Effects of coconut oil long-term supplementation in Wistar rats during metabolic syndrome - regulation of metabolic conditions involving glucose homeostasis, inflammatory signals, and oxidative stress.

This study was designed to evaluate the long-term effects of Fructose (20%) feeding in rats, simulating metabolic syndrome (MetS), and the effects of coconut oil (C.O.) supplementation when administered in a MetS context. MetS is a cluster of systemic conditions that represent an increased chance of developing cardiovascular diseases and type 2 diabetes in the future. C.O. has been the target of media speculation, and recent studies report inconsistent results. C.O. improved glucose homeostasis and reduced fat accumulation in Fructose-fed rats while decreasing the levels of triglycerides (TGs) in the liver. C.O. supplementation also increased TGs levels and fructosamine in serum during MetS, possibly due to white adipose tissue breakdown and high fructose feeding. Pro-inflammatory cytokines IL-1ß and TNF-α were also increased in rats treated with Fructose and C.O. Oxidative stress marker nitrotyrosine is increased in fructose-fed animals, and C.O. treatment did not prevent this damage. No significant changes were observed in lipoperoxidation marker 4-Hydroxynonenal; however, fructose feeding increased total conjugated dienes and caused conjugated dienes to switch their conformation from cis-trans to trans-trans, which was not prevented by C.O. treatment. Potential benefits of C.O. have been reported with inconsistent results, and indeed we observed some benefits of C.O. supplementation in aiding weight loss, fat accumulation, and improving glucose homeostasis. Nonetheless, we also demonstrated that long-term C.O. supplementation could present some problematic effects with higher risk for individuals suffering MetS, including increased TGs and fructosamine levels and conformational changes in dienes.

High fat diet-induced obesity causes a reduction in brain tyrosine hydroxylase levels and non-motor features in rats through metabolic dysfunction, neuroinflammation and oxidative stress.

Obesity is a health problem that has been associated with neuroinflammation, decreased cognitive functions and development of neurodegenerative diseases. Parkinson's disease (PD) is a chronic neurodegenerative condition characterized by motor and non-motor abnormalities, increased brain inflammation, α-synuclein protein aggregation and dopaminergic neuron loss that is associated with decreased levels of tyrosine hydroxylase (TH) in the brain. Diet-induced obesity is a global epidemic and its role as a risk factor for PD is not clear. Herein, we showed that 25 weeks on a high-fat diet (HFD) promotes significant alterations in the nigrostriatal axis of Wistar rats. Obesity induced by HFD exposure caused a reduction in TH levels and increased TH phosphorylation at serine 40 in the ventral tegmental area. These effects were associated with insulin resistance, increased tumor necrosis factor-α levels, oxidative stress, astrogliosis and microglia activation. No difference was detected in the levels of α-synuclein. Obesity also induced impairment of locomotor activity, total mobility and anxiety-related behaviors that were identified in the open-field and light/dark tasks. There were no changes in motor coordination or memory. Together, these data suggest that the reduction of TH levels in the nigrostriatal axis occurs through an α-synuclein-independent pathway and can be attributed to brain inflammation, oxidative/nitrosative stress and metabolic disorders induced by obesity.

Guarana supplementation attenuated obesity, insulin resistance, and adipokines dysregulation induced by a standardized human Western diet via brown adipose tissue activation.

Obesity is a metabolic disorder associated with adverse health consequences that has increased worldwide at an epidemic rate. This has encouraged many people to utilize nonprescription herbal supplements for weight loss without knowledge of their safety or efficacy. However, mounting evidence has shown that some herbal supplements used for weight loss are associated with adverse effects. Guarana seed powder is a popular nonprescription dietary herb supplement marketed for weight loss, but no study has demonstrated its efficacy or safety when administered alone. Wistar rats were fed four different diets (low-fat diet and Western diet with or without guarana supplementation) for 18 weeks. Metabolic parameters, gut microbiota changes, and toxicity were then characterized. Guarana seed powder supplementation prevented weight gain, insulin resistance, and adipokine dysregulation induced by Western diet compared with the control diet. Guarana induced brown adipose tissue expansion, mitochondrial biogenesis, uncoupling protein-1 overexpression, AMPK activation, and minor changes in gut microbiota. Molecular docking suggested a direct activation of AMPK by four guarana compounds tested here. We propose that brown adipose tissue activation is one of the action mechanisms involved in guarana supplementation-induced weight loss and that direct AMPK activation may underlie this mechanism. In summary, guarana is an attractive potential therapeutic agent to treat obesity.

Oral administration of carvacrol/ß-cyclodextrin complex protects against 6-hydroxydopamine-induced dopaminergic denervation.

Carvacrol (CARV) presents valuable biological properties such as anti-inflammatory and antioxidant activities. However, pharmacological uses of CARV are largely limited due to disadvantages related to solubility, bioavailability, preparation and storage processes. The complexation of monoterpenes with ß-cyclodextrin (ß-CD) increases their stability, solubility and oral bioavailability. Here, the protective effect of oral treatment with CARV/ß-CD complex (25 µg/kg/day) against dopaminergic (DA) denervation induced by unilateral intranigral injection of 6-hydroxydopamine (6-OHDA - 10 µg per rat) was analyzed, in order to evaluate a putative application in the development of neuroprotective therapies for Parkinson's disease (PD). Pretreatment with CARV/ß-CD for 15 days prevented the loss of DA neurons induced by 6-OHDA in adult Wistar rats. This effect may occur through CARV anti-inflammatory and antioxidant properties, as the pretreatment with CARV/ß-CD inhibited the release of IL-1ß and TNF-α; besides, CARV prevented the increase of mitochondrial superoxide production induced by 6-OHDA in cultured SH-SY5Y cells. Importantly, hepatotoxicity or alterations in blood cell profile were not observed with oral administration of CARV/ß-CD. Therefore, this study showed a potential pharmacological application of CARV/ß-CD in PD using a non-invasive route of drug delivery, i.e., oral administration.

The effects of retinol oral supplementation in 6-hydroxydopamine dopaminergic denervation model in Wistar rats.

Vitamin A (retinol) is involved in signaling pathways regulating gene expression and was postulated to be a major antioxidant and anti-inflammatory compound of the diet. Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by loss of nigral dopaminergic neurons, involving oxidative stress and pro-inflammatory activation. The aim of the present study was to evaluate the neuroprotective effects of retinol oral supplementation against 6-hydroxydopamine (6-OHDA, 12 µg per rat) nigrostriatal dopaminergic denervation in Wistar rats. Animals supplemented with retinol (retinyl palmitate, 3000 IU/kg/day) during 28 days exhibited increased retinol content in liver, although circulating retinol levels (serum) were unaltered. Retinol supplementation did not protect against the loss of dopaminergic neurons (assessed through tyrosine hydroxylase immunofluorescence and Western blot). Retinol supplementation prevented the effect of 6-OHDA on Iba-1 levels but had no effect on 6-OHDA-induced GFAP increase. Moreover, GFAP levels were increased by retinol supplementation alone. Rats pre-treated with retinol did not present oxidative damage or thiol redox modifications in liver, and the circulating levels of TNF-α, IL-1ß, IL-6 and IL-10 were unaltered by retinol supplementation, demonstrating that the protocol used here did not cause systemic toxicity to animals. Our results indicate that oral retinol supplementation is not able to protect against 6-OHDA-induced dopaminergic denervation, and it may actually stimulate astrocyte reactivity without altering parameters of systemic toxicity.

Systemic Inflammation Changes the Site of RAGE Expression from Endothelial Cells to Neurons in Different Brain Areas.

The receptor for advanced glycation endproducts (RAGE) is a transmembrane, immunoglobulin-like receptor that interacts with a broad repertoire of extracellular ligands. RAGE belongs to a family of cell adhesion molecules and is considered a key receptor in the inflammation axis and a potential contributor to the neurodegeneration. The present study aimed to investigate the content and cell localization of RAGE in the brain of Wistar rats subjected to systemic inflammation induced by a single dose of lipopolysaccharide (LPS, 5 mg/kg, i.p.). Fifteen days after LPS administration, the content of RAGE was analyzed in the prefrontal cortex (PFC), hippocampus (HIPP), cerebellum (CB), and substantia nigra (SN) were investigated. RAGE levels increased in all structures, except HIPP; however, immunohistochemistry analysis demonstrated that the cell site of RAGE expression changed from blood vessel-like structures to neuronal cells in all brain areas. Besides, the highest level of RAGE expression was found in SN. Immunofluorescence analysis in SN confirmed that RAGE expression was mainly co-localized in endothelial cells (RAGE/PECAM-1 co-staining) in untreated animals, while LPS-treated animals had RAGE expression predominantly in dopaminergic neurons (RAGE/TH co-staining). Decreased TH levels, as well as increased pro-inflammatory markers (TNF-α, IL-1ß, Iba-1, GFAP, and phosphorylated ERK1/2) in SN, occurred concomitantly to RAGE stimulation in the same site. These results suggest a role for RAGE in the establishment of a neuroinflammation-neurodegeneration axis that develops as a long-term response to systemic inflammation by LPS.

TRF1 as a major contributor for telomeres' shortening in the context of obesity.

Obesity is a prevalent multifactorial chronic disorder characterized by metabolic dysregulation. Sustained pro-oxidative mediators trigger harmful consequences that reflect at systemic level and contribute for the establishment of a premature senescent phenotype associated with macromolecular damage (DNA, protein, and lipids). Telomeres are structures that protect chromosome ends and are associated with a six-protein complex called the shelterin complex and subject to regulation. Under pro-oxidant conditions, telomere attrition and the altered expression of the shelterin proteins are central for the establishment of many pathophysiological conditions such as obesity. Thus, considering that individuals with obesity display a systemic oxidative stress profile that may compromise the telomeres length or its regulation, the aim of this study was to investigate telomere homeostasis in patients with obesity and explore broad/systemic associations with the expression of shelterin genes and the plasma redox state. We performed a cross-sectional study in 39 patients with obesity and 27 eutrophic subjects. Telomere length (T/S ratio) and gene expression of shelterin components were performed in peripheral blood mononuclear cells by qPCR. The oxidative damage (lipid peroxidation and protein carbonylation) and non-enzymatic antioxidant system (total radical-trapping antioxidant potential/reactivity, sulfhydryl and GSH content) were evaluated in plasma. Our results demonstrate that independently of comorbidities, individuals with obesity had significantly shorter telomeres, augmented expression of negative regulators of the shelterin complex, increased lipid peroxidation and higher oxidized protein levels associated with increased non-enzymatic antioxidant defenses. Principal component analysis revealed TRF1 as a major contributor for firstly telomeres shortening. In conclusion, our study is first showing a comprehensive analysis of telomeres in the context of obesity, associated with dysregulation of the shelterin components that was partially explained by TRF1 upregulation that could not be reversed by the observed adaptive non-enzymatic antioxidant response.

Obese rats are more vulnerable to inflammation, genotoxicity and oxidative stress induced by coal dust inhalation than non-obese rats.

Obesity is an important nutritional disorder worldwide. Its association with environmental pollution may trigger an increase in oxidative stress and inflammatory parameters. Coal is a resource used throughout the world as an important fuel source for generating electricity. The ashes released by the coal combustion cause serious problems for human health due to their high toxicity and their capacity to bioaccumulate. The aim of this work was to investigate the effects of coal dust inhalation in the organs of obese and non-obese Wistar rats. Pro-inflammatory cytokines, oxidative stress, oxidative damage, histological analysis, comet assay, and micronuclei were investigated. Both obesity and coal dust inhalation increased the pro-inflammatory cytokines IL-1ß and TNF-α and decreased HSP70 levels in serum, however, in obese animals that inhaled coal dust these changes were more pronounced. Liver histological analysis showed severe microvesicular steatosis in obese animals that inhaled coal dust. Lung histologic investigation showed abnormalities in lung structure of animals exposed to coal dust and showed severe lung distensibility in obese animals exposed to coal dust. The comet assay showed DNA damage in animals subjected to coal. In addition, there were modulations in enzymatic activities and damage to protein and lipids. Based on our results, the coal dust inhalation can potentiate the pro-inflammatory profile present in obese rats. We also observed an increase in the protein oxidative damage in obese rats that inhaled coal dust. Taken together, our results suggest that the combination of obesity and coal inhalation increased the risks of the development of diseases related to oxidative stress and inflammation.

Effects of the consumption of polyunsaturated fatty acids on the oxidative status of adult dogs.

The present study evaluated the alterations of the oxidative stress markers in adult dogs fed with high levels of PUFA from the mixture of soybean oil enriched with docosahexaenoic acid (DHA) and supplemented with a natural algae-based antioxidant (AOX). Twelve healthy adult (2 years old) Beagle dogs (6 males and 6 females, 11.20 ± 1.92 kg BW), were distributed in 2 completely randomized blocks design and fed with 4 experimental diets coated with 2 lipid sources: saturated (13% bovine tallow) or unsaturated (13% soybean oil enriched with DHA), supplemented or not with 500 mg of AOX for 4 wk, intercalated with a 4-wk adaptation period. Blood samples were collected on days 0, 15, and 30 of each block. Glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), sulfhydryl group (SH), protein carbonylation, thiobarbituric acid reactive substances (TBARS), and total reactive antioxidant potential (TRAP) were evaluated in the serum, while GSH-Px, SOD, glutathione S-transferase (GST), catalase (CAT), SH, and TBARS were measured in erythrocytes. There was no significant difference in most of the oxidative markers evaluated. In contrast, GST activity in erythrocytes was greater in the animals that consumed the diets coated with bovine tallow compared to dogs that consumed diets coated with soybean oil enriched with DHA (P < 0.05). Serum from dogs fed on diets supplemented with AOX presented greater TRAP values (P < 0.05). These data demonstrate that the concentrations of unsaturated fatty acids (UNS) used in the diets for dogs were not sufficient to cause large changes in the oxidative status. It was not possible to evaluate the efficiency of the natural antioxidant in maintaining the oxidative balance of the animals as it appears that the oxidative status of the dogs was not challenged by the unsaturated diets. Our findings also suggest that dogs, as descendants from carrion carnivores, may have some natural protection against oxidation.

Supplementation with Achyrocline satureioides Inflorescence Extracts to Pregnant and Breastfeeding Rats Induces Tissue-Specific Changes in Enzymatic Activity and Lower Neonatal Survival.

Achyrocline satureioides (AS, family Asteraceae) is a plant widely used in traditional medicine for stomach, digestive, and gastrointestinal disorders during pregnancy. Studies regarding the indiscriminate use of plant infusions during pregnancy are limited. Recent reports have shown that chronic flavonoid supplementation induces toxicity in vivo and raises the mortality rates of healthy subjects. Therefore, we investigated whether supplementation of pregnant and lactating Wistar rats with two AS inflorescence extracts, consisting of an aqueous (AQ) extract similar to a tea (47 mg·kg-1·day) and a hydroethanolic (HA) extract (35 mg·kg-1·day-1) with a higher flavonoid content, could induce redox-related side effects. Total reactive antioxidant potential (TRAP), thiobarbituric reactive species (TBARS), and total reduced thiol (SH) content were evaluated. Superoxide dismutase (SOD) and catalase (CAT) activities were additionally quantified. Our data suggest that both AQ and HA of AS inflorescence extracts may induce symptoms of toxicity in concentrations of (47 mg·kg-1·day) and (35 mg·kg-1·day-1), respectively, in mothers regarding the delivery index and further decrease of neonatal survival. Of note, significant tissue-specific changes in maternal (liver, kidney, heart, and hippocampus) and pups (liver and kidney) biochemical oxidative parameters were observed. Our findings provide evidence that may support the need to control supplementation with the AQ of AS inflorescence extracts during gestation due to potential toxicity in vivo, which might be related, at least in part, to changes in tissue-specific redox homeostasis and enzymatic activity.

Characterization of dietary constituents and antioxidant capacity of Tropaeolum pentaphyllum Lam.

The aim of this study was to evaluate the nutritional content and antioxidant capacity of the tubers, leaves and, flowers of the species Tropaeolum pentaphyllum Lam. The three parts of the plant were analyzed by physicochemical methods, atomic absorption spectrometry, spectrophotometric and chromatographic techniques. The tubers, leaves, and flowers exhibited significant differences in all parameters evaluated. The leaves showed significantly higher values of protein (16.28 ± 0.02 g/100 g), total dietary fiber (27.78 ± 0.15 g/100 g) and quercetin (3798.61 ± 37.57 µg/g) when compared to the tubers and flowers. The study revealed a potential content of the protein, dietary fiber, and flavonoids the species Tropaeolum pentaphyllum, when compared with the sweet potatoes leaves (Ipomoea batatas L.). In addition, the antioxidant activities of leaves and flowers were also higher measured by ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid), DPPH (2,2-diphenyl-1-picrylhydrazyl), and TRAP (total radical-trapping antioxidant potential) methods. Tropaeolum pentaphyllum have high nutritional potential that can be exploited to improve nutritional value of various food products.

Targeted inhibition of RAGE in substantia nigra of rats blocks 6-OHDA-induced dopaminergic denervation.

The receptor for advanced glycation endproducts (RAGE) is a pattern-recognition receptor associated with inflammation in most cell types. RAGE up-regulates the expression of proinflammatory mediators and its own expression via activation of NF-kB. Recent works have proposed a role for RAGE in Parkinson's disease (PD). In this study, we used the multimodal blocker of RAGE FPS-ZM1, which has become available recently, to selectively inhibit RAGE in the substantia nigra (SN) of rats intracranially injected with 6-hydroxydopamine (6-OHDA). FPS-ZM1 (40 µg per rat), injected concomitantly with 6-OHDA (10 µg per rat) into the SN, inhibited the increase in RAGE, activation of ERK1/2, Src and nuclear translocation of NF-kB p65 subunit in the SN. RAGE inhibition blocked glial fibrillary acidic protein and Iba-1 upregulation as well as associated astrocyte and microglia activation. Circulating cytokines in serum and CSF were also decreased by FPS-ZM1 injection. The loss of tyrosine hydroxylase and NeuN-positive neurons was significantly inhibited by RAGE blocking. Finally, FPS-ZM1 attenuated locomotory and exploratory deficits induced by 6-OHDA. Our results demonstrate that RAGE is an essential component in the neuroinflammation and dopaminergic denervation induced by 6-OHDA in the SN. Selective inhibition of RAGE may offer perspectives for therapeutic approaches.

Role of vitamin A oral supplementation on oxidative stress and inflammatory response in the liver of trained rats.

The use of dietary supplements to enhance the benefit of exercise training is a common practice. The liver is the organ where all substances are metabolized, and certain supplements have been associated with liver injury. Vitamin A (VA), a liposoluble vitamin stored in the liver, is commonly used as an antioxidant supplement. Here, we evaluated the effect of chronic VA supplementation on oxidative damage and stress parameters in trained rats. Animals were divided into the following groups: sedentary (SE), sedentary/VA (SE+VA), exercise training (ET), and exercise training/VA (ET+VA). During 8 weeks, animals were subjected to swimming (0%, 2%, 4%, 6% body weight) for 5 days/week and a VA daily intake of 450 retinol equivalents/day. Parameters were evaluated by enzymatic activity analysis, ELISA, and Western blotting. VA caused liver lipid peroxidation and protein damage in exercised rats and inhibited the increase in HSP70 expression acquired with exercise alone. The ET group showed higher levels of antioxidant enzyme activity, and VA inhibited this adaptation. Expression of the pro-inflammatory cytokines, interleukin (IL)-1ß, and tumor necrosis factor-α was reduced in the ET+VA group, while the anti-inflammatory cytokine, IL-10, was increased. Western blotting showed that both exercised groups had lower levels of the receptor for advanced glycation end products, suggesting that VA did not affect this receptor. Our study demonstrated that, although VA caused oxidative damage, a controlled administration might exert anti-inflammatory effects. Further studies with higher VA doses and longer ET interventions would elucidate more the effects of the supplementation and exercise on liver parameters.

Vitamin A Oral Supplementation Induces Oxidative Stress and Suppresses IL-10 and HSP70 in Skeletal Muscle of Trained Rats.

Exercise training intensity is the major variant that influences the relationship between exercise, redox balance, and immune response. Supplement intake is a common practice for oxidative stress prevention; the effects of vitamin A (VA) on exercise training are not yet described, even though this molecule exhibits antioxidant properties. We investigated the role of VA supplementation on redox and immune responses of adult Wistar rats subjected to swimming training. Animals were divided into four groups: sedentary, sedentary + VA, exercise training, and exercise training + VA. Over eight weeks, animals were submitted to intense swimming 5 times/week and a VA daily intake of 450 retinol equivalents/day. VA impaired the total serum antioxidant capacity acquired by exercise, with no change in interleukin-1ß and tumor necrosis factor-α levels. In skeletal muscle, VA caused lipid peroxidation and protein damage without differences in antioxidant enzyme activities; however, Western blot analysis showed that expression of superoxide dismutase-1 was downregulated, and upregulation of superoxide dismutase-2 induced by exercise was blunted by VA. Furthermore, VA supplementation decreased anti-inflammatory interleukin-10 and heat shock protein 70 expression, important factors for positive exercise adaptations and tissue damage prevention. Our data showed that VA supplementation did not confer any antioxidative and/or protective effects, attenuating exercise-acquired benefits in the skeletal muscle.

Sperm quality and oxidative status as affected by homogenization of liquid-stored boar semen diluted in short- and long-term extenders.

Homogenization of diluted boar semen during storage has for a long time been regarded as beneficial. Recent studies indicated an adverse effect of homogenization on sperm quality for yet unknown reasons. This study aimed to verify the effect of homogenization on sperm parameters and to elucidate the impact of oxidative stress. Twenty-one normospermic ejaculates (21 boars) were diluted with Androstar® Plus (AND) and Beltsville Thawing Solution (BTS). Semen doses were submitted to no-homogenization (NoHom) or twice-a-day manual homogenization (2xHom) during storage at 17°C for 168h. NoHom and 2xHom were similar (P>0.05) for both short- and long-term extenders with respect to motility and kinematics parameters (CASA system), membrane viability (SYBR-14/PI), acrosome integrity, lipid peroxidation, protein oxidation, intracellular reactive oxygen species, sulfhydryl content, and total radical-trapping antioxidant potential. 2xHom reduced sperm motility and motion kinematics (VCL, VSL, VAP, BCF, and ALH) following the thermoresistance test and presented with a slight increase in pH along the storage (P=0.05) as compared to NoHom. Furthermore, 2xHom semen doses presented with a constant SOD and GSH-Px activity during storage whereas enzymatic activity increased for NoHom at the end of the storage. These findings confirm that homogenization of semen doses is detrimental to sperm quality. Moreover, it is shown that the effect of homogenization is unlikely to be primarily related to oxidative stress. Homogenization is not recommended for storage of liquid boar semen for up to 168h in both short- and long-term extenders.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis.

BACKGROUND: Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES: To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS: Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS: In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1ß, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS: L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

N-acetyl-cysteine inhibits liver oxidative stress markers in BALB/c mice infected with Leishmania amazonensis

BACKGROUND Leishmaniasis is a parasitosis caused by several species of the genus Leishmania. These parasites present high resistance against oxidative stress generated by inflammatory cells. OBJECTIVES To investigate oxidative stress and molecular inflammatory markers in BALB/c mice infected with L. amazonensis and the effect of antioxidant treatment on these parameters. METHODS Four months after infection, oxidative and inflammatory parameters of liver, kidneys, spleen, heart and lungs from BALB/c mice were assessed. FINDINGS In liver, L. amazonensis caused thiol oxidation and nitrotyrosine formation; SOD activity and SOD2 protein content were increased while SOD1 protein content decreased. The content of the cytokines IL-1β, IL-6, TNF-α, and the receptor of advanced glycation endproducts (RAGE) increased in liver. Treatment with the antioxidant N-acetyl-cysteine (20 mg/kg b.w) for five days inhibited oxidative stress parameters. MAIN CONCLUSIONS L. amazonensis induces significant alterations in the redox status of liver but not in other organs. Acute antioxidant treatment alleviates oxidative stress in liver, but it had no effect on pro-inflammatory markers. These results indicate that the pathobiology of leishmaniasis is not restricted to the cutaneous manifestations and open perspectives for the development of new therapeutic approaches to the disease, especially for liver function.

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.

Effects of Freeze-Thaw and Storage on Enzymatic Activities, Protein Oxidative Damage, and Immunocontent of the Blood, Liver, and Brain of Rats.

Most scientific studies are too long to be conducted in a single day or even in a few days. Thus, there is a need to store samples for subsequent investigations. There is sparse information about specific sample storage protocols that minimize analytical error and variability in evaluations of redox parameters. Therefore, the effects of storage temperature and freezing time on enzymatic activities, protein oxidative damage, and CAT (catalase) and SOD1 (superoxide dismutase) immunocontent of blood, liver, and brain from rats were determined for two different sample forms (frozen homogenized tissue or frozen intact tissue). Superoxide dismutase activity was drastically decreased in blood and liver with an increase in freezing time, but not in brain. Catalase activity showed a decrease only in intact liver at -20 and -80°C. In contrast, in blood it showed an increase in intact tissue at -20 and -80°C. Reduced thiol groups generally decreased with freezing time, but showed an increase in intact blood at -20 and -80°C, probably because of color interference. Carbonyl groups in homogenized liver and brain, and in intact blood (except at 80°C) drastically increased with freezing time. Freezing time did not modulate the immunocontent of CAT and SOD1 levels in any tissue. In conclusion, our results indicate that storage at -20°C affects redox parameters more than storage at -80°C. Storage for a long time may compromise the samples, leading to changing parameters due to oxidative stress. Thus, we suggest processing the samples as soon as possible. However, if this is not possible, then material can be aliquoted into different tubes to prevent the effect of refreezing of samples.

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.