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.

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.

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.

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.

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.

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.