Influence of seasonality and habitat on chemical composition, cytotoxicity and antimicrobial properties of the Libidibia ferrea.

Libidibia ferrea Mart, belonging to the Fabacee family, is a medicinal plant known for its biological properties and production of phenolic compounds. Previous studies reveal the biological activity of its phenolic constituents, making it very promising for the development of new medicines. Seasonality and geographic distribution of species can modify the production of secondary metabolites in Fabaceae species in terms of the preferentially activated metabolic pathways and, consequently, interfere with the medicinal properties of these species. Studying the influence of seasonality on the production of phenolic constituents is essential to establish conditions for "cultivation," species collection, standardization, production, and safety in traditional medicine. This unprecedented study proposed to evaluate the influence of seasonal variations and habitat on the production of phenolic compounds and biological properties of the ethanolic extracts of the stem bark from L. ferrea, whose specimens were collected from the Caatinga and the Atlantic Forest, biomes of Brazil. Antimicrobial activity was determined by broth microdilution. Cytotoxicity was evaluated through a colorimetric assay using MTT. ABTS and DPPH radical reduction methods estimated antioxidant capacities. Folin-Ciocalteu and AlCl3 spectrophotometric methods quantified total phenolics and flavonoids, respectively. In turn, radial diffusion quantified tannin content. PCA score plot and HCA dendogram were obtained by multivariate analysis of 1H NMR data. The cytotoxicity against C6 glioma cells was observed only for Atlantic Forest extracts (EC50 = 0.13-0.5 mg mL-1). These extracts also showed selectivity against Gram-positive bacteria Bacillus subtilis (ATCC 6633) [MICs 500-2000 µg mL-1], B. cereus CCT 0096) [MIC = 250 µg mL-1], Staphylococcus aureus (ATCC 6538) [MICs = 250-500 µg mL-1], S. epidermidis (ATCC 12228) [62.5-1000 µg mL-1], mainly to Staphylococcus sp. Caatinga extracts showed higher production of flavonoids and antioxidants in the summer [7.36 ± 0.19 µg QE mg-1 extract; IC50ABTS = 4.86 ± 0.05 µg mL-1], spring [5.96 ± 0.10 µg QE mg-1 extract; IC50ABTS = 5.96 ± 0.08 µg mL-1 ], winter [4.89 ± 0.25 µg QE mg-1 extract; IC50ABTS = 6.72 ± 0.08 µg mL-1 ]. Regarding habitat, two discriminating compound patterns in the studied biomes were revealed by NMR. The results indicated that the Caatinga biome offers better conditions for activating the production of phenolics [336.34 ± 18.1 µgGAE mg-1 extract], tannins [328.38 ± 30.19 µgTAE mg-1 extract] in the summer and flavonoids in winter, spring, and summer. The extracts that showed the best antioxidant activities were also those from the Caatinga. In turn, extracts from the Atlantic Forest are more promising for discovering antibacterial compounds against Staphylococcus sp and cytotoxic for C6 glioma cells. These findings corroborated the traditional use of L. ferrea bark powder for treating skin wounds and suggest the cytotoxic potential of these extracts for glioblastoma cell lines.

Astrocyte Reaction to Catechol-Induced Cytotoxicity Relies on the Contact with Microglia Before Isolation.

Astrocytes preserve the brain microenvironment homeostasis in order to protect other brain cells, mainly neurons, against damages. Glial cells have specific functions that are important in the context of neuronal survival in different models of central nervous system (CNS) diseases. Microglia are among these cells, secreting several molecules that can modulate astrocyte functions. Although 1,2-dihydroxybenzene (catechol) is a neurotoxic monoaromatic compound of exogenous origin, several endogenous molecules also present the catechol group. This study compared two methods to obtain astrocyte-enriched cultures from newborn Wistar rats of both sexes. In the first technique (P1), microglial cells began to be removed early 48 h after primary mixed glial cultures were plated. In the second one (P2), microglial cells were late removed 7 to 10 days after plating. Both cultures were exposed to catechol for 72 h. Catechol was more cytotoxic to P1 cultures than to P2, decreasing cellularity and changing the cell morphology. Microglial-conditioned medium (MCM) protected P1 cultures and inhibited the catechol autoxidation. P2 cultures, as well as P1 in the presence of 20% MCM, presented long, dense, and fibrillary processes positive for glial fibrillary acidic protein, which retracted the cytoplasm when exposed to catechol. The Ngf and Il1beta transcription increased in P1, meanwhile astrocytes expressed more Il10 in P2. Catechol decreased Bdnf and Il10 in P2 cultures, and it decreased the expression of Il1beta in both conditions. A prolonged contact with microglia before isolation of astrocyte-enriched cultures modifies astrocyte functions and morphology, protecting these cells against catechol-induced cytotoxicity.

Valeriana officinalis Counteracts Rotenone Effects on Spreading Depression in the Rat Brain in vivo and Protects Against Rotenone Cytotoxicity Toward Rat Glioma C6 Cells in vitro.

Astrocytes can protect neurons against oxidative stress and excitability-dependent disorders, such as epilepsy. Valeriana officinalis has been used as anticonvulsant and can exert an antioxidant effect, which may underlie its opposing action against the toxic effects of the pesticide rotenone. We investigated the V. officinalis /rotenone interaction in the cortical spreading depression (CSD), a phenomenon that depends upon brain excitability (in vivo model). In addition, we analyzed the protective action of V. officinalis against the cytotoxic effects of rotenone in cultures of rat C6 glioma cells (in vitro model). For the CSD study, Wistar rats received either V. officinalis (250 mg/kg/day via gavage for 15 days; n = 8) or 10 mg/kg/day rotenone via subcutaneous injections for 7 days (n = 7), or they received both substances (n = 5). Two control groups received either saline (vehicle for V. officinalis; n = 8) or 1% Tween-80 aqueous solution (vehicle for rotenone; n = 9). After treatment, CSD was recorded for 4 h. The rotenone- and V. officinalis-treated groups presented, respectively, with lower (2.96 ± 0.14 mm/min), and higher CSD propagation velocity (3.81 ± 0.10 mm/min) when compared with the controls (Tween-80, 3.37 ± 0.06 mm/min and saline, 3.35 ± 0.08 mm/min; p < 0.05). The rotenone plus V. officinalis-treated group displayed a CSD velocity (3.38 ± 0.07 mm/min) that was similar to controls. In line with these results, in vitro experiments on rat glioma C6 cells revealed a protective effect (MTT assay) of V. officinalis against rotenone-induced cytotoxicity. These results suggest the therapeutic potential of V. officinalis for treating neurological diseases involving redox imbalance and astrocyte dysfunction.

PDGF-BB protects mitochondria from rotenone in T98G cells.

Rotenone is one of the most-studied neurotoxic substances as it induces oxidative stress processes both in cellular and animal models. Rotenone affects ATP generation, reactive oxygen species (ROS) production, and mitochondrial membrane potential in neurons and astrocyte-like cells. Previous epidemiologic studies have supported the role of neurotrophic factors such as BDNF and GDNF in neuroprotection mainly in neurons; however, only very few studies have focused on the importance of astrocytic protection in neurodegenerative models. In the present study, we assessed the neuroprotective effects of PDGF-BB against toxicity induced by rotenone in the astrocytic-like model of T98G human glioblastoma cell line. Our results demonstrated that pretreatment with PDGF-BB for 24 h increased cell viability, preserved nuclear morphology and mitochondrial membrane potential following stimulation with rotenone, and reduced ROS production nearly to control conditions. These observations were accompanied by important morphological changes induced by rotenone and that PDGF-BB was able to preserve cellular morphology under this toxic stimuli. These findings indicated that PDGF-BB protects mitochondrial functions, and may serve as a potential therapeutic strategy in rotenone-induced oxidative damage in astrocytes.

Role of IFN-γ and LPS on neuron/glial co-cultures infected by Neospora caninum.

Neospora caninum causes cattle abortion and neurological symptoms in dogs. Although infection is usually asymptomatic, classical neurological symptoms of neosporosis may be associated with encephalitis. This parasite can grow in brain endothelial cells without markedly damages, but it can modulate the cellular environment to promote its survival in the brain. In previous studies, we described that IFN-γ decreased the parasite proliferation and down regulated nitric oxide (NO) production in astrocyte/microglia cultures. However, it remains unclear how glial cells respond to N. caninum in the presence of neurons. Therefore, we evaluated the effect of 300 IU/mL IFN-γ or 1.0 mg/mL of LPS on infected rat neuron/glial co-cultures. After 72 h of infection, LPS did not affect the mitochondrial dehydrogenase activity. However, IFN-γ decreased this parameter by 15.5 and 12.0% in uninfected and infected cells, respectively. The number of tachyzoites decreased 54.1 and 44.3% in cells stimulated with IFN-γ and LPS, respectively. Infection or LPS treatment did not change NO production. On the other hand, IFN-γ induced increased nitrite release in 55.7%, but the infection reverted this induction. IL-10 levels increased only in infected cultures (treated or not), meanwhile PGE2 release was improved in IFN-γ/infected or LPS/infected cells. Although IFN-γ significantly reduced the neurite length in uninfected cultures (42.64%; p < 0.001), this inflammatory cytokine reverted the impairment of neurite outgrowth induced by the infection (81.39%). The results suggest a neuroprotective potential response of glia to N. caninum infection under IFN-γ stimulus. This observation contributes to understand the immune mediated mechanisms of neosporosis in central nervous system (CNS).

8-Methoxypsoralen is a competitive inhibitor of glutathione S-transferase P1-1.

The blood-brain barrier (BBB) is known to protect healthy brain cells from potentially dangerous chemical agents, but there are many evidences supporting the idea that this protective action is extended to tumor cells. Since the process of angiogenesis in brain tumors leads to BBB breakdown, biochemical characteristics of the BBB seem to be more relevant than physical barriers to protect tumor cells from chemotherapy. In fact, a number of resistance related factors were already demonstrated to be component of both BBB and tumor cells. The enzyme glutathione S-transferases (GST) detoxify electrophilic xenobiotics and endogenous secondary metabolites formed during oxidative stress. A role has been attributed to GST in the resistance of cancer cells to chemotherapeutic agents. This study characterized 8-methoxypsoralen (8-MOP) as a human GST P1-1 (hGST P1-1) inhibitor. To identify and characterize the potential inhibitory activity of 8-MOP, we studied the enzyme kinetics of the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) with GSH catalyzed by hGST P1-1. We report here that 8-MOP competitively inhibited hGST P1-1 relative to CDNB, but there was an uncompetitive inhibition relative to GSH. Chromatographic analyses suggest that 8-MOP is not a substrate. Molecular docking simulations suggest that 8-MOP binds to the active site, but its position prevents the GSH conjugation. Thus, we conclude that 8-MOP is a promising prototype for new GST inhibitors pharmacologically useful in the treatment of neurodegenerative disorders and the resistance of cancer to chemotherapy.

Astrocytic modulation of blood brain barrier: perspectives on Parkinson's disease.

The blood-brain barrier (BBB) is a tightly regulated interface in the Central Nervous System (CNS) that regulates the exchange of molecules in and out from the brain thus maintaining the CNS homeostasis. It is mainly composed of endothelial cells (ECs), pericytes and astrocytes that create a neurovascular unit (NVU) with the adjacent neurons. Astrocytes are essential for the formation and maintenance of the BBB by providing secreted factors that lead to the adequate association between the cells of the BBB and the formation of strong tight junctions. Under neurological disorders, such as chronic cerebral ischemia, brain trauma, Epilepsy, Alzheimer and Parkinson's Diseases, a disruption of the BBB takes place, involving a lost in the permeability of the barrier and phenotypical changes in both the ECs and astrocytes. In this aspect, it has been established that the process of reactive gliosis is a common feature of astrocytes during BBB disruption, which has a detrimental effect on the barrier function and a subsequent damage in neuronal survival. In this review we discuss the implications of astrocyte functions in the protection of the BBB, and in the development of Parkinson's disease (PD) and related disorders. Additionally, we highlight the current and future strategies in astrocyte protection aimed at the development of restorative therapies for the BBB in pathological conditions.

Mitochondrial functions in astrocytes: neuroprotective implications from oxidative damage by rotenone.

Mitochondria are critical for cell survival and normal development, as they provide energy to the cell, buffer intracellular calcium, and regulate apoptosis. They are also major targets of oxidative stress, which causes bioenergetics failure in astrocytes through the activation of different mechanisms and production of oxidative molecules. This review provides an insightful overview of the recent discoveries and strategies for mitochondrial protection in astrocytes. We also discuss the importance of rotenone as an experimental approach for assessing oxidative stress in the brain and delineate some molecular strategies that enhance mitochondrial function in astrocytes as a promising strategy against brain damage.

Brain rust: recent discoveries on the role of oxidative stress in neurodegenerative diseases.

Oxidative stress (OS) and damages due to excessive reactive oxygen species (ROS) are common causes of injuries to cells and organisms. The prevalence of neurodegenerative diseases (ND) increases with aging and much of the research involving ROS and OS has emerged from works in this field. This text reviews some recent published articles about the role of OS in ND. Since there are many reviews in this field, the focus was centered in articles published recently. The Scientific Journals Directory supported by the Brazilian Ministry of Education Office for the Coordination of Higher Educational Personnel Improvement (CAPES) was used to search, download, and review articles. The search engine looked for the terms 'oxidative stress AND neurodegenerative diseases AND nutrition' in 10 different scientific collections. Biochemical markers for ND lack sensitivity or specificity for diagnosis or for tracking response to therapy today. OS has an intimate connection with ND, albeit low levels of ROS seem to protect the brain. Deleterious changes in mitochondria, OS, calcium, glucocorticoids, inflammation, trace metals, insulin, cell cycle, protein aggregation, and hundreds to thousands of genes occur in ND. The interaction of genes with their environment, may explain ND. Although OS has received much attention over the years, which increased the number of scientific works on antioxidant interventions, no one knows how to stop or delay ND at present. Interventions in vitro, in vivo, and in humans will continue to contribute for a better understanding of these pathologies.

Investigation of toxic factors affecting cells of rat brains exposed to 3-methylcatechol

The aim of this work was to study the effects of 3MC on the peroxidation of biomolecules in nuclear fractions and nonsynaptic mitochondrial respiration in organelles obtained from rat brains. The cytotoxicity towards rat primary astrocytes in vitro was also tested. 3MC at 1mM oxidized consuming oxygen at a rate of 1.98 ± 0.19 µM.min-1 and formed reactive quinones. At the same concentration, 3MC induced peroxidation of biomolecules in nuclear fractions obtained from rat brain homogenates and inhibited state 2 FADH2-linked respiration in nonsynaptic mitochondria. Furthermore, 3MC oxidized in the culture medium, leading to the formation of quinones. This toluene metabolite was cytotoxic to rat primary astrocytes. The concentration that killed 50 percent of cells after 72 h was 107 mM. The results of the study indicated a direct relationship between cytotoxicity and 3MC oxidation.

O 3-metilcatecol (3MC) é um metabólito do tolueno. Para esclarecer se o 3MC seria tóxico para o sistema nervoso central, examinou-se seus efeitos sobre a peroxidação de biomoléculas em frações nucleares e a respiração mitocondrial em organelas obtidas de cérebros de ratos. Também se testou a citotoxicidade para astrócitos primários de ratos. O 3MC a 1mM oxida-se consumindo oxigênio a uma taxa de 1,98 ± 0,19 mM.min-1, formando quinonas reativas. Nessa mesma concentração o 3MC peroxidou biomoléculas nas frações nucleares. Esse composto também inibiu o estado 2 da respiração mitocondrial associada ao FADH2. Além disso, o 3MC também se oxida em meio de cultura levando à formação de quinonas. Esse metabólito do tolueno foi citotóxico para astrócitos de ratos. A concentração que matou 50 por cento das células após 72 horas foi 107 mM. Os resultados desse estudo indicam uma relação direta entre a citotoxicidade e a oxidação do 3MC.