João Paulo Almeida dos Santos

Redox characterization of usnic acid and its cytotoxic effect on human neuron-like cells (SH-SY5Y)

Usnic acid (UA) is the most common and abundant lichenic secondary metabolite with potential therapeutic application. Anti-inflammatory and antitumour properties have already been reported and UA-enriched extracts are widely used to treat several diseases in the folk medicine. First, we performed in silico evaluation of UA interactions with genes/proteins and important compounds for cellular redox balance and NO pathway. Then, we assessed UA redox properties against different reactive species (RS) generated in vitro, and evaluated its action on SH-SY5Y neuronal like cells upon hydrogen peroxide (H(2)O(2)), since no in vitro neurotoxicological data has been reported so far. Total reactive antioxidant potential index (TRAP) showed a significant antioxidant capacity of UA at the highest tested concentration; UA was also effective against hydroxyl radicals and reduced the formation of nitric oxide. In vitro, lipoperoxidation was enhanced by UA and changed the cellular viability at highest concentration of 20μg/mL for 1 and 4h, as well as 2 and 20μg/mL for 24h of treatment, according to MTT reduction assay. Moreover, UA did not display protective effects against H(2)O(2)-induced cell death in any case. Evaluation of intracellular RS production by the DCFH-based assay indicated that UA was able to induce changes in basal RS production at concentration of 20μg/mL for 1h and from 2ng/mL to 20μg/mL for 4 and 24h. In conclusion, UA could display variable redox-active properties, according to different system conditions and/or cellular environment. Moreover, our results suggest that potential neurotoxicological effects of UA should be further studied by additional approaches; for instance, in vivo and clinical studies.

Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation endproduct (RAGE) and tau levels in multiple organs in mice.

Schistosomiasis is a parasitic disease caused by trematode worms from the Schistosoma genus and is characterized by high rates of morbidity. The main organs affected in this pathology, such as liver, kidneys and spleen, are shifted to a pro-oxidant state in the course of the infection. Here, we compared oxidative stress parameters of liver, kidney and spleen with other organs affected by schistosomiasis - heart, brain cortex and lungs. The results demonstrated that mice infected with Schistosoma mansoni had altered non-enzymatic antioxidant status in lungs and brain, increased carbonyl levels in lungs, and a moderate level of oxidative stress in heart. A severe redox imbalance in liver and kidneys and decreased non-enzymatic antioxidant capacity in spleen were also observed. Superoxide dismutase and catalase activities were differently modulated in liver, kidney and heart, and we found that differences in Superoxide dismutase 2 and catalase protein content may be responsible for these differences. Lungs had decreased receptor for advanced glycation endproduct expression and the brain cortex presented altered tau expression and phosphorylation levels, suggesting important molecular changes in these tissues, as homeostasis of these proteins is widely associated with the normal function of their respective organs. We believe that these results demonstrate for the first time that changes in the redox profile and expression of tissue-specific proteins of organs such as heart, lungs and brain are observed in early stages of S. mansoni infection.

Acute exercise induce endothelial nitric oxide synthase phosphorylation via Akt and AMP-activated protein kinase in aorta of rats: Role of reactive oxygen species

BACKGROUND Acute exercise increases reactive oxygen species (ROS) levels, including hydrogen peroxide (H2O2). H2O2 promotes endothelial nitric oxide synthase (eNOS) activation and phosphorylation in endothelial cells. With this in mind, the present study was designed to evaluate ex vivo eNOS phosphorylation in rat aortas incubated with H2O2 and to test this hypothesis in vivo in the aortas of rats submitted to acute exercise. METHODS For ex vivo studies, six groups of aortic tissue were formed: control, H2O2, N-acetylcysteine (NAC), LY294002, compound C, and LY294002 plus compound C. While incubation with H2O2 increased Akt, AMPK and eNOS phosphorylation, pre-incubation with NAC strongly reduced the phosphorylation of these enzymes. For in vivo studies, male Wistar rats were divided into four groups: control, cont+NAC, exercise, and exer+NAC. After a 3h swimming session, animals were decapitated and aortas were excised for biochemical and immunoblotting analysis. RESULTS Acute exercise increased superoxide levels and dichlorofluorescein (DCF) concentrations, and this increase was related to phosphorylation of Akt, AMPK and eNOS. On the other hand, use of NAC reduced superoxide levels and DCF concentration. Reduced superoxide levels and DCF in the exer+NAC group were associated with decreased Akt, AMPK and eNOS phosphorylation. These results appear to be connected with vascular function because VASP phosphorylation increased in acute exercise and decreased in exer+NAC. CONCLUSION Our results indicate that ROS induced by acute exercise play the important role of activating eNOS, a process apparently mediated by Akt and AMPK.

Intracerebroventricular administration of okadaic acid induces hippocampal glucose uptake dysfunction and tau phosphorylation.

Intraneuronal aggregates of neurofibrillary tangles (NFTs), together with beta-amyloid plaques and astrogliosis, are histological markers of Alzheimers disease (AD). The underlying mechanism of sporadic AD remains poorly understood, but abnormal hyperphosphorylation of tau protein is suggested to have a role in NFTs genesis, which leads to neuronal dysfunction and death. Okadaic acid (OKA), a strong inhibitor of protein phosphatase 2A, has been used to induce dementia similar to AD in rats. We herein investigated the effect of intracerebroventricular (ICV) infusion of OKA (100 and 200ng) on hippocampal tau phosphorylation at Ser396, which is considered an important fibrillogenic tau protein site, and on glucose uptake, which is reduced early in AD. ICV infusion of OKA (at 200ng) induced a spatial cognitive deficit, hippocampal astrogliosis (based on GFAP increment) and increase in tau phosphorylation at site 396 in this model. Moreover, we observed a decreased glucose uptake in the hippocampal slices of OKA-treated rats. In vitro exposure of hippocampal slices to OKA altered tau phosphorylation at site 396, without any associated change in glucose uptake activity. Taken together, these findings further our understanding of OKA neurotoxicity, in vivo and vitro, particularly with regard to the role of tau phosphorylation, and reinforce the importance of the OKA dementia model for studying the neurochemical alterations that may occur in AD, such as NFTs and glucose hypometabolism.

Phytochemical screening, antinociceptive and anti-inflammatory activities of Chrysopogon zizanioides essential oil

Chrysopogon zizanioides (L.) Roberty, Poaceae, is a plant widely used in northeast Brazil in folk medicine for the treatment of various pathological conditions, including inflammatory pain. The present study evaluated the antinociceptive and anti-inflammatory effects of C. zizanioides essential oil (EO) in rodents. EO was further characterized by GC/MS. The major components of EO were identified as khusimol (19.57%), E-isovalencenol (13.24%), α-vetivone (5.25%), β-vetivone (4.87%) and hydroxy-valencene (4.64%). Following intraperitoneal injection (i.p.), EO at 50 and 100 mg/kg significantly reduced the number of writhes (51.9 and 64.9%, respectively) and the number of paw licks during phase 2 (56.7 and 86.2%, respectively) of a formalin model when compared to control group animals. However, EO-treated mice were ineffective at all doses in hot-plate and rota-rod tests. The EO inhibited the carrageenan-induced leukocyte migration to the peritoneal cavity in a dose-dependent manner (34.7, 35.4, and 62.5% at doses of 25, 50 and 100 mg/kg, respectively). In the paw edema test, the EO (100 mg/kg) inhibited all three phases of the edema equally well, suggesting that the EO has a non-selective inhibitory effect on the release or actions of these mediators. Our results suggest possible antinociceptive and anti-inflammatory effects of the EO.

Apoptotic signaling pathways induced by acute administration of branched-chain amino acids in an animal model of maple syrup urine disease

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of the branched-chain α-keto acid dehydrogenase complex activity. This blockage leads to accumulation of the branched-chain amino acids leucine, isoleucine and valine, as well as their corresponding α-keto acids and α-hydroxy acids. The affected patients present severe neurological symptoms, such as coma and seizures, as well as edema and cerebral atrophy. Considering that the mechanisms of the neurological symptoms presented by MSUD patients are still poorly understood, in this study, protein levels of apoptotic factors are measured, such as Bcl-2, Bcl-xL, Bax, caspase-3 and −8 in hippocampus and cerebral cortex of rats submitted to acute administration of branched-chain amino acids during their development. The results in this study demonstrated that BCAA acute exposure during the early postnatal period did not significantly change Bcl-2, Bcl-xL, Bax and caspase-8 protein levels. However, the Bax/Bcl-2 ratio and procaspase-3 protein levels were decreased in hippocampus. On the other hand, acute administration of BCAA in 30-day-old rats increase in Bax/Bcl-2 ratio followed by an increased caspase-3 activity in cerebral cortex, whereas BCAA induces apoptosis in hippocampus through activation and cleavage of caspase-3 and −8 without changing the Bax/Bcl-2 ratio. In conclusion, the results suggest that apoptosis could be of pivotal importance in the developmental neurotoxic effects of BCAA. In addition, the current studies also suggest that multiple mechanisms may be involved in BCAA-induced apoptosis in the cerebral cortex and hippocampus.

Hippocampal changes in STZ-model of Alzheimer's disease are dependent on sex.

HighlightsAlzheimer’s disease (AD) has prevalence dependent on gender.We investigated alterations dependent on sex in a streptozotocin model of AD in rats.Hippocampal parameters were investigated 2, 4 and 8 weeks after streptozotocin.Cholinergic neurons and glucose uptake decrease were dependent on sex.S100B alteration in the hippocampus were also dependent on sex, but not GFAP. ABSTRACT The majority of Alzheimer’s disease (AD) cases are sporadic and aging is the major risk factor for developing the disease, affecting more women than men. In spite of different gender prevalence, most experimental studies in animal models have been performed in male. This study investigates the streptozotocin (STZ)‐induced AD model at three different times (2, 4 and 8 weeks afterwards) and in male and female rats, evaluating cognitive deficit, cholinergic neurotransmission, glucose uptake, glutathione content and specific glial markers (GFAP and S100B protein) in the hippocampus of the rat. Our data reinforce the relevance of alterations in STZ model of dementia, reported in the genesis and/or progression of AD such as cholinergic deficit and glucose uptake decrease. All alterations in these parameters (except GFAP) were dependent on sex. It is unclear, at this moment, which alterations are due to sex steroid modulation. In spite of limitations of this experimental model, these data may contribute to understand AD susceptibility and progression dependent on sex.