Leila Zanatta

Flavonoids: Prospective Drug Candidates

The purpose of this review is to discuss the recent developments related to the chemistry and medicinal properties of flavonoids. Major flavonoids that show well categorized structures and well defined structure function-relationships are: flavans, flavanones, flavones, flavanonols, flavonols, catechins, anthocyanidins and isoflavone. The biological properties of flavonoids include antioxidant, anti-inflamatory, antitumoral, antiviral and antibacterial, as well as a direct cytoprotective effect on coronary and vascular systems, the pancreas and the liver. These characteristics place them among the most attractive natural substances available to enrich the current therapy options.

Flavonoids: Cellular and Molecular Mechanism of Action in Glucose Homeostasis

The purpose of this review is to discuss the cellular and molecular mechanisms of action of flavonoids focusing on carbohydrate metabolism. The beneficial effects of flavonoids have been studied in relation to diabetes mellitus, either through their capacity to avoid glucose absorption or to improve glucose tolerance. Furthermore, flavonoids stimulate glucose uptake in peripheral tissues, regulate the activity and/or expression of the rate-limiting enzymes in the carbohydrate metabolism pathway and act per se as insulin secretagogues or insulin mimetics, probably, by influencing the pleiotropic mechanisms of insulin signaling, to ameliorate the diabetes status.

Age- and cell-related gene expression of aromatase and estrogen receptors in the rat testis.

Spermatogenesis is a complex and coordinated process leading to the formation of spermatozoa. This event, which is under the control of numerous endocrine and paracrine factors, seems to also be controlled by estrogens which exert their effects via nuclear estrogen receptors (ESRs) ESR1 and ESR2. Estrogens are synthesized by aromatase which is biologically expressed in the rat testis. The objective of our study was to clarify the gene expression patterns of aromatase and ESRs according to age and in the two compartments of the adult rat testis. In the adult, transcripts of aromatase vary according to the germ cell type and to the stages of seminiferous epithelium, a maximum being observed at stage I. The ESR1 gene is highly expressed in the adult testis and in stages from VIIc-d to XIV. Moreover, both ESR mRNA levels are higher in purified round spermatids than in pachytene spermatocytes, suggesting a putative role of estrogens in the haploid steps of spermatogenesis. The variability of the results in the expression of both ESRs led us to explore the putative presence of variants in the rat testis. Concerning ESR1, we have shown the presence of the full-length form and of one isoform with exon 4 deleted. For ESR2, besides the wild type, three isoforms were observed: one with exon 3 deleted, another with an insertion of 54 nucleotides, and the last one with both modifications. Therefore, the stage-regulated expression of aromatase and ESR1 genes in the rat testis suggests a likely role of estrogens in spermatogenesis.

Rutin potentiates calcium uptake via voltage-dependent calcium channel associated with stimulation of glucose uptake in skeletal muscle.

Rutin is a flavonoid with several pharmacological properties and it has been demonstrated that rutin can modulate glucose homeostasis. In skeletal muscle, an increase in intracellular calcium concentration may induce glucose transporter-4 (GLUT-4) translocation with consequent glucose uptake. The aim of this study was to investigate the effect of rutin and intracellular pathways on calcium uptake as well as the involvement of calcium in glucose uptake in skeletal muscle. The results show that rutin significantly stimulated calcium uptake through voltage-dependent calcium channels as well as mitogen-activated kinase (MEK) and protein kinase A (PKA) signaling pathways. Also, rutin stimulated glucose uptake in the soleus muscle and this effect was mediated by extracellular calcium and calcium-calmodulin-dependent protein kinase II (CaMKII) activation. In conclusion, rutin significantly stimulates calcium uptake in rat soleus muscles. Furthermore, the increase in intracellular calcium concentration is involved in DNA activation by rutin. Also, rutin-induced glucose uptake via CaMKII may result in GLUT-4 translocation to the plasma membrane, characterizing an insulin-independent pathway. These findings indicate that rutin is a potential drug candidate for diabetes therapy.

Prevention of unpredictable chronic stress-related phenomena in zebrafish exposed to bromazepam, fluoxetine and nortriptyline

RationaleSeveral model organisms have been employed to study the impacts of stress on biological systems. Different models of unpredictable chronic stress (UCS) have been established in rodents; however, these protocols are expensive, long-lasting, and require a large physical structure. Our group has recently reported an UCS protocol in zebrafish with several advantages compared to rodent models. We observed that UCS induced behavioral, biochemical, and molecular changes similar to those observed in depressed patients, supporting the translational relevance of the protocol.ObjectivesConsidering that a pharmacological assessment is lacking in this zebrafish model, our aim was to evaluate the effects of anxiolytic (bromazepam) and antidepressant drugs (fluoxetine and nortriptyline) on behavioral (novel tank test), biochemical (whole-body cortisol), and molecular parameters (cox-2, tnf-α, il-6, and il-10 gene expression) in zebrafish subjected to UCS.ResultsWe replicated previous data showing that UCS induces behavioral and neuroendocrine alterations in zebrafish, and we show for the first time that anxiolytic and antidepressant drugs are able to prevent such effects. Furthermore, we extended the molecular characterization of the model, revealing that UCS increases expression of the pro-inflammatory markers cox-2 and il-6, which was also prevented by the drugs tested.ConclusionsThis study reinforces the use of zebrafish as a model organism to study the behavioral and physiological effects of stress. The UCS protocol may also serve as a screening tool for evaluating new drugs that can be used to treat psychiatric disorders with stress-related etiologies.

Subchronic atrazine exposure changes defensive behaviour profile and disrupts brain acetylcholinesterase activity of zebrafish

Animal behaviour is the interaction between environment and an individual organism, which also can be influenced by its neighbours. Variations in environmental conditions, as those caused by contaminants, may lead to neurochemical impairments altering the pattern of the behavioural repertoire of the species. Atrazine (ATZ) is an herbicide widely used in agriculture that is frequently detected in surface water, affecting non-target species. The zebrafish is a valuable model organism to assess behavioural and neurochemical effects of different contaminants since it presents a robust behavioural repertoire and also all major neurotransmitter systems described for mammalian species. The goal of this study was to evaluate the effects of subchronic ATZ exposure in defensive behaviours of zebrafish (shoaling, thigmotaxis, and depth preference) using the split depth tank. Furthermore, to investigate a putative role of cholinergic signalling on ATZ-mediated effects, we tested whether this herbicide alters acetylcholinesterase (AChE) activity in brain and muscle preparations. Fish were exposed to ATZ for 14days and the following groups were tested: control (0.2% acetone) and ATZ (10 and 1000μg/L). The behaviour of four animals in the same tank was recorded for 6min and biological samples were prepared. Our results showed that 1000μg/L ATZ significantly increased the inter-fish distance, as well as the nearest and farthest neighbour distances. This group also presented an increase in the shoal area with decreased social interaction. No significant differences were detected for the number of animals in the shallow area, latency to enter the shallow and time spent in shallow and deep areas of the apparatus, but the ATZ 1000 group spent significantly more time near the walls. Although ATZ did not affect muscular AChE, it significantly reduced AChE activity in brain. Exposure to 10μg/L ATZ did not affect behaviour or AChE activity. These data suggest that ATZ impairs defensive behaviours of zebrafish, which could be related to its action on brain cholinergic neurotransmission. Moreover, the use of the split depth tank could be an alternative strategy to assess group behaviour and depth preference after exposure to chemical compounds.

N-acetylcysteine prevents stress-induced anxiety behavior in zebrafish

Despite the recent advances in understanding the pathophysiology of anxiety disorders, the pharmacological treatments currently available are limited in efficacy and induce serious side effects. A possible strategy to achieve clinical benefits is drug repurposing, i.e., discovery of novel applications for old drugs, bringing new treatment options to the market and to the patients who need them. N-acetylcysteine (NAC), a commonly used mucolytic and paracetamol antidote, has emerged as a promising molecule for the treatment of several neuropsychiatric disorders. The mechanism of action of this drug is complex, and involves modulation of antioxidant, inflammatory, neurotrophic and glutamate pathways. Here we evaluated the effects of NAC on behavioral parameters relevant to anxiety in zebrafish. NAC did not alter behavioral parameters in the novel tank test, prevented the anxiety-like behaviors induced by an acute stressor (net chasing), and increased the time zebrafish spent in the lit side in the light/dark test. These data may indicate that NAC presents an anti-stress effect, with the potential to prevent stress-induced psychiatric disorders such as anxiety and depression. The considerable homology between mammalian and zebrafish genomes invests the current data with translational validity for the further clinical trials needed to substantiate the use of NAC in anxiety disorders.

1α,25-Dihydroxyvitamin D 3 Signaling Pathways on Calcium Uptake in 30-Day-Old Rat Sertoli Cells

1α,25-Dihydroxyvitamin D(3) (1,25D(3)) is the active metabolite of vitamin D(3) and the major calcium regulatory hormone in tissues. The aim of this work was to investigate the mechanism of action of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells from 30-day-old rats. Results showed that 10(-9) and 10(-12) M 1,25D(3) increased the rate of (45)Ca(2+) uptake 5 and 15 min after hormone exposure and that 1α,25(OH)(2) lumisterol(3) (JN) produced a similar effect suggesting that 1,25D(3) action occurs via a putative membrane receptor. The involvement of voltage-dependent calcium channels (VDCC) in 1,25D(3) action was evidenced by using nifedipine, while the use of Bapta-AM demonstrated that intracellular calcium was not implicated. Moreover, the incubation with ouabain and digoxin increased the rate of (45)Ca(2+) uptake, indicating that the effect of 1,25D(3) may also result from Na(+)/K(+)-ATPase inhibition. In addition, we demonstrated that the mechanism underlying the hormone action involved extracellular signal-regulated kinase (ERK) and protein kinase C (PKC) activation in a phospholipase C-independent way. Furthermore, a local elevation of the level of cAMP, as demonstrated by incubating cells with dibutyryl cAMP or a phosphodiesterase inhibitor, produced an effect similar to that of 1,25D(3), and the inhibition of protein kinase A (PKA) nullified the hormone action. In conclusion, the stimulatory effect of 1,25D(3) on (45)Ca(2+) uptake in Sertoli cells occurs via VDCC, as well as PKA, PKC, and ERK activation. These protein kinases seem to act by inhibiting Na(+)/K(+)-ATPase or directly phosphorylating calcium channels. The Na(+)/K(+)-ATPase inhibition may result in Na(+)/Ca(2+) exchanger activation in reverse mode and consequently induce the uptake of calcium into the cells.

Acute effect of β-sitosterol on calcium uptake mediates anti-inflammatory effect in murine activated neutrophils.

Objectives  To evaluate the effect of β‐sitosterol on 45Ca2+ uptake in activated murine neutrophils, and upon myeloperoxidase and adenosine deaminase activity, and interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) levels, in carrageenan‐induced inflammation in the mouse air pouch model.

Rapid Responses to Reverse T3 Hormone in Immature Rat Sertoli Cells: Calcium Uptake and Exocytosis Mediated by Integrin

There is increasing experimental evidence of the nongenomic action of thyroid hormones mediated by receptors located in the plasma membrane or inside cells. The aim of this work was to characterize the reverse T3 (rT3) action on calcium uptake and its involvement in immature rat Sertoli cell secretion. The results presented herein show that very low concentrations of rT3 are able to increase calcium uptake after 1 min of exposure. The implication of T-type voltage-dependent calcium channels and chloride channels in the effect of rT3 was evidenced using flunarizine and 9-anthracene, respectively. Also, the rT3-induced calcium uptake was blocked in the presence of the RGD peptide (an inhibitor of integrin-ligand interactions). Therefore, our findings suggest that calcium uptake stimulated by rT3 may be mediated by integrin αvβ3. In addition, it was demonstrated that calcium uptake stimulated by rT3 is PKC and ERK-dependent. Furthermore, the outcomes indicate that rT3 also stimulates cellular secretion since the cells manifested a loss of fluorescence after 4 min incubation, indicating an exocytic quinacrine release that seems to be mediated by the integrin receptor. These findings indicate that rT3 modulates the calcium entry and cellular secretion, which might play a role in the regulation of a plethora of intracellular processes involved in male reproductive physiology.