Ravikanth Velagapudi

Tiliroside, a dietary glycosidic flavonoid, inhibits TRAF-6/NF-κB/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.

Punicalagin inhibits neuroinflammation in LPS-activated rat primary microglia.

SCOPE In this study, the effects of punicalagin on neuroinflammation in LPS-activated microglia were investigated. METHODS AND RESULTS The ability of punicalagin to reduce the production of TNF-α, IL-6 and prostaglandin E2 was measured in culture medium using enzyme immunoassay. TNF-α and IL-6 gene expression in mouse hippocampal slices was measured with PCR. cyclooxygenase-2 and microsomal prostaglandin E synthase 1 protein and mRNA were evaluated with Western blotting and PCR, respectively. Further experiments to investigate effects of punicalagin on protein expressions of inflammatory targets were also determined with Western blotting. Pretreatment of rat primary microglia with punicalagin (5-40 μM) prior to LPS (10 ng/mL) stimulation produced a significant (p < 0.05) inhibition of TNF-α, IL-6 and prostaglandin E2 production. Punicalagin completely abolished TNF-α and IL-6 gene expression in LPS-stimulated hippocampal slices. Protein and mRNA expressions of cyclooxygenase-2 and microsomal prostaglandin E synthase 1 were also reduced by punicalagin pretreatment. Results show that punicalagin interferes with NF-κB signalling through attenuation of NF-κB-driven luciferase expression, as well as inhibition of IκB phosphorylation and nuclear translocation of p65 subunit in the microglia. CONCLUSION These results suggest that punicalagin inhibits neuroinflammation in LPS-activated microglia through interference with NF-κB signalling, suggesting its potential as a nutritional preventive strategy 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 biflavonoids 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-κB and Nrf2/ARE signalling pathways were analysed by immunoblotting, binding assays and reporter assays. 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 pro-inflammatory cytokines (TNFα and IL-6) in LPS-stimulated microglia. Further mechanistic studies showed that kolaviron inhibited neuroinflammation by inhibiting IκB/NF-κB signalling pathway in LPS-activated BV2 microglia. Kolaviron produced antioxidant effect in BV2 microglia by increasing HO-1 via the Nrf2/antioxidant response element pathway. RNAi experiments revealed that Nrf2 is needed for the anti-inflammatory effects 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.

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

Abstract 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‐&kgr;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‐&kgr;B‐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‐&kgr;B‐mediated neuroinflammation. Graphical abstract Figure. No caption available. HighlightsThymoquinone inhibits NF‐&kgr;B‐mediated neuroinflammation.Thymoquinone activates the Nrf2/ARE protective mechanisms in the microglia.Nrf2 is required for inhibition of neuroinflammation by thymoquinone.

Picralima nitida seeds suppress PGE2 production by interfering with multiple signalling pathways in IL 1β-stimulated SK-N-SH neuronal cells

ETHNOPHARMACOLOGICAL RELEVANCE The dried seed of Picralima nitida is used in rheumatic fever and as an antipyretic in West Africa. In this study we have investigated the effects of an extract obtained from the seeds of Picralima nitida (PNE) on PGE2 production in IL-1β-stimulated cells. MATERIALS AND METHODS Prostaglandin E2 (PGE2) was measured in supernatants of IL-1β-stimulated SK-N-SH cells using enzyme immunoassay (EIA) for PGE2. In Cell ELISA and western blot were used to evaluate the effects of PNE on protein expressions of COX-2, mPGES-1, IκB and IKK. To determine the effect of the extract on NF-κB transactivation, a reporter gene assay was carried out in HEK293 cells stimulated with TNFα. An ELISA was used to measure the roles of p38, ERK1/2 and JNK Mitogen Activated Protein Kinases (MAPKs) on anti-neuroinflammatory actions of PNE. RESULTS Results show that PNE significantly inhibited PGE2 production, as well as COX-2 and mPGES-1 protein expressions in IL-1β-stimulated SK-N-SH cells. Molecular targeting experiments showed that PNE interfered with NF-κB signalling pathway through attenuation of TNFα-stimulated NF-κB transcriptional activation in HEK 293 cells. Furthermore, IL-1β-mediated phosphorylation of IκB and IKK were inhibited in SK-N-SH cells. PNE (50-200 μg/ml) also produced significant inhibition of IL-1β-induced p38 MAPK phosphorylation in SK-N-SH cells. However, phosphorylation of ERK1/2 and JNK MAPKs were achieved at 100 and 200 μg/ml of the extract. CONCLUSIONS Taken together, these results clearly demonstrate that Picralima nitida suppresses PGE2 production by targeting multiple pathways involving NF-κB and MAPK signalling in IL-1β-stimulated SK-N-SH neuronal cells.

Agathisflavone isolated from Anacardium occidentale suppresses SIRT1‐mediated neuroinflammation in BV2 microglia and neurotoxicity in APPSwe‐transfected SH‐SY5Y cells

Agathisflavone is a bioactive compound in Anacardium occidentale. In this study, we investigated inhibition neuroinflammation in BV2 microglia by agathisflavone. Neuroprotective activity of the compound was investigated in differentiated SH‐SY5Y cells. Experiments in lipopolysaccharide (LPS)‐activated BV2 microglia showed that pretreatment with agathisflavone (5–20 μM) produced significant reduction in the release of tumour necrosis factor‐α, interleukin‐6, interleukin‐1β, NO, and PGE2 from the cells. Immunoblotting experiments also revealed that agathisflavone reduced levels of iNOS and COX‐2 protein. Further studies revealed that agathisflavone reduced neuroinflammation by targeting critical steps in NF‐κB signalling in BV2 microglia. Treatment of SH‐SY5Y cells with conditioned medium from LPS‐activated BV2 microglia produced a significant reduction in neuronal viability. However, conditioned medium from BV2 cells that were stimulated with LPS in the presence of agathisflavone did not induce neurotoxicity. Agathisflavone also produced neuroprotection in APPSwe plasmid‐transfected SH‐SY5Y neurons. The compound further attenuated LPS‐induced and APPSwe plasmid‐induced reduction in SIRT1 in BV2 microglia and SH‐SY5Y, respectively. In the presence of EX527, agathisflavone lost its anti‐inflammatory and neuroprotective activities. Our results suggest that agathisflavone inhibits neuroinflammation in BV2 microglia by targeting NF‐κB signalling pathway. The compound also reduces neurotoxicity through mechanisms that are possibly linked to SIRT1 in the microglia and neurons.