The Phytochemical Agathisflavone Modulates miR146a and miR155 in Activated Microglia Involving STAT3 Signaling.

MicroRNAs (miRs) act as important post-transcriptional regulators of gene expression in glial cells and have been shown to be involved in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease (AD). Here, we investigated the effects of agathisflavone, a biflavonoid purified from the leaves of Cenostigma pyramidale (Tul.), on modulating the expression of miRs and inflammatory mediators in activated microglia. C20 human microglia were exposed to oligomers of the ß-amyloid peptide (Aß, 500 nM) for 4 h or to lipopolysaccharide (LPS, 1 µg/mL) for 24 h and then treated or not with agathisflavone (1 µM) for 24 h. We observed that ß-amyloid and LPS activated microglia to an inflammatory state, with increased expression of miR-146a, miR-155, IL1-ß, IL-6, and NOS2. Treatment with agathisflavone resulted in a significant reduction in miR146a and miR-155 induced by LPS or Aß, as well as inflammatory cytokines IL1-ß, IL-6, and NOS2. In cells stimulated with Aß, there was an increase in p-STAT3 expression that was reduced by agathisflavone treatment. These data identify a role for miRs in the anti-inflammatory effect of agathisflavone on microglia in models of neuroinflammation and AD.

Amburana cearensis seed extract stimulates astrocyte glutamate homeostatic mechanisms in hippocampal brain slices and protects oligodendrocytes against ischemia.

BACKGROUND: Stroke is a leading cause of death and disability worldwide. A major factor in brain damage following ischemia is excitotoxicity caused by elevated levels of the neurotransmitter glutamate. In the brain, glutamate homeostasis is a primary function of astrocytes. Amburana cearensis has long been used in folk medicine and seed extract obtained with dichloromethane (EDAC) have previously been shown to exhibit cytoprotective activity in vitro. The aim of the present study was to analyse the activity of EDAC in hippocampal brain slices. METHODS: We prepared a dichloromethane extract (EDAC) from A. cearensis seeds and characterized the chemical constituents by 1H and 13C-NMR. Hippocampal slices from P6-8 or P90 Wistar rats were used for cell viability assay or glutamate uptake test. Hippocampal slices from P10-12 transgenic mice SOX10-EGFP and GFAP-EGFP and immunofluorescence for GS, GLAST and GLT1 were used to study oligodendrocytes and astrocytes. RESULTS: Astrocytes play a critical role in glutamate homeostasis and we provide immunohistochemical evidence that in excitotoxicity EDAC increased expression of glutamate transporters and glutamine synthetase, which is essential for detoxifying glutamate. Next, we directly examined astrocytes using transgenic mice in which glial fibrillary acidic protein (GFAP) drives expression of enhanced green fluorescence protein (EGFP) and show that glutamate excitotoxicity caused a decrease in GFAP-EGFP and that EDAC protected against this loss. This was examined further in the oxygen-glucose deprivation (OGD) model of ischemia, where EDAC caused an increase in astrocytic process branching, resulting in an increase in GFAP-EGFP. Using SOX10-EGFP reporter mice, we show that the acute response of oligodendrocytes to OGD in hippocampal slices is a marked loss of their processes and EDAC protected oligodendrocytes against this damage. CONCLUSION: This study provides evidence that EDAC is cytoprotective against ischemia and glutamate excitotoxicity by modulating astrocyte responses and stimulating their glutamate homeostatic mechanisms.

The Flavonoid Agathisflavone Directs Brain Microglia/Macrophages to a Neuroprotective Anti-Inflammatory and Antioxidant State via Regulation of NLRP3 Inflammasome.

Agathisflavone, purified from Cenostigma pyramidale (Tul.) has been shown to be neuroprotective in in vitro models of glutamate-induced excitotoxicity and inflammatory damage. However, the potential role of microglial regulation by agathisflavone in these neuroprotective effects is unclear. Here we investigated the effects of agathisflavone in microglia submitted to inflammatory stimulus in view of elucidating mechanisms of neuroprotection. Microglia isolated from cortices of newborn Wistar rats were exposed to Escherichia coli lipopolysaccharide (LPS, 1 µg/mL) and treated or not with agathisflavone (1 µM). Neuronal PC12 cells were exposed to a conditioned medium from microglia (MCM) treated or not with agathisflavone. We observed that LPS induced microglia to assume an activated inflammatory state (increased CD68, more rounded/amoeboid phenotype). However, most microglia exposed to LPS and agathisflavone, presented an anti-inflammatory profile (increased CD206 and branched-phenotype), associated with the reduction in NO, GSH mRNA for NRLP3 inflammasome, IL1-ß, IL-6, IL-18, TNF, CCL5, and CCL2. Molecular docking also showed that agathisflavone bound at the NLRP3 NACTH inhibitory domain. Moreover, in PC12 cell cultures exposed to the MCM previously treated with the flavonoid most cells preserved neurites and increased expression of ß-tubulin III. Thus, these data reinforce the anti-inflammatory activity and the neuroprotective effect of agathisflavone, effects associated with the control of NLRP3 inflammasome, standing out it as a promising molecule for the treatment or prevention of neurodegenerative diseases.

Targeting the Subventricular Zone to Promote Myelin Repair in the Aging Brain.

The subventricular zone (SVZ) is the largest and most active germinal zone in the adult forebrain. Neural stem cells (NSCs) of the SVZ generate olfactory interneurons throughout life and retain the intrinsic ability to generate oligodendrocytes (OLs), the myelinating cells of the central nervous system. OLs and myelin are targets in demyelinating diseases such as multiple sclerosis (MS). Remyelination is dependent on the ability of oligodendrocyte progenitor cells (OPCs) to proliferate, migrate, and terminally differentiate into myelinating OLs. During aging, there is a gradual decrease in the regenerative capacity of OPCs, and the consequent loss of OLs and myelin is a contributing factor in cognitive decline and the failure of remyelination in MS and other pathologies with aging contexts, including Alzheimer's disease (AD) and stroke. The age-related decrease in oligodendrogenesis has not been fully characterised but is known to reflect changes in intrinsic and environmental factors affecting the ability of OPCs to respond to pro-differentiation stimuli. Notably, SVZ-derived OPCs are an important source of remyelinating OLs in addition to parenchymal OPCs. In this mini-review, we briefly discuss differences between SVZ-derived and parenchymal OPCs in their responses to demyelination and highlight challenges associated with their study in vivo and how they can be targeted for regenerative therapies in the aged brain.

Agathisflavone Modifies Microglial Activation State and Myelination in Organotypic Cerebellar Slices Culture.

Oligodendrocytes produce the myelin that is critical for rapid neuronal transmission in the central nervous system (CNS). Disruption of myelin has devastating effects on CNS function, as in the demyelinating disease multiple sclerosis (MS). Microglia are the endogenous immune cells of the CNS and play a central role in demyelination and repair. There is a need for new potential therapies that regulate myelination and microglia to promote repair. Agathisflavone (FAB) is a non-toxic flavonoid that is known for its anti-inflammatory and neuroprotective properties. Here, we examined the effects of FAB (5-50 µM) on myelination and microglia in organotypic cerebellar slices prepared from P10-P12 Sox10-EGFP and Plp1-DsRed transgenic mice. Immunofluorescence labeling for myelin basic protein (MBP) and neurofilament (NF) demonstrates that FAB significantly increased the proportion of MBP + /NF + axons but did not affect the overall number of oligodendroglia or axons, or the expression of oligodendroglial proteins CNPase and MBP. FAB is known to be a phytoestrogen, but blockade of α- or ß- estrogen receptors (ER) indicated the myelination promoting effects of FAB were not mediated by ER. Examination of microglial responses by Iba1 immunohistochemistry demonstrated that FAB markedly altered microglial morphology, characterized by smaller somata and reduced branching of their processes, consistent with a decreased state of activation, and increased Iba1 protein expression. The results provide evidence that FAB increases the extent of axonal coverage by MBP immunopositive oligodendroglial processes and has a modulatory effect upon microglial cells, which are important therapeutic strategies in multiple neuropathologies.

Amburana cearensis: Pharmacological and Neuroprotective Effects of Its Compounds.

Amburana cearensis A.C. Smith is an endemic tree from Northeastern Brazil used in folk medicine as teas, decocts and syrups for the treatment of various respiratory and inflammatory diseases, since therapeutic properties have been attributed to compounds from its stem bark and seeds. Numerous pharmacological properties of semi-purified extracts and isolated compounds from A. cearensis have been described in several biological systems, ranging from antimicrobial to anti-inflammatory effects. Some of these activities are attributed to coumarins and phenolic compounds, the major compounds present in A. cearensis seed extracts. Multiple lines of research demonstrate these compounds reduce oxidative stress, inflammation and neuronal death induced by glutamate excitotoxicity, events central to most neuropathologies, including Alzheimer's disease (AD) and Parkinson's Disease (PD). This review focuses on the botanical aspects, folk medicine use, biological effects and pharmacological activities of A. cearensis compounds and their potential as novel non-toxic drugs for the treatment of neurodegenerative diseases.

The flavonoid agathisflavone modulates the microglial neuroinflammatory response and enhances remyelination.

Myelin loss is the hallmark of the demyelinating disease multiple sclerosis (MS) and plays a significant role in multiple neurodegenerative diseases. A common factor in all neuropathologies is the central role of microglia, the intrinsic immune cells of the central nervous system (CNS). Microglia are activated in pathology and can have both pro- and anti-inflammatory functions. Here, we examined the effects of the flavonoid agathisflavone on microglia and remyelination in the cerebellar slice model following lysolecithin induced demyelination. Notably, agathisflavone enhances remyelination and alters microglial activation state, as determined by their morphology and cytokine profile. Furthermore, these effects of agathisflavone on remyelination and microglial activation were inhibited by blockade of estrogen receptor α. Thus, our results identify agathisflavone as a novel compound that may act via ER to regulate microglial activation and enhance remyelination and repair.

Neuroimmunomodulatory and Neuroprotective Effects of the Flavonoid Apigenin in in vitro Models of Neuroinflammation Associated With Alzheimer's Disease.

Neurodegenerative disorders (ND) are characterized by the progressive and irreversible loss of neurons. Alzheimer's Disease (AD) is the most incident age-related ND, in which the presence of a chronic inflammatory compound seems to be related to its pathogenesis. Different stimuli in the central nervous system (CNS) can induce activation, proliferation, and changes in phenotype and glial function, which can be modulated by anti-inflammatory agents. Apigenin (4,5,7-trihydroxyflavone) is a flavonoid found in abundance in many fruits and vegetables, that has shown important effects upon controlling the inflammatory response. This study evaluated the neuroprotective and neuroimmunomodulatory potential of apigenin using in vitro models of neuroinflammation associated with AD. Co-cultures of neurons and glial cells were obtained from the cortex of newborn and embryonic Wistar rats. After 26 days in vitro, cultures were exposed to lipopolysaccharide (LPS; 1 µg/ml), or IL-1ß (10 ng/ml) for 24 h, or to Aß oligomers (500 nM) for 4 h, and then treated with apigenin (1 µM) for further 24 h. It was observed that the treatment with apigenin preserved neurons and astrocytes integrity, determined by Rosenfeld's staining and immunocytochemistry for ß-tubulin III and GFAP, respectively. Moreover, it was observed by Fluoro-Jade-B and caspase-3 immunostaining that apigenin was not neurotoxic and has a neuroprotective effect against inflammatory damage. Additionally, 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-1ß increased the mRNA expression of IL-6, IL-1ß, and CCL5, and decreased the mRNA expression of IL-10. Contrary, after treatment with apigenin in inflammatory stimuli (IL-1ß 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-1ß 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.

Agathisflavone modulates astrocytic responses and increases the population of neurons in an in vitro model of traumatic brain injury.

Traumatic brain injury (TBI) is a critical health problem worldwide, with a high incidence rate and potentially severe long-term consequences. Depending on the level of mechanical stress, astrocytes react with complex morphological and functional changes known as reactive astrogliosis. In cases of severe tissue injury, astrocytes proliferate in the area immediately adjacent to the lesion to form the glial scar, which is a major barrier to neuronal regeneration in the central nervous system. The flavonoid agathisflavone has been shown to have neuroprotective, neurogenic, and immunomodulatory effects and could have beneficial effects in situations of TBI. In this study, we investigated the effects of agathisflavone on modulating the responses of astrocytes and neurons to injury, using the in vitro scratch wound model of TBI in primary cultures of rat cerebral cortex. In control conditions, the scratch wound induced an astroglial injury response, characterized by upregulation of glial fibrillary acidic protein (GFAP) and hypertrophy, together with the reduction in proportion of neurons within the lesion site. Treatment with agathisflavone (1 µM) decreased astroglial GFAP expression and hypertrophy and induced an increase in the number of neurons and neurite outgrowth into the lesion site. Agathisflavone also induced increased expression of the neurotrophic factors NGF and GDNF, which are associated with the neuroprotective profile of glial cells. These results demonstrate that in an in vitro model of TBI, the flavonoid agathisflavone modulates the astrocytic injury response and glial scar formation, stimulating neural recomposition.

Phytoestrogen Agathisflavone Ameliorates Neuroinflammation-Induced by LPS and IL-1ß and Protects Neurons in Cocultures of Glia/Neurons.

Inflammation and oxidative stress are common aspects of most neurodegenerative diseases in the central nervous system. In this context, microglia and astrocytes are central to mediating the balance between neuroprotective and neurodestructive mechanisms. Flavonoids have potent anti-inflammatory and antioxidant properties. Here, we have examined the anti-inflammatory and neuroprotective potential of the flavonoid agathisflavone (FAB), which is derived from the Brazilian plant Poincianella pyramidalis, in in vitro models of neuroinflammation. Cocultures of neurons/glial cells were exposed to lipopolysaccharide (LPS, 1 µg/mL) or interleukin (IL)-1ß (10 ng/mL) for 24 h and treated with FAB (0.1 and 1 µM, 24 h). FAB displayed a significant neuroprotective effect, as measured by nitric oxide (NO) production, Fluoro-Jade B (FJ-B) staining, and immunocytochemistry (ICC) for the neuronal marker ß-tubulin and the cell death marker caspase-3, preserving neuronal soma and increasing neurite outgrowth. FAB significantly decreased the LPS-induced microglial proliferation, identified by ICC for Iba-1/bromodeoxyuridine (BrdU) and CD68 (microglia M1 profile marker). In contrast, FAB had no apparent effect on astrocytes, as determined by ICC for glial fibrillary acidic protein (GFAP). Furthermore, FAB protected against the cytodestructive and proinflammatory effects of IL-1ß, a key cytokine that is released by activated microglia and astrocytes, and ICC showed that combined treatment of FAB with α and ß estrogen receptor antagonists did not affect NF-κB expression. In addition, qPCR analysis demonstrated that FAB decreased the expression of proinflammatory molecules TNF-α, IL-1ß, and connexins CCL5 and CCL2, as well as increased the expression of the regulatory molecule IL-10. Together, these findings indicate that FAB has a significant neuroprotective and anti-inflammatory effect in vitro, which may be considered as an adjuvant for the treatment of neurodegenerative diseases.

Agathisflavone, a flavonoid derived from Poincianella pyramidalis (Tul.), enhances neuronal population and protects against glutamate excitotoxicity.

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 ERß). 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, IL1ß 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.