Treatment Of BV2 Cells Research Articles

Tangeretin alleviates inflammation and oxidative response induced by spinal cord injury by activating the Sesn2/Keap1/Nrf2 pathway.

Spinal cord injury (SCI) is a severe disabling disease that is characterized by inflammation and oxidative reactions. Tangeretin has been shown to possess significant antioxidant and anti-inflammatory activities. The Keap1/Nrf2 pathway, downstream of the Sesn2 gene, is involved in regulating the inflammation and oxidative response. The main objective of this study was to investigate the effect of tangeretin on SCI and its possible mechanism through cell and animal models. A T9 clamp injury was used for the mouse model and the LPS-induced stimulation of BV-2 cells was used for the cell model. The improvement of motor function after SCI was assessed by open field, swimming, and footprint experiments. The morphological characteristics of mouse spinal cord tissue and the levels of INOS, Sesn2, TNF-α, Keap1, Nrf2, IL-10, and reactive oxygen species (ROS) in vivo and in vitro were measured by several methods including western blotting, qPCR, immunofluorescence, HE, and Nissl staining. In vivo data showed that tangeretin can improve motor function recovery and reduce neuron loss and injury size in mice with SCI. Simultaneously, the in vitro findings suggested that treatment of BV-2 cells with tangeretin after LPS stimulation reduced the production of inflammatory factors and ROS, and could convert BV-2 cells from the M1 to the M2 type. Furthermore, Sesn2 knockout suppressed Keap1/Nrf2, inflammatory factors, ROS levels, and the M1 to M2 transition. Tangeretin can alleviate the inflammation and oxidative response induced by SCI by activating the Sesn2/Keap1/Nrf2 pathway.

Sulforaphane reverses Aβ fiber-mediated M1 type microglia polarization and neuroinflammation-mediated necroptosis of neural stem cells by downregulating the MAPK/NF-κB signaling pathways

To explore the effects of sulforaphane (SFN) and Aβ25-35 fibers (fAβ25-35) on M1/M2 polarization of BV-2 cells and neuroinflammation-mediated programmed necrosis of neural stem cells. BV-2 cells treated with different concentrations of fAβ25-35 and SFN were examined for changes in cell viability using the CCK-8 kit. The effect of fAβ25-35 alone or in combination with SFN or SB203580 on expressions of IL-6 and TNF-α mRNA and proteins were assessed in BV-2 cells using RT-qPCR and ELISA. CD16/32 and CD206 in the treated cells were analyzed with flow cytometry, and the cellular expressions of p-p38 and p-p65 protein were detected with Western blotting. C17.2 cells co-cultured with BV-2 cells for 24 h were examined for p-mlkl protein expression using Western blotting. fAβ25-35 at the concentration of 6.25 μmol/L significantly increased the viability of BV-2 cells (P < 0.01) whereas fAβ25-35 beyond 50 μmol/L decreased the cell viability (P < 0.0001). Treatment of BV-2 cells with SFN below 10 μmol/L for 24 h did not significant affect the cell viability (P > 0.05). BV-2 cells treated with fAβ25-35 alone, as compared with the cells in the other 3 groups, showed significantly increased IL-6 and TNF-α mRNA and protein expressions (P < 0.001), enhanced CD16/32 expression (P < 0.05), lowered CD206 expression (P < 0.01), and increased protein expressions of p-p38 and p- p65 (P < 0.01). C17.2 cells co-cultured with BV-2 cells treated with fAβ25-35, compared with the combined treatments, showed a significant reduction in the protein expression of p-mlkl (P < 0.05). SFN reverses M1 type microglia polarization and neuroinflammation-mediated programmed necrosis of neural stem cells by downregulating the MAPK/NF-κB signaling pathway in Aβ25-35-activated BV-2 cells.

Discovery of a NADPH oxidase inhibitor, (E)-3-cyclohexyl-5-(4-((2-hydroxyethyl)(methyl)amino)benzylidene)-1-methyl-2-thioxoimidazolidin-4-oneone, as a novel therapeutic for Parkinson's disease

Several lines of evidence indicated that generation of NADPH oxidase (Nox)-mediated reactive oxygen species are associated with neuronal inflammation, leading to Parkinson's disease (PD). Novel benzylidene-1-methyl-2-thioxoimidazolidin-one derivatives as Nox inhibitors were designed and synthesized in order to increase blood-brain barrier (BBB) permeability to target Nox in brain cells. In lucigenin chemiluminescence assay, eight compounds showed excellent inhibition activity against NADPH oxidases and parallel artificial membrane permeability assay (PAMPA) identified compound 11 with high passive permeability. To validate the effect of compound 11 on neuronal inflammation, we tested the regulatory activity of compound 11 in lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in BV-2 microglial cells and LPS-mediated microglial migration. Treatment of BV2 cells with compound 11 resulted in suppressed production of pro-inflammatory cytokines and migration activity of BV2 cells in response to LPS. To evaluate the therapeutic efficacy of compound 11 in PD animal model, compound 11 was applied to MPTP-induced PD mouse model. Oral administration of compound 11 (30 mg/kg/daily, 4 weeks) into the mice resulted in suppression of dopaminergic neuronal death in substantia nigra (SN) and in striatum as well as inhibition of microglial migration into SN. These results implicate compound 11 as a novel therapeutic agent for the treatment of PD.

Double-negative T cells mediate M1 polarization of microglial cells via TNF-α-NLRP3 to aggravate neuroinflammation and cognitive impairment in Alzheimer's disease mice.

We mainly study the role and regulatory mechanism of double-negative T cells(DNTs) in Alzheimer's disease(AD). The mice splenic DNTs were separated and amplified by Rosettesep antibody adsorption method and Easysep magnetic activated cell sorting. DNTs were intraperitoneally injected into the APP/PS1-AD mice model, which was found to aggravate cognitive impairment in mice. DNTs secreted tumor necrosis factor α (TNF-α) to promote the activation of NLRP3 and the M1 polarization of microglial cells, and silencing NLRP3 with small interfering RNA (siRNA) suppressed the effect of DNTs. DNTs were later cocultured with mice microglial cell line BV2, then fluorescence staining was conducted to detect NLRP3 expression, and enzyme-linked immunoassay was performed to measure the expression of inflammatory factors. Moreover, the levels of NLRP3, ASC, and TNFR1 proteins were detected by western-blot assay, and the proportion of F4/80 + CD11b + M1 cells was detected by flow cytometry. DNTs promoted the M1 polarization of BV2 cells and the activation of NLRP3 inflammasome. After treatment of BV2 cells with NLRP3 inhibitor, the effect of DNTs was weakened. Later, TNF-α siRNA was transfected into DNTs, and it was found that DNTs with TNF-α silencing had markedly weakened polarization effect on BV2 cells. We discovered that the proportion of DNTs increased in AD patients. DNTs secreted TNF-α to regulate the activation of NLRP3 inflammasome and the M1 polarization of microglial cells, thus promoting the central inflammatory response and aggravating the cognitive impairment in AD mice.

Inhibition of PDE1-B by Vinpocetine Regulates Microglial Exosomes and Polarization Through Enhancing Autophagic Flux for Neuroprotection Against Ischemic Stroke.

Exosomes contribute to cell–cell communications. Emerging evidence has shown that microglial exosomes may play crucial role in regulation of neuronal functions under ischemic conditions. However, the underlying mechanisms of microglia-derived exosome biosynthesis are largely unknown. Herein, we reported that the microglial PDE1-B expression was progressively elevated in the peri-infarct region after focal middle cerebral artery occlusion. By an oxygen-glucose-deprivation (OGD) ischemic model in cells, we found that inhibition of PDE1-B by vinpocetine in the microglial cells promoted M2 and inhibited M1 phenotype. In addition, knockdown or inhibition of PDE1-B significantly enhanced the autophagic flux in BV2 cells, and vinpocetine-mediated suppression of M1 phenotype was dependent on autophagy in ischemic conditions. Co-culture of BV2 cells and neurons revealed that vinpocetine-treated BV2 cells alleviated OGD-induced neuronal damage, and treatment of BV2 cells with 3-MA abolished the observed effects of vinpocetine. We further demonstrated that ischemia and vinpocetine treatment significantly altered microglial exosome biogenesis and release, which could be taken up by recipient neurons and regulated neuronal damage. Finally, we showed that the isolated exosome per se from conditioned BV2 cells is sufficient to regulate cortical neuronal survival in vivo. Taken together, these results revealed a novel microglia-neuron interaction mediated by microglia-derived exosomes under ischemic conditions. Our findings further suggest that PDE1-B regulates autophagic flux and exosome biogenesis in microglia which plays a crucial role in neuronal survival under cerebral ischemic conditions.

NQO1 mediates the anti-inflammatory effects of nootkatone in lipopolysaccharide-induced neuroinflammation by modulating the AMPK signaling pathway

Neuroinflammation and oxidative stress play key roles in the progression of neurodegenerative diseases. Thus, the use of potent anti-inflammatory/antioxidant agents has been suggested as a promising therapeutic strategy for neurodegenerative diseases. In the present study, we investigated the anti-inflammatory and antioxidant effects of nootkatone (NKT), a sesquiterpenoid compound isolated from grapefruit, in in vitro and in vivo models of neuroinflammation. In lipopolysaccharide (LPS)-stimulated BV2 microglial cells, NKT inhibited the expression of iNOS, COX-2, and pro-inflammatory cytokines, and increased the expression of the anti-inflammatory cytokine, IL-10. In addition, NKT inhibited reactive oxygen species (ROS) production and upregulated the expression of antioxidant enzymes, such as NQO1 and HO-1. Molecular mechanistic studies showed that NKT inhibited Akt, p38 MAPK, and NF-κB activities, while increasing AMPK, PKA/CREB, and Nrf2/ARE signaling in LPS-stimulated BV2 cells. Since NKT dramatically increased NQO1 expression, we investigated the role of this enzyme using pharmacological inhibition or knockdown experiments. Treatment of BV2 cells with the NQO1-specific inhibitor, dicoumarol, or with NQO1 siRNA significantly blocked NKT-mediated inhibition of NO, ROS, TNF-α, IL-1β, and upregulation of IL-10. Furthermore, NQO1 inhibition reversed the effects of NKT on pro- and anti-inflammatory signaling molecules. Intriguingly, we found that the AMPK inhibitor, compound C, mimicked the effects of dicoumarol, suggesting the presence of a crosstalk between NQO1 and AMPK. Finally, we demonstrated that NKT inhibited microglial activation, lipid peroxidation, and the expression of pro-inflammatory markers in the brains of LPS-injected mice, which was also reversed by dicoumarol. These data collectively suggest that NQO1 plays a critical role in mediating the anti-inflammatory and antioxidant effects of NKT in LPS-induced neuroinflammation by modulating AMPK and its downstream signaling pathways.

Stimulation of toll-like receptor 4 downregulates the expression of α7 nicotinic acetylcholine receptors via histone deacetylase in rodent microglia

Microglia have both protective and degenerative roles in the central nervous system. The α7 nicotinic acetylcholine receptor (nAChR) is crucial in the regulation of the neuroprotective role in microglia. Recent studies have demonstrated decreased expression of α7 nAChR in brain in response to neuroinflammation, but the mechanism mediating the downregulation of the α7 nAChR has yet to be elaborated. Treatment of microglial cell line BV2 cells or rat primary cultured microglia with the inflammogen lipopolysaccharide (LPS) significantly decreased the expression of α7 nAChR mRNA in a time and concentration-dependent manner. The effects of LPS were prevented by pretreatment with TAK-242, a toll-like receptor 4 (TLR4) blocker. The LPS-induced downregulation of α7 nAChR was also prevented with trichostatin A, a histone deacetylase (HDAC) inhibitor, but not 5-aza-2′-deoxycytidine, a DNA methyltransferase inhibitor. Further pharmacological probing revealed that HDAC2 and HDAC3 were involved in the effects of LPS. Treatment of BV2 cells with LPS significantly reduced acetylation of histone H3 at lysine 9 of the α7 nAChR promoter. The current findings demonstrate that inflammation-evoked activation of TLR4 leads to the reduction of the neuroprotective function of microglia through the downregulation of the α7 nAChR. Also, histone modification could be crucial in the regulation of the neuroprotective role of microglia during neuroinflammatory states.

Advanced oxidation protein products induce microglia-mediated neuroinflammation via MAPKs-NF-\u03baB signaling pathway and pyroptosis after secondary spinal cord injury

BackgroundInflammatory response mediated by oxidative stress is considered as an important pathogenesis of spinal cord injury (SCI). Advanced oxidation protein products (AOPPs) are novel markers of oxidative stress and their role in inflammatory response after SCI remained unclear. This study aimed to investigate the role of AOPPs in SCI pathogenesis and explore the possible underlying mechanisms.MethodsA C5 hemi-contusion injury was induced in Sprague-Dawley rats to confirm the involvement of AOPPs after SCI. For in vivo study, apocynin, the NADPH oxidase inhibitor was used to study the neuroprotective effects after SCI. For in vitro study, the BV2 microglia cell lines were pretreated with or without the inhibitor or transfected with or without small interference RNA (siRNA) and then stimulated with AOPPs. A combination of molecular and histological methods was used to clarify the mechanism and explore the signaling pathway both in vivo and in vitro. One-way analysis of variance (ANOVA) was conducted with Bonferroni post hoc tests to examine the differences between groups.ResultsThe levels of AOPPs in plasma and cerebrospinal fluid as well as the contents in the spinal cord showed significant increase after SCI. Meanwhile, apocynin ameliorated tissue damage in the spinal cord after SCI, improving the functional recovery. Immunofluorescence staining and western blot analysis showed activation of microglia after SCI, which was in turn inhibited by apocynin. Pretreated BV2 cells with AOPPs triggered excessive generation of reactive oxygen species (ROS) by activating NADPH oxidase. Increased ROS induced p38 MAPK and JNK phosphorylation, subsequently triggering nuclear translocation of NF-κB p65 to express pro-inflammatory cytokines. Also, treatment of BV2 cells with AOPPs induced NLRP3 inflammasome activation and cleavage of Gasdermin-d (GSDMD), causing pyroptosis. This was confirmed by cleavage of caspase-1, production of downstream mature interleukin (IL)-1β and IL-18 as well as rupture of rapid cell membrane.ConclusionsCollectively, these data indicated AOPPs as biomarkers of oxidative stress, modulating inflammatory response in SCI by multiple signaling pathways, which also included the induction of NADPH oxidase dependent ROS, and NLRP3-mediated pyroptosis, and activation of MAPKs and NF-κB.

Antioxidant activity and inhibitory effect on nitric oxide production of rang chuet (Thunbergia laurifolia Lindl.) leaf extracts in lipopolysaccharide-stimulated BV2 microglial cells

Background: Oxidative stress and neuroinflammation mediated by microglial activation play a significant role in the pathogenesis of neurodegenerative diseases. Therefore, negative regulators of microglial activation have been identified as potential therapeutic candidates for the treatment of such diseases. Objectives: The aim of this study was to investigate the effects of Thunbergia laurifolia Lindl. or Rang Chuet (RC) extracts on reactive oxygen species (ROS) and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV2 microglia cells. Materials and Methods: BV2 cells were treated with LPS in the presence or absence of RC extract, and the levels of ROS and NO were measured using CM-H2DCFDA and Griess reagent assay, respectively. The nuclear levels of nuclear factor kappa B (NF-kB) p65 and the activation of Akt and the extracellular signal-regulated kinase 1/2 (ERK1/2) were detected using immunofluorescence and western blotting assay, respectively. Results: The treatment of BV2 cells with RC concentration-dependently suppressed the LPS-induced ROS production compared to the control group. RC treatment also significantly decreased LPS-induced NO production in a concentration-dependent manner. In addition, RC treatment also suppressed NF-kB p65 translocation and the activation of Akt and the ERK1/2 pathway in LPS-stimulated BV2 cells. Conclusion: Taken together, these results demonstrate that RC exerts anti-oxidative activity by suppressing ROS production and that it exerts antioxidative activity by suppressing NO in LPS-stimulated BV2 cells. The mechanisms by which RC suppresses NO and ROS production may occur through inhibition of Akt and the ERK1/2-mediated NF-κB pathway. The results suggest that RC may be useful in treating neurodegenerative diseases mediated by microglial cells.

Soluble iron accumulation induces microglial glutamate release in the spinal cord of sporadic amyotrophic lateral sclerosis.

Previous studies on sporadic amyotrophic lateral sclerosis (SALS) demonstrated iron accumulation in the spinal cord and increased glutamate concentration in the cerebrospinal fluid. To clarify the relationship between the two phenomena, we first performed quantitative and morphological analyses of substances related to iron and glutamate metabolism using spinal cords obtained at autopsy from 12 SALS patients and 12 age-matched control subjects. Soluble iron content determined by the Ferrozine method as well as ferritin (Ft) and glutaminase C (GLS-C) expression levels on Western blots were significantly higher in the SALS group than in the control group, while ferroportin (FPN) levels on Western blots were significantly reduced in the SALS group as compared to the control group. There was no significant difference in aconitase 1 (ACO1) and tumor necrosis factor-alpha (TNFα)-converting enzyme (TACE) levels on Western blots between the two groups. Immunohistochemically, Ft, ACO1, TACE, TNFα, and GLS-C were proven to be selectively expressed in microglia. Immunoreactivities for FPN and hepcidin were localized in neuronal and glial cells. Based on these observations, it is predicted that soluble iron may stimulate microglial glutamate release. To address this issue, cell culture experiments were carried out on a microglial cell line (BV-2). Treatment of BV-2 cells with ferric ammonium citrate (FAC) brought about significant increases in intracellular soluble iron and Ft expression levels and conditioned medium glutamate and TNFα concentrations. Glutamate concentration was also significantly increased in conditioned media of TNFα-treated BV-2 cells. While the FAC-driven increases in glutamate and TNFα release were completely canceled by pretreatment with ACO1 and TACE inhibitors, respectively, the TNFα-driven increase in glutamate release was completely canceled by GLS-C inhibitor pretreatment. Moreover, treatment of BV-2 cells with hepcidin resulted in a significant reduction in FPN expression levels on Western blots of the intracellular total protein extracts. The present results provide in vivo and in vitro evidence that microglial glutamate release in SALS spinal cords is enhanced by intracellular soluble iron accumulation-induced activation of ACO1 and TACE and by increased extracellular TNFα-stimulated GLS-C upregulation, and suggest a positive feedback mechanism to maintain increased intracellular soluble iron levels, involving TNFα, hepcidin, and FPN.

A Fragment of Apolipoprotein E4 Leads to the Downregulation of a CXorf56 Homologue, a Novel ER-Associated Protein, and Activation of BV2 Microglial Cells.

Despite the fact that harboring the apolipoprotein E4 (APOE4) allele represents the single greatest risk factor for late-onset Alzheimer's disease (AD), the exact mechanism by which apoE4 contributes to disease progression remains unknown. Recently, we demonstrated that a 151 amino-terminal fragment of apoE4 (nApoE41-151) localizes within the nucleus of microglia in the human AD brain, suggesting a potential role in gene expression. In the present study, we investigated this possibility utilizing BV2 microglia cells treated exogenously with nApoE41-151. The results indicated that nApoE41-151 leads to morphological activation of microglia cells through, at least in part, the downregulation of a novel ER-associated protein, CXorf56. Moreover, treatment of BV2 cells with nApoE41-151 resulted in a 68-fold increase in the expression of the inflammatory cytokine, TNFα, a key trigger of microglia activation. In this regard, we also observed a specific binding interaction of nApoE41-151 with the TNFα promoter region. Collectively, these data identify a novel gene-regulatory pathway involving CXorf56 that may link apoE4 to microglia activation and inflammation associated with AD.

Complement protein C5a enhances the β-amyloid-induced neuro-inflammatory response in microglia in Alzheimer’s disease

The dysregulation of neuro-inflammation is one of the attributes of the pathogenesis of Alzheimer's disease (AD). Over-expression of complement proteins co-localizes with neurofibrillary tangles, thereby indicating that a complement system may be involved in neuro-inflammation. Here, we report the influence of complement activation on the neuro-inflammation using a microglial cell line. first, we performed a cytotoxic assay using the microglial cells BV-2. Second, after treatment of BV-2 cells with Aβ42 and/ or C5a, the anaphylatoxin derived from C5, we determined the expression levels of the pro-inflammatory factors TNF-α, IL-1β, and IL-6. Finally, we explored whether this neuroinflammatory response was mediated by JAK/ STAT3 signaling. C5a had an enhanced effect on the neural cell viability of BV-2 cells treated with Aβ42. In addition, C5a also increased the Aβ-induced neuro-inflammatory response, and these effects were blocked by the C5aR antagonist, PMX205. Finally, we demonstrated that the neuro-inflammatory responses induced by Aβ and C5a were mediated through JAK/STAT3 signaling. By blocking this pathway with an antagonist, AG490, the expression of TNF-α, IL-1β, and IL-6 was alleviated. The complement protein C5a could exaggerate the Aβ-induced neuroinflammatory response in microglia, and C5aR may be a potential therapeutic tool for AD treatment.

LRRK2 Kinase Activity Induces Mitochondrial Fission in Microglia via Drp1 and Modulates Neuroinflammation

Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD). LRRK2 contains a functional kinase domain and G2019S, the most prevalent LRRK2 pathogenic mutation, increases its kinase activity. LRRK2 regulates mitochondria morphology and autophagy in neurons. LPS treatment increases LRRK2 protein level and mitochondrial fission in microglia, and down-regulation of LRRK2 expression or inhibition of its kinase activity attenuates microglia activation. Here, we evaluated the direct role of LRRK2 G2019S in mitochondrial dynamics in microglia. Initial observation of microglia in G2019S transgenic mice revealed a decrease in mitochondrial area and shortage of microglial processes compared with their littermates. Next, we elucidated the molecular mechanisms of these phenotypes. Treatment of BV2 cells and primary microglia with LPS enhanced mitochondrial fission and increased Drp1, a mitochondrial fission marker, as previously reported. Importantly, both phenotypes were rescued by treatment with GSK2578215A, a LRRK2 kinase inhibitor. Finally, the protein levels of CD68, an active microglia marker, Drp1 and TNF-α were significantly higher in brain lysates of G2019S transgenic mice compared with the levels in their littermates. Taken together, our data suggest that LRRK2 could promote microglial mitochondrial alteration via Drp1 in a kinase-dependent manner, resulting in stimulation of pro-inflammatory responses. This mechanism in microglia might be a potential target to develop PD therapy since neuroinflammation by active microglia is a major symptom of PD.

Micheliolide suppresses LPS-induced neuroinflammatory responses.

Microglia-involved neuroinflammation is thought to promote brain damage in various neurodegenerative disorders. Thus, inhibition of microglial over-activation may have a therapeutic benefit for the treatment of neurodegenerative disorders. Micheliolide (MCL) is a sesquiterpene lactone which inhibits various inflammatory response. However, whether MCL can inhibit neuroinflammation caused by LPS-activated BV2 microglia has not yet been explored. In this study, we demonstrated that treatment of BV2 cells with MCL significantly repressed LPS-stimulated nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression, as well as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and nitric oxide (NO) induction. MCL also attenuated mRNA levels of multiple pro-inflammatory cytokines and mediators such as iNOS, COX-2, TNF-α, IL-6 and IL-1β. Mechanistic studies revealed that MCL suppressed LPS-stimulated the activation of IκBα/NF-κB pathway and Akt pathway. Moreover, MCL inhibited LPS-induced the activition of c-Jun N-terminal kinase (JNK), p38 MAPK kinase, and extracellular signal-regulated kinases 1/2 (ERK1/2). Meanwhile, MCL markedly promoted antioxidant protein heme oxygenase-1 (HO-1) expression by enhancing NF-E2-related factor 2 (Nrf2) activity. Together, our results imply that MCL may serve as a neuroprotective agent in neuroinflammation-related neurodegenerative disorders.

Okra (&lt;i&gt;Abelmoschus esculentus&lt;/i&gt; Linn) inhibits lipopolysaccharide-induced inflammatory mediators in BV2 microglial cells

Purpose: To investigate the inhibitory effects of okra (Abelmoschus esculentus Linn.) extract on the production of reactive oxygen species (ROS) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglia.Methods: Okra was extracted with ethanol by Soxhlet extraction. Non-cytotoxic doses of okra at concentrations of 50, 100 and 200 μg/mL were used in this study. BV2 cells were cultured and treated with LPS in the presence or absence of okra at the concentrations indicated above. ROS, nitric oxide (NO), tumor necrotic factor alpha (TNF-α), interleukin 1 beta (IL-1β), phosphorylation levels of nuclear factor-kappa B (NF-kB) p65 and Akt were determined.Results: Treatment of BV2 cells with okra concentrations of 50, 100 and 200μg/mL significantly suppressed LPS-induced NO as well as ROS compared to untreated cells. There was also a significant decrease in the production of TNF-α and IL-1β in okra-treated BV2 microglia cells. The level of LPSinduced NF-kB p65 phosphorylation was significantly decreased by okra treatment. In addition, okra inhibited LPS-induced Akt phosphorylation, which is an upstream molecule of NF-kB.Conclusion: Okra exerts anti-oxidative and anti-inflammatory effects in LPS-stimulated BV2 microglial cells by suppressing Akt-mediated NF-κB pathway. This suggests that okra might be a valuable agent for the treatment of anti-neuroinflammatory diseases mediated by microglial cells.Keywords: Abelmoschus esculentus Linn, Inflammatory cytokines, Lipopolysaccharide, Neuroinflammation, Microglia, Reactive oxygen species

Excretory and Secretory Proteins of Naegleria fowleri Induce Inflammatory Responses in BV-2 Microglial Cells.

Naegleria fowleri, a free-living amoeba that is found in diverse environmental habitats, can cause a type of fulminating hemorrhagic meningoencephalitis, primary amoebic meningoencephalitis (PAM), in humans. The pathogenesis of PAM is not fully understood, but it is likely to be primarily caused by disruption of the host's nervous system via a direct phagocytic mechanism by the amoeba. Naegleria fowleri trophozoites are known to secrete diverse proteins that may indirectly contribute to the pathogenic function of the amoeba, but this factor is not clearly understood. In this study, we analyzed the inflammatory responses in BV-2 microglial cells induced by excretory and secretory proteins of N.fowleri (NfESP). Treatment of BV-2 cells with NfESP induced the expression of various cytokines and chemokines, including the proinflammatory cytokines IL-1α and TNF-α. NfESP-induced IL-1α and TNF-α expression in BV-2 cells were regulated by p38, JNK, and ERK MAPKs. NfESP-induced IL-1α and TNF-α production in BV-2 cells were effectively downregulated by inhibition of NF-kB and AP-1. These results collectively suggest that NfESP stimulates BV-2 cells to release IL-1α and TNF-α via NF-kB- and AP-1-dependent MAPK signaling pathways. The released cytokines may contribute to inflammatory responses in microglia and other cell types in the brain during N.fowleri infection.

Mesenchymal stem cells enhance α-synuclein clearance via M2 microglia polarization in experimental and human parkinsonian disorder.

Microglia in the brain show distinctive phenotypes that serve different functions. In particular, M2-polarized microglia are anti-inflammatory and phagocytic cells that serve a restorative function. In this study, we investigated whether mesenchymal stem cells (MSCs) enhance the phagocytic clearance of α-synuclein via M2 microglia polarization, and thereby exert neuroprotective effects in α-synuclein-enriched experimental models and patients with multiple system atrophy (MSA). Treatment of BV2 cells with α-synuclein induced an inflammatory phenotype, whereas co-culture of α-synuclein-treated BV2 cells with MSCs induced an anti-inflammatory M2 phenotype, with decreased α-synuclein levels and increased lysosomal activity, leading to greater viability of neuronal cells co-cultured with BV2 cells. Using IL-4 receptor siRNA in BV2 cells and IL-4 siRNA in MSCs, we found that M2 microglia polarization was induced by IL-4 secreted from MSCs. In α-synuclein-inoculated mice, MSC treatment induced M2 microglia polarization decreased α-synuclein levels, and had a prosurvival effect on neurons. Using IL-4 and IL-4 receptor knockout mice, we further confirmed that IL-4 secreted from MSCs induced phagocytic clearance of α-synuclein through M2 microglia polarization. Next, we found that the cerebrospinal fluid (CSF) from MSC-transplanted MSA patients induced microglia M2 polarization and had a prosurvival effect via enhanced clearance of α-synuclein in α-synuclein-treated BV2 cells. Finally, a serial CSF study demonstrated that changes in oligomeric α-synuclein from baseline to 1-year follow-up were greater in the CSF of MSC-transplanted MSA patients than in placebo-transplanted MSA patients. These findings indicate that MSCs exert a neuroprotective effect via the clearance of extracellular α-synuclein by controlling microglia M2 polarization, suggesting that MSCs could be used as a disease-modifying therapy for patients with α-synucleinopathies.

Anti-inflammatory Mechanism of Ginseng Saponin Metabolite Rh3 in Lipopolysaccharide-Stimulated Microglia: Critical Role of 5′-Adenosine Monophosphate-Activated Protein Kinase Signaling Pathway

Ginsenoside Rh3 is a bacterial metabolite of Rg5, which is the main constituent of heat-processed ginseng. The present study was undertaken to examine the anti-inflammatory effect of ginsenoside Rh3 in lipopolysaccharide (LPS)-stimulated microglia. Rh3 inhibits the expressions of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6, at mRNA and protein levels, while Rh3 enhanced anti-inflammatory hemeoxygenase-1 expression. Moreover, Rh3 inhibited nuclear factor-κB (NF-κB) by upregulation of sirtuin 1 (SIRT1) and enhanced Nrf2 DNA-binding activities. Analysis of signaling pathways revealed that Rh3 enhanced the phosphorylation of 5'-adenosine monophosphate-activated protein kinase (AMPK) and inhibited Akt and janus kinase 1 (JAK1)/signal transducer and activator of transcription 1 (STAT1) induced by LPS. By treatment of BV2 cells with AICAR (a pharmacological activator of AMPK), we found that AMPK is an upstream regulator of phosphatidylinositol 3-kinase (PI3K)/Akt and JAK1/STAT1. Furthermore, AMPK knockdown experiments demonstrated the anti-inflammatory role of AMPK in LPS/Rh3-treated BV2 microglia. Our data collectively suggest that Rh3 exerts an anti-inflammatory effect in microglia by modulating AMPK and its downstream signaling pathways.

Inducible nitric oxide synthase is key to peroxynitrite-mediated, LPS-induced protein radical formation in murine microglial BV2 cells

Microglia are the resident immune cells in the brain. Microglial activation is characteristic of several inflammatory and neurodegenerative diseases including Alzheimer's disease, multiple sclerosis, and Parkinson's disease. Though lipopolysaccharide (LPS)-induced microglial activation in models of Parkinson's disease is well documented, the free radical-mediated protein radical formation and its underlying mechanism during LPS-induced microglial activation are not known. Here we have used immuno-spin trapping and RNA interference to investigate the role of inducible nitric oxide synthase (iNOS) in peroxynitrite-mediated protein radical formation in murine microglial BV2 cells treated with LPS. Treatment of BV2 cells with LPS resulted in morphological changes, induction of iNOS, and increased protein radical formation. Pretreatments with FeTPPS (a peroxynitrite decomposition catalyst), L-NAME (total NOS inhibitor), 1400W (iNOS inhibitor), and apocynin significantly attenuated LPS-induced protein radical formation and tyrosine nitration. Results obtained with coumarin-7-boronic acid, a highly specific probe for peroxynitrite detection, correlated with LPS-induced tyrosine nitration, which demonstrated involvement of peroxynitrite in protein radical formation. A similar degree of protection conferred by 1400W and L-NAME led us to conclude that only iNOS, and no other forms of NOS, is involved in LPS-induced peroxynitrite formation. Subsequently, siRNA for iNOS, the iNOS-specific inhibitor 1400W, the NF-κB inhibitor PDTC, and the p38 MAPK inhibitor SB202190 was used to inhibit iNOS directly or indirectly. Inhibition of iNOS precisely correlated with decreased protein radical formation in LPS-treated BV2 cells. The time course of protein radical formation also matched the time course of iNOS expression. Taken together, these results prove the role of iNOS in peroxynitrite-mediated protein radical formation in LPS-treated microglial BV2 cells.

Anthocyanin-rich Açai (Euterpe oleracea Mart.) Fruit Pulp Fractions Attenuate Inflammatory Stress Signaling in Mouse Brain BV-2 Microglial Cells

Age-related diseases of the brain compromise memory, learning, and movement and are directly linked with increases in oxidative stress and inflammation. Previous research has shown that supplementation with berries can modulate signaling in primary hippocampal neurons or BV-2 mouse microglial cells. Because of their high polyphenolic content, fruit pulp fractions of açai ( Euterpe oleracea Mart.) were explored for their protective effect on BV-2 mouse microglial cells. Freeze-dried açai pulp was fractionated using solvents with different polarities and analyzed using HPLC for major anthocyanins and other phenolics. Fractions extracted using methanol (MEOH) and ethanol (ETOH) were particularly rich in anthocyanins such as cyanidin, delphinidin, malvidin, pelargonidin, and peonidin, whereas the fraction extracted using acetone (ACE) was rich in other phenolics such as catechin, ferulic acid, quercetin, resveratrol, and synergic and vanillic acids. Studies were conducted to investigate the mitigating effects of açai pulp extracts on lipopolysaccharide (LPS, 100 ng/mL) induced oxidative stress and inflammation; treatment of BV-2 cells with acai fractions resulted in significant (p < 0.05) decreases in nitrite production, accompanied by a reduction in inducible nitric oxide synthase (iNOS) expression. The inhibition pattern was emulated with the ferulic acid content among the fractions. The protection of microglial cells by açai pulp extracts, particularly that of MEOH, ETOH, and ACE fractions, was also accompanied by a significant concentration-dependent reduction in cyclooxygenase-2 (COX-2), p38 mitogen-activated protein kinase (p38-MAPK), tumor necrosis factor-α (TNFα), and nuclear factor κB (NF-κB). The current study offers valuable insights into the protective effects of açai pulp fractions on brain cells, which could have implications for improved cognitive and motor functions.