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-β, 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 β-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.

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 β-amyloid peptide (Aβ, 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 β-amyloid and LPS activated microglia to an inflammatory state, with increased expression of miR-146a, miR-155, IL1-β, IL-6, and NOS2. Treatment with agathisflavone resulted in a significant reduction in miR146a and miR-155 induced by LPS or Aβ, as well as inflammatory cytokines IL1-β, IL-6, and NOS2. In cells stimulated with Aβ, 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.

Agathisflavone is a flavonoid that exhibits anti-inflammatory and anti-oxidative properties. Here, we investigated the neuroprotective effects of agathisflavone on central nervous system (CNS) neurons and glia in the cerebellar slice ex vivo model of neonatal ischemia. Cerebellar slices from neonatal mice, in which glial fibrillary acidic protein (GFAP) and SOX10 drive expression of enhanced green fluorescent protein (EGFP), were used to identify astrocytes and oligodendrocytes, respectively. Agathisflavone (10 μM) was administered preventively for 60 min before inducing ischemia by oxygen and glucose deprivation (OGD) for 60 min and compared to controls maintained in normal oxygen and glucose (OGN). The density of SOX-10⁺ oligodendrocyte lineage cells and NG2 immunopositive oligodendrocyte progenitor cells (OPCs) were not altered in OGD, but it resulted in significant oligodendroglial cell atrophy marked by the retraction of their processes, and this was prevented by agathisflavone. OGD caused marked axonal demyelination, determined by myelin basic protein (MBP) and neurofilament (NF70) immunofluorescence, and this was blocked by agathisflavone preventative treatment. OGD also resulted in astrocyte reactivity, exhibited by increased GFAP-EGFP fluorescence and decreased expression of glutamate synthetase (GS), and this was prevented by agathisflavone pretreatment. In addition, agathisflavone protected Purkinje neurons from ischemic damage, assessed by calbindin (CB) immunofluorescence. The results demonstrate that agathisflavone protects neuronal and myelin integrity in ischemia, which is associated with the modulation of glial responses in the face of ischemic damage.

The imbalance between the overproduction of reactive species and antioxidant mechanisms can result in astrogliosis and oxidative stress associated with neurodegeneration. Based on the described antioxidant activity of naturally occurring flavonoids, this study evaluated the antioxidant mechanisms of the flavonoid naringenin and the senecioic acid ester derivatives in cortical astrocytes. Naringenin and (S)-naringenin were purified from Citrus paradisi, and from them 7,4-O-disenecioic ester naringenin, (S)-7,4-O-disenecioic ester naringenin, and 7-O-senecioic ester naringenin were synthesized and tested for antioxidant activity by the free-radical scavenging reaction with DPPH. The flavonoids’ toxicity and glutathione (GS) depletion were determined in rat astrocyte cultures; the effects on the astrocytes’ reactivity was determined by the expression of the glial fibrillary acidic protein (GFAP) and by measuring nitric oxide (NO) production in astrocytes treated with lipopolysaccharide (LPS, 1 µg/mL/24 h). The compounds (1–10 μM) presented antioxidant effects, and the (S)-7,4′-O-disenecioic ester naringenin was the most effective. The compounds (1–100 μM) were not toxic to the astrocytes, also promoting an antioxidant effect by increasing GSH. Moreover, naringenin, (S)-7,4′-O-disenecioic ester naringenin, and 7-O-senecioc ester naringenin mitigated the astrocyte reactivity induced by LPS, reducing GFAP expression and NO production. These findings indicate that naringenin and senecioic acid ester derivatives present a pharmacological potential as antioxidant and anti-inflammatory compounds for brain diseases via the modulation of astrocyte response.

Background: Apigenin, as a flavonoid, can be protective against oxidative damage in hypoxic events due to its antioxidant properties. Here, we have investigated the neuroprotective effects of apigenin in an ex vivo model of ischemic damage, using cerebellar slices from postnatal day (P)8–12 reporter mice to identify oligodendrocytes (SOX10-EGFP) and astrocytes (GFAP-EGFP). Methods: Apigenin (10 and 20 μM) was administered preventively at 60 min prior to and during inducing ischemic damage by oxygen and glucose deprivation (OGD); controls were maintained with glucose and normoxia (OGN). Results: OGD induced a marked retraction of oligodendroglial processes without reducing the oligodendrocyte number. This structural disruption was prevented by apigenin; notably, 10 μM apigenin blocked process retraction, whereas 20 μM did not, indicating a dose-dependent effect on the oligodendroglial morphology. Consistent with this, MBP and NF70 immunofluorescence analyses of axonal myelination demonstrated that OGD caused a significant loss of myelin sheaths, and this was prevented by pre-treatment with apigenin. In addition, apigenin prevented astrocyte reactivity induced by OGD, as assessed by increased GFAP-EGFP expression and decreased expression of glutamine synthetase. Moreover, immunofluorescence for calbindin indicated that apigenin protected Purkinje neurons from ischemic damage. Conclusions: These results demonstrate that apigenin is neuroprotective in ischemia and this is associated with modulation of astrocyte reactivity and maintenance of oligodendrocyte and myelin integrity.