Artemether, a lipid-soluble derivative of artemisinin has been reported to possess anti-inflammatory properties. In this study, we have investigated the molecular mechanisms involved in the inhibition of neuroinflammation by the drug. The effects of artemether on neuroinflammation-mediated HT22 neuronal toxicity were also investigated in a BV2 microglia/HT22 neuron co-culture. To investigate effects on neuroinflammation, we used LPS-stimulated BV2 microglia treated with artemether (5-40µM) for 24 hours. ELISAs and western blotting were used to detect pro-inflammatory cytokines, nitric oxide, PGE2, iNOS, COX-2 and mPGES-1. BACE-1 activity and Aβ levels were measured with ELISA kits. Protein levels of targets in NF-κB and p38 MAPK signalling, as well as HO-1, NQO1 and Nrf2 were also measured with western blot. NF-κB binding to the DNA was investigated using EMSA. MTT, DNA fragmentation and ROS assays in BV2-HT22 neuronal co-culture were used to evaluate the effects of artemether on neuroinflammation-induced neuronal death. The role of Nrf2 in the anti-inflammatory activity of artemether was investigated in BV2 cells transfected with Nrf2 siRNA. Artemether significantly suppressed pro-inflammatory mediators (NO/iNOS, PGE2/COX-2/mPGES-1, TNFα, and IL-6), Aβ and BACE-1 in BV2 cells following LPS stimulation. These effects of artemether were shown to be mediated through inhibition of NF-κB and p38MAPK signalling. Artemether produced increased levels of HO-1, NQO1 and GSH in BV2 microglia. The drug activated Nrf2 activity by increasing nuclear translocation of Nrf2 and its binding to antioxidant response elements in BV2 cells. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of both anti-inflammatory and neuroprotective activities of artemether. We conclude that artemether induces Nrf2 expression and suggest that Nrf2 mediates the anti-inflammatory effect of artemether in BV2 microglia. Our results suggest that this drug has a therapeutic potential in neurodegenerative disorders. 3 KeywordsArtemether, neuroinflammation, BV2 microglia, HT22 hippocampal neurons, NF-κB, Nrf2 4 BackgroundIn the central nervous system (CNS), the microglia plays an important role in immune defence and tissue repair [1]. Under normal conditions, these cells provide surveillance whilst maintaining homeostasis in the brain [2]. In response to injury, harmful toxins, infection or inflammation, microglial cells become activated, secreting pro-inflammatory mediators such as nitric oxide (NO), prostaglandin E2 (PGE2), reactive oxygen/nitrogen species and pro-inflammatory cytokines including IL-6 and TNFα [3,4]. These pro-inflammatory mediators are mainly regulated by the transcription factor NF-κB [5]. NF-κB binds to the DNA and its transcriptional activity regulates several genes, which promote neuroinflammation. The p38 mitogenactivated protein kinase (p38 MAPK) signalling pathway also plays an important role in the expression and activity of pro-inflammatory cytokines in microglial cells [6][7][8].Excessive production o...

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.

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.

Kolaviron is a mixture of bioflavonoids found in the nut of the West African edible seed Garcinia kola, and it has been reported to exhibit a wide range of pharmacological activities. In this study, we investigated the effects of kolaviron in

Hyperactivated microglia plays a key role in regulating neuroinflammatory responses which cause damage to neurons. In recent years, substantial attention has been paid in identifying new strategies to abrogate neuroinflammation. Tiliroside, a natural dietary glycosidic flavonoid, is known to inhibit neuroinflammation. This study was aimed at investigating the molecular mechanisms involved in the inhibition of neuroinflammation and neurotoxicity by tiliroside. The effects of tiliroside on Nrf2 and SIRT1 activities in BV2 microglia and HT22 hippocampal neurons were investigated using immunoblotting and DNA binding assays. The roles of Nrf2 and SIRT1 in the anti-inflammatory activity of tiliroside were further investigated using RNA interference experiments. HT22 neuronal viability was determined by XTT, calcium influx, DNA fragmentation assays. The effect of tiliroside on MAP2 protein expression in HT22 neurons was investigated using western blotting and immunofluorescence. We also studied the impact of tiliroside on DNA fragmentation and ROS generation in APPSwe-transfected 3D neuronal stem cells. Results show that tiliroside increased protein levels of Nrf2, HO-1 and NQO1, indicating an activation of the Nrf2 protective mechanisms in the microglia. Furthermore, transfection of BV2 cells with Nrf2 siRNA resulted in the loss of anti-inflammatory activity by tiliroside. Tiliroside reduced protein levels of acetylated-NF-κB-p65, and increased SIRT1 in LPS/IFNγ-activated BV2 microglia. RNAi experiments revealed that inhibition of neuroinflammation by tiliroside was not affected by silencing SIRT1 gene. Results of neurotoxicity experiments revealed that neuroinflammation-induced toxicity, DNA fragmentation, ROS generation and calcium accumulation in HT22 neurons were significantly reduced by tiliroside treatment. In addition, the compound also protected differentiated human neural progenitor cells by blocking ROS generation and DNA fragmentation. Overall, this study has established that tiliroside protected BV2 microglia from LPS/IFNγ-induced neuroinflammation and HT22 neuronal toxicity by targeting Nrf2 antioxidant mechanisms. The compound also produced inhibition of NF-κB acetylation through activation of SIRT1, as well as increasing SIRT1 activity in mouse hippocampal neurons. Results from this study have further established the mechanisms involved in the anti-neuroinflammatory and neuroprotective activities of tiliroside.Electronic supplementary materialThe online version of this article (10.1007/s12035-018-0975-2) contains supplementary material, which is available to authorized users.

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-transfectedReNcell 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.