The tiliroside derivative, 3-O-[(E)-(2-oxo-4-(p-tolyl) but-3-en-1-yl] kaempferol produced inhibition of neuroinflammation and activation of AMPK and Nrf2/HO-1 pathways in BV-2 microglia.

Neuroinflammation is now widely accepted as an important pathophysiological mechanism in neurodegenerative disorders, thus providing a critical target for novel compounds. In this study, 3-O-[(E)-(2-oxo-4-(p-tolyl)but-3-en-1-yl] kaempferol (OTBK) prevented the production of pro-inflammatory mediators TNFα, IL-6, PGE2 and nitrite from BV-2 microglia activated with LPS and IFNγ. These effects were accompanied by reduction in the levels of pro-inflammatory proteins COX-2 and iNOS. Involvement of NF-κB in the anti-inflammatory activity of OTBK was evaluated in experiments showing that the compound prevented phosphorylation, nuclear accumulation and DNA binding of p65 sub-unit induced by stimulation of BV-2 microglia with LPS and IFNγ. Exposure of mouse hippocampal HT22 neurons to conditioned media from LPS + IFNγ-stimulated BV-2 cells resulted in reduced cell viability and generation of cellular reactive oxygen species. Interestingly, conditioned media from LPS/IFNγ-stimulated BV-2 cells which were treated with OTBK did not induce neuronal damage or oxidative stress. OTBK was shown to increase protein levels of phospho-AMPKα, Nrf2 and HO-1 in BV-2 microglia. It was further revealed that OTBK treatment increased Nrf2 DNA binding in BV-2 microglia. The actions of the compound on AMPKα and Nrf2 were shown to contribute to its anti-inflammatory activity as demonstrated by diminished activity in the presence of the AMPK antagonist dorsomorphin and Nrf2 inhibitor trigonelline. These results suggest that OTBK inhibits neuroinflammation through mechanisms that may involve activation of AMPKα and Nrf2 in BV-2 microglia.

Induction of Autophagy and Activation of SIRT-1 Deacetylation Mechanisms Mediate Neuroprotection by the Pomegranate Metabolite Urolithin A in BV2 Microglia and Differentiated 3D Human Neural Progenitor Cells.

SCOPE: Urolithin A is an anti-inflammatory and neuroprotective gut-derived metabolite from ellagitannins and ellagic acid in pomegranate, berries, and nuts. The roles of SIRT-1 and autophagy in the neuroprotective activity of urolithin A are investigated. METHODS AND RESULTS: Analyses of culture supernatants from lipopolysaccharide-stimulated BV2 microglia show that urolithin A (2.5-10 µm) produced significant reduction in the production of nitrite, tumor necrosis factor (TNF)-α and IL-6. The anti-inflammatory effect of the compound is reversed in the presence of sirtuin (SIRT)-1 and the autophagy inhibitors EX527 and chloroquine, respectively. Protein analyses reveal reduction in p65 and acetyl-p65 protein. Treatment of BV2 microglia with urolithin A results in increased SIRT-1 activity and nuclear protein, while induction of autophagy by the compound is demonstrated using autophagy fluorescent and autophagy LC3 HiBiT reporter assays. Viability assays reveal that urolithin A produces a neuroprotective effect in APPSwe-transfected ReNcell VM human neural cells, which is reversed in the presence of EX527 and chloroquine. Increase in both SIRT-1 and autophagic activities are also detected in these cells following treatment with urolithin A. CONCLUSIONS: It has been proposed that SIRT-1 activation and induction of autophagy are involved in the neuroprotective activity of urolithin A in brain cells.

Inhibition of neuroinflammation by thymoquinone requires activation of Nrf2/ARE signalling.

Thymoquinone is an antioxidant phytochemical that has been shown to inhibit neuroinflammation. However, little is known about the potential roles of intracellular antioxidant signalling pathways in its anti-inflammatory activity. The objective of this study was to elucidate the roles played by activation of the Nrf2/ARE antioxidant mechanisms in the anti-inflammatory activity of this compound. Thymoquinone inhibited lipopolysaccharide (LPS)-induced neuroinflammation through interference with NF-κB signalling in BV2 microglia. Thymoquinone also activated Nrf2/ARE signalling by increasing nuclear localisation, DNA binding and transcriptional activity of Nrf2, as well as increasing protein levels of HO-1 and NQO1. Suppression of Nrf2 activity through siRNA or with the use of trigonelline resulted in the loss of anti-inflammatory activity by thymoquinone. Taken together, our studies show that thymoquinone inhibits NF-κB-dependent neuroinflammation in BV2 microglia, by targeting antioxidant pathway involving activation of both Nrf2/ARE. We propose that activation of Nrf2/ARE signalling pathway by thymoquinone probably results in inhibition of NF-κB-mediated neuroinflammation.

AMPK and SIRT1 activation contribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia.

Thymoquinone is a known inhibitor of neuroinflammation. However, the mechanism(s) involved in its action remain largely unknown. In this study, we investigated the roles of cellular reactive oxygen species (ROS), 5' AMP-activated protein kinase (AMPK) and sirtuin 1 (SIRT1) in the anti-neuroinflammatory activity of thymoquinone. We investigated effects of the compound on ROS generation in LPS-activated microglia using the fluorescent 2',7'-dichlorofluorescin diacetate (DCFDA)-cellular ROS detection. Immunoblotting was used to detect protein levels of p40phox, gp91phox, AMPK, LKB1 and SIRT1. Additionally, ELISA and immunofluorescence were used to detect nuclear accumulation of SIRT1. NAD+/NADH assay was also performed. The roles of AMPK and SIRT1 in anti-inflammatory activity of thymoquinone were investigated using RNAi and pharmacological inhibition. Our results show that thymoquinone reduced cellular ROS generation, possibly through inhibition of p40phox and gp91phox protein. Treatment of BV2 microglia with thymoquinone also resulted in elevation in the levels of LKB1 and phospho-AMPK proteins. We further observed that thymoquinone reduced cytoplasmic levels and increased nuclear accumulation of SIRT1 protein and increased levels of NAD+. Results also show that the anti-inflammatory activity of thymoquinone was abolished when the expressions of AMPK and SIRT1 were suppressed by RNAi or pharmacological antagonists. Pharmacological antagonism of AMPK reversed thymoquinone-induced increase in SIRT1. Taken together, we propose that thymoquinone inhibits cellular ROS generation in LPS-activated BV2 microglia. It is also suggested that activation of both AMPK and NAD+/SIRT1 may contribute to the anti-inflammatory, but not antioxidant activity of the compound in BV2 microglia.