Microglial Cell Line BV-2 Research Articles

Neuroprotective Effects of Annexin A1 Tripeptide after Deep Hypothermic Circulatory Arrest in Rats.

Resolution agonists, including lipid mediators and peptides such as annexin A1 (ANXA1), are providing novel approaches to treat inflammatory conditions. Surgical trauma exerts a significant burden on the immune system that can affect and impair multiple organs. Perioperative cerebral injury after cardiac surgery is associated with significant adverse neurological outcomes such as delirium and postoperative cognitive dysfunction. Using a clinically relevant rat model of cardiopulmonary bypass (CPB) with deep hypothermic circulatory arrest (DHCA), we tested the pro-resolving effects of a novel bioactive ANXA1 tripeptide (ANXA1sp) on neuroinflammation and cognition. Male rats underwent 2 h CPB with 1 h DHCA at 18°C, and received vehicle or ANXA1sp followed by timed reperfusion up to postoperative day 7. Immortalized murine microglial cell line BV2 were treated with vehicle or ANXA1sp and subjected to 2 h oxygen–glucose deprivation followed by timed reoxygenation. Microglial activation, cell death, neuroinflammation, and NF-κB activation were assessed in tissue samples and cell cultures. Rats exposed to CPB and DHCA had evident neuroinflammation in various brain areas. However, in ANXA1sp-treated rats, microglial activation and cell death (apoptosis and necrosis) were reduced at 24 h and 7 days after surgery. This was associated with a reduction in key pro-inflammatory cytokines due to inhibition of NF-κB activation in the brain and systemically. Treated rats also had improved neurologic scores and shorter latency in the Morris water maze. In BV2 cells treated with ANXA1sp, similar protective effects were observed including decreased pro-inflammatory cytokines and cell death. Notably, we also found increased expression of ANXA1, which binds to NF-κB p65 and thereby inhibits its transcriptional activity. Our findings provide evidence that treatment with a novel pro-resolving ANXA1 tripeptide is neuroprotective after cardiac surgery in rats by attenuating neuroinflammation and may prevent postoperative neurologic complications.

Heme Oxygenase-1-Inducing Activity of 4-Methoxydalbergione and 4'-Hydroxy-4-methoxydalbergione from Dalbergia odorifera and Their Anti-inflammatory and Cytoprotective Effects in Murine Hippocampal and BV2 Microglial Cell Line and Primary Rat Microglial Cells.

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

Molecular Diversity and Potential Anti-neuroinflammatory Activities of Cyathane Diterpenoids from the Basidiomycete Cyathus africanus

Ten new polyoxygenated cyathane diterpenoids, named neocyathins A–J (1–10), together with four known diterpenes (11–14), were isolated from the liquid culture of the medicinal basidiomycete fungus Cyathus africanus. The structures and configurations of these new compounds were elucidated through comprehensive spectroscopic analyses including 1D NMR, 2D NMR (HSQC, HMBC, NOESY) and HRESIMS, and electronic circular dichroism (ECD) data. Neuroinflammation is implicated in the pathogenesis of various neurodegenerative diseases, such as Alzheimers’ disease (AD). All isolated compounds were evaluated for the potential anti-neuroinflammatory activities in BV2 microglia cells. Several compounds showed differential effects on the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated and Aβ1–42-treated mouse microglia cell line BV-2. Molecular docking revealed that bioactive compounds (e.g., 11) could interact with iNOS protein other than COX-2 protein. Collectively, our results suggested that this class of cyathane diterpenoids might serve as important lead compounds for drug discovery against neuroinflammation in AD.

HsdS, Belonging to the Type I Restriction-Modification System, Contributes to the Streptococcus suis Serotype 2 Survival Ability in Phagocytes.

Streptococcus suis serotype 2 (SS2) is an important zoonotic agent in swine and humans. Anti-phagocytosis and survival in phagocytic cells and whole blood is essential for bacteria to be pathogenic. In this study, the host specificity determinant specificity subunit (coded by hsdS) of the Type I Restriction-Modification system and two peptidoglycan-binding proteins (coded by lysM and lysM′, respectively), which were simultaneously found to be subjected to transcript-level influence by hsdS, were identified to facilitate the anti-phagocytosis of SS2 to a microglia cell line BV2. Furthermore, they significantly enhanced its survival in BV2, whole blood, and a peroxidation environment (H2O2) (p < 0.05), yet not in the acidic condition based on statistical analysis of the characteristic differences between gene mutants and wild-type SS2. In contrast, another specificity subunit, coded by hsdS′, that belonged to the same Type I Restriction-Modification system, only significantly reduced the survival ability of SS2 in the acidic condition when in the form of a gene-deleted mutant (p < 0.05), but it did not significantly influence the survival ability in other conditions mentioned above or have enhanced anti-phagocytosis action when compared with wild-type SS2. In addition, the mutation of hsdS significantly enhanced the secretion of nitric oxide and TNF-α by BV2 with SS2 incubation (p < 0.05). The SS2 was tested, and it failed to stimulate BV2 to produce IFN-γ. These results demonstrated that hsdS contributed to bacterial anti-phagocytosis and survival in adverse host environments through positively impacting the transcription of two peptidoglycan-binding protein genes, enhancing resistance to reactive oxygen species, and reducing the secretion of TNF-α and nitric oxide by phagocytes. These findings revealed new mechanisms of SS2 pathogenesis.

Curcumin Protects against Ischemic Stroke by Titrating Microglia/Macrophage Polarization.

Stroke is the most common type of cerebrovascular disease and is a leading cause of disability and death. Ischemic stroke accounts for approximately 80% of all strokes. The remaining 20% of strokes are hemorrhagic in nature. To date, therapeutic options for acute ischemic stroke are very limited. Recent research suggests that shifting microglial phenotype from the pro-inflammatory M1 state toward the anti-inflammatory and tissue-reparative M2 phenotype may be an effective therapeutic strategy for ischemic stroke. The dietary phytochemical curcumin has shown promise in experimental stroke models, but its effects on microglial polarization and long-term recovery after stroke are unknown. Here we address these gaps by subjecting mice to distal middle cerebral artery occlusion (dMCAO) and administering curcumin intraperitoneally (150 mg/kg) immediately after ischemia and 24 h later. Histological studies revealed that curcumin post-treatment significantly reduced cerebral ischemic damage 3 days after dMCAO. Sensorimotor functions—as measured by the adhesive removal test and modified Garcia scores—were superior in curcumin-treated mice at 3, 5, 7 and 10 days after stroke. RT-PCR measurements revealed an elevation of M2 microglia/macrophage phenotypic markers and a reduction in M1 markers in curcumin-treated brains 3 days after dMCAO. Immunofluorescent staining further showed that curcumin treatment significantly increased the number of CD206+Iba1+ M2 microglia/macrophages and reduced the number of CD16+Iba1+ M1 cells 10 days after stroke. In vitro studies using the BV2 microglial cell line confirmed that curcumin inhibited lipopolysaccharide (LPS) and interferon-γ (IFN-γ)-induced M1 polarization. Curcumin treatment concentration-dependently reduced the expression of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-12p70, in the absence of any toxic effect on microglial cell survival. In conclusion, we demonstrate that curcumin has a profound regulatory effect on microglial responses, promoting M2 microglial polarization and inhibiting microglia-mediated pro-inflammatory responses. Curcumin post-treatment reduces ischemic stroke-induced brain damage and improves functional outcomes, providing new evidence that curcumin might be a promising therapeutic strategy for stroke.

Interleukin-6 regulates iron-related proteins through c-Jun N-terminal kinase activation in BV2 microglial cell lines.

Parkinson’s disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic (DA) neurons in the substantia nigra (SN) and subsequent DA depletion in the striatum. Microglia activation and nigral iron accumulation play important roles in the pathogenesis of PD. Activated microglia show increased iron deposits. However, the relationship between microglia activation and iron accumulation remains unclear. In the present study, we aimed to determine how iron levels affect interleukin-6 (IL-6) synthesis, and the effect of IL-6 on cellular iron metabolism in BV2 microglial cells.IL-6 mRNA was up-regulated after FAC treatment for 12 h in BV2 cells. Iron regulatory protein 1 (IRP1) and divalent metal transporter 1 (DMT1) were up-regulated and iron exporter ferroportin 1 (FPN1) was down-regulated in BV2 cells after 24 h of IL-6 treatment. Phosphorylated JNK increased significantly compared to the control after BV2 cells were treated with IL-6 for 1 h. Pretreatment with SP600125 attenuated the up-regulation of IRP1 and DMT1 and down-regulation of FPN1 (compared to IL-6-treated group). These results suggest that iron load could increase IL-6 mRNA expression in BV2 cells. Further, IL-6 likely up-regulates IRP1 and DMT1 expression and down-regulates FPN1 expression in BV2 microglial cells through JNK signaling pathways.

A split-luciferase complementation, real-time reporting assay enables monitoring of the disease-associated transmembrane protein TREM2 in live cells

Triggering receptor expressed on myeloid cells 2 (TREM2) is a single transmembrane molecule uniquely expressed in microglia. TREM2 mutations are genetically linked to Nasu-Hakola disease and associated with multiple neurodegenerative disorders, including Alzheimer's disease. TREM2 may regulate microglial inflammation and phagocytosis through coupling to the adaptor protein TYRO protein-tyrosine kinase-binding protein (TYROBP). However, there is no functional system for monitoring this protein-protein interaction. We developed a luciferase-based modality for real-time monitoring of TREM2-TYROBP coupling in live cells that utilizes split-luciferase complementation technology based on TREM2 and TYROBP fusion to the C- or N-terminal portion of the Renilla luciferase gene. Transient transfection of human embryonic kidney 293 cells with this reporter vector increased luciferase activity upon stimulation with an anti-TREM2 antibody, which induces their homodimerization. This was confirmed by ELISA-based analysis of the TREM2-TYROBP interaction. Antibody-mediated TREM2 stimulation enhanced spleen tyrosine kinase (SYK) activity and uptake of Staphylococcus aureus in microglial cell line BV-2 in a kinase-dependent manner. Interestingly, the TREM2 T66M mutation significantly enhanced luciferase activity without stimulation, indicating constitutive coupling to TYROBP. Finally, flow cytometry analyses indicated significantly lower surface expression of T66M TREM2 variant than wild type or other TREM2 variants. These results demonstrate that our TREM2 reporter vector is a novel tool for monitoring the TREM2-TYROBP interaction in real time.

Implicating Receptor Activator of NF-κB (RANK)/RANK Ligand Signalling in Microglial Responses to Toll-Like Receptor Stimuli

Inflammation in the perinatal brain caused by maternal or intrauterine fetal infection is now well established as an important contributor to the development of perinatal brain injury. Exposure to inflammatory products can impair perinatal brain development and act as a risk factor for neurological dysfunction, cognitive disorders, cerebral palsy, or preterm birth. Pre-exposure to inflammation significantly exacerbates brain injury caused by hypoxic/ischaemic insult. Tumour necrosis factor (TNF) is a family of cytokines largely involved in inflammation signalling. In our previous study, we identified the importance of TNF-related apoptosis-inducing ligand (TRAIL) signalling in the development of perinatal brain injury. We observed a significant increase in the expression levels of a soluble decoy receptor for TRAIL, osteoprotegerin (OPG). Besides TRAIL, OPG is able to bind the receptor activator of the NF-κB (RANK) ligand (RANKL) and inhibit its signalling. The function of the RANK/RANKL/OPG system in the brain has not come under much scrutiny. The aim of this research study was to elucidate the role of RANK, RANKL, and OPG in microglial responses to the proinflammatory stimuli lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (Poly I:C). Here, we show that RANK signalling is important for regulating the activation of the BV2 microglial cell line. We found that LPS treatment causes a significant decrease in the expression of RANK in the BV2 cell line while significantly increasing the expression of OPG, Toll-like receptor (TLR)3, and the adaptor proteins MyD88 and TRIF. We found that pretreatment of BV2 cells with RANKL for 24 h before the LPS or Poly I:C exposure decreases the expression of inflammatory markers such as inducible nitric oxide synthase and cyclooxygenase. This is accompanied by a decreased expression of the TLR adaptor proteins MyD88 and TRIF, which we observed after RANKL treatment. Similar results were obtained in our experiments with primary mouse microglia. Using recently developed CRISPR/Cas9 technology, we generated a BV2 cell line lacking RANK (RANK^-/- BV2). We showed that most effects of RANKL pretreatment were abolished, thereby proving the specificity of this effect. Taken together, these findings suggest that RANK signalling is important for modulating the inflammatory activation of microglial cells to a moderate level, and that RANK attenuates TLR3/TLR4 signalling.

A dibenzoylmethane derivative inhibits lipopolysaccharide-induced NO production in mouse microglial cell line BV-2

Microglial activation has been suggested to play important roles in various neurodegenerative diseases by phagocytosis and producing various factors such as nitric oxide (NO), proinflammatory cytokines. Excessive production of NO, as a consequence of increased inducible nitric oxide synthase (iNOS) in microglia, contributes to the neurodegeneration. During a search for compounds that regulate endoplasmic reticulum (ER) stress, a dibenzoylmethane derivative, 2,2’-dimethoxydibenzoylmethane (DBM 14–26) was identified as a novel neuroprotective agent (Takano et al., Am. J. Physiol. Cell Physiol. 293, C1884-1894, 2007). We previously reported in cultured astrocytes that DBM 14–26 protected hydrogen peroxide-induced cell death and inhibited lipopolysaccharide (LPS)-induced NO production (Takano et al., J. Neurosci. Res. 89, 955–965, 2011). In the present study, we assessed the effects of DBM 14–26 on microglia using the mouse cell line BV-2 and found that DBM 14–26 inhibited LPS-induced iNOS expression and NO production also in microglia. DBM 14–26 also suppressed LPS-induced IL-1β expression. Conditioned medium of BV-2 cells stimulated by LPS significantly decreased cell viability of neuron (human neuroblastoma SH-SY5Y cells) compared with the absence of LPS. Conditioned medium of BV-2 cells stimulated by LPS in the presence of DBM 14–26 did not significantly decreased cell viability of neuron. These results indicate that microglial activation by LPS causes neuronal cell death and DBM 14–26 protect neuron through the inhibition of microglial activation. Functional regulation of microglia by DBM 14–26 could be a therapeutic candidate for the treatment of neurodegenerative diseases.

Three new coumarin glycosides from the stems of Hydrangea paniculata

Three new coumarin glycosides (1-3), together with three known compounds (4-6), have been obtained from the stems of Hydrangea paniculata Sieb. Their structures were elucidated based on spectroscopic data and chemical evidence. In addition, compounds 1-3 were screened for their neuroprotective effects against serum deprivation-induced PC12 cell damage, hepatoprotective activities against DL-galactosamine-induced toxicity in HL-7702 cells and their ability to inhibit LPS-induced nitric oxide production in the murine microglia BV2 cell line, but they were inactive.

Dengue virus infection increases microglial cell migration

Activated microglial cells are present in dengue virus (DENV)-infected brains; however, the possible effects of DENV on microglia remain unclear. Here, we demonstrated DENV caused infection, including viral entry, RNA replication, viral protein expression, and virus release, in the murine microglial cell line BV2. DENV infection caused an increase in the formation of the multipolar phenotype in vitro and in vivo without affecting cell growth and cytotoxicity. DENV infection considerably increased cell motility and disrupting either actin filaments or clathrin retarded such effect. Increase in cell migration was only occurred by DENV infection following a clathrin-regulated endocytosis of DENV entry. Ultraviolet-inactivated DENV did not affect cell migration, and pharmacologically blocking toll-like receptor (TLR) 3 and TLR3-related signaling pathways reduced the DENV-induced increase in cell migration. These results demonstrate an advanced effect of DENV infection on microglial migration via a mechanism involving viral entry, RNA release, and TLR3 signal activation.

Neuroprotective erythropoietin attenuates microglial activation, including morphological changes, phagocytosis, and cytokine production

Erythropoietin (EPO), a hematopoietic hormonal cytokine induced in response to hypoxia, has neuroprotective effects. EPO receptor (EPOR) is expressed in microglia, resident immune cells in the brain. However, the effect of EPO on microglial activation is not clear. In the present study, we demonstrated that the EPOR is highly expressed in microglia, rather than in neurons or astrocytes, in in vitro experiments. Therefore, we investigated whether EPO could attenuate lipopolysaccharide (LPS)-mediated activation of microglia in vitro. The BV-2 microglial cell line was treated with LPS in the absence or presence of EPO. In the presence of EPO, microglial expression of LPS-induced inflammatory cytokine genes was significantly decreased. In addition, EPO suppressed the LPS-induced phagocytic activity of BV-2 cells towards fluorescent beads, as well as induction of inducible nitric oxide synthase. In in vivo experiments, EPO significantly decreased the LPS-induced expression of inflammatory cytokine genes in mouse brains. Furthermore, morphological analysis of cortical microglia in the brains of mice stimulated with LPS revealed that combined treatment with EPO alleviated LPS-induced morphological changes in the microglia. These data indicate that EPO attenuates microglial activation, including morphological changes in vivo, phagocytosis in vitro, and the production of inflammatory cytokines in vivo and in vitro. Further investigation of EPO modulation of LPS-induced microglial activation may contribute to the development of novel neuroprotective therapies.

Amphotericin B Increases Transglutaminase 2 Expression Associated with Upregulation of Endocytotic Activity in Mouse Microglial Cell Line BV-2.

Amphotericin B (AmB), a polyene antibiotic, is reported to cause the microglial activation to induce nitric oxide (NO) production and proinflammatory cytokines expression, and change neurotrophic factors expression in cultured microglia (Motoyoshi et al. in Neurochem Int 52:1290-1296, 2008). On the other hand, tissue-type transglutaminase (TG2) is involved in connection to phagocytes with apoptotic cells. Engulfment of neurons by activated microglia is thought to cause neurodegenerative diseases but detail is unclear, and involvement of TG2 in phagocytosis has been reported in our previous study using lipopolysaccharide-stimulated BV-2 cells (Kawabe et al. in Neuroimmunomodulation 22(4):243-249, 2015). In the present study, we examined the changes of TG2 expression, phagocytosis and pinocytosis in BV-2 cells stimulated by AmB. AmB stimulation increased TG2 expression and TG activity. Phagocytosis of dead cells and pinocytosis of fluorescent microbeads were also up-regulated by AmB stimulation in BV-2 cells. Blockade of TG activity by cystamine, an inhibitor of TGs, suppressed AmB-enhanced TG2 expression, TG activity, NO production, phagocytosis and pinocytosis. Excessive NO production from microglia and/or facilitation of phagocytosis might be involved in neuronal death. To control TG activity might make possible to protect neurons and care for CNS diseases.

Regulatory role of cytosolic phospholipase A2 alpha in the induction of CD40 in microglia

BackgroundThe aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A2 alpha (cPLA2α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA2α has a direct, participatory role in the molecular events leading to CD40 induction.MethodsCultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA2α in the events leading to CD40 protein induction.ResultsAddition of LPS or IFNγ caused a significant upregulation of cPLA2α and of CD40, while prevention of cPLA2α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA2α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA2α detected by its phosphorylated form, while addition of IFNγ induced cPLA2α activation at a later time scale (4 h). The activation of cPLA2α is mediated by ERK activity. Suppression of cPLA2α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-κB detected by the phosphorylation of p65 on serine 536 at 15 min by LPS and at 4 h by IFNγ. Inhibition of NOX2 prevented NF-κB activation and CD40 induction but did not affect cPLA2α activation, suggesting cPLA2α is located upstream to NOX2 and NF-κB. The activation of cPLA2 by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA2α by IFNγ was mediated by the secreted TNF-α at 4 h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA2α inhibitors, indicating that STAT1α is not under cPLA2α regulation.ConclusionsOur results show for the first time that cPLA2 upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-κB. Cumulatively, our results indicate that cPLA2α may serve as a pivotal amplifier of the inflammatory response in the CNS.

Down-Regulation of miRNA-128 Contributes to Neuropathic Pain Following Spinal Cord Injury via Activation of P38.

BackgroundNeuropathic pain (NPP) arises from a lesion or dysfunction of the somatosensory nervous system. Recent studies have demonstrated multiple microRNAs (miRNAs) play key roles in NPP development. This study aimed to investigate the effects of miR-128 on microglial cells.Material/MethodsWe established a compressive spinal cord injury (SCI) model and collected the spinal cord segment-derived conditioned medium (CM). We then measured the expression of miR-128 in the murine microglial cell line BV2 treated with CM-SCI or CM obtained from control (CM-NC). Furthermore, lentivirus production of miR-128 and scrambled control were transfected into BV2 cells, which were first treated with CM-SCI or CM-NC. Moreover, the effects of miR-128 on cell viability, M1/M2 microglial gene expression, inflammatory cytokines concentration, and the protein expression of P38 and phosphorylated P38 (P-P38) were investigated.ResultsThe expression of miR-128 was downregulated in murine microglial BV2 cells treated with CM-SCI. Overexpression of miR-128 markedly promoted the viability of murine microglial cells. In addition, miR-128 overexpression significantly decreased the expression levels of microglial M1 phenotypic markers CD86 and CD32, and increased the expression levels of M2 phenotypic markers Arg1 and CD206. Furthermore, miR-128 overexpression obviously decreased the concentration of TNF-α, IL-1β, and IL-6. We found that miR-128 overexpression significantly downregulated the expression levels of P38 andP-P38.ConclusionsOur findings indicate that down-regulation of miR-128 in murine microglial cells may contribute to the development of NPP following SCI via activation of P38. MiR-128 may be a potential intervention target for NPP.

Bioassay-guided fractionation of ethyl acetate extract from Armillaria mellea attenuates inflammatory response in lipopolysaccharide (LPS) stimulated BV-2 microglia

BackgroundArmillaria mellea (A. mellea) is a traditional Chinese medicinal and edible mushroom, which is proved to possess a lot of biological activities, including anti-oxidation, immunopotentiation, anti-vertigo and anti-aging activities. However, little information is available in regard to its neuroprotection activity in inflammation-mediated neurodegenerative diseases. PurposeWe have found that A. mellea has an anti-inflammatory activity in LPS-induced RAW264.7 cells in our previous study. The objective of this study is to investigate the anti-neuroinflammatory mechanism of a bioassay-guided fractionation (Fr.2) and its active components/compounds. MethodsCompounds were isolated by preparative high performance liquid chromatography (pre-HPLC) and their structures were established by mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopic analyses. The anti-neuroinflammatory effect of Fr.2 and each compounds were investigated in lipopolysaccharide (LPS)-stimulated murine microglia cell lineBV-2. ResultsWe demonstrated that Fr.2 significantly decreased the production of inflammation mediator nitric oxide (NO) and inflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1beta (IL-1β) in a dose-dependent manner (10, 30, 100µg/ml). In addition, Fr.2 markedly down-regulated the phosphorylation levels of nuclear factor kappa B p65 (NF-κB p65), inhibitory κB-α (IκB-α) and c-Jun N-terminal kinases (JNKs) pathways. Sevens compounds were isolated from Fr.2, among them, three compounds, 5-hydroxymethylfurfural (CP1), vanillic acid (CP4) and syringate (CP5) were reported for the first time in A. mellea. NO and inflammatory cytokines (TNF-α, IL-6, IL-1β) secretion indicated that daidzein (CP6) and genistein (CP7) showed a more outstanding anti-inflammation potential at non-toxic concentrations (10, 30, 100µM) than the other five compounds. ConclusionsIn conclusion, Fr.2 may have therapeutic potential for neurodegenerative diseases by inhibiting inflammatory mediators and suppress inflammation pathway in activated microglia. Daidzein and genistein may serve as the effective anti-inflammation compounds of Fr.2.

Andrographolide Activates Keap1/Nrf2/ARE/HO-1 Pathway in HT22 Cells and Suppresses Microglial Activation by Aβ42 through Nrf2-Related Inflammatory Response.

Therapeutic approach of Alzheimer's disease (AD) has been gradually diversified. We examined the therapeutic and preventive potential of andrographolide, which is a lactone diterpenoid from Andrographis paniculata, and focused on the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated heme oxygenase (HO)-1-inducing effects and the inhibitory activity of amyloid beta (Aβ)42-induced microglial activation related to Nrf2 and nuclear factor κB (NF-κB)-mediated inflammatory responses. Andrographolide induced the expression and translocation of Nrf2 from the cytoplasm to the nucleus, thereby activating antioxidant response element (ARE) gene transcription and HO-1 expression in murine hippocampal HT22 cells. Andrographolide eliminated intracellular Aβ42 in BV-2 cells and decreased the production of interleukin (IL)-6, IL-1β, prostaglandin (PG)E2, and nitric oxide (NO) because of artificial phagocytic Aβ42. It decreased pNF-κB accumulation in the nucleus and the expression of inducible nitric oxide synthase (i-NOS) and cyclooxygenase II (COX-II) in the microglial BV-2 cell line. In summary, andrographolide activates Nrf2-mediated HO-1 expression and inhibits Aβ42-overexpressed microglial BV-2 cell activation. These results suggested that andrographolide might have the potential for further examination of the therapeutics of AD.

Spike-In SILAC Approach for Proteomic Analysis of Ex Vivo Microglia.

Stable isotope labeling by amino acids in cell culture (SILAC) is a versatile mass spectrometry-based proteomic approach that can achieve accurate relative protein quantitation on a global scale. In this approach, proteins are labeled while being synthesized by the cell due to the presence of certain amino acids exclusively as heavier mass analogs than their regular (light) counterparts. This differential labeling allows for the identification of heavy and light forms of each peptide corresponding to two or more different experimental groups upon mass spectrometric analysis, the intensities of which reflect their abundance in the sample analyzed. Relative quantitation is straightforward when SILAC labeling efficiency is high (>99%) and the same cell proteome is used as the quantitation reference, which is typically the case for immortalized cell lines. However, the SILAC methodology for the proteomic analysis of primary cells isolated after in vivo experimentation is more challenging given the low labeling efficiency that would be achieved post-isolation. Alternatively, a stable-isotope-labeled cell line representing the cell type can be used as an internal standard (spike-in SILAC); however, adequate representation of the primary cell proteome with the stable-isotope-labeled internal standard may limit overall protein quantitation, especially for cell types that exhibit a broad range of phenotypes such as microglia, the resident immune cells in the brain. Here, we present a way to circumvent this limitation by combining multiple phenotypes of a single-cell type (the immortalized mouse BV2 microglial cell line) into a single spike-in standard using primary mouse microglia as our model system. We describe the preparation of media, incorporation of labels, induction of four different activation states (plus resting), isolation of primary microglia from adult mice brains, preparation of lysates for analysis, and general guidelines for data processing.

The protein-tyrosine phosphatase DEP-1 promotes migration and phagocytic activity of microglial cells in part through negative regulation of fyn tyrosine kinase.

Microglia cells are brain macrophages whose proper functioning is essential for maintenance and repair processes of the central nervous system (CNS). Migration and phagocytosis are critical aspects of microglial activity. By using genetically modified cell lines and knockout mice we demonstrate here that the receptor protein-tyrosine phosphatase (PTP) DEP-1 (also known as PTPRJ or CD148) acts as a positive regulator of both processes in vitro and in vivo. Notably, reduced microglial migration was detectable in brains of Ptprj-/- mice using a wounding assay. Mechanistically, density-enhanced phosphatase-1 (DEP-1) may in part function by inhibiting the activity of the Src family kinase Fyn. In the microglial cell line BV2 DEP-1 depletion by shRNA-mediated knockdown resulted in enhanced phosphorylation of the Fyn activating tyrosine (Tyr420 ) and elevated specific Fyn-kinase activity in immunoprecipitates. Moreover, Fyn mRNA and protein levels were reduced in DEP-1 deficient microglia cells. Consistent with a negative regulatory role of Fyn for microglial functions, which is inhibited by DEP-1, microglial cells from Fyn-/- mice exhibited elevated migration and phagocytosis. Enhanced microglia migration to a site of injury was also observed in Fyn-/- mice in vivo. Taken together our data revealed a previously unrecognized role of DEP-1 and suggest the existence of a potential DEP-1-Fyn axis in the regulation of microglial functions. GLIA 2017;65:416-428.

Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation.

BackgroundTolerance seriously impedes the application of morphine in clinical medicine. Thus, it is necessary to investigate the exact mechanisms and efficient treatment. Microglial activation and neuroinflammation in the spinal cord are thought to play pivotal roles on the genesis and maintaining of morphine tolerance. Activation of adenosine monophosphate-activated kinase (AMPK) has been associated with the inhibition of inflammatory nociception. Metformin, a biguanide class of antidiabetic drugs and activator of AMPK, has a potential anti-inflammatory effect. The present study evaluated the effects and potential mechanisms of metformin in inhibiting microglial activation and alleviating the antinociceptive tolerance of morphine.MethodsThe microglial cell line BV-2 cells and mouse brain-derived endothelial cell line bEnd3 cells were used. Cytokine expression was measured using quantitative polymerase chain reaction. Cell signaling was assayed by western blot and immunohistochemistry. The antinociception and morphine tolerance were assessed in CD-1 mice using tail-flick tests.ResultsWe found that morphine-activated BV-2 cells, including the upregulation of p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation, pro-inflammatory cytokines, and Toll-like receptor-4 (TLR-4) mRNA expression, which was inhibited by metformin. Metformin suppressed morphine-induced BV-2 cells activation through increasing AMPK phosphorylation, which was reversed by the AMPK inhibitor compound C. Additionally, in BV-2 cells, morphine did not affect the cell viability and the mRNA expression of anti-inflammatory cytokines. In bEnd3 cells, morphine did not affect the mRNA expression of interleukin-1β (IL-1β), but increased IL-6 and tumor necrosis factor-α (TNF-α) mRNA expression; the effect was inhibited by metformin. Morphine also did not affect the mRNA expression of TLR-4 and chemokine ligand 2 (CCL2). Furthermore, systemic administration of metformin significantly blocked morphine-induced microglial activation in the spinal cord and then attenuated the development of chronic morphine tolerance in mice.ConclusionsMetformin significantly attenuated morphine antinociceptive tolerance by suppressing morphine-induced microglial activation through increasing AMPK phosphorylation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0754-9) contains supplementary material, which is available to authorized users.