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Neurodegenerative disorders have a common characteristic that is the involvement of different cell types, typically the reactivity of astrocytes and microglia, characterizing gliosis, which in turn contributes to the neuronal dysfunction and or death. Flavonoids are secondary metabolites of plant origin widely investigated at present and represent one of the most important and diversified among natural products phenolic groups. Several biological activities are attributed to this class of polyphenols, such as antitumor activity, antioxidant, antiviral, and anti-inflammatory, among others, which give significant pharmacological importance. Our group have observed that flavonoids derived from Brazilian plants Dimorphandra mollis Bent., Croton betulaster Müll. Arg., e Poincianella pyramidalis Tul., botanical synonymous Caesalpinia pyramidalis Tul. also elicit a broad spectrum of responses in astrocytes and neurons in culture as activation of astrocytes and microglia, astrocyte associated protection of neuronal progenitor cells, neuronal differentiation and neuritogenesis. It was observed the flavonoids also induced neuronal differentiation of mouse embryonic stem cells and human pluripotent stem cells. Moreover, with the objective of seeking preclinical pharmacological evidence of these molecules, in order to assess its future use in the treatment of neurodegenerative disorders, we have evaluated the effects of flavonoids in preclinical in vitro models of neuroinflammation associated with Parkinson's disease and glutamate toxicity associated with ischemia. In particular, our efforts have been directed to identify mechanisms involved in the changes in viability, morphology, and glial cell function induced by flavonoids in cultures of glial cells and neuronal cells alone or in interactions and clarify the relation with their neuroprotective and morphogetic effects.

Prosopis juliflora is used for feeding cattle and humans. Intoxication with the plant has been reported, and is characterized by neuromuscular alterations and gliosis. Total alkaloidal extract (TAE) was obtained using acid/basic-modified extraction and was fractionated. TAE and seven alkaloidal fractions, at concentrations ranging 0.03-30 microg/ml, were tested for 24h on astrocyte primary cultures derived from the cortex of newborn Wistar rats. The MTT test and the measure of LDH activity on the culture medium, revealed that TAE and fractions F29/30, F31/33, F32 and F34/35 were cytotoxic to astrocytes. The EC(50) values for the most toxic compounds, TAE, F31/33 and F32 were 2.87 2.82 and 3.01 microg/ml, respectively. Morphological changes and glial cells activation were investigated through Rosenfeld's staining, by immunocytochemistry for the protein OX-42, specific of activated microglia, by immunocytochemistry and western immunoblot for GFAP, the marker of reactive and mature astrocytes, and by the production of nitric oxide (NO). We observed that astrocytes exposed to 3 microg/ml TAE, F29/30 or F31/33 developed compact cell body with many processes overexpressing GFAP. Treatment with 30 microg/ml TAE and fractions, induced cytotoxicity characterized by a strong cell body contraction, very thin and long processes and condensed chromatin. We also observed that when compared with the control (+/-1.34%), the proportion of OX-42 positive cells was increased in cultures treated with 30 microg/ml TAE or F29/30, F31/33, F32 and F34/35, with values raging from 7.27% to 28.74%. Moreover, incubation with 3 microg/ml F32, 30 microg/ml TAE, F29/30, F31/33 or F34/35 induced accumulation of nitrite in culture medium indicating induction of NO production. Taken together these results show that TAE and fractionated alkaloids from P. juliflora act directly on glial cells, inducing activation and/or cytotoxicity, stimulating NO production, and may have an impact on neuronal damages observed on intoxicated animals.

Apigenin Modulates Glia and Protects Neurons apigenin reduced microglial activation, characterized by inhibition of proliferation (BrdU+ cells) and modulation of microglia morphology (Iba-1 + cells), and decreased the expression of the M1 inflammatory marker CD68. Moreover, as determined by RT-qPCR, inflammatory stimuli induced by IL-1β increased the mRNA expression of IL-6, IL-1β, and CCL5, and decreased the mRNA expression of IL-10. Contrary, after treatment with apigenin in inflammatory stimuli (IL-1β or LPS) there was a modulation of the mRNA expression of inflammatory cytokines, and reduced expression of OX42, IL-6 and gp130. Moreover, apigenin alone and after an inflammatory stimulus with IL-1β also induced the increase in the expression of brain-derived neurotrophic factor (BDNF), an effect that may be associated with anti-inflammatory and neuroprotective effects. Together these data demonstrate that apigenin presents neuroprotective and anti-inflammatory effects in vitro and might represent an important neuroimmunomodulatory agent for the treatment of neurodegenerative conditions.

Flavonoids are bioactive compounds that are known to be neuroprotective against glutamate-mediated excitotoxicity, one of the major causes of neurodegeneration. The mechanisms underlying these effects are unresolved, but recent evidence indicates flavonoids may modulate estrogen signaling, which can delay the onset and ameliorate the severity of neurodegenerative disorders. Furthermore, the roles played by glial cells in the neuroprotective effects of flavonoids are poorly understood. The aim of this study was to investigate the effects of the flavonoid agathisflavone (FAB) in primary neuron-glial co-cultures from postnatal rat cerebral cortex. Compared to controls, treatment with FAB significantly increased the number of neuronal progenitors and mature neurons, without increasing astrocytes or microglia. These pro-neuronal effects of FAB were suppressed by antagonists of estrogen receptors (ERα and ERβ). In addition, treatment with FAB significantly reduced cell death induced by glutamate and this was associated with reduced expression levels of pro-inflammatory (M1) microglial cytokines, including TNFα, IL1β and IL6, which are associated with neurotoxicity, and increased expression of IL10 and Arginase 1, which are associated with anti-inflammatory (M2) neuroprotective microglia. We also observed that FAB increased neuroprotective trophic factors, such as BDNF, NGF, NT4 and GDNF. The neuroprotective effects of FAB were also associated with increased expression of glutamate regulatory proteins in astrocytes, namely glutamine synthetase (GS) and Excitatory Amino Acid Transporter 1 (EAAT1). These findings indicate that FAB acting via estrogen signaling stimulates production of neurons in vitro and enhances the neuroprotective properties of microglia and astrocytes to significantly ameliorate glutamate-mediated neurotoxicity.

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Prosopis juliflora was introduced in northeastern Brazil in the 1940s, and since then, it has been available as an alternative for animal nutrition. However, the consumption of P. juliflora as main or sole source of food causes an illness in animals known locally as "cara torta" disease. Cattle and goats experimentally intoxicated presents neurotoxic damage in the central nervous system. Histologic lesions were mainly characterized by vacuolation and loss of neurons in trigeminal motor nuclei. Furthermore, mitochondrial damage in neurons and gliosis was reported in trigeminal nuclei of intoxicated cattle. Studies, using neural cell cultures, have reproduced the main cellular alterations visualized in cara torta disease and contributed to understanding the mechanism of action piperidine alkaloids, the main neurotoxic compound in P. juliflora leaves and pods. Here, we will present aspects of the biological and toxicological properties of P. juliflora and its pharmacologically active compounds.

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