LPS-stimulated BV2 Microglial Cells Research Articles

2-Hydroxy-4-Methylbenzoic Anhydride Inhibits Neuroinflammation in Cellular and Experimental Animal Models of Parkinson's Disease.

Microglia-mediated neuroinflammation is one of the key mechanisms involved in acute brain injury and chronic neurodegeneration. This study investigated the inhibitory effects of 2-hydroxy-4-methylbenzoic anhydride (HMA), a novel synthetic derivative of HTB (3-hydroxy-4-trifluoromethylbenzoic acid) on neuroinflammation and underlying mechanisms in activated microglia in vitro and an in vivo mouse model of Parkinson’s disease (PD). In vitro studies revealed that HMA significantly inhibited lipopolysaccharide (LPS)-stimulated excessive release of nitric oxide (NO) in a concentration dependent manner. In addition, HMA significantly suppressed both inducible NO synthase and cyclooxygenase-2 (COX-2) at the mRNA and protein levels in LPS-stimulated BV-2 microglia cells. Moreover, HMA significantly inhibited the proinflammatory cytokines such as interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha in LPS-stimulated BV-2 microglial cells. Furthermore, mechanistic studies ensured that the potent anti-neuroinflammatory effects of HMA (0.1, 1.0, and 10 μM) were mediated by phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) in LPS-stimulated BV-2 cells. In vivo evaluations revealed that intraperitoneal administration of potent neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, 20 mg/kg, four times a 1 day) in mice resulted in activation of microglia in the brain in association with severe behavioral deficits as assessed using a pole test. However, prevention of microglial activation and attenuation of Parkinson’s disease (PD)-like behavioral changes was obtained by oral administration of HMA (30 mg/kg) for 14 days. Considering the overall results, our study showed that HMA exhibited strong anti-neuroinflammatory effects at lower concentrations than its parent compound. Further work is warranted in other animal and genetic models of PD for evaluating the efficacy of HMA to develop a potential therapeutic agent in the treatment of microglia-mediated neuroinflammatory disorders, including PD.

Nitric Oxide Inhibitory Carbazole Alkaloids from the Folk Medicine Murraya tetramera C.C. Huang.

The phytochemical investigation of the leaves and stems of Murraya tetramera C.C. Huang, a traditional folk medicine used as an anti-inflammatory agent, yielded 19 simple carbazole alkaloids, two of which (1-ethoxy-3-methyl-9H-carbazol-2-ol (1) and 7-hydroxy-2,8-dimethoxy-6-methyl-9H-carbazole-1-carbaldehyde (2)) are new ones. The structures of the new compounds were determined by extensive spectroscopic analysis including NMR and HR-EI-MS experiments, as well as comparison with the reported data. Most of the isolates showed potent inhibitory effects on NO production in LPS-stimulated BV-2 microglial cells with IC50 values ranging from 5.1 to 15.1 μM.

Mucuna pruriens (L.) DC. seed extract inhibits lipopolysaccharide-induced inflammatory responses in BV2 microglial cells

Ethnopharmacological relevanceInflammation caused by activated microglia is known to be associated with neurodegenerative diseases, e.g., Parkinson's disease (PD) and Alzheimer's disease (AD). Inhibiting the inflammatory process can be considered a potential strategy for the treatment of inflammation-associated diseases. Mucuna pruriens (L.) DC. (Leguminosae) has long been used in Thailand, India, China and other tropical countries to treat several diseases including PD. M. pruriens seeds have been found to possess a variety of pharmacological properties including antioxidant and anti-Parkinsonism effects. However, the anti-inflammatory effects of M. pruriens seeds during microglial activation have yet to be reported. Aim of the studyThe present study was performed to evaluate the anti-inflammatory effects of M. pruriens seed extract and elucidate its underlying mechanism using lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Materials and methodsBV2 microglial cells were pretreated with various concentrations of M. pruriens seed extract before being stimulated with LPS. The levels of inflammatory mediators were analyzed by Griess assay and enzyme-linked immunoassay (ELISA). The protein expression levels of inflammatory cytokines were determined by Western blot analysis. The translocation of nuclear factor-kappa B (NF-κB) was assessed by immunofluorescence microscopy. ResultsM. pruriens seed extract significantly inhibited the release of inflammatory mediators including nitric oxide (NO), IL-1β, IL-6, and TNF-α in LPS-stimulated BV2 microglial cells. The extract also decreased the protein expression of IL-1β, IL-6, and TNF-α. Moreover, M. pruriens seed extract inhibited the translocation of NF-κB. ConclusionsM. pruriens seed extract could suppress inflammatory responses in LPS-activated BV2 microglial cells by inhibiting the NF-κB signaling pathway. These findings support the use of M. pruriens seeds in traditional and alternative medicine for the treatment of PD and other inflammation-associated diseases.

Phenolic glycosides from Oroxylum indicum

Two new phenolic glycosides, oroxylumosides A (1) and B (2), along with four known compounds darendoside A (3), leucosceptoside A (4), acteoside (5) and decaffeoylacteoside (6) were isolated from the stem bark of Oroxylum indicum. Their structures were elucidated by extensive analysis of the 1 D and 2 D NMR as well as HR-ESI-QTOF-MS. In addition, compounds 1 − 4 exhibited inhibitory effects on NO production in LPS-stimulated BV2 microglial cell line with IC50 values of 58.2 ± 2.9, 70.6 ± 3.5, 56.8 ± 2.8 and 61.1 ± 3.1 µM, respectively.

Oroxindols A and B, two novel secoabietane diterpenoids from Oroxylum indicum

Abstract Two novel secoabietane diterpenoids, oroxindols A (1) and B (2), along with two known flavones oroxylin A (3) and hispidulin (4) were isolated from the stem bark of Oroxylum indicum. Their structures were elucidated by extensive analysis of the 1D and 2D NMR as well as HR-ESI-QTOF mass spectra. Noteworthily, the isolation of compounds 1 and 2 represents the first case of 11β,13;12,13-diepoxy-12,13-secoabietane-type diterpene obtaining reported to date. In addition, compound 3 exhibited significant inhibitory effects on nitric oxide (NO) production in LPS-stimulated BV2 microglial cell line, with an IC50 value of 21.5 ± 1.1 μM.

The Efficacy of the Novel TSPO Ligands 2-Cl-MGV-1 and 2,4-Di-Cl-MGV-1 Compared to the Classical TSPO Ligand PK 11195 to Counteract the Release of Chemokines from LPS-Stimulated BV-2 Microglial Cells.

The impact of ligands of the 18 kDa translocator protein (TSPO) on the release of chemokines is not vastly investigated. In the present study, we assessed the effect of our novel TSPO ligands 2-Cl-MGV-1 and 2,4-Di-Cl-MGV-1 compared to the classical TSPO ligand PK 11195 on chemokine release in LPS-stimulated BV-2 microglial cells. As per the effect of 2-Cl-MGV-1, CCL2, CCL3, and CCL5 were inhibited by 90%, CCL8 by 97%, and IL-2 by 77% (p < 0.05 for all). 2,4-Di-Cl-MGV-1 inhibited CCL2 release by 92%, CCL3 by 91%, CCL5 by 90%, CCL8 by 89%, and IL-2 by 80% (p < 0.05 for all). PK 11195 exhibited weaker inhibitory effects: CCL2 by 22%, CCL3 by 83%, CCL5 by 34%, CCL8 by 41%, and the cytokine IL-2 by 14% (p < 0.05 for all). Thus, it appears that the novel TSPO ligands are potent suppressors of LPS-stimulated BV-2 microglial cells, and their inhibitory effect is larger than that of PK 11195. Such immunomodulatory effects on microglial cells may be relevant to the treatment of neurodegenerative and neuroinflammatory diseases.

Antineuroinflammatory Effects of 7,3',4'-Trihydroxyisoflavone in Lipopolysaccharide-Stimulated BV2 Microglial Cells through MAPK and NF-κB Signaling Suppression.

Neuroinflammation—a common pathological feature of neurodegenerative disorders such as Alzheimer’s disease—is mediated by microglial activation. Thus, inhibiting microglial activation is vital for treating various neurological disorders. 7,3’,4’-Trihydroxyisoflavone (THIF)—a secondary metabolite of the soybean compound daidzein—possesses antioxidant and anticancer properties. However, the effects of 7,3’,4’-THIF on microglial activation have not been explored. In this study, antineuroinflammatory effects of 7,3’,4’-THIF in lipopolysaccharide (LPS)-stimulated BV2 microglial cells were examined. 7,3’,4’-THIF significantly suppressed the production of the proinflammatory mediators nitric oxide (NO), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) as well as of the proinflammatory cytokine interleukin-6 (IL-6) in LPS-stimulated BV2 microglial cells. Moreover, 7,3’,4’-THIF markedly inhibited reactive oxygen species (ROS) generation. Western blotting revealed that 7,3’,4’-THIF diminished LPS-induced phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), glycogen synthase kinase-3β (GSK-3β), and nuclear factor kappa B (NF-κB). Overall, 7,3’,4’-THIF exerts antineuroinflammatory effects against LPS-induced microglial activation by suppressing mitogen-activated protein kinase (MAPK) and NF-κB signaling, ultimately reducing proinflammatory responses. Therefore, these antineuroinflammatory effects of 7,3’,4’-THIF suggest its potential as a therapeutic agent for neurodegenerative disorders.

Iridoid glucosides and phenylethanoid glycosides from Plantago major

Abstract Using various chromatographic separations, five iridoid glucosides (1―5) and five phenylethanoid glycosides (6―10) were isolated from the Plantago major whole plants and included two new compounds: 10-acetylarborescoside (1) and plantainoside G (6). Their structures were elucidated by detailed analysis of 1D and 2D NMR data, HR QTOF MS data, and experimental and calculated ECD data. The isolated compounds exhibited moderate inhibitory effects on NO overproduction in LPS-stimulated BV2 microglial cells, with IC50 values ranging from 44.2–62.3 μM.

Glutamine Cooperatively Upregulates Lipopolysaccharide-Induced Nitric Oxide Production in BV2 Microglial Cells through the ERK and Nrf-2/HO-1 Signaling Pathway

Glutamine (Gln) is a nonessential α-amino acid for protein biosynthesis. However, the mechanism through which Gln regulates NO production in microglial cells is still unclear. In this study, we investigated whether the presence or absence of Gln affects NO production in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Our data revealed that Gln depletion decreased cell viability accompanied by mild cytotoxicity, and blocked LPS-induced NO production concomitant with a significant decrease in inducible NO synthase (iNOS) expression. Additionally, Gln depletion for 24 h blocked the restoration of LPS-mediated NO production in the presence of Gln, suggesting that Gln depletion caused long-term immune deprivation. In particular, sodium-coupled amino acid transporter 1 and 2 (SNAT1 and SNAT2), which are the main Gln transporters, were highly upregulated in LPS-stimulated BV2 microglial cells, in the presence of Gln accompanied by NO production. Regardless of the presence of Gln, LPS positively stimulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) expression, and transient Nrf2 knockdown and HO-1 inhibition stimulated LPS-induced NO production and iNOS expression; however, transient Nrf2 knockdown did not affect SNAT1 and SNAT2 expression, indicating that Gln transporters, SNAT1 and SNAT2, were not regulated by Nrf2, which downregulated the HO-1-mediated NO production. Moreover, Gln depletion significantly reduced LPS-induced extracellular signal-regulated kinase (ERK) phosphorylation; furthermore, a specific ERK inhibitor, PD98059, and transient ERK knockdown attenuated LPS-stimulated NO production and iNOS expression, in the presence of Gln, accompanied by downregulation of SNAT1 and SNAT2, suggesting that the ERK signaling pathway was related to LPS-mediated NO production via SNAT1 and SNAT2. Altogether, our data indicated that extracellular Gln is vital for NO production from microglia in inflammatory conditions.

Dynorphin activation of kappa opioid receptor promotes microglial polarization toward M2 phenotype via TLR4/NF-\u03baB pathway

BackgroundMicroglia-mediated neuroinflammation is associated with epilepsy. Switching microglial polarization from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype represents a novel therapeutic strategy for mitigating epileptogenesis. We previously found that dynorphins protected against epilepsy via activation of kappa opioid receptor (KOR). Here, this study aims to investigate the role and the mechanism of dynorphin in regulating microglial polarization.MethodsA pilocarpine-induced rat model of epilepsy was established and lipopolysaccharide (LPS)-activated BV-2 microglial cells were used as an inflammatory model to explore the mechanism of dynorphin regulating microglial polarization.ResultsOverexpression of the dynorphin precursor protein prodynorphin (PDYN) alleviated the pilocarpine-induced neuronal apoptosis, promoted microglial polarization to the M2 phenotype, and inhibited pilocarpine-induced Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) pathway in the hippocampi of epileptic rats. Dynorphin activation of KOR promoted microglial M2 polarization via inhibiting TLR4/NF-κB pathway in LPS-stimulated BV-2 microglial cells. Moreover, dynorphin/KOR regulated microglial M2 polarization inhibited apoptosis of the primary mouse hippocampal neurons.ConclusionIn conclusion, dynorphin activation of KOR promotes microglia polarization toward M2 phenotype via inhibiting TLR4/NF-κB pathway.

Longxuetongluo capsule alleviates lipopolysaccharide-induced neuroinflammation by regulating multiple signaling pathways in BV2 microglia cells.

Longxuetongluo capsule (LTC), derived from the total phenolic compounds of Chinese dragon's blood, is now used in the treatment of ischemic stroke in convalescence. The aim of this study is to explore the neuroprotective effect of LTC from the perspective of neuroinflammation. Cell viability and lactate dehydrogenase (LDH) release were measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) and LDH assay kit. Proinflammatory mediators and cytokines production including Nitric Oxide (NO), prostaglandin E2, (PGE2), interleukin (IL-β), IL-6, and tumor necrosis factor-α (TNF-α) were detected by enzyme-linked immunosorbent assay (ELISA) assay. In addition, western blot was used to detect the expression of inflammatory proteins associated with the mitogen-activated protein kinases (MAPKs), janus kinase/signal transducer and activator of tranions (JAK/STAT), nuclear transcription factor κB (NF-κB), and nuclear factor erythroid-2-related actor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathways. Moreover, immunofluorescence assay and electrophoretic mobility shift assays (EMSA) were performed to determine the Nrf2 translocation and the binding-DNA activity of NF-κB, respectively. LTC at 0.5 to 2 μg/mL significantly increased cell viability and decreased LDH, NO, PGE2, IL-1β, IL-6, and TNF-α production in oxygen-glucose deprivation/reoxygenation (OGD/R) and lipopolysaccharide (LPS)-induced BV2 microglia cells. Meanwhile, LTC not only decreased the protein expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) but also down-regulated phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38, and up-regulated HO-1 expression via nuclear translocation of Nrf2. LTC can significantly inhibit the phosphorylation of JAK1/STAT3 and reduce the translocation of NF-κB from cytosol to nucleus as well as the binding-DNA activity. PC12 cell pretreated with LTC-condition medium (CM) significantly alleviated LPS-induced neurotoxicity and increased PC12 cell viability in a dose-dependent manner. The present study showed that LTC exhibited a strong antineuroinflammatory activity and neuroprotective effects on LPS-stimulated BV2 microglial cells and PC12 cells.

Inhibition of inflammatory mediators and cell migration by 1,2,3,4-tetrahydroquinoline derivatives in LPS-stimulated BV2 microglial cells via suppression of NF-κB and JNK pathway

Novel 1,2,3,4-tetrahydroquinoline derivatives with N-alkanoyl, N-benzoyl, or chlorobenzoyl substituents were designed and synthesized to inhibit nuclear factor-kappa B (NF-κB) known to be involved in the regulation of many immune and inflammatory responses. These compounds have been previously reported to inhibit NF-κB transcriptional activity in Raw 267.4 macrophage cells and exhibit cytotoxicities to several human cancer cell lines (Jo et al., ACS Med. Chem. Lett. 7 (2016) 385–390). Accumulating evidence indicated that NF-κB is also involved in neuroinflammation implicated in many neurodegenerative diseases. Thus, the present study investigated effects of 1,2,3,4-tetrahydroquinoline derivatives on LPS-stimulated inflammatory mediators and cell migration using BV2 microglial cells as a model. We found that seven compounds tested in this study inhibited LPS-induced pro-inflammatory mediators including interleukin-6, tumor necrosis factor-α, and nitric oxide in concentration-dependent manners. Among these compounds, ELC-D-2 exhibited the most potent inhibition without showing significant cytotoxicity. We also found that ELC-D-2 attenuated levels of LPS-induced inducible nitric oxide synthase and cyclooxygenase-2. Moreover, ELC-D-2 inhibited nuclear translocation of NF-κB by suppressing inhibitor of kappa Bα phosphorylation. Furthermore, ELC-D-2 inhibited LPS-induced activation of c-Jun N-terminal kinase (JNK), which was associated with suppression of inflammatory mediators and migration of LPS-treated BV2 cells. Collectively, our findings demonstrate that ELC-D-2 inhibits LPS-induced pro-inflammatory mediators and cell migration by suppressing NF-κB translocation and JNK phosphorylation in BV2 microglial cells. These results suggest that ELC-D-2 might have a beneficial impact on various brain disorders in which neuroinflammation involving microglial activation plays a crucial role in the pathogenesis of these diseases.

Dexamethasone sodium phosphate attenuates lipopolysaccharide-induced neuroinflammation in microglia BV2 cells.

Abnormal neuroinflammation ignited by overproduction of chemokines and cytokines via microglial cells can induce the occurrence and development of neurodegenerative disorders. The aim of this study is to investigate the effects of dexamethasone sodium phosphate (Dex-SP) on chemokine and cytokine secretion in lipopolysaccharide (LPS)-activated microglial cells. LPS markedly enhanced the secretion of pro-inflammatory factors such as regulated on activation, normal T cell expressed and secreted (RANTES), transforming growth factor beta-β1 (TGF-β1) and nitric oxide (NO), but decreased the production of macrophage inflammatory protein-1α (MIP-1α) and interleukin 10 (IL-10) in BV-2 microglial cells. Furthermore, LPS increased BV-2 microglial cell migration. However, Dex-SP treatment had the opposite effect, dampening the secretion of RANTES, TGF-β1, and NO, while increasing the production of MIP-1α and IL-10 and blocking migration of LPS-stimulated BV-2 microglial cells. Furthermore, Dex-SP markedly suppressed the LPS-induced degradation of IRAK-1 and IRAK-4, and blocked the activation in TRAF6, p-TAK1, and p-JNK in BV-2 microglial cells. These results showed that Dex-SP inhibited the neuroinflammatory response and migration in LPS-activated BV-2 microglia by inhibiting the secretion of RANTES, TGF-β1, and NO and increasing the production of MIP-1α and IL-10. The molecular mechanism of Dex-SP may be associated with inhibition of TRAF6/TAK-1/JNK signaling pathways mediated by IRAK-1 and IRAK-4.

Inhibitory Effects of Asparagus cochinchinensis in LPS-Stimulated BV-2 Microglial Cells through Regulation of Neuroinflammatory Mediators, the MAP Kinase Pathway, and the Cell Cycle

Inhibitory Effects of Asparagus cochinchinensis in LPS-Stimulated BV-2 Microglial Cells through Regulation of Neuroinflammatory Mediators, the MAP Kinase Pathway, and the Cell Cycle

Rhein Suppresses Neuroinflammation via Multiple Signaling Pathways in LPS‐Stimulated BV2 Microglia Cells

As a bioactive absorbed compound of rhubarb, Rhein is applied for the treatment of brain injury. However, the underlying pharmacological mechanisms remain unclear. In this study, we aimed to explore antineuroinflammatory functions and underlying mechanisms of Rhein in vitro. BV2 microglia cells were chosen and irritated by LPS. The influence of Rhein on cell viability was determined using MTT assay. We finely gauged the proinflammatory cytokines of TNF-α and IL-1β through tests of immunofluorescence staining, ELISA, RT-qPCR, and western blot. Additionally, mediators including IL-6, IL-12, iNOS, and IL-10 were surveyed by ELISA. Furthermore, protein levels of the underlying signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) were tested adopting western blot. We found that Rhein reduced the secretion of pivotal indicators including TNF-α and IL-1β, effectively restraining their mRNA and protein expression in LPS-activated BV2 microglial cells. Besides, Rhein treatment demoted the production of IL-6, IL-12, and iNOS and promoted the excretion of IL-10. Subsequent mechanistic experiments revealed that Rhein obviously downregulated the phosphorylation levels of PI3K, Akt, p38, and ERK1/2 and simultaneously upregulated the PTEN expression. In addition, Rhein antagonized the increase of TLR4, p-IκBα, and NF-κB. In summary, Rhein suppresses neuroinflammation via multiple signaling pathways (PI3K/Akt, p38, ERK1/2, and TLR4/NF-κB) in LPS-stimulated BV2 microglia cells. This study highlights a natural agent for prevention and treatment of neuroinflammation.

Anti-Amyloidogenic Effects of Asarone Derivatives From Perilla frutescens Leaves against Beta-Amyloid Aggregation and Nitric Oxide Production.

Alzheimer’s disease (AD) is a progressive, neurodegenerative brain disorder associated with loss of memory and cognitive function. Beta-amyloid (Aβ) aggregates, in particular, are known to be highly neurotoxic and lead to neurodegeneration. Therefore, blockade or reduction of Aβ aggregation is a promising therapeutic approach in AD. We have previously reported an inhibitory effect of the methanol extract of Perilla frutescens (L.) Britton (Lamiaceae) and its hexane fraction on Aβ aggregation. Here, the hexane fraction of P. frutescens was subjected to diverse column chromatography based on activity-guided isolation methodology. This approach identified five asarone derivatives including 2,3-dimethoxy-5-(1E)-1-propen-1-yl-phenol (1), β-asarone (2), 3-(2,4,5-trimethoxyphenyl)-(2E)-2-propen-1-ol (3), asaronealdehyde (4), and α-asarone (5). All five asarone derivatives efficiently reduced the aggregation of Aβ and disaggregated preformed Aβ aggregates in a dose-dependent manner as determined by a Thioflavin T (ThT) fluorescence assay. Furthermore, asarone derivatives protected PC12 cells from Aβ aggregate-induced toxicity by reducing the aggregation of Aβ, and significantly reduced NO production from LPS-stimulated BV2 microglial cells. Taken together, these results suggest that asarone derivatives derived from P. frutescens are neuroprotective and have the prophylactic and therapeutic potential in AD.

Dexmedetomidine Attenuates Neuroinflammation In LPS-Stimulated BV2 Microglia Cells Through Upregulation Of miR-340.

BackgroundDexmedetomidine (Dex) was reported to exhibit anti-inflammatory effect in the nervous system. However, the mechanism by which Dex exhibits anti-inflammation effects on LPS-stimulated BV2 microglia cells remains unclear. Thus, this study aimed to investigate the role of Dex in LPS-stimulated BV2 cells.MethodsThe BV2 cells were stimulated by lipopolysaccharides (LPS). BV2 cells were infected with short-hairpin RNAs targeting NF-κB (NF-κB-shRNAs) and NF-κB overexpression lentivirus, respectively. In addition, miR-340 mimics or miR-340 inhibitor was transfected into BV2 cells, respectively. Meanwhile, the dual-luciferase reporter system assay was used to explore the interaction of miR-340 and NF-κB in BV2 cells. CCK-8 was used to detect the viability of BV2 cells. In addition, Western blotting was used to detect the level of NF-κB in LPS-stimulated BV2 cells. The levels of TNF-α, IL-6, IL-1β, IL-2, IL-12, IL-10 and MCP-1 in LPS-stimulated BV2 cells were measured with ELISA.ResultsThe level of miR-340 was significantly upregulated in Dex-treated BV2 cells. Meanwhile, the level of NF-κB was significantly increased in BV2 cells following infection with lenti-NF-κB, which was markedly reversed by Dex. LPS markedly increased the expression of NF-κB and proinflammatory cytokines in BV2 cells, which were reversed in the presence of Dex. Moreover, miR-340 mimics enhanced the anti-inflammatory effects of Dex in LPS-stimulated BV2 cells via inhibiting NF-κB and proinflammatory cytokines. Furthermore, Dex obviously inhibited LPS-induced phagocytosis in BV2 cells.ConclusionTaken together, our results suggested that Dex might exert anti-inflammatory effects in LPS-stimulated BV2 cells via upregulation of miR-340. Therefore, Dex might serve as a potential agent for the treatment of neuroinflammation.

The Methanol Extract of Allium cepa L. Protects Inflammatory Markers in LPS-Induced BV-2 Microglial Cells and Upregulates the Antiapoptotic Gene and Antioxidant Enzymes in N27-A Cells.

Neuroinflammation, apoptosis, and oxidative stress are connected to the pathogenesis of neurodegenerative diseases (NDDs). Targeting these three factors, the intervention of neuroprotective agents may have great potential in the treatment of NDDs. In the current study, the anti-inflammatory effects of the methanol extract of Allium cepa (MEAC) in lipopolysaccharide (LPS)-induced BV-2 microglial cells were investigated. MEAC has been studied in regard to the regulation of the antiapoptotic gene (Bcl-2) and various antioxidant enzyme (HO-1, NQO-1, and catalase) expressions in N27-A cells. Additionally, the protective action of MEAC has also been studied against MPP+-induced death in N27-A cells. The results suggest that MEAC is significantly protected from NO release and increase iNOS expression at the mRNA and protein levels in LPS-stimulated BV-2 microglial cells. MEAC treatment also protects COX-2 expression at the mRNA and protein levels. Furthermore, MEAC treatment prevents LPS-stimulated increases of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β. In N27-A cells, MEAC treatment significantly upregulates antiapoptotic gene (Bcl-2) and antioxidant enzyme (HO-1, NQO1, and catalase) expressions. Moreover, MEAC treatment protects against MPP+-induced death in N27-A cells. To conclude, A cepa extract takes protective action against LPS and MPP+, and upregulates the antioxidant enzymes that could potentially be used in the therapy of NDDs.

MiR-124/VAMP3 is a novel therapeutic target for mitigation of surgical trauma-induced microglial activation

Activation of microglia and the subsequently elevated inflammatory cytokine release in the brain during surgery predispose individuals to cognitive dysfunction, also known as postoperative cognitive dysfunction (POCD). miR-124 is one of the most abundant microRNAs in the brain that regulates microglial function. Elucidating the role of miR-124 in microglial activation in the context of surgery may therefore promote understanding of as well as therapeutic development for post-surgical disorders involving microglial activation. The downstream targets of miR-124 were investigated using bioinformatic screening and dual-luciferase reporter assay validation, and vesicle-associated membrane protein 3 (VAMP3) was identified as a potential target. The kinetics of miR-124/VAMP3 expression was first examined in vitro in microglial cells (primary microglia and BV2 microglial cells) following lipopolysaccharide (LPS) stimulation. LPS induced a time-dependent decrease of miR-124 and upregulated the expression of VAMP3. Manipulating miR-124/VAMP3 expression by using miR-124 mimics or VAMP3-specific siRNA in LPS-stimulated BV2 microglial cells inhibited BV2 microglial activation-associated inflammatory cytokine release. To further examine the role of miR-124/VAMP3 in a surgical setting, we employed a rat surgical trauma model. Significant microglial activation and altered miR-124/VAMP3 expression were observed following surgical trauma. We also altered miR-124/VAMP3 expression in the rat surgical trauma model by administration of exogenous miR-124 and by using electroacupuncture, which is a clinically applicable treatment that modulates microglial function and minimizes postoperative disorders. We determined that electroacupuncture treatment specifically increases the expression of miR-124 in the hypothalamus and hippocampus. Increased miR-124 expression with a concomitant decrease in VAMP3 expression resulted in decreased inflammatory cytokine release related to microglial activation post-surgery. Our study indicates that miR-124/VAMP3 is involved in surgery-induced microglial activation and that targeting miR-124/VAMP3 could be a potential therapeutic strategy for postoperative disorders involving microglial activation.

Hesperetin inhibits neuroinflammation on microglia by suppressing inflammatory cytokines and MAPK pathways.

Neuroinflammation is a specific or nonspecific immunological reaction in the central nervous system that is induced by microglia activation. Appropriate regulation of activated microglial cells is therefore important for inhibiting neuroinflammation. Hesperetin is a natural flavanone and an aglycone of hesperidin that is found in citrus fruits. Hesperetin reportedly possesses anti-inflammatory, anti-cancer, and antioxidant effects. However, the anti-neuroinflammatory effects of hesperetin on microglia are still unknown. Here, we investigated the anti-neuroinflammatory effects of hesperetin on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We found that hesperetin strongly inhibited nitric oxide production and expression of inducible nitric oxide synthase in LPS-stimulated BV-2 microglial cells. Hesperetin also significantly reduced secretion of inflammatory cytokines including interleukin (IL)-1β and IL-6. Furthermore, hesperetin down-regulated the phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase, exerting anti-inflammatory effects. Hesperetin suppressed astrocyte and microglia activation in the LPS-challenged mouse brain. Collectively, our findings indicate that hesperetin inhibits microglia-mediated neuroinflammation and could be a prophylactic treatment for neurodegenerative diseases.