Nimbolide Targets Multiple Signalling Pathways to Reduce Neuroinflammation in BV-2 Microglia.

Nimbolide, a limonoid compound found in the neem plant, was investigated for effects on neuroinflammation in BV-2 microglia activated with lipopolysaccharide (LPS). Cultured BV-2 cells were treated with nimbolide (125, 250 and 500 nM) followed by stimulation with LPS (100 ng/ml). Results showed that nimbolide caused a significant reduction in the levels of TNFα, IL-6, IFNγ, NO/iNOS and PGE2/COX-2 in LPS-activated BV-2 cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-IκBα proteins were reduced in the presence of nimbolide. Also, LPS-induced NF-κB acetylation, increased binding to consensus sites and transactivation, as well as phosphorylation of p38 and JNK MAPKs were reduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by a reduction in gp91phox protein levels, while antioxidant effects were also observed through elevation in protein levels of HO-1 and NQO-1. It was observed that treatment of BV-2 microglia with nimbolide resulted in reduced levels of cytoplasmic Nrf2, which was accompanied by increased levels in the nucleus. Furthermore, treatment with this compound resulted in increased binding of Nrf2 to antioxidant responsive element (ARE) consensus sites accompanied by enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatory activity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclear accumulation of SIRT-1, while siRNA knockdown of SIRT-1 resulted in the reversal of anti-inflammatory activity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV-2 microglia through mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. It is also proposed that activation of Nrf2 antioxidant mechanisms may be contributing to its anti-inflammatory activity.

Neuroprotection by Alstonia boonei De Wild., Anacardium occidentale L., Azadirachta indica A.Juss. and Mangifera indica L.

ETHNOPHARMACOLOGY RELEVANCE: Alstonia boonei De Wild. (stem bark), Anacardium occidentale L. (stem bark), Azadirachta indica A.Juss (leaves), Enantia chlorantha Oliv. (stem bark), Khaya senegalensis A.Juss (stem bark) Mangifera indica L. (stem bark), and Nauclea latifolia Sm. (stem bark) are used for treating malaria in southwest Nigeria. Surveys revealed that these plants are also employed for treating symptoms of malaria and cerebral malaria in the region. AIM OF THE STUDY: In this study, the effects of freeze-dried extracts of these plants were investigated on synthetic hemozoin (HZ)-induced neuroinflammation, neuronal damage, and increased permeability of brain microvascular endothelial cells. MATERIALS AND METHODS: Effects of freeze-dried plant extracts were investigated on neuroinflammation by measuring levels of pro-inflammatory mediators in culture supernatants, while in-cell western assays were used to measure protein levels of iNOS and NLRP3. Effects on HZ-induced neurotoxicity and ROS generation was measured using MTT and DCFDA assays, respectively. HZ-induced permeability of hCMEC/D3 endothelial cells was determined using the in vitro vascular permeability assay kit. RESULTS: The extracts produced significant (p < 0.05) reduction in TNFα, IL-6, IL-1ß, MCP-1, RANTES and iNOS/NO production in HZ-stimulated BV-2 microglia. Pre-treatment with 50 µg/mL of A. boonei, A. indica, A. occidentale, E. chlorantha and M. indica also resulted in the inhibition of NF-κB activation. Pre-treatment with A. indica produced, A. occidentale, M. indica and A. boonei reduced HZ-induced increased NLRP3 protein expression. HZ-induced increased caspase-1 activity was also reduced by A. boonei, A. occidentale, A. indica, E. chlorantha, and M. indica. Freeze-dried extracts of A. boonei, A. occidentale, A. indica and M. indica produced neuroprotective effect in HT-22 neuronal cells incubated with HZ by preventing HZ-induced neurotoxicity, ROS generation, DNA fragmentation and caspase 3/7 activity. Inhibition of HZ-induced increase in permeability of human hCMEC/D3 brain endothelial cells was also observed with A. boonei, A. occidentale, A. indica and M. indica, while reducing the release of TNFα and MMP-9. CONCLUSIONS: These results suggest that A. boonei, A. occidentale, A. indica and M. indica are neuroprotective through inhibition of neuroinflammation, neuronal damage and increased permeability of blood brain barrier. The outcome of the study provides pharmacological evidence for the potential benefits of plants as herbal treatments for cerebral malaria symptoms.

Neuroprotection by Skimmianine in Lipopolysaccharide-Activated BV-2 Microglia.

Skimmianine is a furoquinoline alkaloid which is found in the Zanthoxylum genus and also in other plants of the Rutaceae family. This study evaluated the effects of skimmianine on the production of pro-inflammatory mediators in LPS-activated BV-2 microglia. Cultured BV-2 cells were treated with skimmianine (10, 20 and 30 µM), followed by stimulation with LPS (100 ng/mL). Levels of TNFα and IL-6 in cell supernatants were measured using ELISA, while NO and PGE2 levels were evaluated with Griess assay and EIA, respectively. Western blotting was used to determine the protein expression of iNOS, COX-2, phospho-p65 and phospho-IκBα. Results showed that Skimmianine reduced LPS-induced elevated the secretion of TNFα, IL-6, NO, and PGE2, as well as the increased protein expression of iNOS and COX-2. Experiments to elucidate the mechanisms of the anti-neuroinflammatory activity of skimmianine revealed the significant inhibition of LPS-induced increased NF-κB-mediated luciferase activity. Pre-treatment with skimmianine also reduced LPS-induced the increased phosphorylation of NF-κB/p65 and IκBα proteins. Furthermore, skimmianine interfered with the binding capacity of NF-κB to consensus sites. Skimmianine pre-treatment protected HT-22 cells from toxicity induced by microglia-conditioned media, as well as increasing MAP-2 expression. The results of this study suggest that skimmianine inhibits neuroinflammation in LPS-activated microglia by targeting the NF-κB activation pathway. Skimmianine also produced neuroprotection against neurotoxicity induced by microglia-conditioned media.

Iminosugar Amino Acid idoBR1 Reduces Inflammatory Responses in Microglia.

(1) Background. Inflammation is reported to be a key factor in neurodegeneration. The microglia are immune cells present in the central nervous system; their activation results in the release of inflammatory cytokines and is thought to be related to aging and neurodegenerative disorders, such as Alzheimer's disease. (2) Methods. A mouse BV-2 microglia cell line was activated using LPS and the anti-inflammatory cucumber-derived iminosugar amino acid idoBR1, (2R,3R,4R,5S)-3,4,5-trihydroxypiperidine-2-carboxylic acid, was used alongside dexamethasone as the control to determine whether it could reduce the inflammatory responses. (3) Results. A dose-dependent reduction in the LPS-induced production of the proinflammatory factors TNFα, IL-6, and nitric oxide and the transcription factor NF-κB was found. (4) Conclusions. Further investigations of the anti-inflammatory effects of idoBR1 in other models of neurodegenerative diseases are warranted.

SARS-CoV-2 Spike Glycoprotein S1 Induces Neuroinflammation in BV-2 Microglia.

In addition to respiratory complications produced by SARS-CoV-2, accumulating evidence suggests that some neurological symptoms are associated with the disease caused by this coronavirus. In this study, we investigated the effects of the SARS-CoV-2 spike protein S1 stimulation on neuroinflammation in BV-2 microglia. Analyses of culture supernatants revealed an increase in the production of TNF-α, IL-6, IL-1ß and iNOS/NO. S1 also increased protein levels of phospho-p65 and phospho-IκBα, as well as enhanced DNA binding and transcriptional activity of NF-κB. These effects of the protein were blocked in the presence of BAY11-7082 (1 µM). Exposure of S1 to BV-2 microglia also increased the protein levels of NLRP3 inflammasome and enhanced caspase-1 activity. Increased protein levels of p38 MAPK was observed in BV-2 microglia stimulated with the spike protein S1 (100 ng/ml), an action that was reduced in the presence of SKF 86,002 (1 µM). Results of immunofluorescence microscopy showed an increase in TLR4 protein expression in S1-stimulated BV-2 microglia. Furthermore, pharmacological inhibition with TAK 242 (1 µM) and transfection with TLR4 small interfering RNA resulted in significant reduction in TNF-α and IL-6 production in S1-stimulated BV-2 microglia. These results have provided the first evidence demonstrating S1-induced neuroinflammation in BV-2 microglia. We propose that induction of neuroinflammation by this protein in the microglia is mediated through activation of NF-κB and p38 MAPK, possibly as a result of TLR4 activation. These results contribute to our understanding of some of the mechanisms involved in CNS pathologies of SARS-CoV-2.

Garcinia kola and garcinoic acid suppress SARS-CoV-2 spike glycoprotein S1-induced hyper-inflammation in human PBMCs through inhibition of NF-κB activation.

Symptoms and complications associated with severe SARS-CoV-2 infection such as acute respiratory distress syndrome (ARDS) and organ damage have been linked to SARS-CoV-2 spike protein S1-induced increased production of pro-inflammatory cytokines by immune cells. In this study, the effects of an extract of Garcinia kola seeds and garcinoic acid were investigated in SARS-CoV-2 spike protein S1-stimulated human PBMCs. Results of ELISA experiments revealed that Garcinia kola extract (6.25, 12.5, and 25 µg/ml) and garcinoic acid (1.25, 2.5, and 5 µM) significantly reduced SARS-CoV-2 spike protein S1-induced secretion of TNFα, IL-6, IL-1ß, and IL-8 in PBMCs. In-cell western assays showed that pre-treatment with Garcinia kola extract and garcinoic acid reduced expressions of both phospho-p65 and phospho-IκBα proteins, as well as NF-κB DNA binding capacity and NF-κB-driven luciferase expression following stimulation of PBMCs with spike protein S1. Furthermore, pre-treatment of PBMCs with Garcinia kola extract prior to stimulation with SARS-CoV-2 spike protein S1 resulted in reduced damage to adjacent A549 lung epithelial cells. These results suggest that the seed of Garcinia kola and garcinoic acid are natural products which may possess pharmacological/therapeutic benefits in reducing cytokine storm in severe SARS-CoV-2 and other coronavirus infections.

Induction of Exaggerated Cytokine Production in Human Peripheral Blood Mononuclear Cells by a Recombinant SARS-CoV-2 Spike Glycoprotein S1 and Its Inhibition by Dexamethasone.

An understanding of the pathological inflammatory mechanisms involved in SARS-CoV-2 virus infection is necessary in order to discover new molecular pharmacological targets for SARS-CoV-2 cytokine storm. In this study, the effects of a recombinant SARS-CoV-2 spike glycoprotein S1 was investigated in human peripheral blood mononuclear cells (PBMCs). Stimulation of PBMCs with spike glycoprotein S1 (100 ng/mL) resulted in significant elevation in the production of TNFα, IL-6, IL-1ß and IL-8. However, pre-treatment with dexamethasone (100 nM) caused significant reduction in the release of these cytokines. Further experiments revealed that S1 stimulation of PBMCs increased phosphorylation of NF-κB p65 and IκBα, and IκBα degradation. DNA binding of NF-κB p65 was also significantly increased following stimulation with spike glycoprotein S1. Treatment of PBMCs with dexamethasone (100 nM) or BAY11-7082 (1 µM) resulted in inhibition of spike glycoprotein S1-induced NF-κB activation. Activation of p38 MAPK by S1 was blocked in the presence of dexamethasone and SKF 86002. CRID3, but not dexamethasone pre-treatment, produced significant inhibition of S1-induced activation of NLRP3/caspase-1. Further experiments revealed that S1-induced increase in the production of TNFα, IL-6, IL-1ß and IL-8 was reduced in the presence of BAY11-7082 and SKF 86002, while CRID3 pre-treatment resulted in the reduction of IL-1ß production. These results suggest that SARS-CoV-2 spike glycoprotein S1 stimulated PBMCs to release pro-inflammatory cytokines through mechanisms involving activation of NF-κB, p38 MAPK and NLRP3 inflammasome. It is proposed that the clinical benefits of dexamethasone in COVID-19 are possibly due to its anti-inflammatory activity in reducing SARS-CoV-2 cytokine storm.

Zanthoxylum zanthoxyloides inhibits lipopolysaccharide- and synthetic hemozoin-induced neuroinflammation in BV-2 microglia: roles of NF-κB transcription factor and NLRP3 inflammasome activation.

OBJECTIVES: The effects of a root extract of Zanthoxylum zanthoxyloides on neuroinflammation in BV-2 microglia stimulated with LPS and hemozoin were investigated. METHODS: ELISA, enzyme immunoassay and Griess assay were used to evaluate levels of cytokines, PGE2 and NO in culture supernatants, respectively. Microglia-mediated neurotoxicity was evaluated using a BV-2 microglia-HT-22 neuron transwell co-culture. KEY FINDINGS: Treatment with Z. zanthoxyloides caused reduced elevated levels of TNFα, IL-6, IL-1ß, NO and PGE2, while increasing the levels of IL-10. In addition, there were reduced levels of iNOS and COX-2 proteins. This was accompanied by a prevention of microglia-mediated damage to HT-22 mouse hippocampal neurons. Z. zanthoxyloides reduced elevated levels of phospho-IκB and phospho-p65, while preventing degradation of IκB protein and DNA binding of p65. Further mechanistic studies revealed that Z. zanthoxyloides reduced the levels of pro-IL-1ß and IL-1ß in hemozoin-activated BV-2 microglia. This was accompanied by a reduction in caspase-1 activity and NLRP3 protein expression. Bioassay-guided fractionation resulted in the isolation of skimmianine as an anti-inflammatory compound in Z. zanthoxyloides. CONCLUSION: This is the first report showing the inhibition of neuroinflammation in LPS- and hemozoin-activated BV-2 microglia by the root extract of Z. zanthoxyloides by targeting the activation of both NF-κB and NLRP3 inflammasome.

New cytotoxic compounds from the leaves of Caesalpinia benthamiana (Baill.) Herend. & Zarucchi (Fabaceae).

OBJECTIVE: The incidence of multi-drug resistant cancer and the adverse effects associated with available chemotherapy have necessitated the search for new drug candidates. This study investigates the cytotoxic activity of Caesalpinia benthamiana. MATERIALS AND METHODS: Column chromatography (CC) and preparative thin layer chromatography (PTLC) were used to isolate compounds. Structural elucidation was done by spectroscopic analysis. MTT assay was used to evaluate cytotoxicity of the compounds against three human adenocarcinoma cells, using methotrexate and dimethyl sulfoxide (DMSO) as positive and negative controls, respectively. CyQuant direct cell proliferation and caspase-3/7 green detection assays were used to investigate the dichloromethane fraction. IC50 values of isolated compounds were determined from sigmoidal dose-response curve. RESULTS: Four new cytotoxic compounds, benthamianoate (2), benthamiacone (3), benthamianin (5) and benthamianol (6), and two known compounds, methyl gallate (1) and 2-methoxyacrylic acid (4) were identified. All the compounds were active with the new monoterpenoid characterized as benthamiacone exhibiting the highest activity (IC50 13.23-21.97 µg/ml) across cancer cell lines investigated. CyQuant direct cell proliferation assay showed significant reduction in the number of live carcinoma cells, while caspase-3/7 green detection assay showed significant increase in the number of dead carcinoma cells. CONCLUSION: This study revealed potential cytotoxic compounds which are here reported for the first time from C. benthamiana.

Synthesis of novel isoflavone/benzo-δ-sultam hybrids as potential anti-inflammatory drugs.

A small series of novel isoflavone/benzo-δ-sultam hybrids was synthesised and evaluated as potential anti-inflammatory and neuroprotective drugs in LPS-activated BV2 microglia. The benzo-δ-sultam core was constructed in a two-step reaction by coupling 2-halobenzenesulfonamide derivatives with terminal alkynes, followed by a 6-endo-dig cyclisation. The synthesised compounds, including precursors and hybrids, were tested for their ability to inhibit NO and TNF-α production in LPS-stimulated BV2 microglial cells, and the results are promising. The most potent hybrid reduces the NO production to 41%, and the TNF-α to 34% at 20 µM final concentration in the well.

Alzheimer's disease: natural products as inhibitors of neuroinflammation.

Alzheimer's disease (AD) is the most common form of dementia and affects 44 million people worldwide. New emerging evidence from pre-clinical and clinical investigations shows that neuroinflammation is a major pathological component of AD suggesting that anti-inflammatory strategies are important in delaying the onset or slowing the progression of the disease. However, efforts to employ current anti-inflammatory agents in AD clinical trials have produced limited success. Consequently, there is a need to explore anti-inflammatory natural products, which target neuroinflammatory pathways relevant to AD pathogenesis. This review summarises important druggable molecular targets of neuroinflammation and presents classes of anti-neuroinflammatory natural products with potentials for preventing and reducing symptoms of AD.

Methyl 3,4,5-trimethoxycinnamate suppresses inflammation in RAW264.7 macrophages and blocks macrophage-adipocyte interaction.

Methyl 3,4,5-trimethoxycinnamate (MTC) is a bioactive natural phenylpropanoid. We evaluated anti-inflammatory effects of synthetic MTC in RAW264.7 macrophages and RAW264.7-3T3-L1 adipocytes co-culture. Levels of cytokines and chemokines, as well as NO and PGE2 in cell supernatants were analysed using ELISAs, Griess assay and enzyme immunoassays, respectively. In-cell cytoblot was used to assess levels of proteins; while DNA binding and reporter gene assays were used to measure transcription factor DNA binding and transcriptional activities, respectively. Glucose uptake in adipocytes was evaluated with 2-deoxy-2-[(7-nitro-2, 1, 3-benzoxadiazol-4-yl) amino]-D-glucose uptake. MTC (5-20 µM) suppressed LPS + IFNγ-induced release of TNFα, IL-6 and IL-1ß, as well as NO/iNOS and PGE2/COX-2 levels in RAW264.7 cells. Furthermore, there was a reduction in phospho-IκB and phospho-p65 proteins, accompanied by a reduction in total IκB in RAW264.7 cells. Further studies showed that MTC also produced a reduction in NF-κB DNA binding and luciferase activity. Treatment of RAW264.7 cells with MTC (5-20 µM) resulted in enhanced DNA binding of Nrf2 and an increase in ARE-luciferase activity. In a macrophage-adipocyte co-culture, the compound reduced the release of TNFα, IL-6, IL-1ß, MCP-1 and RANTES, while enhancing glucose uptake and activation of AMPKα. Our results suggest that MTC produced anti-inflammatory and antioxidant activities in macrophages. MTC also prevented inflammation in macrophage-adipocyte co-culture. The effect of MTC on glucose uptake in adipocytes is proposed to be linked to activation of AMPK.

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 Neuroinflammation and Neurotoxicity by Synthetic Hemozoin.

Hemozoin produced by Plasmodium falciparum during malaria infection has been linked to the neurological dysfunction in cerebral malaria. In this study, we determined whether a synthetic form of hemozoin (sHZ) produces neuroinflammation and neurotoxicity in cellular models. Incubation of BV-2 microglia with sHZ (200 and 400 µg/ml) induced significant elevation in the levels of TNFα, IL-6, IL-1ß, NO/iNOS, phospho-p65, accompanied by an increase in DNA binding of NF-κB. Treatment of BV-2 microglia with sHZ increased protein levels of NLRP3 with accompanying increase in caspase-1 activity. In the presence of NF-κB inhibitor BAY11-7082 (10 µM), there was attenuation of sHZ-induced release of pro-inflammatory cytokines, NO/iNOS. In addition, increase in caspase-1/NLRP3 inflammasome activation was blocked by BAY11-7082. Pre-treatment with BAY11-7082 also reduced both phosphorylation and DNA binding of the p65 sub-unit. The NLRP3 inhibitor CRID3 (100 µM) did not prevent sHZ-induced release of TNFα and IL-6. However, production of IL-1ß, NO/iNOS as well as caspase-1/NLRP3 activity was significantly reduced in the presence of CRID3. Incubation of differentiated neural progenitor (ReNcell VM) cells with sHZ resulted in a reduction in cell viability, accompanied by significant generation of cellular ROS and increased activity of caspase-6, while sHZ-induced neurotoxicity was prevented by N-acetylcysteine and Z-VEID-FMK. Taken together, this study shows that the synthetic form of hemozoin induces neuroinflammation through the activation of NF-κB and NLRP3 inflammasome. It is also proposed that sHZ induces ROS- and caspase-6-mediated neurotoxicity. These results have thrown more light on the actions of malarial hemozoin in the neurobiology of cerebral malaria.

Chemical Compositions of Metals in Bhasmas and Tibetan Zuotai Are a Major Determinant of Their Therapeutic Effects and Toxicity.

Minerals are alchemically processed as Bhasmas in Ayurvedic medicines or as Zuotai in Tibetan medicines. Ayurveda is a knowledge system of longevity and considers the mineral elixir made from "nature" capable of giving humans perpetual life. Herbo-metallic preparations have a long history in the treatment of various diseases in India, China, and around the world. Their disposition, pharmacology, efficacy, and safety require scientific evaluation. This review discusses the Bhasmas in Ayurvedic medicines and Zuotai in Tibetan medicines for their occurrence, bioaccessibility, therapeutic use, pharmacology, toxicity, and research perspectives. A literature search on Mineral, Bhasma, Ayurvedic medicine, Zuotai, Tibetan medicine, and Metals/metalloids from PubMed, Google and other sources was carried out, and the relevant papers on their traditional use, pharmacology, and toxicity were selected and analyzed. Minerals are processed to form Bhasma or Zuotai to alter their physiochemical properties distinguishing them from environmental metals. The metals found in Ayurveda are mainly from the intentional addition in the form of Bhasma or Zuotai. Bhasma and Zuotai are often used in combination with other herbals and/or animal-based products as mixtures. The advanced technologies are now utilized to characterize herbo-metallic preparations as Quality Assurance/Quality Control. The bioaccessibility, absorption, distribution, metabolism, and elimination of herbo-metallic preparations are different from environmental metals. The pharmacological basis of Bhasma in Ayurveda and Zuotai in Tibetan medicines and their interactions with drugs require scientific research. Although the toxic potentials of Bhasma and Zuotai differ from environmental metals, the metal poisoning case reports, especially lead (Pb), mercury (Hg), and arsenic (As) from inappropriate use of traditional medicines, are increasing, and pharmacovigilance is desired. In risk assessment, chemical forms of metals in Bhasma and Zuotai should be considered for their disposition, efficacy, and toxicity.

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.

Formononetin inhibits neuroinflammation and increases estrogen receptor beta (ERß) protein expression in BV2 microglia.

Formononetin is a bioactive non-steroidal polyphenol found in a variety of plants. In this study we evaluated the effects of formononetin on neuroinflammation in LPS-stimulated BV2 microglia. Results showed that formononetin significantly reduced the production of TNF-α, IL-6 and IL-1ß, nitrite and PGE2, as well as protein levels of iNOS and COX-2. Reporter gene assays showed that formononetin produced inhibition of NF-κB luciferase activity in HEK293 cells stimulated with TNF-α. Immunoblotting experiments revealed an inhibition of IKKα phosphorylation, with the resultant attenuation of phosphorylation and degradation of IκBα following LPS stimulation. Formononetin also produced an inhibition of nuclear translocation and DNA binding by NF-κB following LPS stimulation. RNAi experiments showed that transfection of BV2 microglia with ERß siRNA resulted in the loss of anti-inflammatory action of formononetin. MTT assay and MAP2 immunoreactivity experiments showed that formononetin produced significant neuroprotective activity by preventing BV2 microglia conditioned media-induced toxicity to HT22 neurons. Investigations on the effect of formononetin on MCF7 breast cancer cells revealed that, while the compound significantly increased ER-luciferase activity, its effects on proliferation were modest. This study has established that formononetin inhibits neuroinflammation by targeting NF-κB signalling pathway in BV2 microglia, possibly through mechanisms involving ERß. Formononetin appears to modulate ERß in MCF7 breast cancer cells with limited proliferative effect. Formononetin could therefore serve as a chemical scaffold for the development of novel compounds which have selective neuroprotective actions in the brain.

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-1ß, 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.

Activation of Nrf2 Pathway Contributes to Neuroprotection by the Dietary Flavonoid Tiliroside.

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.

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.