Inflammatory Responses In Microglial Cells Research Articles

Withania somnifera and Its Withanolides Attenuate Oxidative and Inflammatory Responses and Up-Regulate Antioxidant Responses in BV-2 Microglial Cells.

Withania somnifera (L.) Dunal, commonly known as Ashwagandha, has been used in Ayurvedic medicine for promoting health and quality of life. Recent clinical trials together with experimental studies indicated significant neuroprotective effects of Ashwagandha and its constituents. This study is aimed to investigate anti-inflammatory and anti-oxidative properties of this botanical and its two withanolide constituents, namely, Withaferin A and Withanolide A, using the murine immortalized BV-2 microglial cells. Ashwagandha extracts not only effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) and reactive oxygen species (ROS) production in BV-2 cells, but also stimulates the Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) pathway, leading to induction of heme oxygenase-1 (HO-1), both in the presence and absence of LPS. Although the withanolides were also capable of inhibiting LPS-induced NO production and stimulating Nrf2/HO-1 pathway, Withaferin A was tenfold more effective than Withanolide A. In serum-free culture, LPS can also induce production of long thin processes (filopodia) between 4 and 8h in BV-2 cells. This morphological change was significantly suppressed by Ashwagandha and both withanolides at concentrations for suppressing LPS-induced NO production. Taken together, these results suggest an immunomodulatory role for Ashwagandha and its withanolides, and their ability to suppress oxidative and inflammatory responses in microglial cells by simultaneously down-regulating the NF-kB and upregulating the Nrf2 pathways.

PI3k/Akt signalling pathway plays a crucial role in the anti-inflammatory effects of curcumin in LPS-activated microglia

Microglia are resident macrophages in the central nervous system (CNS) deputed to defend against pathogens. Persistent or acute inflammation of microglia leads to CNS disorders, so regulation of pro-inflammatory responses of microglial cells is thought to be a promising therapeutic strategy to attenuate abnormal inflammatory responses observed in neurodegenerative disease. We hypothesized that curcumin supplementation could reduce the inflammatory responses of activated microglial cells modulating PI3K/Akt pathway. Different curcumin concentrations were administered as BV-2 microglia pre-treatment 1h prior to LPS stimulation. Nitric oxide (NO) and inducible nitric oxide synthase (iNOS) expression were determined by Griess reagent and western blotting, respectively. Inflammatory cytokines release was evaluated by ELISA and qRT-PCR. PI3K/Akt expression was analyzed by western blotting analysis. Curcumin significantly attenuated, in a dose-dependent manner, LPS-induced release of NO and pro-inflammatory cytokines, as well as iNOS expression. Interestingly, curcumin was able to reduce, again in a dose-dependent manner, PI3K/Akt phosphorylation as well as NF-κB activation in LPS-activated microglial cells. Overall these results suggest that curcumin plays an important role in the attenuation of LPS-induced inflammatory responses in microglial cells and that the mechanisms involve down-regulation of the PI3K/Akt signalling.

CD200Fc reduces TLR4-mediated inflammatory responses in LPS-induced rat primary microglial cells via inhibition of the NF-κB pathway.

Based on recent information, CD200Fc, a CD200R1 agonist, could attenuate the inflammatory response of microglial cells in autoimmune diseases and neuro-degeneration. However, the exact molecular mechanisms responsible for the anti-inflammatory activity of CD200Fc in microglial cells have not been elucidated. In the present study, we investigated the anti-inflammatory effects and the molecular mechanisms of CD200Fc in lipopolysaccharide (LPS)-stimulated rat primary microglial cells. The cell viability was measured by MTT assay. The LPS-induced cytokines release (IL-1β, IL-6, TNF-α, iNOS, MCP-1, and COX-2) was monitored by ELISA or real-time PCR, while NF-κB-related signals (MyD88, p-TAK1, TRIF, p-TBK1, p-IRF3, p-IκB, and NF-κB-P65) were assessed by real-time PCR, western blot and/or Immunofluorescent staining. CD200Fc and/or LPS exerted no significant cytotoxicity on microglial cells. LPS reduced the CD200R1 expression in microglial cells, and this effect was attenuated by CD200Fc. In addition, CD200Fc inhibited LPS-induced expression of TLR4 and its adapter molecules (MyD88 and p-TAK1, TRIF, p-TBK1, and p-IRF3), and abolished its interactions with MyD88, TAK1, and TRIF in microglial cells. CD200Fc also attenuated LPS-induced protein expression of p-IκB and NF-κB-P65 translocation to nucleus in microglial cells. Moreover, CD200Fc suppressed the LPS-induced release of inflammatory mediators in microglial cells, including IL-1β, IL-6, TNF-α, iNOS, MCP-1, and COX-2. These results indicated that CD200Fc displayed an anti-inflammatory effect in LPS-induced microglial cells by blocking TLR4-mediated NF-κB activation.

Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1.

A large group of flavonoids found in fruits and vegetables have been suggested to elicit health benefits due mainly to their anti-oxidative and anti-inflammatory properties. Recent studies with immune cells have demonstrated inhibition of these inflammatory responses through down-regulation of the pro-inflammatory pathway involving NF-κB and up-regulation of the anti-oxidative pathway involving Nrf2. In the present study, the murine BV-2 microglial cells were used to compare anti-inflammatory activity of quercetin and cyanidin, two flavonoids differing by their alpha, beta keto carbonyl group. Quercetin was 10 folds more potent than cyanidin in inhibition of lipopolysaccharide (LPS)-induced NO production as well as stimulation of Nrf2-induced heme-oxygenase-1 (HO-1) protein expression. In addition, quercetin demonstrated enhanced ability to stimulate HO-1 protein expression when cells were treated with LPS. In an attempt to unveil mechanism(s) for quercetin to enhance Nrf2/HO-1 activity under endotoxic stress, results pointed to an increase in phospho-p38MAPK expression upon addition of quercetin to LPS. In addition, pharmacological inhibitors for phospho-p38MAPK and MEK1/2 for ERK1/2 further showed that these MAPKs target different sites of the Nrf2 pathway that regulates HO-1 expression. However, inhibition of LPS-induced NO by quercetin was not fully reversed by TinPPIX, a specific inhibitor for HO-1 activity. Taken together, results suggest an important role of quercetin to regulate inflammatory responses in microglial cells and its ability to upregulate HO-1 against endotoxic stress through involvement of MAPKs.

Astragalus polysaccharide attenuates lipopolysaccharide-induced inflammatory responses in microglial cells: regulation of protein kinase B and nuclear factor-κB signaling.

Microglia play an important role in immune and inflammatory responses in the central nervous system. Astragalus polysaccharide (APS) has been reported as an immune stimulant for various inflammation-associated diseases in vivo. The present study investigated the effects of APS on lipopolysaccharide-stimulated inflammatory responses in microglial cells. Cultured BV2 microglial cells were pre-treated with APS (0-200 μg/ml) prior to lipopolysaccharide (50 ng/ml) stimulation. The production of proinflammatory mediators including inducible nitric oxide synthase (iNOS)/nitric oxide (NO), cyclooxygenase-2 (COX-2)/prostaglandin E (PGE2), tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were evaluated. APS dose-dependently reduced lipopolysaccharide stimulated nitric oxide and PGE2 production, as well as iNOS and cyclooxygenase-2 gene expression. It also attenuated proinflammatory cytokines IL-1β and TNF-α generation. In addition, APS inhibited nuclear factor-κB translocation by blockade of IκB degradation and suppressed protein kinase B phosphorylation in lipopolysaccharide-stimulated cells. The inhibitory effects of APS on lipopolysaccharide-stimulated inflammatory mediator production in microglia are associated with suppression of nuclear factor-κB and protein kinase B signaling pathways. APS may offer therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied with microglial activation.

Effects of postconditioning with propofol on lipopolysaccharide-induced inflammatory responses of microglial cells in rat brain tissues

Objective To evaluate the effects of postconditioning with propofol on lipopolysaccharide (LPS)-induced inflammatory responses of microglial cells in rat brain tissues.Methods The primary cultured microglial cells in brain tissues of Sprague-Dawley rats were seeded in 24 multi-well plates at a density of 1 × 105 cells/ml,and the microglial cells of 100 wells were randomly divided into 5 groups (n =20 each) using a random number table:control group (group C),LPS group (group L),and propofol 25,50 and 100 μmol/L groups (P25,P50,P100 groups).The cells were cultured routinely in group C.LPS 1 μg/ml was added and the cells were incubated for 24 h in group L.In P25,P20,and P100 groups,when the cells were incubated for 24 h with LPS 1 μg/ml,propofol with the final concentrations of 25,50 and 100 μmol/L was added,respectively.The cells were collected at 1 h of incubation with propofol for determination of the expression of inducible nitric oxide synthase (iNOS) mRNA,cyclooxygenase-2 (COX-2) mRNA,tumor necrosis factor-α (TNF-α) mRNA and interleukin-1β (IL-1β) mRNA (by RT-PCR).The supematant was separated for determination of the concentrations of nitric oxide (NO) (by Griess method) and pmstaglandin E2 (PGE2),TNF-α and IL-1β (by ELISA).Results Compared with group C,the expression of iNOS mRNA,COX-2 mRNA,TNF-α mRNA and IL-1β mRNA was significantly up-regulated and the concentrations of NO,PGE2,TNF-α and IL-1β in the supematant were increased in group L (P ＜ 0.01).Compared with group L,the expression of iNOS mRNA,COX-2 mRNA,TNF-α mRNA and IL-1β mRNA was significantly down-regulated and the concentrations of NO,PGE2,TNF-α and IL-1β in the supernatant were decreased in P50 and P100 groups (P ＜ 0.05 or 0.01),while no significant change in the indexes mentioned above was found in P25 group (P ＞ 0.05).Conclusion Postconditioning with propofol 50 and 100 μmol/L can inhibit LPS-induced inflammatory responses of microglial cells in rat brain tissues. Key words: Propofol; Microglia; Endotoxemia; Inflammation

Surfactin exhibits neuroprotective effects by inhibiting amyloid β-mediated microglial activation

Microglial-mediated neuroinflammation and neurotoxicity contribute to the pathogenesis of neurodegenerative diseases including Alzheimer's disease; therefore, control of microglial activation and subsequent suppression of neurotoxic pro-inflammatory molecules could provide a potential therapeutic approach for the treatment of such diseases. In this study, we investigated the effects of surfactin, a surfactant from Bacillus subtilis, on oligomeric amyloid β (Aβ)-induced microglial activation and neurotoxicity. Surfactin significantly suppressed expression of MMP-9, iNOS and COX-2, as well as production of ROS, NO, PGE2, TNF-α, IL-1β, IL-6 and MCP-1 in Aβ-stimulated BV-2 microglial cells. Moreover, surfactin markedly inhibited Aβ-induced nuclear translocation and activation of NF-κB as well as phosphorylation of JNK and p38 MAPK. Furthermore, surfactin protected hippocampal HT22 cells from indirect neuronal toxicity mediated by Aβ-treated microglial cells, but had no effect on Aβ-induced direct toxicity to HT22 cells. These results suggest that surfactin impairs the Aβ-induced inflammatory response of microglial cells and confers protection against indirect neurotoxicity to hippocampal cells. Our findings indicate that surfactin may have therapeutic potential for ameliorating Alzheimer's disease as well as other neurodegenerative disorders which involve neuroinflammation.

Prion Peptide Uptake in Microglial Cells – The Effect of Naturally Occurring Autoantibodies against Prion Protein

In prion disease, a profound microglial activation that precedes neurodegeneration has been observed in the CNS. It is still not fully elucidated whether microglial activation has beneficial effects in terms of prion clearance or whether microglial cells have a mainly detrimental function through the release of pro-inflammatory cytokines. To date, no disease-modifying therapy exists. Several immunization attempts have been performed as one therapeutic approach. Recently, naturally occurring autoantibodies against the prion protein (nAbs-PrP) have been detected. These autoantibodies are able to break down fibrils of the most commonly used mutant prion variant PrP106-126 A117V and prevent PrP106-126 A117V-induced toxicity in primary neurons. In this study, we examined the phagocytosis of the prion peptide PrP106-126 A117V by primary microglial cells and the effect of nAbs-PrP on microglia. nAbs-PrP considerably enhanced the uptake of PrP106-126 A117V without inducing an inflammatory response in microglial cells. PrP106-126 A117V uptake was at least partially mediated through scavenger receptors. Phagocytosis of PrP106-126 A117V with nAbs-PrP was inhibited by wortmannin, a potent phosphatidylinositol 3-kinase inhibitor, indicating a separate uptake mechanism for nAbs-PrP mediated phagocytosis. These data suggest the possible mechanisms of action of nAbs-PrP in prion disease.

Ethanol induces TLR4/TLR2 association, triggering an inflammatory response in microglial cells

Alcohol consumption can induce brain damage, demyelination, and neuronal death, although the mechanisms are poorly understood. Toll-like receptors are sensors of the innate immune system and their activation induces inflammatory processes. We have reported that ethanol activates and recruits Toll-like receptor (TLR)4 receptors within the lipid rafts of glial cells, triggering the production of inflammatory mediators and causing neuroinflammation. Since TLR2 can also participate in the glial response and in the neuroinflammation, we investigate the effects of ethanol on TLR4/TLR2 responses. Here, we demonstrate that ethanol up-regulates TLR4 and TLR2 expression in microglial cells, inducing the production of inflammatory mediators which triggers reactive oxygen species generation and neuronal apoptosis. Ethanol also promotes TLR4/TLR2 recruitment into lipid rafts-caveolae, mimicking their activation by their ligands, lipopolysaccharide, and lipoteichoic acid (LTA). Immunoprecipitation and confocal microscopy studies reveal that ethanol induces a physical association between TLR2 and TLR4 receptors, suggesting the formation of heterodimers. Using microglia from either TLR2 or TLR4 knockout mice, we show that TLR2 potentiates the effects of ethanol on the TLR4 response reflected by the activation of MAPKs and inducible NO synthase. In summary, we provide evidence for a mechanism by which ethanol triggers TLR4/TLR2 association contributing to the neuroinflammation and neurodegeneration associated with alcohol abuse.

Gua Lou Gui Zhi decoction suppresses LPS-induced activation of the TLR4/NF-κB pathway in BV-2 murine microglial cells

Toll-like receptor4 (TLR4)/nuclear factor-κB (NF-κB) signaling-mediated neuroinflammation contributes to secondary brain damage in ischemic stroke; therefore, anti-inflammatory therapy via suppression of the TLR4/NF-κB pathway could be a promising strategy for the treatment of stroke and post-stroke disabilities. Gua Lou Gui Zhi decoction (GLGZD) has long been used in China to clinically treat dysfunction after stroke such as muscular spasticity, but the precise mechanisms are largely unknown. In the present study, we evaluated the anti-inflammatory effect of GLGZD and investigated the underlying molecular mechanisms using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells as an invitro inflammatory model of neural cells. We found that GLGZD inhibited the inflammatory response in microglial cells as it significantly reduced LPS-induced expression of pro-inflammatory nitric oxide, tumour necrosis factor-α, interleukin (IL)-6 and IL-1β in BV-2 cells, in a dose-dependent manner. In addition, GLGZD treatment significantly decreased the protein expression of TLR4 and myeloid differentiation factor88, inhibited the phosphorylation of IκB and blocked the nuclear translocation of NF-κB in BV-2 cells, demonstrating its inhibitory effect on the activation of TLR4/NF-κB signaling. Collectively, our findings suggest that inhibition of the inflammatory response via suppression of the TLR4/NF-κB pathway may be one of the mechanisms through which GLGZD ameliorates the damage in ischemic cerebral tissues.

Magnolia polyphenols attenuate oxidative and inflammatory responses in neurons and microglial cells

BackgroundThe bark of magnolia has been used in Oriental medicine to treat a variety of remedies, including some neurological disorders. Magnolol (Mag) and honokiol (Hon) are isomers of polyphenolic compounds from the bark of Magnolia officinalis, and have been identified as major active components exhibiting anti-oxidative, anti-inflammatory, and neuroprotective effects. In this study, we investigate the ability of these isomers to suppress oxidative stress in neurons stimulated by the ionotropic glutamate receptor agonist N-methyl-D-aspartate (NMDA) and oxidative and inflammatory responses in microglial cells activated by interferon-γ (IFNγ) and lipopolysaccharide (LPS). We also attempt to elucidate the mechanism and signaling pathways involved in cytokine-induced production of reactive oxygen species (ROS) in microglial cells.MethodsDihydroethidium (DHE) was used to assay superoxide production in neurons, while CM-H2DCF-DA was used to test for ROS production in murine (BV-2) and rat (HAPI) immortalized microglial cells. NADPH oxidase inhibitors (for example, diphenyleneiodonium (DPI), AEBSF, and apocynin) and immunocytochemistry targeting p47phox and gp91phox were used to assess the involvement of NADPH oxidase. Western blotting was used to assess iNOS and ERK1/2 expression, and the Griess reaction protocol was employed to determine nitric oxide (NO) concentration.ResultsExposure of Hon and Mag (1–10 μM) to neurons for 24 h did not alter neuronal viability, but both compounds (10 μM) inhibited NMDA-stimulated superoxide production, a pathway known to involve NADPH oxidase. In microglial cells, Hon and Mag inhibited IFNγ±LPS-induced iNOS expression, NO, and ROS production. Studies with inhibitors and immunocytochemical assay further demonstrated the important role of IFNγ activating the NADPH oxidase through the p-ERK-dependent pathway. Hon and, to a lesser extent, Mag inhibited IFNγ-induced p-ERK1/2 and its downstream pathway for ROS and NO production.ConclusionThis study highlights the important role of NADPH oxidase in mediating oxidative stress in neurons and microglial cells and has unveiled the role of IFNγ in stimulating the MAPK/ERK1/2 signaling pathway for activation of NADPH oxidase in microglial cells. Hon and Mag offer anti-oxidative or anti-inflammatory effects, at least in part, through suppressing IFNγ-induced p-ERK1/2 and its downstream pathway.

Activation of KCNN3/SK3/KCa2.3 channels attenuates enhanced calcium influx and inflammatory cytokine production in activated microglia

In neurons, small-conductance calcium-activated potassium (KCNN/SK/K(Ca)2) channels maintain calcium homeostasis after N-methyl-D-aspartate (NMDA) receptor activation, thereby preventing excitotoxic neuronal death. So far, little is known about the function of KCNN/SK/K(Ca)2 channels in non-neuronal cells, such as microglial cells. In this study, we addressed the question whether KCNN/SK/K(Ca)2 channels activation affected inflammatory responses of primary mouse microglial cells upon lipopolysaccharide (LPS) stimulation. We found that N-cyclohexyl-N-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-4-pyrimidinamine (CyPPA), a positive pharmacological activator of KCNN/SK/K(Ca)2 channels, significantly reduced LPS-stimulated activation of microglia in a concentration-dependent manner. The general KCNN/SK/K(Ca)2 channel blocker apamin reverted these effects of CyPPA on microglial proliferation. Since calcium plays a central role in microglial activation, we further addressed whether KCNN/SK/K(Ca)2 channel activation affected the changes of intracellular calcium levels, [Ca(2+)](i), in microglial cells. Our data show that LPS-induced elevation of [Ca(2+)](i) was attenuated following activation of KCNN2/3/K(Ca)2.2/K(Ca)2.3 channels by CyPPA. Furthermore, CyPPA reduced downstream events including tumor necrosis factor alpha and interleukin 6 cytokine production and nitric oxide release in activated microglia. Further, we applied specific peptide inhibitors of the KCNN/SK/K(Ca)2 channel subtypes to identify which particular channel subtype mediated the observed anti-inflammatory effects. Only inhibitory peptides targeting KCNN3/SK3/K(Ca)2.3 channels, but not KCNN2/SK2/K(Ca)2.2 channel inhibition, reversed the CyPPA-effects on LPS-induced microglial proliferation. These findings revealed that KCNN3/SK3/K(Ca)2.3 channels can modulate the LPS-induced inflammatory responses in microglial cells. Thus, KCNN3/SK3/K(Ca)2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in the central nervous system.

Anti-inflammatory effects of aromatic-turmerone through blocking of NF-κB, JNK, and p38 MAPK signaling pathways in amyloid β-stimulated microglia

Amyloid β (Aβ) induces the production of neuroinflammatory molecules, which may contribute to the pathogenesis of numerous neurodegenerative diseases. Therefore, suppression of neuroinflammatory molecules could be developed as a therapeutic method. Aromatic (ar)-turmerone, turmeric oil isolated from Curcuma longa, has long been used in Southeast Asia as both a remedy and a food. In this study, we investigated the anti-inflammatory effects of ar-turmerone in BV2 microglial cells. Aβ-stimulated microglial cells were tested for the expression and activation of MMP-9, iNOS, and COX-2, the production of proinflammatory cytokines, chemokines, and ROS, as well as the underlying signaling pathways. Ar-turmerone significantly suppressed Aβ-induced expression and activation of MMP-9, iNOS, and COX-2, but not MMP-2. Ar-turmerone also reduced TNF-α, IL-1β, IL-6, and MCP-1 production in Aβ-stimulated microglial cells. Further, ar-turmerone markedly inhibited the production of ROS. Impaired translocation and activation of NF-κB were observed in Aβ-stimulated microglial cells exposed to ar-turmerone. Furthermore, ar-turmerone inhibited the phosphorylation and degradation of IκB-α as well as the phosphorylation of JNK and p38 MAPK. These results suggest that ar-turmerone impaired the Aβ-induced inflammatory response of microglial cells by inhibiting the NF-κB, JNK, and p38 MAPK signaling pathways. Lastly, ar-turmerone protected hippocampal HT-22 cells from indirect neuronal toxicity induced by activated microglial cells. These novel findings provide new insights into the development of ar-turmerone as a therapeutic agent for the treatment of neurodegenerative disorders.

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.

Licochalcone E activates Nrf2/antioxidant response element signaling pathway in both neuronal and microglial cells: therapeutic relevance to neurodegenerative disease

Oxidative stress and neuroinflammation are hallmarks of neurodegenerative diseases, which do not play independently but work synergistically through complex interactions exacerbating neurodegeneration. Therefore, the mechanism that is directly implicated in controlling oxidative stress and inflammatory response could be an attractive strategy to prevent the onset and/or delay the progression of neurodegenerative diseases. The transcription factor nuclear factor-E2-related factor-2 (Nrf2) is the guardian of redox homeostasis by regulating a battery of antioxidant and phase II detoxification genes, which are relevant to defense mechanism against oxidative stress and inflammatory responses. In this study, we show that a recently identified Glycyrrhiza-inflata-derived chalcone, licochalcone E (Lico-E), attenuates lipopolysaccharide-induced inflammatory responses in microglial BV2 cells and protects dopaminergic SH-SY5Y cells from 6-hydroxydopamine cytotoxicity. Lico-E activates Nrf2-antioxidant response element (ARE) system and up-regulates downstream NAD(P)H:quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). Anti-inflammatory and cytoprotective effects of Lico-E are attenuated in siRNA-mediated Nrf2-silencing cells as well as in the presence with specific inhibitor of HO-1 or NQO1, respectively. Lico-E also has neuroprotective effect against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal dopaminergic neurodegeneration in mice, with up-regulation of HO-1 and NQO1 in the substantia nigra of the brain. This study demonstrates that Lico-E is a potential activator of the Nrf2/ARE-dependent pathway and is therapeutically relevant not only to oxidative-stress-related neurodegeneration but also inflammatory responses of microglial cells both in vitro and in vivo.

Artesunate activates Nrf2 pathway-driven anti-inflammatory potential through ERK signaling in microglial BV2 cells

Chronic inflammatory response in the brain is a characteristic etiopathology of various neurodegenerative diseases; consequently increasing the intrinsic anti-inflammatory potency could be an especially desirable strategy to prevent inflammation-related neuronal injuries. Transcription factor NF-E2-related factor-2 (Nrf2)-mediated control of redox homeostasis may participate in the modulation of microglial responses by regulating expression of important antioxidant and phase II detoxification genes. In our present work, we show that artesunate, a semi-synthetic derivative of anti-malarial agent artemisinin, attenuates LPS-induced inflammatory responses in microglial BV2 cells. Artesunate activates Nrf2-ARE system, and leads to an increase in the level of downstream heme oxygenase-1. Artesunate also activates PI3K/Akt, ERK, and JNK MAPKs signaling, but artesunate-induced activation of Nrf2 signaling and up-regulation of heme oxygenase-1 are ERK pathway-dependent. Collectively, this study demonstrates that artesunate is a potential activator of the Nrf2/ARE-dependent pathway and is therapeutically relevant to inflammatory responses of microglial cells.

Rough around the Edges: The Inflammatory Response of Microglial Cells to Spiky Nanoparticles

The versatility of nanoparticle design has established nanotechnology as a potential "one-stop solution" to many biological and medical applications. The capacity to control nanoparticle size, shape, and surface chemistry has enabled their use as imaging contrast agents or carriers for drugs and other compounds. However, concerns of nanoparticle toxicity have surfaced that could limit their clinical translation. In order to overcome this challenge, researchers are starting to characterize how particle properties influence their interactions with biological systems. By identifying the specific nanoparticle parameters responsible for toxicity, it may be possible to engineer safer and nontoxic nanoparticles.

Stimuli-responsive star poly(ethylene glycol) drug conjugates for improved intracellular delivery of the drug in neuroinflammation

N-Acetyl cysteine (NAC) is a vital drug currently under clinical trials for the treatment of neuroinflammation in maternal–fetal applications. The free sulfhydryl groups in NAC lead to high plasma protein binding, resulting in low bioavailability. Preparation and activity of conjugates of NAC with thiol terminated multi-arm (6 and 8) poly(ethylene-glycol) (PEG) with disulfide linkages involving sulfhydryls of NAC are reported. Multiple copies (5 and 7) of NAC were conjugated on 6 and 8-arm-PEG respectively. Both the conjugates released 74% of NAC within 2 h by thiol exchange reactions in the redox environment provided by glutathione (GSH) intracellularly (2–10 mM). At physiological extracellular glutathione concentration (2 µM) both the conjugates were stable and did not release NAC. MTT assay showed comparable cell viability for unmodified PEGs and both the PEG–S–S–NAC conjugates. The conjugates were readily endocytosed by cells, as confirmed by flow cytometry and confocal microscopy. Efficacy of 6 and 8-arm-PEG–S–S–NAC conjugates was evaluated on activated microglial cells (the target cells, in vivo) by monitoring cytokine release in lipopolysaccharide (LPS) induced inflammatory response in microglial cells using the reactive oxygen species (ROS), free radical nitrile (NO), anti-inflammatory activity and GSH depletion. The conjugates showed significant increase in antioxidant activity (more than a factor of 2) compared to free drug as seen from the inhibition of LPS induced ROS, NO, GSH and tumor necrosis factor-alpha (TNF-α) release in microglial cells.

Tripchlorolide protects neuronal cells from microglia‐mediated β‐amyloid neurotoxicity through inhibiting NF‐κB and JNK signaling

Recent research has focused on soluble oligomeric assemblies of beta-amyloid peptides (Abeta) as the proximate cause of neuroinflammation, synaptic loss, and the eventual dementia associated with Alzheimer's disease (AD). In this study, tripchlorolide (T4), an extract of Tripterygium wilfordii Hook. F (TWHF), was studied as a novel agent to suppress neuroinflammatory process in microglial cells and to protect neuronal cells against microglia-mediated oligomeric Abeta toxicity. T4 significantly attenuated oligomeric Abeta(1-42)-induced release of inflammatory productions such as tumor necrosis factor-alpha, interleukin-1beta, nitric oxide (NO), and prostaglandin E2. It also downregulated the protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in microglial cells. Further molecular mechanism study demonstrated that T4 inhibited the nuclear translocation of nuclear factor-kappaB (NF-kappaB) without affecting I-kappaBalpha phosphorylation. It repressed Abeta-induced JNK phosphorylation but not ERK or p38 MAPK. The inhibition of NF-kappaB and JNK by T4 is correlated with the suppression of inflammatory mediators in Abeta-stimulated microglial cells. These results suggest that T4 protects neuronal cells by blocking inflammatory responses of microglial cells to oligomeric Abeta(1-42) and that T4 acts on the signaling of NF-kappaB and JNK, which are involved in the modulation of inflammatory response. Therefore, T4 may be an effective agent in modulating neuroinflammatory process in AD.

Gene expression analysis of the murine model of amyotrophic lateral sclerosis: Studies of the Leu126delTT mutation in SOD1

The pathogenic events that lead to amyotrophic lateral sclerosis (ALS) have not been elucidated. We previously described familial amyotrophic lateral sclerosis (FALS) caused by a Leu126delTT mutation in the Cu/Zn superoxide dismutase gene (SOD1) and have produced transgenic mice (TgM) carrying the same mutation (SOD1 L126delTT TgM), which exhibited distinct ALS-like motor symptoms and pathological findings. In this study, we analyzed gene expression in the spinal cord of SOD1 L126delTT TgM by cDNA microarray. Eleven genes were upregulated and two genes downregulated in pre-symptomatic TgM. In post-symptomatic TgM, 54 genes were upregulated and four genes downregulated. We performed real-time polymerase chain reaction (PCR) analysis of 10 of the 54 upregulated genes in the post-symptomatic TgM. The results of real-time PCR were consistent with those obtained by microarray for μ-crystallin (Crym), heat shock protein 1 (Hspb1/HSP27), serine proteinase inhibitor clade A member 3N (Serpina3n), complement component 1q subcomponent beta polypeptide (C1qb), cathepsin H (Ctsh) and polyadenylate binding protein-interacting protein 1 (Paip1). In immunohistochemical analysis, Hsbp1/HSP27 and Ctsh expression levels were increased in reactive astrocytes at the ventral horn of the spinal cord in post-symptomatic TgM, as were Crym, some of Ctsh and Paip1 in microglial cells. Increased expression of those genes was not observed in the control mice. These four genes may be related to the pathogenesis of FALS, especially with regard to the progression of reactive astrocytes and the inflammatory response of microglial cells.