation by retinol is mediated by the free radical-dependent activation of p38 and Akt.

Serum heat shock protein 70 levels, oxidant status, and mortality in sepsis.

Animal studies as well as prospective randomized clinical trials associated sepsis with redox imbalance and oxidative stress, but other studies failed to establish a correlation between antioxidant-based therapies and improvement of sepsis condition. This is also true for studies on the role of the chaperone heat shock protein 70 (HSP70), which is increased in serum during sepsis. Heat shock protein 70 is affected at several levels by oxidative stress, but this relationship has never been studied in sepsis. Here, we evaluated the relationship between serum HSP70 immunocontent and oxidant status in sepsis. Patients with severe sepsis were followed up for 28 days after diagnosis, or until death. Up to a maximum of 12 h after sepsis diagnosis, serum was collected for determination of HSP70 immunocontent by Western blot and evaluation of oxidative parameters (TRAP [total radical-trapping antioxidant parameter], TBARSs [thiobarbituric acid-reactive substances], and carbonyl levels). Serum of sepsis patients presented enhanced HSP70 levels. Analysis of oxidative parameters revealed that septic patients with pronounced oxidative damage in serum had also increased HSP70 serum levels. Sepsis patients in whom serum oxidative stress markers were not different from control presented normal serum HSP70. Analysis of septic patients according to survival outcome also indicated that patients with increased HSP70 serum levels presented increased mortality. We concluded that serum HSP70 levels are modulated according to the patient oxidant status, and increased serum HSP70 is associated to mortality in sepsis.