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

Modulation of multiple neuroinflammatory signalling pathways by the dietary glycosidic flavonoid tiliroside

Hyperactivated microglia plays a key role in regulating neuroinflammatory responses which propagate damage to neurons. In recent years, substantial attention has been paid in identifying new strategies to abrogate neuroinflammation. Tiliroside, a dietary glycosidic flavonoid found in several medicinal and dietary plants is known to possess anti-inflammatory and antioxidant activities. This study is aimed at investigating the molecular mechanisms involved in the inhibition of neuroinflammation by the tiliroside. Neuroinflammation inhibitory effects of tiliroside (2-6 μM) were investigated in BV2 microglia stimulated with a combination of LPS (100 ng/ml) and IFN (5 ng/ml). Results show that tiliroside significantly reduced the production of pro-inflammatory cytokines IL-6, TNFα, IL-1β while increasing the production of anti-inflammatory cytokine IL-10 in LPS/IFN-stimulated BV2 microglia. The compound reduced NO production in LPS/IFN-stimulated BV2 cells through inhibition of iNOS protein expression. Tiliroside also suppressed COX-2 protein expression and inhibited PGE2 production in activated microglia. Western blotting and functional experiments revealed that inhibition of neuroinflammation by tiliroside was shown to be mediated through inhibition of NF-B and p38 MAPK signalling pathways. Also, the compound activated SIRT1 and inhibited the expression of acetylated-NF-B-p65 in LPS/IFN-activated BV2 microglia. Further experiments revealed that inhibition of neuroinflammation by tiliroside is not dependent on SIRT1. Tiliroside increased the levels of Nrf2, HO-1 and NQO1 antioxidant proteins, 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 activities of tiliroside. Results of neurotoxicity experiments showed that neuroinflammation-induced neurodegeneration, DNA fragmentation, ROS generation and calcium accumulation were significantly reduced in HT22 neurons when exposed to conditioned medium from BV2 microglia that were pre-treated with tiliroside prior to stimulation with LPS/IFN. Results from this study suggest that tiliroside inhibits neuroinflammation in LPS/IFN-activated BV2 microglia by targeting NF-B and p38 MAPK signalling pathways. Furthermore, the compound activated Nrf2 antioxidant mechanisms in the microglia, which appears to contribute to its anti-inflammatory activity. The study also established that tiliroside protects HT22 neurons from neuroinflammation-induced toxicity

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-B 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-kappaB-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-kappaB-mediated neuroinflammation

Antimalarial drug artemether inhibits neuroinflammation in BV2 microglia through Nrf2-dependent mechanisms

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-kappaB and p38 MAPK signalling, as well as HO-1, NQO1 and Nrf2 were also measured with western blot. NF-kappaB 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-kappaB 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

Inhibition of neuroinflammation in BV2 microglia by the biflavonoid kolaviron is dependent on the Nrf2/ARE antioxidant protective mechanism

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 neuroinflammation. The effects of kolaviron on the expression of nitric oxide/inducible nitric oxide synthase (iNOS), prostaglandin E2 (PGE2)/cyclooxygenase-2, cellular reactive oxygen species (ROS) and the pro-inflammatory cytokines were examined in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Molecular mechanisms of the effects of kolaviron on NF-B and Nrf2/ARE signalling pathways were analysed by immunoblotting, binding assay, and reporter assay. RNA interference was used to investigate the role of Nrf2 in the anti-inflammatory effect of kolaviron. Neuroprotective effect of kolaviron was assessed in a BV2 microglia/HT22 hippocampal neuron co-culture. Kolaviron inhibited the protein levels of NO/iNOS, PGE2/COX-2, cellular ROS and the proinflammatory cytokines (TNFα and IL-6) in LPS-stimulated microglia. Further mechanistic studies showed that kolaviron inhibited neuroinflammation by inhibiting IB/NF-B signalling pathway in LPS-activated BV2 microglia. Kolaviron produced antioxidant effect in BV2 microglia by increasing HO-1 via the Nrf2/ antioxidant response element (ARE) pathway. RNAi experiments revealed that Nrf2 is need for the anti-inflammatory effect of kolaviron. Kolaviron protected HT22 neurons from neuroinflammation-induced toxicity. Kolaviron inhibits neuroinflammation through Nrf2-dependent mechanisms. This compound may therefore be beneficial in neuroinflammation-related neurodegenerative disorders

Tiliroside Produced Anti-Neuroinflammatory Effects Through Interference With NF-κB And MAPK Signalling In LPS+ IFN-γ Stimulated BV-2 Microglia.

Tiliroside is a glycosidic flavonoid, which possesses anti-inflammatory, antioxidant, anticarcinogenic and hepatoprotective activities. It is contained in several dietary plants like linden, rosehip, raspberry and strawberry [1, 2]. In this study the effects of tiliroside on the production of prostaglandin E2 (PGE2) and nitric oxide (NO) from LPS+ IFN-γ stimulated BV-2 microglia as well as its interference with NF-κB and MAP kinase signaling cascades were investigated.BV-2 cells were stimulated with LPS (100ng/ml) and IFN-γ (5ng/ml) in the presence or absence of tiliroside (2-6µM). After 24 hours, supernatants were collected to measure PGE2 and NO production. MTT assay was used to determine the effect of tiliroside on BV-2 microglia viability. Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) protein expressions were evaluated in LPS+ IFN-γ activated BV-2 microglia by western blot. NF-κB transcriptional activity was evaluated using the luciferase reporter gene assay. Protein expressions of phosphorylated IκB, IKK, p38 and MAPKAPK2 in the presence or absence of tiliroside were evaluated using western blots after one hour stimulation with LPS (100ng/ml) and IFN-γ (5ng/ml). Tiliroside (2-6µM) dose dependently (p<0.05) inhibited PGE2 and NO production without effecting viability of BV-2 cells. Tiliroside (6µM) caused a significant (p<0.05) inhibition of COX-2 expression by 27±4.3% and iNOS protein expression by 60.3±1.2% compared to LPS+ IFN-γ control. Further experiments revealed significant (p<0.05) inhibition of nuclear translocation of activated NF-κB by 26.3±3.1% with 6µM tiliroside. The compound (6µM) produced significant (p<0.05) inhibition of IκB and IKK phosphorylation by 51.9±3% and 54.9±4.1%. At 6µM, tiliroside significantly (p<0.05) inhibited p38 phosphorylation by 65.8±2%. Further, tiliroside (6µM) inhibited MAPKAPK2 phosphorylation by 39.9±1%. Taken together, these results suggest that tiliroside suppresses ne

Tiliroside, a dietary glycosidic flavonoid inhibits TRAF-6/NF-kB/p38-mediated neuroinflammation in activated BV2 microglia

Background Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action. Methods BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE2 production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot. Results Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE2 production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system. Conclusions Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound. General Significance Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders