Murine Microglial BV-2 Cells Research Articles

Pu'er tea water extract protects against cognitive impairment in a mouse model of lipopolysaccharide-induced neuroinflammation

BackgroundPu'er tea, a type of post-fermented tea made from Camellia sinensis leaves, has long been widely used in East Asian countries. It is mainly produced in southern China and is effective in preventing obesity due to its ability to break down fat. However, the effects of Pu'er tea on cognitive impairment or neuroinflammation by endotoxin have not yet been studied. PurposeHere, we assessed the inhibitory activity of Pu'er tea hot water extract (PTW) on neuroinflammation and cognitive impairment and explored its mechanism. Study designThe ability of PTW to inhibit cognitive impairment was investigated in a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation and murine microglia BV2 cells. MethodsWe examined whether oral administration of PTW prevented cognitive impairment and LPS-induced neuroinflammation using behavioral tests, Nissl staining, immunohistochemistry, western blotting, real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), Griess assay, and enzyme-linked immunosorbent assay (ELISA). ResultsFirst, Morris water maze (MWM) and passive avoidance (PA) tests demonstrated that oral administration of PTW effectively attenuated LPS-induced spatial memory loss and inhibited neuronal damage of mouse brains. Histopathological analysis showed that PTW repressed LPS-induced expression of the activation markers ionized calcium-binding adaptor molecule-1 (Iba-1) and glial fibrillary acidic protein (GFAP). Furthermore, PTW inhibited the expression of amyloidogenesis proteins such as amyloid-β precursor protein (APP), C99, and β-secretase-1 (BACE-1); production of inflammatory proteins such as Iba-1, GFAP, inducible nitric oxide synthase (iNOS), and cyclooxygenase (COX)-2; activation of inflammatory pathways; and expression of inflammatory mediator mRNAs in hippocampal tissue. In cultured microglia, PTW treatment inhibited the generation of various inflammatory factors activated by LPS. ConclusionOur results in vivo and in vitro demonstrate that PTW effectively prevents cognitive impairment caused by neuroinflammation and is, therefore, a potential candidate for the development of a therapeutic agent for neurodegenerative diseases.

Equol, a Blood-Brain Barrier Permeable Gut Microbial Metabolite of Dietary Isoflavone Daidzein, Exhibits Neuroprotective Effects against Neurotoxins Induced Toxicity in Human Neuroblastoma SH-SY5Y Cells and Caenorhabditis elegans.

Emerging data support that plant food based isoflavones have ameliorating effects on a variety of neurodegenerative diseases including Parkinson's disease (PD). Our previous investigation revealed that dietary isoflavones including genistein (GEN), daidzein (DAI), and equol (EQL; a gut microbial metabolite of DAI) showed promising blood-brain barrier permeability and anti-neuroinflammatory activity in murine microglial BV2 cells. However, the neuroprotective effects of EQL against neurotoxins induced toxicity in PD related models remains unclear. Herein, EQL, along with GEN and DAI, were evaluated for their cytoprotective effect in a non-contact co-culture model with LPS-BV2-conditioned media and human neuroblastoma SH-SY5Y cells. In addition, their neuroprotective effects against PD related neurotoxins including 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) induced cytotoxicity were evaluated in SH-SY5Y cells. Furthermore, EQL was evaluated for its neuroprotective effects against MPP+ induced neurotoxicity using in vivo PD model including Caenorhabditis elegans lifespan assay. DAI (10μM) and EQL (10 and 20μM) showed cytoprotective effects by decreasing LPS-BV2-conditioned media induced cytotoxicity in SH-SY5Y cells by 29.2, 32.4 and 27.2%, respectively. EQL (10 and 20μM) also showed neuroprotective effects by decreasing 6-OHDA and MPP+ induced cytotoxicity in SH-SY5Y cells by 30.6-34.5 and 17.9-18.9%, respectively. Additionally, data from the in vivo assay supported EQL's neuroprotective effect as it increases survival of C. elegans exposed to MPP+ from 72 to 108h. Our findings support a growing body of evidence of the neuroprotective effects of dietary isoflavones and further studies are warranted to elucidate their mechanisms of action.

Nitric oxide inhibitory iridoids as potential anti-inflammatory agents from Valeriana jatamansi.

Five new iridoids, jatadomins A-E (1-5), together with six known analogues (6-11) and one known sesquiterpenoid (12), were isolated from the roots of Valeriana jatamansi Jones. Their structures were determined by analysis of their NMR, HRESIMS, and electronic circular dichroism calculations (ECD) data. The biological evaluation revealed that compounds 1-6 had anti-inflammatory activities by inhibiting nitric oxide (NO) release in LPS-induced murine microglial BV-2 cells, with IC50 values of 24.4, 9.2, 21.2, 25.9, 30.6, and 0.4μM, respectively. Further molecular docking studies revealed a potential mechanism for NO inhibition by the bioactive compounds.

Engeletin Attenuates Aβ1-42-Induced Oxidative Stress and Neuroinflammation by Keap1/Nrf2 Pathway.

Alzheimer's disease (AD) is a serious neuropathologic disease characterized by aggregation of amyloid-β (Aβ) peptide. Aβ-mediated oxidative stress and neuroinflammation play crucial role in the development of AD. Engeletin is a flavononol glycoside that possesses anti-inflammatory effect. However, the effects of engeletin on AD have not been investigated. In the present study, we investigated the role of engeletin in AD using an in vitro AD model. Murine microglia BV-2 cells were stimulated with Aβ1-42 (5μM) for 24h to induce oxidative stress and inflammation. Our results showed that treatment with engeletin suppressed Aβ1-42-induced viability reduction and lactate dehydrogenase (LDH) release in BV-2 cells. Engeletin attenuated Aβ1-42-induced oxidative stress in BV-2 cells, as proved by decreased production of reactive oxygen species (ROS) and malonaldehyde (MDA) and increased glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities. Aβ1-42-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression were inhibited by engeletin treatment. Besides, engeletin inhibited Aβ1-42-induced production and mRNA levels of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6). Engeletin enhanced Aβ1-42-induced activation of Kelch-like ECH-associated protein 1 (Keap1)/nuclear transcription factor E2-related factor 2 (Nrf2) signaling pathway in BV-2 cells. Inhibition of Keap1/Nrf2 signaling pathway reversed the inhibitory effects of engeletin on Aβ1-42-induced oxidative stress and inflammation in BV-2 cells. Taken together, engeletin attenuated Aβ1-42-induced oxidative stress and inflammation in BV-2 cells via regulating the of Keap1/Nrf2 pathway. These findings indicated that engeletin might be served as a therapeutic agent for the treatment of AD.

Urban airborne PM2.5-activated microglia mediate neurotoxicity through glutaminase-containing extracellular vesicles in olfactory bulb

Emerging evidence has showed that exposure to airborne particulate matter (PM) with an aerodynamic diameter less than 2.5 μm (PM2.5) is associated with neurodegeneration. Our previous studies in vitro found that PM2.5 exposure causes primary neurons damage through activating microglia. However, the molecular mechanism of microglia-mediated neurotoxicity remains to elucidate. In this study, five groups (N = 13 or 10) of six-week-old male C57BL/6 mice were daily exposed to PM2.5 (0.1 or 1 mg/kg/day body weight), Chelex-treated PM2.5 (1 mg/kg/day body weight), PM2.5 (1 mg/kg/day body weight) plus CB-839 (glutaminase inhibitor), or deionized water by intranasal instillation for 28 days, respectively. Compared with the control groups, We found that PM2.5 triggered reactive oxygen species (ROS) generation and microglia activation evidenced by significant increase of ionized calcium binding adaptor molecule-1 (IBa-1) staining in the mouse olfactory bulbs (OB). Data from transmission electron microscope (TEM) images and Western blot analysis showed that PM2.5 significantly increased extracellular vesicles (EVs) release from OB or murine microglial line BV2 cells, and glutaminase C (GAC) expression and glutamate generation in isolated OB and BV2 cells. However, treatment with N-acetylcysteine (NAC) or CB-839 significantly diminished the number of EVs and the expression of GAC and abolished PM2.5-induced neurotoxicity. These findings provide new insights that PM2.5 induces oxidative stress and microglia activation through its metal contents and glutaminase-containing EVs in OBs, which may serve as a potential pathway/mechanism of excessive glutamate generation in PM2.5-induced neurotoxicity.

6,7‐dihydroxy‐2,4‐dimethoxy phenanthrene isolated from Dioscorea batatas peel suppresses microglia‐associated neuroinflammation in vitro

Inflammation occured in the neural tissue is a cause of neurodegenerative diseases. Our previous study showed that the ethanolic extract of Dioscorea batatas Decne peel promoted antioxidant mechanism in colonic epithelial cells and attenuated inflammatory response in activated macrophages. Recently, 6, 7‐dihydroxy 2, 4‐dimethoxy phenanthrene (DDP) has been identified as one of the bioactive compounds having antioxidative and anti‐inflammatory activities. The current study aimed at examining the neuroprotective potential of DDP by staving off neuroinflammation. Our data demonstrated that (1) DDP decreased glutamate‐induced murine hippocampal neuronal cell death; hlowever, there was no significant increase in ARE‐luciferase activity, (2) DDP decreased the expressions of nuclear NF‐κB and its downstream proteins in BV2 murine microglial cells and thereby lowered NO production and cytokine secretion, and (3) DDP increased the production of antioxidant enzyme, heme oxygenase 1(HO‐1) in BV2 murine microglial cell. These results indicate that DDP suppressed microglial activation which generally causes neuroinflammation. In consideration of our observations implying that DDP may not directly stimulate the antioxidant mechanism in neuronal cells for its in vitro neuroprotective effect, DDP would possibly enhance neuronal cell survival due to its anti‐neuroinflammatory capability in microglia. The interaction between neuronal cells and microglia cells in culture as well as under physiological conditions awaits further study.

A Noncompetitive and Selective Prostaglandin EP2 Antagonist with Anti‐inflammatory Property

The prostaglandin E2 (PGE2) receptor subtype EP2 promotes chronic inflammation, modulates tumorigenesis, and regulates immune function. Inhibition of EP2 receptors reduces inflammation in rodent models of epilepsy, endometriosis, and arthritis, and decreases tumor cell growth. Therefore, inhibiting EP2 receptors could be a promising method for treatment of inflammatory diseases. To date all reported EP2 antagonists have a purely competitive mode of action, which can be overcome by high concentrations of agonist. Herein, we report the characterization of a novel compound belongs to substituted pyrroloquinoxaline scaffold as the first EP2 antagonist that features a noncompetitive mode of action. Cell‐based time‐resolved fluorescence resonance energy transfer (TR‐FRET) assays were developed in 384‐well format to measure the accumulation of cyclic adenosine monophosphate (cAMP) induced by PGE2, on the basis of the fact that activation of the Gas‐coupled EP2 receptor induces cAMP formation. At a low concentration (3 μM) of the pyrroloquinoxaline antagonist, a saturating concentration of PGE2 (3 μM) could reverse the inhibition of EP2 receptor activation in human EP2‐overexpressing C6 glioma (C6G‐EP2) cells. At higher antagonist concentrations (10–30 μM), the inhibition of EP2 receptors could not be fully overcome by the agonist. Meanwhile, the maximal agonist response inhibition by the antagonist increased with increasing concentration. The maximum effect (cAMP induction) by PGE2 was decreased to 81.9 ±1.44%, unlike 100% for a typical competitive antagonist. The antagonist itself had no effect on the TR‐FRET signal since there was no change in the TR‐FRET signal in the absence of C6G‐EP2 cells. The antagonist was reversible as demonstrated by washing out the antagonist three times each at 80% efficiency. After washing out the antagonist, the maximal response of C6G‐EP2 cells to PGE2 increased to 97.6 ± 0.69% from 86.4 ± 0.85% (p < 0.0001, one‐way ANOVA with Bonferroni). Furthermore, the antagonist exhibited little to no effect on the EC50 or maximal response of other Gas‐coupled prostanoid receptors (EP4, IP, and DP1). The agonist EC50s for EP2, EP4, IP and DP1 receptors were shifted 13.2, 2.2, 1.1 and 0.95‐fold to the right, respectively, in 30 μM antagonist. To determine whether the noncompetitive antagonist exerts an anti‐inflammatory action we studied the expression of IL‐6 and IL‐1β in the murine BV2 microglial cell line stably expressing human EP2 receptors (BV2‐hEP2). Treatment of these cells with a >EC90 concentration (30 nM) of the EP2 selective agonist, ONO‐AE1‐259, caused elevated mRNA levels of the two cytokines (measured by qRT‐PCR) that was suppressed by the antagonist (20 μM). Thus, the pyrroloquinoxaline is the first noncompetitive EP2 receptor antagonist. This compound may provide a basis for further development of novel noncompetitive EP2 antagonists.Support or Funding InformationThis work was supported by the National Institutes of Health [Grant Numbers 1R01NS097776 (RD), 1 U01 AG052460 (TG)]. C.J. is a member of the joint training program between Emory University School of Medicine and Xiangya School of Medicine, Central South University, and is supported by the China Scholarship Council. We gratefully acknowledge the gift of ONO‐AE1‐259 by Ono Pharmaceuticals.

Two new ditetrahydrofuran lignans from Artemisia sieversiana

Two new ditetrahydrofuran lignans, named sieverlignans A and B (1 and 2), together with six known ones (3–8), were isolated from the aerial parts of Artemisia sieversiana. Their structures were established on the basis of spectroscopic analysis including HRMS, NMR spectra and circular dichroism experiments. All the compounds were evaluated for their anti-neuroinflammatory effects on the lipopolysaccharides (LPS)-induced nitric oxide production in BV-2 murine microglial cells. Compound 2 exhibited the significant activity with its IC50 value of 11.9 ± 0.8 μM, respectively, compared to a positive control quercetin with its IC50 value of 16.0 ± 1.1 μM.

VGF-derived peptide TLQP-21 modulates microglial function through C3aR1 signaling pathways and reduces neuropathology in 5xFAD mice

BackgroundMultiomic studies by several groups in the NIH Accelerating Medicines Partnership for Alzheimer’s Disease (AMP-AD) identified VGF as a major driver of Alzheimer’s disease (AD), also finding that reduced VGF levels correlate with mean amyloid plaque density, Clinical Dementia Rating (CDR) and Braak scores. VGF-derived peptide TLQP-21 activates the complement C3a receptor-1 (C3aR1), predominantly expressed in the brain on microglia. However, it is unclear how mouse or human TLQP-21, which are not identical, modulate microglial function and/or AD progression.MethodsWe performed phagocytic/migration assays and RNA sequencing on BV2 microglial cells and primary microglia isolated from wild-type or C3aR1-null mice following treatment with TLQP-21 or C3a super agonist (C3aSA). Effects of intracerebroventricular TLQP-21 delivery were evaluated in 5xFAD mice, a mouse amyloidosis model of AD. Finally, the human HMC3 microglial cell line was treated with human TLQP-21 to determine whether specific peptide functions are conserved from mouse to human.ResultsWe demonstrate that TLQP-21 increases motility and phagocytic capacity in murine BV2 microglial cells, and in primary wild-type but not in C3aR1-null murine microglia, which under basal conditions have impaired phagocytic function compared to wild-type. RNA sequencing of primary microglia revealed overlapping transcriptomic changes induced by treatment with TLQP-21 or C3a super agonist (C3aSA). There were no transcriptomic changes in C3aR1-null or wild-type microglia exposed to the mutant peptide TLQP-R21A, which does not activate C3aR1. Most of the C3aSA- and TLQP-21-induced differentially expressed genes were linked to cell migration and proliferation. Intracerebroventricular TLQP-21 administration for 28 days via implanted osmotic pump resulted in a reduction of amyloid plaques and associated dystrophic neurites and restored expression of subsets of Alzheimer-associated microglial genes. Finally, we found that human TLQP-21 activates human microglia in a fashion similar to activation of murine microglia by mouse TLQP-21.ConclusionsThese data provide molecular and functional evidence suggesting that mouse and human TLQP-21 modulate microglial function, with potential implications for the progression of AD-related neuropathology.

Attenuation of paraquat-induced inflammation by inhibitors of phosphorylation of mitogen-activated protein kinases in BV2 microglial cells

Paraquat has dopaminergic neurotoxicity and potentially contributes to Parkinson's disease (PD) as a risk factor. However, the cellular and molecular mechanisms of PQ-induced neurodegeneration have not been clearly elucidated. Studies have shown that PQ induces microglial neuroinflammation through toll-like receptor 4 (TLR4)-nuclear factor-κB pathway, resulting in neuronal cell loss. Mitogen-activated protein kinases (MAPKs) are involved in the production of pro-inflammatory cytokines in microglia, and in this study, the role of MAPKs in PQ-activated microglial inflammation was investigated. Murine BV2 microglial cells were treated with 40 μM of PQ following pretreatment of the cells with selective inhibitor of MAPKs phosphorylation for blockage of the phosphorylation of ERK, JNK and P38, or a specific TLR4 inhibitor for blocking the activation of TLR4. The protein expression of phosphorylated ERK, JNK and p38, and the transcription expression of pro-inflammatory mediators were assessed with Western blotting and qRT-PCR technique, respectively. The results indicated that PQ significantly induced the phosphorylation of ERK, JNK and P38 in microglia, while MAPKs inhibitors suppressed PQ-induced phosphorylation of ERK, JNK and P38, and reduced the transcription level of pro-inflammatory cytokines. PQ-stimulated phosphorylation of ERK, JNK and P38 was also reduced by TLR4 inhibitor. The inhibited intensity in the level of pro-inflammatory cytokine transcription was obviously greater in TLR4 inhibitor + PQ group than in each MAPK inhibitor + PQ group. Taken together, inhibition of MAPKs phosphorylation partially attenuates PQ-induced microglial inflammation, which may become a potential intervention strategy for PQ neurotoxicity.

Harpagophytum procumbens Extract Ameliorates Allodynia and Modulates Oxidative and Antioxidant Stress Pathways in a Rat Model of Spinal Cord Injury.

Spinal cord injury (SCI) isa deliberating disorder with impairments in locomotor deficits and incapacitating sensory abnormalities. Harpagophytum procumbens (Hp) is a botanical widely used for treating inflammation and pain related to various inflammatory and musculoskeletal conditions. Using a modified rodent contusion model of SCI, we explored the effects of this botanical on locomotor function and responses to mechanical stimuli, and examined possible neurochemical changes associated with SCI-induced allodynia. Following spinal cord contusion at T10 level, Hp (300mg/kg, p.o.) or vehicle (water) was administered daily starting 24h post-surgery, and behavioral measurements made every-other day until sacrifice (Day 21). Hp treatment markedly ameliorated the contusion-induced decrease in locomotor function and increased sensitivity to mechanical stimuli. Determination of Iba1 expression in spinal cord tissues indicated microglial infiltration starting 3days post-injury. SCI results in increased levels of 4-hydroxynonenal, an oxidative stress product and proalgesic, which was diminished at 7days by treatment with Hp. SCI also enhanced antioxidant heme oxygenase-1 (HO-1) expression. Concurrent studies of cultured murine BV-2 microglial cells revealed that Hp suppressed oxidative/nitrosative stress and inflammatory responses, including production of nitric oxide and reactive oxygen species, phosphorylation of cytosolic phospholipases A2, and upregulation of the antioxidative stress pathway involving the nuclear factor erythroid 2-related factor 2 and HO-1. These results support the use of Hp for management of allodynia by providing resilience against the neuroinflammation and pain associated with SCI and other neuropathological conditions.

Deacetyl Ganoderic Acid F Inhibits LPS-Induced Neural Inflammation via NF-κB Pathway Both In Vitro and In Vivo.

Microglia mediated neuronal inflammation has been widely reported to be responsible for neurodegenerative disease. Deacetyl ganoderic acid F (DeGA F) is a triterpenoid isolated from Ganoderma lucidum, which is a famous edible and medicinal mushroom used for treatment of dizziness and insomnia in traditional medicine for a long time. In this study the inhibitory effects and mechanisms of DeGA F against lipopolysaccharide (LPS)-induced inflammation both in vitro and in vivo were investigated. On murine microglial cell line BV-2 cells, DeGA F treatment inhibited LPS-triggered NO production and iNOS expression and affected the secretion and mRNA levels of relative inflammatory cytokines. DeGA F inhibited LPS-induced activation of the NF-κB pathway, as evidenced by decreased phosphorylation of IKK and IκB and the nuclear translocation of P65. In vivo, DeGA F treatment effectively inhibited NO production in zebrafish embryos. Moreover, DeGA F suppressed the serum levels of pro-inflammatory cytokines, including TNF-α and IL-6 in LPS-stimulated mice model. DeGA F reduced inflammatory response by suppressing microglia and astrocytes activation and also suppressed LPS-induced NF-κB activation in mice brains. Taken together, DeGA F exhibited remarkable anti-inflammatory effects and promising therapeutic potential for neural inflammation associated diseases.

Dopamine attenuates lipopolysaccharide-induced expression of proinflammatory cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglia.

Many reports have indicated that dopamine has immunomodulatory effects on peripheral immune cells. The purpose of this study was to reveal the immunomodulatory effect of dopamine on the expression of proinflammatory cytokines in microglial cells, which are the immune cells of the central nervous system. In murine microglial cell line BV-2 cells, pretreatment with dopamine for 24 h attenuated the lipopolysaccharide (LPS)-induced expression of proinflammatory cytokines such as tumor-necrosis factor-α, interleukin-1β, and interleukin-6. Neither (5R)-8-chloro-3-methyl-5-phenyl-1,2,4,5-tetrahydro-3-benzazepin-7-ol; hydrochloride (SCH-23390) nor sulpiride, which are dopamine D1-like and D2-like receptor antagonists, respectively, affected the attenuation of LPS-induced expression of cytokines by dopamine. In addition, pretreatment with neither (−)-(6aR,12bR)-4,6,6a,7,8,12b-Hexahydro-7-methylindolo[4,3-a]phenanthridin (CY208-243) nor bromocriptine, dopamine D1-like and D2-like receptor agonists, respectively, was effective in doing so. However, N-acetylcysteine (NAC), which inhibits dopamine oxidation to dopamine quinone, did inhibit this attenuated expression. Dopamine increased the level of quinoproteins, and this increase was inhibited by NAC. Western blot and immunocytochemical analyses revealed that dopamine inhibited LPS-induced nuclear translocation of nuclear factor-kappa B (NF-κB) p65. Dopamine also attenuated the expression of cytokines and the nuclear translocation of NF-κB p65 induced by LPS in mouse microglial cells in primary culture. These results suggest that dopamine attenuated LPS-induced expression of cytokines by inhibiting the nuclear translocation of NF-κB p65 through the formation of dopamine quinone in microglial cells.

Neuroprotective effect of biosynthesised gold nanoparticles synthesised from root extract of Paeonia moutan against Parkinson disease – In vitro &In vivo model

Parkinson disease is one of the most common neurological movement disorders affecting geriatric population. Biosynthesized gold nanoparticles are the ideal alternatives spotlighted by many researchers to treat various diseases. In the present study we synthesized gold nanoparticles using the root extract of Paeonia mountan, woody trees which are used in traditional Chinese medicine to be prescribed for diverse diseases. The synthesis of gold nanoparticles was confirmed with UV-Vis spectroscopic analysis and characterized using FTIR, HR-TEM, EDAX and XRD analysis. The cytotoxicity property of synthesized gold nanoparticles was assessed using MTT assay in the murine microglial BV2 cells. The neuroprotective effect of synthesized gold nanoparticles in inflammatory agent lipopolysaccharides triggered murine microglial BV2 cells was evaluated using nitric oxide, prostaglandin E2 and inflammatory cytokines assays such as IL-6&IL-1β. Further to confirm in vivo effect of synthesized nanoparticles, the nanoparticles were treated to Parkinson induced C57BL/6 mice. Behavioral, biochemical and molecular analysis were performed to estimate the potency of synthesized gold nanoparticles against the Parkinson induction in mice model. Our characterization results prove the gold nanoparticles synthesized using Paeonia mountan fulfills the requirement of ideal nanodrug and it potentially inhibited the inflammation in in vitro murine microglial BV2. The results of in vivo experiments authentically confirm gold nanoparticles synthesized using Paeonia mountan alleviates the neuroinflammation and improves the motor coordination in Parkinson induced mice.

Novel Microglia Cell Line Expressing the Human EP2 Receptor.

Recently, EP2 signaling pathways were shown to regulate the classical activation and death of microglia in rat primary microglial culture. The study of microglial cells has been challenging because they are time-consuming to isolate in culture, they are demanding in their growth requirements, and they have a limited lifespan. To circumvent these difficulties, we created a murine BV2 microglial cell line stably expressing human EP2 receptors (BV2-hEP2) and further explored EP2 modulation of microglial functions. The BV2-hEP2 cells displayed cAMP elevation when exposed to the selective EP2 receptor agonists (ONO-AE1-259-1 and CP544326), and this response was competitively inhibited by TG4-155, a selective EP2 antagonist (Schild KB = 2.6 nM). By contrast, untransfected BV2 cells were unresponsive to selective EP2 agonists. Similar to the case of rat primary microglia, BV2-hEP2 microglia treated with lipopolysaccharide (LPS) (100 ng/mL) displayed rapid and robust induction of the inflammatory mediators COX-2, IL-1β, TNFα, and IL-6. EP2 activation depressed TNFα induction but exacerbated that of the other inflammatory mediators. Like primary microglia, classically activated BV2 microglia phagocytose fluorescent-labeled latex microspheres. The presence of EP2, but not its activation by agonists, in BV2-hEP2 microglia reduced phagocytosis and proliferation by 65% and 32%, respectively, compared to BV2 microglia. Thus, BV2-hEP2 is the first microglial cell line that retains the EP2 modulation of immune regulation and phagocytic ability of native microglia. Suppression of phagocytosis by the EP2 protein appears unrelated to classical EP2 signaling pathways, which has implications for therapeutic development of EP2 antagonists.

A novel target to reduce microglial inflammation and neuronal damage after deep hypothermic circulatory arrest

BackgroundNeuroinflammation acts as a contributor to neurologic deficits after deep hypothermic circulatory arrest. However, the molecular mechanism remains unclear. This study postulates that cold-inducible RNA-binding protein can promote deep hypothermic circulatory arrest–induced neuroinflammation. MethodsRats were randomly assigned into 3 groups (n = 5, each group): sham group, deep hypothermic circulatory arrest group, and deep hypothermic circulatory arrest + Cirp−/− group (Cirp−/− group). Murine microglial BV2 cells were administered by adeno-associated viral vectors containing cold-inducible RNA-binding protein small interference RNA or negative control small interference RNA at 2 days before 4-hour oxygen–glucose deprivation at 18°C. Microglial activation, cell death, neuroinflammation, and related protein expression were assessed in tissue samples and cell cultures. ResultsCold-inducible RNA-binding protein was elevated along with evident neuroinflammation and neuronal damage in rats exposed to deep hypothermic circulatory arrest. In Cirp−/− rats, histologic injury (3.00 [interquartile range, 2.00-3.00] vs 1.00 [interquartile range, 1.00-1.50] neuropathological score, P < .001) and microglial activation (40 ± 4 vs 13 ± 7 CA1 area, P < .001) were alleviated after deep hypothermic circulatory arrest. With RNA-sequencing analysis, this associated with reduction of key proinflammatory cytokines induced by inhibiting Brd2-NF-κB signals. In BV2 cells treated with small interference RNA-cold-inducible RNA-binding protein, similar protective effects were observed, including decreased proinflammatory cytokines and cytotoxicity. Brd2-NF-κB signals were confirmed by the addition of Brd2 inhibitor JQ1. Notably, the conditioned medium from BV2 cells transfected with small interference RNA cold-inducible RNA-binding protein significantly reduced apoptosis in neural SH-SY5Y cells after oxygen–glucose deprivation, which was similar to that after JQ1 administration. ConclusionsEnhanced cold-inducible RNA-binding protein in microglia aggravates neuronal injury by promoting the release of proinflammatory cytokines, which might be mediated through Brd2-NF-κB signals during deep hypothermic circulatory arrest.

Vitexnegheteroin M, a new phenolic glycoside from Vitex negundo var. heterophylla

The investigation of chemical constituents from the leaves and seeds of Vitex negundo var. heterophylla resulted in the isolation of one new phenolic glycoside, vitexnegheteroin M (1), together with eleven known compounds (2-12). Their structures were elucidated through extensive spectroscopic analyses including 1D, 2D NMR, HR-ESI-MS, and by comparison with the literature. Among them, compound 3 was obtained from the Vitex genus for the first time, and compounds 4-8, and 10-12 were isolated from this plant for the first time. Their anti-inflammatory activities were investigated by measuring nitric oxide (NO) production in lipopolysaccharide (LPS)-induced murine microglial BV-2 cells. As the obtained results, compound 9 showed the strongest inhibitory activity against LPS-stimulated NO production in BV-2 cells with IC50 value ranging from 8.65 ± 0.67 μM.

Human Urinary Kallidinogenase Reduces Lipopolysaccharide-Induced Neuroinflammation and Oxidative Stress in BV-2 Cells.

Migraine is one of the most common neurological disorders which poses significant socioeconomic burden worldwide. Neuroinflammation and oxidative stress both play important roles in the pathogenesis of migraine. Human urinary kallidinogenase (UK) is a tissue kallikrein derived from human urine. Increasing evidence suggests that UK may protect against ischemic stroke, but UK's treatment potential against migraine remains to be explored. Immortal BV-2 murine microglial cells were treated with UK (125 nM, 250 nM, and 500 nM) and then given lipopolysaccharides (LPS, 1000 ng/mL). Cell viability of BV-2 cells was tested by the CCK-8 assay. Expressions of tumor necrosis factor-α (TNFα), prostaglandin E2 (PGE2), interleukin-6 (IL-6), and interleukin-1β (IL-1β) were examined with the ELISA method and western blot. Intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) were measured to determine oxidative stress. Our results showed that LPS administration increased the levels of proinflammatory cytokines (TNFα, PGE2, IL-6, and IL-1β) and oxidative stress (ROS and MDA) when compared with the control group and decreased significantly upon introduction with UK. Taken together, UK treatment reduced LPS-induced neuroinflammation and oxidative stress in a dose-dependent manner, which might be a potential treatment of migraine.

Curcumin Regulates Anti-Inflammatory Responses by JAK/STAT/SOCS Signaling Pathway in BV-2 Microglial Cells.

Microglia play important physiological roles in central nervous system (CNS) homeostasis and in the pathogenesis of inflammatory brain diseases. Inflammation stimulates microglia to secrete cytokines and chemokines that guide immune cells to sites of injury/inflammation. Neuroinflammation is also strongly implicated in the pathogenesis of a number of neurodegenerative diseases, including Alzheimer’s disease and Parkinson’s disease, for which nutritional intervention could represent a benefit due to a lack of clinically efficacious drugs. To this end, the anti-inflammatory mechanisms of several phytochemicals, including curcumin, have been extensively studied. The present experiments show that the administration of curcumin is able to increase the production of the anti-inflammatory cytokines, IL-4 and IL-10, in murine BV-2 microglial cells treated with lipopolysaccharide (LPS). Consistent with these data, curcumin stimulation upregulates the expression of Suppressors of cytokine signaling (SOCS)-1, whereas phosphorylation of the JAK2 and STAT3 was reduced. Taken together, these results provide evidence that curcumin is able to regulate neuroinflammatory reactions by eliciting anti-inflammatory responses in microglia through JAK/STAT/SOCS signaling pathway modulation.

Resveratrol Suppresses Aβ-Induced Microglial Activation Through the TXNIP/TRX/NLRP3 Signaling Pathway.

Microglia-mediated neuroinflammation plays an important role in Alzheimer's disease development. Resveratrol, a natural polyphenol from the Japanese knotweed (Polygonum cuspidatumand), is known to protect against neuroinflammation, but the mechanism remains unclear. To begin to explore potential mechanisms, we created a model of inflammatory injury in BV-2 murine microglial cells based on the induction of amyloid-β. We found that resveratrol (10 and 50 nM) significantly inhibited Aβ-induced proliferation and activation of BV-2 cells, as well as their release of the proinflammatory cytokines, IL-6 and TNF-α. Resveratrol also suppressed the overexpression of cleaved caspase-1 and IL-1β, and decreased Aβ-stimulated degradation of IkBα and phosphorylation of NF-κB phosphorylation. Western blot analysis showed that Aβ upregulated the TXNIP/TRX/NLRP3 pathway, while resveratrol treatment inhibited it. We conclude that resveratrol protects microglia from Aβ-stimulated inflammation by suppressing the inflammatory response, at least in part by inhibiting the TXNIP/TRX/NLRP3 signaling pathway.