Alice Kunzler

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.

Preventive supplementation with fresh and preserved peach attenuates CCl4-induced oxidative stress, inflammation and tissue damage

The present study was elaborated to comparatively evaluate the preventive effect of different peach-derived products obtained from preserved fruits (Syrup and Preserve Pulp Peach [PPP]) and from fresh peels and pulps (Peel and Fresh Pulp Peach [FPP]) in a model of liver/renal toxicity and inflammation induced by carbon tetrachloride (CCl4) in rats. Tissue damage (carbonyl, thiobarbituric acid reactive species and sulfhydril), antioxidant enzymes activity (catalase and superoxide dismutase) and inflammatory parameters [tumor necrosis factor (TNF)-α and interleukin (IL)-1β levels, and receptor for advanced glycation end-products (RAGE) and nuclear factor (NF)κB-p65 immunocontent] were investigated. Our findings demonstrated that Peel, PPP and FPP (200 or 400 mg/kg) daily administration by oral gavage for 30 days conferred a significant protection against CCl4-induced antioxidant enzymes activation and, most importantly, oxidative damage to lipids and proteins as well as blocked induction of inflammatory mediators such as TNF-α, IL-1β, RAGE and NFκB. This antioxidant/anti-inflammatory effect seems to be associated with the abundance of carotenoids and polyphenols present in peach-derived products, which are enriched in fresh-fruit-derived preparations (Peel and FPP) but are also present in PPP. The Syrup - which was the least enriched in antioxidants - displayed no protective effect in our experiments. These effects cumulated in decreased levels of transaminases and lactate dehydrogenase leakage into serum and maintenance of organ architecture. Therefore, the herein presented results show evidence that supplementation with peach products may be protective against organ damage caused by oxidative stress, being interesting candidates for production of antioxidant-enriched functional foods.

Increased tau phosphorylation and receptor for advanced glycation endproducts (RAGE) in the brain of mice infected with Leishmania amazonensis

Leishmaniasis is a parasitosis caused by several species of the genus Leishmania, an obligate intramacrophagic parasite. Although neurologic symptoms have been observed in human cases of leishmaniasis, the manifestation of neurodegenerative processes is poorly studied. The aim of the present work was to investigate if peripheral infection of BALB/c mice with Leishmania amazonensis affects tau phosphorylation and RAGE protein content in the brain, which represent biochemical markers of neurodegenerative processes observed in diseases with a pro-inflammatory component, including Alzheimers disease and Down syndrome. Four months after a single right hind footpad subcutaneous injection of L. amazonensis, the brain cortex of BALB/c mice was isolated. Western blot analysis indicated an increase in tau phosphorylation (Ser(396)) and RAGE immunocontent in infected animals. Brain tissue TNF-α, IL-1β, and IL-6 levels were not different from control animals; however, increased protein carbonylation, decreased IFN-γ levels and impairment in antioxidant defenses were detected. Systemic antioxidant treatment (NAC 20mg/kg, i.p.) inhibited tau phosphorylation and recovered IFN-γ levels. These data, altogether, indicate an association between impaired redox state, tau phosphorylation and RAGE up-regulation in the brain cortex of animals infected with L. amazonensis. In this context, it is possible that neurologic symptoms associated to chronic leishmaniasis are associated to disruptions in the homeostasis of CNS proteins, such as tau and RAGE, as consequence of oxidative stress. This is the first demonstration of alterations in biochemical parameters of neurodegeneration in an experimental model of Leishmania infection.

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 β -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.

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.

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-1β 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.

Effects of methylmercury and retinol palmitate co-administration in rats during pregnancy and breastfeeding: Metabolic and redox parameters in dams and their offspring

Ubiquitous low-dose methylmercury (MeHg) exposure through an increased fish consumption represents a global public health problem, especially among pregnant women. A plethora of micronutrients presented in fish affects MeHg uptake/distribution, but limited data is available. Vitamin A (VitA), another fish micronutrient is used in nutritional supplementation, especially during pregnancy. However, there is no information about the health effects arising from their combined exposure. Therefore, the present study aimed to examine the effects of both MeHg and retinyl palmitate administered on pregnant and lactating rats in metabolic and redox parameters from dams and their offspring. Thirty Wistar female rats were orally supplemented with MeHg (0,5 mg/kg/day) and retinyl palmitate (7500 µg RAE/kg/day) via gavage, either individually or in combination from the gestational day 0 to weaning. For dams (150 days old) and their offspring (31 days old), glycogen accumulation (hepatic and cardiac) and retinoid contents (plasma and liver) were analyzed. Hg deposition in liver tissue was quantified. Redox parameters (liver, kidney, and heart) were evaluated for both animals. Cytogenetic damage was analyzed with micronucleus test. Our results showed no general toxic or metabolic alterations in dams and their offspring by MeHg-VitA co-administration during pregnancy and lactation. However, increased lipoperoxidation in maternal liver and a disrupted pro-oxidant response in the heart of male pups was encountered, with apparently no particular effects in the antioxidant response in female offspring. GST activity in dam kidney was altered leading to possible redox disruption of this tissue with no alterations in offspring. Finally, the genomic damage was exacerbated in both male and female pups. In conclusion, low-dose MeHg exposure and retinyl palmitate supplementation during gestation and lactation produced a potentiated pro-oxidant effect, which was tissue-specific. Although this is a pre-clinical approach, we recommend precaution for pregnant women regarding food consumption, and we encourage more epidemiological studies to assess possible modulations effects of MeHg-VitA co-administration at safe or inadvertently used doses in humans, which may be related to specific pathologies in mothers and their children.

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.