Rosiglitazone protects dopaminergic neurons against lipopolysaccharide-induced neurotoxicity through inhibition of microglia activation

Recent evidence has suggested that microglia activation plays an important role in the pathogenesis of Parkinson's disease (PD). Activated microglia secrete various proinflammatory cytokines and neurotoxic mediators, which may contribute to the development of PD. Thus, the inhibition of microglia activation may have a therapeutic benefit in the treatment of PD. In the present study, using mesencephalic neuron-microglia mixed culture and microglia-enriched culture, we investigated whether rosiglitazone (RGZ), a member of peroxisome proliferator-activated receptor gamma (PPARγ) agonists, could inhibit microglia activation. Our results showed that RGZ significantly inhibited lipopolysaccharide (LPS)-induced microglia activation and the production of tumor necrosis factor-alpha (TNF-α), nitric oxide (NO), and superoxide. We further investigated the intracellular signaling pathways regulating the production of TNF-α and NO in LPS-activated microglia. The results showed that RGZ inhibited the phosphorylation and nuclear translocation of the p65 subunit of NF-κB, and the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK). Taken together, our results suggested that the therapeutic effects of RGZ were partially mediated by modulating microglia activation.     

Luteolin reduces primary hippocampal neurons death induced by neuroinflammation

Abstract

Objectives: This study examined whether luteolin may exert an anti-inflammatory effect in microglia and may be neuroprotective by regulating microglia activation.

Methods: We treated BV2 microglia with 1·0 μg/ml lipopolysaccharide (LPS) after incubation with luteolin for 1 hour, the nitric oxide (NO) levels were determined by a Griess reaction, the inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor-alpha (TNF-alpha), and interleukin 1beta (IL-1beta) mRNA expression were determined by real-time PCR analysis, the iNOS and COX-2 protein induction were determined by Western blot analysis, and the levels of prostaglandin E₂ (PGE₂), TNF-alpha, and IL-1beta were determined by enzyme-linked immunosorbent assay (ELISA) kits. Rat primary hippocampal neurons were co-cultured with LPS-activated BV2 microglia with 20 μM luteolin for 24 hours, the hippocampal neurons viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and the number of apoptotic hippocampal neurons was determined by immunofluorescence detection.

Results: Luteolin significantly inhibited the expression of iNOS and COX-2 in LPS-induced BV2 microglia. Moreover, the compound down-regulated the proinflammatory cytokines (TNF-alpha and IL-1beta) as well as the production of NO and PGE₂ in these cells. When hippocampal neurons were co-cultured with LPS-stimulated BV2 microglia, the administration of 20 μM luteolin increased the neurons viability and reduced the number of apoptotic neurons.

Conclusion: These data demonstrate that anti-inflammatory activity of luteolin in microglia contributes to its neuroprotective effect and suggest that it may have a potential therapeutic application in the treatment of neurodegenerative diseases.     

Luteolin inhibits microglial inflammation and improves neuron survival against inflammation

Microglia activation is one of the causative factors for neuroinflammation, which results in brain damage during neurodegenerative disease. Accumulating evidence has shown that the flavonoid luteolin (Lut) possesses potent anti-inflammatory properties; however, its effect on microglia inhibition is currently unknown. Moreover, it is not clear whether Lut also has indirect neuroprotective effects by reducing inflammatory mediators and suppressing microglia activation. In this study, we examined the effects of Lut on lipopolysaccharide (LPS)-induced proinflammatory mediator production and signaling pathways in murine BV2 microglia. In addition, we cocultured microglia and neurons to observe the indirect neuroprotective effects of Lut. Lut inhibited the LPS-stimulated expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor alpha (TNF-α), and interleukin-1β (IL-1β) as well as the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)). Moreover, Lut blocked LPS-induced nuclear factor kappa B (NF-κB) activation. Preincubation of microglia with Lut diminished the neurotoxic effects, owing to the direct anti-inflammatory effects of the compound. Taken together, our findings suggest that Lut may have a potential therapeutic application in the treatment of neuroinflammatory disorders.     

Galectin-1 attenuates neurodegeneration in Parkinson’s disease model by modulating microglial MAPK/IκB/NFκB axis through its carbohydrate-recognition domain

The vicious cycle between the chronic activation of microglia and dopamine neurons degeneration is linked with the progression of Parkinson’s disease (PD). Targeting microglial activation has proven to be a viable option to develop a disease-modified therapy for PD. Galectin-1, which has been reported to have an anti-neuroinflammation effect was used in the present study to evaluate its therapeutic effects on microglia activation and neuronal degeneration in Parkinson’s disease model. It was found that galectin-1 attenuated the inflammatory insult and the apoptosis of SK-N-SH human neuroblastoma cells from conditioned medium of activated microglia induced by Lipopolysaccharides (LPS). Nonetheless, galectin-1 administration (0.5 mg/kg) inhibited the microglia activation, improved the motor deficits in PD mice model induced by MPTP (25 mg/kg weight of mouse, i.p.) and prevented the degeneration of dopaminergic neurons in the substantia nigra. Administration of galectin-1 resulted in p38 and ERK1/2 dephosphorylation followed by IκB/NFκB signaling pathway inhibition. Galectin-1 significantly decreased the secretion of pro-inflammatory cytokines, including interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). The protective effects and modulation of the MAPK/IκB/NFκB signaling pathway were abolished with β-D-galactose which blocked the carbohydrate-recognition domain of galectin-1. The present study demonstrated that galectin-1 inhibited microglia activation and ameliorated neurodegenerative process in PD model by modulating MAPK/IκB/NFκB axis through its carbohydrate-recognition domain.     

Scavenger receptor class A ligands induce secretion of IL1β and exert a modulatory effect on the inflammatory activation of astrocytes in culture

Class-A scavenger receptor (SR-A) is expressed by microglia, and we show here that it is also expressed by astrocytes, where it participates on their inflammatory activation. Astrocytes play a key role on the inflammatory response of the central nervous system, secreting several soluble mediators like cytokines and radical species. Exposure to SR ligands activated MAPKs and NF-κB signaling and increased production of IL1β and nitric oxide (NO). IL1β classically an inflammatory cytokine surprisingly did not increase but inhibited LPS+IFNγ-induced NO production by astrocytes. Our results suggest that SRs expressed by astrocytes participate in the modulation of inflammatory activation.     

Antibody-Mediated Inhibition of Integrin α5β1 Blocks Neurotoxic Prion Peptide PrP(106-126)-Induced Activation of BV2 Microglia

Microglial activation is a characteristic feature of the pathogenesis of prion diseases. The identification of cell surface molecules that mediate the prion protein (PrP) synthetic peptide interaction with microglia is of great significance as it represents potential target molecules to modulate the events leading to the pathophysiology of prion diseases. Here, we carried out in vitro experiments to investigate the involvement of α5β1 integrin in neurotoxic prion peptide PrP(106-126)-induced activation of BV2 microglia. The results showed that the exposure to PrP(106-126) upregulated the mRNA expression of proinflammatory factors (IL-1 β, IL-6, and iNOS) and NALP3 inflammasome components (NALP3 and ASC), increased the release of iNOS and its product nitric oxide, and stimulated NF-κB activation. Blockade of α5β1 integrin with monoclonal antibody BMC5 prior to PrP(106-126) treatment abrogated the upregulation of the mRNA expression of IL-1 β, IL-6, iNOS, and ASC, but had no effect on the mRNA expression of NALP3, blocked the release of iNOS and nitric oxide, and inhibited NF-κB activation. These results suggest that α5β1 integrin is involved in the PrP(106-126)-induced microglial activation through the participation in the activation of NF-κB and NALP3/ASC inflammasome. Our study unveils a previously unidentified role of α5β1 integrin as an intermediate signaling molecule in neurotoxic prion peptides-microglia interactions and identifies a potential molecular target for the modulation of prion-induced microglial activation.     

Combination of EPA with Carotenoids and Polyphenol Synergistically Attenuated the Transformation of Microglia to M1 Phenotype Via Inhibition of NF-κB

Microglia activation toward the M1 phenotype has been reported to contribute to the neurodegenerative processes and cognition alterations due to the release of pro-inflammatory mediators and cytokines. The aim of the present research was to assess the effectiveness of free fatty acids omega-3 preparations: eicosapentaenoic acid (EPA) or/and docosahexaenoic acid (DHA), carotenoids and phenolics combinations, in inhibiting the release of inflammatory mediators from activated microglia. Preincubation of BV-2 microglia cells with each of the FFAs omega-3 preparations in a range of 0.03–2 μM together with Lyc-O-mato^® (0.1 μM), Carnosic acid (0.2 μM) with or without Lutein (0.2 μM), 1 h before addition of lipopolysaccharide (LPS) for 16 h caused a synergistic inhibition of nitric oxide (NO) production with a rank order of EPA > Ropufa (EPA/DHA 2/1) > Krill (EPA/DHA 1.23/1). The optimal inhibitory combinations of EPA (0.125 μM) with the phytonutrients caused a synergistic inhibition of prostaglandin E₂ (PGE₂) release, IL-6 secretion, superoxide and NO production and prevention of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) upregulation and elevated CD40 expression in microglia exposed to LPS or interferon-γ (IFN-γ), representing infection or inflammation, respectively. The presence of the combination caused a synergistic increase in the release of the anti-inflammatory cytokine IL-10. The inhibitory effects by the combinations of EPA with the phytonutrients were mediated by the inhibition of the redox-sensitive NF-κB activation and detected by its phosphorylated p-65 on serine 536 in microglia stimulated by either LPS or IFN-γ. In addition, phosphorylated CREB on serine 133 which was shown to be involved in the induction of iNOS was inhibited by the combinations in stimulated cells. In conclusion, the results suggest that low concentrations of EPA with the phytonutrients are very efficient in inhibiting the transformation of microglia to M1 phenotype and may prevent cognition deficit.     

IL‐12 p40 homodimer,the so‐called biologically inactive molecule,induces nitric oxide synthase in microglia via IL‐12Rβ1

Earlier we have demonstrated that IL‐12 p40 homodimer (p40₂) induces the expression of inducible nitric oxide synthase (iNOS) in microglia. This study was undertaken to investigate underlying mechanisms required for IL‐12 p40₂‐ and IL‐12 p70‐induced expression of iNOS in microglia. IL‐12 p40₂ alone induced the activation of both extracellular signal‐regulated kinase (ERK) and p38 mitogen‐activated protein kinase (MAPK). Interestingly, the ERK pathway coupled p40₂ to iNOS expression via C/EBPβ, but not NF‐κB, whereas the p38 pathway relayed the signal from p40₂ to iNOS expression via both NF‐κB and C/EBPβ. Furthermore, by using microglia from IL‐12Rβ1 (?/?) and IL‐12Rβ2 (?/?) mice or siRNA against IL‐12Rβ1 and IL‐12Rβ2, we demonstrate that p40₂ induced the expression of iNOS in microglia via IL‐12Rβ1–(ERK+p38)–(NF‐κB +C/EBPβ) pathway. In contrast, both IL‐12Rβ1 and IL‐12Rβ2 were involved for IL‐12 p70‐induced microglial expression of iNOS. Although IL‐12Rβ1 coupled p70 to NF‐κB and C/EBPβ, IL‐12Rβ2 was responsible for p70‐mediated activation of GAS. This study delineates a new role of IL‐12Rβ1 and IL‐12Rβ2 for the expression of iNOS and production of NO in microglia that may participate in the pathogenesis of neuroinflammatory diseases. © 2009 Wiley‐Liss, Inc.     

Heme Oxygenase-1-Inducing Activity of 4-Methoxydalbergione and 4’-Hydroxy-4-methoxydalbergione from <Emphasis Type="Italic">Dalbergia odorifera</Emphasis> and Their Anti-inflammatory and Cytoprotective Effects in Murine Hippocampal and BV2 Microglial Cell Line and Primary Rat Microglial Cells

Dalbergia odorifera T. Chen (Leguminosae) grows in Central and South America, Africa, Madagascar, and Southern Asia. D. odorifera possesses many useful pharmacological properties, such as antioxidative and anti-inflammatory activities in various cell types. 4-Methoxydalbergione (MTD) and 4’-hydroxy-4-methoxydalbergione (HMTD) were isolated from the EtOH extract of D. odorifera by several chromatography methods. The chemical structures were elucidated by nuclear magnetic resonance (NMR) and mass spectrum (MS). Anti-inflammatory and cytoprotective effects were examined using BV2 microglial cells and murine hippocampus. MTD and HMTD were demonstrated to induce heme oxygenase (HO)-1 protein levels through the nuclear translocation of nuclear factor E2-related factor 2 (Nrf2) in BV2 microglial cells, while only MTD upregulated HO-1 in HT22 cells. MTD and HMTD induced HO-1 expression through JNK MAPK pathway in BV2 cells, whereas only MTD activated the ERK and p38 pathways in HT22 cells. MTD was also shown to activated MTD and HMTD suppressed lipopolysaccharide-stimulated nitric oxide (NO) and prostaglandin E2 production by inhibiting inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in a dose-dependent manner. Furthermore, MTD and HMTD attenuated pro-inflammatory cytokine productions. These anti-inflammatory effects were found to be mediated through the nuclear factor-kappa B (NF-κB) pathway. MTD exhibited neuroprotective effects on glutamate-induced neurotoxicity by promoting HO-1 in HT22 cells. The anti-inflammatory and cytoprotective effects of MTD and HMTD were partially reversed by an HO inhibitor tin protoporphyrin IX. In addition, MTD and HMTD inhibited pro-inflammatory cytokines and NF-κB pathway in primary rat microglia. These findings suggest that MTD and HMTD have therapeutic potential against neurodegenerative diseases accompanied by microglial activation and/or oxidative cellular injury.     

Trans-Caryophyllene Suppresses Hypoxia-Induced Neuroinflammatory Responses by Inhibiting NF-κB Activation in Microglia

Microglia cells have been reported to mediate hypoxia-induced inflammation through the production of proinflammatory cytokines, including interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNF-α), and IL-6. Given the fact that the activation of the type 2 cannabinoid receptor (CB2R) provides antioxidative and anti-inflammatory results, it is suspected that its selective agonist, trans-caryophyllene (TC), may have protective effects against hypoxia-induced neuroinflammatory responses. In this study, TC was found to significantly inhibit hypoxia-induced cytotoxicity as well as the release of proinflammatory cytokines, including IL-1β, TNF-α, and IL-6, through activation of BV2 microglia following hypoxic exposure (1 % O₂, 24 h). Furthermore, TC significantly inhibited hypoxia-induced generation of reactive oxygen species (ROS) in mitochondria as well as the activation of nuclear factor kappa B (NF-κB) in microglia. Importantly, TC’s effects on inhibiting the activation of NF-κB and the secretion of inflammatory cytokines can be abolished by muting the CB2R using small RNA interference. These observations indicate that TC suppresses the hypoxia-induced neuroinflammatory response through inhibition of NF-κB activation in microglia. Therefore, TC may be beneficial in preventing hypoxia-induced neuroinflammation.     

Astrocytes inhibit nitric oxide‐dependent Ca2+ dynamics in activated microglia: Involvement of ATP released via pannexin 1 channels

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca²⁺ and produce high amounts of nitric oxide (NO). However, whether NO, Ca²⁺ dynamics, and gliotransmitter release are reciprocally modulated is not fully understood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO‐dependent Ca²⁺ dynamics and ATP release in LPS‐stimulated microglia. Griess assays and Fura‐2AM time‐lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca²⁺]_i that depended on the sequential activation of iNOS, COXs, and EP₁ receptor. TGFβ1 released by astrocytes inhibited the abovementioned responses and also abolished LPS‐induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS‐stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS‐stimulated microglia, exogenous ATP triggered activation of purinergic P2Y₁ receptors resulting in Ca²⁺ release from intracellular stores. Interestingly, TGFβ1 released by astrocytes inhibited ATP‐induced Ca²⁺ response in LPS‐stimulated microglia to that observed in control microglia. Finally, COX/EP₁ receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS‐stimulated microglia. Thus, Ca²⁺ dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies. GLIA 2013;61:2023–2037     

NOSH‐aspirin (NBS‐1120), a novel nitric oxide and hydrogen sulfide releasing hybrid,attenuates neuroinflammation induced by microglial and astrocytic activation: A new candidate for treatment of neurodegenerative disorders

Hydrogen sulfide (H₂S) and nitric oxide (NO) have been described as gasotransmitters. Anti‐inflammatory activity in the central and peripheral nervous systems may be one of their functions. Previously we demonstrated that several SH^‐ donors including H₂S‐releasing aspirin (S‐ASA) exhibited anti‐inflammatory and neuroprotective activity in vitro against toxins released by activated microglia and astrocytes. Here we report that NOSH‐ASA, an NO‐ and H₂S‐releasing hybrid of aspirin, has a significantly greater anti‐inflammatory and neuroprotective effect than S‐ASA or NO‐ASA. When activated by LPS/IFNγ, human microglia and THP‐1 cells release materials that are toxic to differentiated SH‐SY5Y cells. These phenomena also occur with IFNγ‐stimulated human astroglia and U373 cells. When the cells were treated with the S‐ASA or NO‐ASA, there was a significant enhancement of neuroprotection. However, NOSH‐ASA had significantly more potent protection properties than NO‐ASA or S‐ASA. The effect was concentration‐dependent, as well as incubation time‐dependent. Such treatment not only reduced the release of the TNFα and IL‐6, but also attenuated activation of P38 MAPK and NFκB proteins. All the compounds tested were not harmful when applied directly to SH‐SY5Y cells. These data suggest that NOSH‐ASA has significant anti‐inflammatory properties and may be a new candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component such as Alzheimer disease and Parkinson disease. GLIA 2013;61:1724–1734     

Resolvin D1 and E1 promote resolution of inflammation in microglial cells in vitro

Sustained inflammation in the brain together with microglia activation can lead to neuronal damage. Hence limiting brain inflammation and activation of microglia is a real therapeutic strategy for inflammatory disease. Resolvin D1 (RvD1) and resolvin E1 (RvE1) derived from n-3 long chain polyunsaturated fatty acids are promising therapeutic compounds since they actively turn off the systemic inflammatory response. We thus evaluated the anti-inflammatory activities of RvD1 and RvE1 in microglia cells in vitro. BV2 cells were pre-incubated with RvD1 or RvE1 before lipopolysaccharide (LPS) treatment. RvD1 and RvE1 both decreased LPS-induced proinflammatory cytokines (TNF-α, IL-6 and IL-1β) gene expression, suggesting their proresolutive activity in microglia. However, the mechanisms involved are distinct as RvE1 regulates NFκB signaling pathway and RvD1 regulates miRNAs expression. Overall, our findings support that pro-resolving lipids are involved in the resolution of brain inflammation and can be considered as promising therapeutic agents for brain inflammation.     

An increase in voltage‐gated sodium channel current elicits microglial activation followed inflammatory responses in vitro and in vivo after spinal cord injury

Inflammation induced by microglial activation plays a pivotal role in progressive degeneration after traumatic spinal cord injury (SCI). Voltage‐gated sodium channels (VGSCs) are also implicated in microglial activation following injury. However, direct evidence that VGSCs are involved in microglial activation after injury has not been demonstrated yet. Here, we show that the increase in VGSC inward current elicited microglial activation followed inflammatory responses, leading to cell death after injury in vitro and in vivo. Isoforms of sodium channel, Na_v1.1, Na_v1.2, and Na_v1.6 were expressed in primary microglia, and the inward current of VGSC was increased by LPS treatment, which was blocked by a sodium channel blocker, tetrodotoxin (TTX). TTX inhibited LPS‐induced NF‐κB activation, expression of TNF‐α, IL‐1β and inducible nitric oxide synthase, and NO production. LPS‐induced p38MAPK activation followed pro‐nerve growth factor (proNGF) production was inhibited by TTX, whereas LPS‐induced JNK activation was not. TTX also inhibited caspase‐3 activation and cell death of primary cortical neurons in neuron/microglia co‐cultures by inhibiting LPS‐induced microglia activation. Furthermore, TTX attenuated caspase‐3 activation and oligodendrocyte cell death at 5 d after SCI by inhibiting microglia activation and p38MAPK activation followed proNGF production, which is known to mediate oligodendrocyte cell death. Our study thus suggests that the increase in inward current of VGSC appears to be an early event required for microglia activation after injury. GLIA 2013;61:1807–1821     

Risperidone significantly inhibits interferon-gamma-induced microglial activation in vitro

Microglia has recently been regarded to be a mediator of neuroinflammation via the release of proinflammatory cytokines, nitric oxide (NO) and reactive oxygen species (ROS) in the central nervous system (CNS). Microglia has thus been reported to play an important role in the pathology of neurodegenerative disease, such as Alzheimer's disease (AD) and Parkinson's disease (PD). The pathological mechanisms of schizophrenia remain unclear while some recent neuroimaging studies suggest even schizophrenia may be a kind of neurodegenerative disease. Risperidone has been reported to decrease the reduction of MRI volume during the clinical course of schizophrenia. Many recent studies have demonstrated that immunological mechanisms via such as interferon (IFN)-gamma and cytokines might be relevant to the pathophysiology of schizophrenia. In the present study, we thus investigated the effects of risperidone on the generation of nitric oxide, inducible NO synthase (iNOS) expression and inflammatory cytokines: interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha by IFN-gamma-activated microglia by using Griess assay, Western blotting and ELISA, respectively. In comparison with haloperidol, risperidone significantly inhibited the production of NO and proinflammatory cytokines by activated microglia. The iNOS levels of risperidone-treated cells were much lower than those of the haloperidol-treated cells. Antipsychotics, especially risperidone may have an anti-inflammatory effect via the inhibition of microglial activation, which is not only directly toxic to neurons but also has an inhibitory effect on neurogenesis and oligodendrogenesis, both of which have been reported to play a crucial role in the pathology of schizophrenia.