Activated BV-2 Microglial Cells Research Articles

Natural product HTP screening for antibacterial (E.coli 0157:H7) and anti-inflammatory agents in (LPS from E. coli O111:B4) activated macrophages and microglial cells; focus on sepsis.

BackgroundAcute systemic inflammatory response syndrome arising from infection can lead to multiple organ failure and death, with greater susceptibility occurring in immunocompromised individuals. Moreover, sub-acute chronic inflammation is a contributor to the pathology of diverse degenerative diseases (Parkinson’s disease, Alzheimer’s disease and arthritis). Given the known limitations in Western medicine to treat a broad range of inflammatory related illness as well as the emergence of antibiotic resistance, there is a renewed interest in complementary and alternative medicines (CAMs) to achieve these means.MethodsA high throughput (HTP) screening of >1400 commonly sold natural products (bulk herbs, cooking spices, teas, leaves, supplement components, nutraceutical food components, fruit and vegetables, rinds, seeds, polyphenolics etc.) was conducted to elucidate anti-inflammatory substances in lipopolysaccharide (LPS) (E. coli serotype O111:B4) monocytes: RAW 264.7 macrophages [peripheral], BV-2 microglia [brain]) relative to hydrocortisone, dexamethasone and L-N6-(1Iminoethyl)lysine (L-NIL). HTP evaluation was also carried out for lethal kill curves against E.coli 0157:H7 1x106 CFU/mL relative to penicillin. Validation studies were performed to assess cytokine profiling using antibody arrays. Findings were corroborated by independent ELISAs and NO2–/iNOS expression quantified using the Griess Reagent and immunocytochemistry, respectively. For robust screening, we developed an in-vitro efficacy paradigm to ensure anti-inflammatory parameters were observed independent of cytotoxicity. This caution was taken given that many plants exert tumoricidal and anti-inflammatory effects at close range through similar signaling pathways, which could lead to false positives.ResultsThe data show that activated BV-2 microglia cells (+ LPS 1μg/ml) release >10-fold greater IL-6, MIP1/2, RANTES and nitric oxide (NO2–), where RAW 264.7 macrophages (+ LPS 1μg/ml) produced > 10-fold rise in sTNFR2, MCP-1, IL-6, GCSF, RANTES and NO2–. Data validation studies establish hydrocortisone and dexamethasone as suppressing multiple pro-inflammatory processes, where L-NIL suppressed NO2–, but had no effect on iNOS expression or IL-6. The screening results demonstrate relative few valid hits with anti-inflammatory effects at < 250μg/ml for the following: Bay Leaf (Laurus nobilis), Elecampagne Root (Inula helenium), Tansy (Tanacetum vulgare),Yerba (Eriodictyon californicum) and Centipeda (Centipeda minima), Ashwagandha (Withania somnifera), Feverfew (Tanacetum parthenium), Rosemary (Rosmarinus officinalis), Turmeric Root (Curcuma Longa), Osha Root (Ligusticum porteri), Green Tea (Camellia sinensis) and constituents: cardamonin, apigenin, quercetin, biochanin A, eupatorin, (-)-epigallocatechin gallate (EGCG) and butein. Natural products lethal against [E. coli 0157:H7] where the LC50 < 100 μg/ml included bioactive silver hydrosol-Argentyn 23, green tea (its constituents EGCG > Polyphenon 60 > (-)-Gallocatechin > Epicatechin > (+)-Catechin), Grapeseed Extract (Vitis vinifera), Chinese Gallnut (its constituents gallic acid > caffeic acid) and gallic acid containing plants such as Babul Chall Bark (Acacia Arabica), Arjun (Terminalia Arjuna) and Bayberry Root Bark (Morella Cerifera). ConclusionsThese findings emphasize and validate the previous work of others and identify the most effective CAM anti-inflammatory, antibacterial compounds using these models. Future work will be required to evaluate potential combination strategies for long-term use to prevent chronic inflammation and possibly lower the risk of sepsis in immunocompromised at risk populations.

Anti-neuroinflammatory effect of Sophoraflavanone G from Sophora alopecuroides in LPS-activated BV2 microglia by MAPK, JAK/STAT and Nrf2/HO-1 signaling pathways

Background: Neuroinflammation plays a vital role in Alzheimer's disease (AD) and other neurodegenerative conditions. Sophora alopecuroides is widely used in traditional Uighur's medicine for the treatment of inflammation. Sophoraflavanone G (SG), a major flavonoid found in the S. alopecuroides, has also been reported to exhibit anti-inflammatory activity both in vitro and in vivo. However, the effect of S. alopecuroides and SG on microglia-mediated neuroinflammation has not been investigated.Purpose: The present study was designed to evaluate the anti-neuroinflammatory effect of S. alopecuroides and SG against lipopolysaccharide (LPS)-activated BV2 microglial cells and to explore the underlying mechanisms.Methods: We measured the production of pro-inflammatory mediators and cytokines, and analyzed relevant mRNA and protein expressions by qRT-PCR and Western Blot.Results: S. alopecuroides extract (SAE) and SG inhibited the LPS-induced release of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β). Additionally, SG reduced gene expressions of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-6 and IL-1β, and further decreased the protein expressions of iNOS and COX-2. Mechanism studies found that SG down-regulated phosphorylated mitogen-activated protein kinases (MAPKs), phosphoinositide-3-kinase (PI3K)/AKT and Janus kinase/signal transducer and activator of transcription (JAK/STAT), and up-regulated heme oxygenase-1 (HO-1) expression via nuclear translocation of nuclear factor E2-related factor 2 (Nrf2). In addition, SG inhibited the cytotoxicity of conditioned medium prepared by LPS-activated BV2 microglia to neuronal PC12 cells and improved cell viability.Conclusion: S. alopecuroides and SG displayed anti-neuroinflammatory activity in LPS-activated BV2 microglia. SG was able to inhibit the neuroinflammation by MAPKs, PI3K/AKT, JAK/STAT and Nrf2/HO-1 signaling pathways and might act as a natural therapeutic agent to be further developed for the treatment of various neuroinflammatory conditions.

Vaccinium bracteatum Thunb. Exerts Anti-Inflammatory Activity by Inhibiting NF-κB Activation in BV-2 Microglial Cells.

This study was designed to evaluate the pharmacological effects of Vaccinium bracteatum Thunb. methanol extract (VBME) on microglial activation and to identify the underlying mechanisms of action of these effects. The anti-inflammatory properties of VBME were studied using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells. We measured the production of nitric oxide (NO), inducible NO synthase (iNOS), cyclooxygenase (COX)-2, prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) as inflammatory parameters. We also examined the effect of VBME on intracellular reactive oxygen species (ROS) production and the activity of nuclear factor-kappa B p65 (NF-κB p65). VBME significantly inhibited LPS-induced production of NO and PGE2 and LPS-mediated upregulation of iNOS and COX-2 expression in a dose-dependent manner; importantly, VBME was not cytotoxic. VBME also significantly reduced the generation of the pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. In addition, VBME significantly dampened intracellular ROS production and suppressed NF-κB p65 translocation by blocking IκB-α phosphorylation and degradation in LPS-stimulated BV2 cells. Our findings indicate that VBME inhibits the production of inflammatory mediators in BV-2 microglial cells by suppressing NF-κB signaling. Thus, VBME may be useful in the treatment of neurodegenerative diseases due to its ability to inhibit inflammatory mediator production in activated BV-2 microglial cells.

Vitamin D2 suppresses amyloid-β 25–35 induced microglial activation in BV2 cells by blocking the NF-κB inflammatory signaling pathway

AimsPresent emerging world is emphasizing the implication of vitamin D deficiency associated with development of inflammation and neurodegenerative disorder like Alzheimer's disease (AD). The chief neuropathological hallmark of AD is aggregation of amyloid-beta (Aβ) peptides surrounding microglial cells in human brain. Microglial activation plays a key role in inflammatory response and neuronal injury. Naturally abundant vitamin D2 (VD2) exhibiting anti-inflammatory activities are yet to explore more. This study has investigated the inhibitory effect of VD2 on inflammatory activities of BV2 microglial cells. Main methodsCellular compatibility of VD2 and Aβ25–35 protein in treated BV2 microglial cells were measured by CCK-8 assay. Induction of iNOS, COX-2 and NF-κB signaling cascade were measured by western blotting, whereas pro-inflammatory cytokines were measured by ELISA. In addition, generation of ROS was detected by fluorescence intensity. Key findingsMorphological observations showed that Aβ25–35 induced BV2 cells stimulation noticeably got reduced in VD2 pre-treated group at 24h time period. Anti-inflammatory activities of VD2 was observed demonstrating the inhibition of up-regulated iNOS and COX-2 protein expression further confirmed by attenuating the activated microglia released pro-inflammatory cytokines IL-1β, IL-6, TNF- α and ROS, while blocking the phosphorylation of NF-κB p65 in nucleus by preventing IκB-α degradation and phosphorylation in cytosol. SignificanceThe present study revealed that VD2 blocked the phosphorylation of NF-κB inflammatory signaling pathway in Aβ25–35 induced activated BV2 microglial cells by suppressing ROS generation and inflammatory cytokines. Our finding suggests that vitamin D2 has therapeutic potential against inflammation and Alzheimer's disease.

Resolvin D1 reduces the toxic effects of active BV-2 microglial cells to PC12 cells

Objective To investigate the effects of Resolvin D1 (RvD1) on lipopolysaccharide (LPS)-induced BV-2 microglial cells activation and neuron-like PC12 cells apoptosis through suppressing activated BV-2 microglial cell-mediated inflammatory reaction. Methods BV-2 microglial cells were divided into four groups: control group, LPS group, RvD1+ LPS group and RvD1group. BV-2 cells in each group were incubated with corresponding drugs for 24 h. The PC12 cells in each group were incubated with corresponding BV-2 cells culture supernatant for 24 h. The apoptotic PC12 cells were observed by 4', 6-diamidino-2-phenylindole (DAPI) staining. The survival rate of PC12 cells was tested by cell counting kit (CCK-8) assay. The apoptosis rate of PC12 cells was examined by Annexin V-fluoresceine isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit. The BV-2 cells culture supernatants were collected after incubating with corresponding drugs for 24 h, and the concentrations of interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) were determined by enzyme linked immunosorbent assay (ELISA). Results Compared to the control group, the PC12 cells presented evident apoptosis features and the cells survival rate (100.00% vs. 84.93%) was significantly reduced (P<0.05) and the apoptosis rate (5.12% vs. 14.42%) was significantly increased (P<0.05) in LPS group. The concentrations of IL-1β, IL-6, and TNF-α [(45.68±4.80) pg/ml vs. (297.97±16.14) pg/ml, (26.97±4.44) pg/ml vs. (297.46±14.21) pg/ml, (80.12±9.34) pg/ml vs. (920.63±25.56) pg/ml]were significantly increased (P<0.05) in LPS group. As compared with the LPS group, the apoptosis of PC12 cells was significantly reduced, the cells survival rate (91.60% vs. 84.93%) was significantly increased (P<0.05) and the apoptosis rate (8.51% vs. 14.42%) was significantly reduced in RvD1+ LPS group. The concentrations of IL-1β, IL-6, and TNF-α [(114.47±9.65) pg/ml vs. (297.97±16.14) pg/ml, (111.10±9.57) pg/ml vs. (297.46±14.21) pg/ml, (320.66±18.21) pg/ml vs. (920.63±25.56) pg/ml)]were significantly reduced (P<0.05) in RvD1+ LPS group. Conclusion RvD1 could reduce the toxic effects of activated BV-2 microglial cells on PC12 cells through reducing activated BV-2 microglial cell-mediated inflammatory factor levels. Key words: Resolvin D1; Lipopolysaccharide; Microglial cells; Inflammation

Ceria/POMs hybrid nanoparticles as a mimicking metallopeptidase for treatment of neurotoxicity of amyloid-β peptide

Protein misfolding to amyloid aggregates is the hallmark for neurodegenerative disease. While much attention has been paid to screen natural proteases that can degrade amyloid-β peptides (Aβ), it is difficult to apply them in the clinics with the intractable problem of immunogenicity in living organisms. Herein, we rationally designed an artificial nanozyme, Ceria/Polyoxometalates hybrid (CeONP@POMs) with both proteolytic and superoxide dismutase (SOD) activities. Our results indicated that CeONP@POMs could efficiently degrade Aβ aggregates and reduce intracellular reactive oxygen species (ROS). More importantly, CeONP@POMD could not only promote PC12 cell proliferation and can cross blood−brain barrier (BBB), but also inhibit Aβ-induced BV2 microglial cell activation which was demonstrated by immunoluorescence assay and flow cytometry measurements. In vivo studies further indicated that CeONP@POMD as nanozyme possessed good biocompatibility, evidenced by a detailed study of their biodistribution, body weight change, and in vivo toxicology. Therefore, our results pave the way for design of multifunctional artificial nanozyme for treatment of neurotoxicity of amyloid-β peptide.

Could Intrathymic Injection of Myelin Basic Protein Suppress Inflammatory Response After Co-culture of T Lymphocytes and BV-2 Microglia Cells?

Background:The interaction between activated microglia and T lymphocytes can yield abundant pro-inflammatory cytokines. Our previous study proved that thymus immune tolerance could alleviate the inflammatory response. This study aimed to investigate whether intrathymic injection of myelin basic protein (MBP) in mice could suppress the inflammatory response after co-culture of T lymphocytes and BV-2 microglia cells.Methods:Totally, 72 male C57BL/6 mice were randomly assigned to three groups (n = 24 in each): Group A: intrathymic injection of 100 μl MBP (1 mg/ml); Group B: intrathymic injection of 100 μl phosphate-buffered saline (PBS); and Group C: sham operation group. Every eight mice in each group were sacrificed to obtain the spleen at postoperative days 3, 7, and 14, respectively. T lymphocytes those were extracted and purified from the spleens were then co-cultured with activated BV-2 microglia cells at a proportion of 1:2 in the medium containing MBP for 3 days. After identified the T lymphocytes by CD3, surface antigens of T lymphocytes (CD4, CD8, CD152, and CD154) and BV-2 microglia cells (CD45 and CD54) were detected by flow cytometry. The expressions of pro-inflammatory factors of BV-2 microglia cells (interleukin [IL]-1β, tumor necrosis factor-α [TNF-α], and inducible nitric oxide synthase [iNOS]) were detected by quantitative real-time polymerase chain reaction (PCR). One-way analysis of variance (ANOVA) and the least significant difference test were used for data analysis.Results:The levels of CD152 in Group A showed an upward trend from the 3rd to 7th day, with a downward trend from the 7th to 14th day (20.12 ± 0.71%, 30.71 ± 1.14%, 13.50 ± 0.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The levels of CD154 in Group A showed a downward trend from the 3rd to 7th day, with an upward trend from the 7th to 14th day (10.00 ± 0.23%, 5.28 ± 0.69%, 14.67 ± 2.71% at postoperative days 3, 7, and 14, respectively, P < 0.05). The ratio of CD4+/CD8 + T in Group A showed a downward trend from the 3rd to 7th day, with the minimum at postoperative day 7, then an upward trend from the 7th to 14th day (P < 0.05). Meanwhile, the levels of CD45 and CD54 in Group A were found as the same trend as the ratio of CD4+/CD8 + T (CD45: 83.39 ± 2.56%, 82.74 ± 2.09%, 87.56 ± 2.11%; CD54: 3.80 ± 0.24%, 0.94 ± 0.40%, 3.41 ± 0.33% at postoperative days 3, 7, and 14, respectively, P < 0.05). The expressions of TNF-α, IL-1β, and iNOS in Group A were significantly lower than those in Groups B and C, and the values at postoperative day 7 were the lowest compared with those at postoperative days 3 and 14 (P < 0.05). No significant difference was found between Groups B and C.Conclusions:Intrathymic injection of MBP could suppress the immune reaction that might reduce the secondary immune injury of brain tissue induced by an inflammatory response.

New Lignans from Antidesma hainanensis Inhibit NO Production in BV2 Microglial Cells.

Two new lignans (7S,7'R,8S,8'R)-3,3'-dimethoxy-7,7'-epoxylignan-4,4',9-triol 4-O-β-D-glucopyranoside (1) and 9-O-formylaviculin (2) together with other thirteen known secondary metabolites were isolated from the leaves of Antidesma hainanensis. Their chemical structures were determined using NMR, electrospray ionization (ESI)-MS, circular dichroism (CD) spectroscopic methods, and as well as by comparison with those reported in the literature. Neuro-inflammatory activity of isolated compounds was evaluated by their inhibition on nitric oxide (NO) production in activated BV2 microglial cells. At concentration of 40 µM, compounds 1-3, 5, 7, 8, 9, 14, and 15 exhibited inhibitory effects over 50%, suggesting that they could be potential candidate drugs for the cure of neuro-inflammation. In addition, compounds 1, 8, 14, and 15 significantly inhibited 16.23, 27.76, 21.23, and 29.44% NO production at diluted concentration as low as 2.5 µM.

Folic Acid Is Able to Polarize the Inflammatory Response in LPS Activated Microglia by Regulating Multiple Signaling Pathways.

We investigated the ability of folic acid to modulate the inflammatory responses of LPS activated BV-2 microglia cells and the signal transduction pathways involved. To this aim, the BV-2 cell line was exposed to LPS as a proinflammatory response inducer, in presence or absence of various concentrations of folic acid. The production of nitric oxide (NO) was determined by the Griess test. The levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and IL-10 were determined by ELISA. Inducible NO synthase (iNOS), nuclear transcription factor-kappa B (NF-κB) p65, MAPKs protein, and suppressors of cytokine signaling (SOCS)1 and SOCS3 were analyzed by western blotting. TNF-α and IL-1β, as well as iNOS dependent NO production, resulted significantly inhibited by folic acid pretreatment in LPS-activated BV-2 cells. We also observed that folic acid dose-dependently upregulated both SOCS1 and SOCS3 expression in BV-2 cells, leading to an increased expression of the anti-inflammatory cytokine IL-10. Finally, p-IκBα, which indirectly reflects NF-κB complex activation, and JNK phosphorylation resulted dose-dependently downregulated by folic acid pretreatment of LPS-activated cells, whereas p38 MAPK phosphorylation resulted significantly upregulated by folic acid treatment. Overall, these results demonstrated that folic acid was able to modulate the inflammatory response in microglia cells, shifting proinflammatory versus anti-inflammatory responses through regulating multiple signaling pathways.

Tiagabine Protects Dopaminergic Neurons against Neurotoxins by Inhibiting Microglial Activation.

Microglial activation and inflammation are associated with progressive neuronal apoptosis in neurodegenerative disorders such as Parkinson’s disease (PD). γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, has recently been shown to play an inhibitory role in the immune system. Tiagabine, a piperidine derivative, enhances GABAergic transmission by inhibiting GABA transporter 1 (GAT 1). In the present study, we found that tiagabine pretreatment attenuated microglial activation, provided partial protection to the nigrostriatal axis and improved motor deficits in a methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. The protective function of tiagabine was abolished in GAT 1 knockout mice that were challenged with MPTP. In an alternative PD model, induced by intranigral infusion of lipopolysaccharide (LPS), microglial suppression and subsequent neuroprotective effects of tiagabine were demonstrated. Furthermore, the LPS-induced inflammatory activation of BV-2 microglial cells and the toxicity of conditioned medium toward SH-SY5Y cells were inhibited by pretreatment with GABAergic drugs. The attenuation of the nuclear translocation of nuclear factor κB (NF-κB) and the inhibition of the generation of inflammatory mediators were the underlying mechanisms. Our results suggest that tiagabine acts as a brake for nigrostriatal microglial activation and that it might be a novel therapeutic approach for PD.

Benfotiamine upregulates antioxidative system in activated BV-2 microglia cells.

Chronic microglial activation and resulting sustained neuroinflammatory reaction are generally associated with neurodegeneration. Activated microglia acquires proinflammatory cellular profile that generates oxidative burst. Their persistent activation exacerbates inflammation, which damages healthy neurons via cytotoxic mediators, such as superoxide radical anion and nitric oxide. In our recent study, we have shown that benfotiamine (S-benzoylthiamine O-monophosphate) possesses anti-inflammatory effects. Here, the effects of benfotiamine on the pro-oxidative component of activity of LPS-stimulated BV-2 cells were investigated. The activation of microglia was accompanied by upregulation of intracellular antioxidative defense, which was further promoted in the presence of benfotiamine. Namely, activated microglia exposed to non-cytotoxic doses of benfotiamine showed increased levels and activities of hydrogen peroxide- and superoxide-removing enzymes—catalase and glutathione system, and superoxide dismutase. In addition, benfotiamine showed the capacity to directly scavenge superoxide radical anion. As a consequence, benfotiamine suppressed the activation of microglia and provoked a decrease in NO and ·O−2 production and lipid peroxidation. In conclusion, benfotiamine might silence pro-oxidative activity of microglia to alleviate/prevent oxidative damage of neighboring CNS cells.

Anti-inflammatory effects of thymoquinone in activated BV-2 microglial cells

Thymoquinone (TQ), the main pharmacological active ingredient within the black cumin seed (Nigella sativa) is believed to be responsible for the therapeutic effects on chronic inflammatory conditions such as arthritis, asthma and neurodegeneration. In this study, we evaluated the potential anti-inflammatory role of TQ in lipopolysaccharide (LPS)-stimulated BV-2 murine microglia cells. The results obtained indicate that TQ was effective in reducing NO2− with an IC50 of 5.04μM, relative to selective iNOS inhibitor LNIL-l-N6-(1-iminoethyl)lysine (IC50 4.09μM). TQ mediated reduction in NO2− was found to parallel the decline of iNOS protein expression as confirmed by immunocytochemistry. In addition, we evaluated the anti-inflammatory effects of TQ on ninety-six (96) cytokines using a RayBio AAM-CYT-3 and 4 cytokine antibody protein array. Data obtained establish a baseline protein expression profile characteristic of resting BV-2 cells in the order of osteopontin>MIP-1alpha>MIP-1g>IGF-1 and MCP-I. In the presence of LPS [1ug/ml], activated BV-2 cells produced a sharp rise in specific pro-inflammatory cytokines/chemokine's IL-6, IL-12p40/70, CCL12 /MCP-5, CCL2/MCP-1, and G-CSF which were attenuated by the addition of TQ (10μM). The TQ mediated attenuation of MCP-5, MCP-1 and IL-6 protein in supernatants from activated BV-2 cells were corroborated by independent ELISA. Moreover, the data obtained from the RT2 PCR demonstrated a similar pattern where the LPS mediated elevation of mRNA for IL-6, CCL12/MCP-5, CCL2/MCP-1 were significantly attenuated by TQ (10μM). Also, in this study, consistent data were obtained for both protein antibody array densitometry and ELISA assays. In addition, TQ was found to reduce LPS mediated elevation in gene expression of Cxcl10 and a number of other cytokines in the panel. These findings demonstrate the significant anti-inflammatory properties of TQ in LPS activated microglial cells. Therefore, the obtained results might indicate the usefulness of TQ in delaying the onset of inflammation-mediated neurodegenerative disorders involving activated microglia cells.

Inhibition of neuroinflammation by synthetic androstene derivatives incorporating amino acid methyl esters on activated BV-2 microglia.

Androstene derivatives incorporating amino acid methyl esters were prepared, and their anti-inflammatory effects were evaluated in lipopolysaccharide (LPS)-activated BV-2 microglial cells. Several compounds exhibited dose-dependent inhibition. The most active compound, methyl ((3S,10R,13S)-3-hydroxy-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-17-carbonyl)-L-phenylalaninate (10) significantly suppressed LPS-induced expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Mechanistic studies revealed that compound 10 markedly inhibits phosphorylation of p38 mitogen-activated protein kinases (MAPKs) and subsequent transcription factor (NF-κB) and activator protein-1 (AP-1) activation. Furthermore, compound 10 decreased LPS-activated microglial neurotoxicity in a condition medium/HT-22 neuroblastoma co-culture model. Taken together, these results suggest 10 is a potential lead compound for the development of a novel therapeutic agent for neurodegenerative diseases.

RNA sequencing reveals distinct mechanisms underlying BET inhibitor JQ1-mediated modulation of the LPS-induced activation of BV-2 microglial cells.

BackgroundMicroglial cells become rapidly activated through interaction with pathogens, and their persistent activation is associated with the production and secretion of various pro-inflammatory genes, cytokines, and chemokines, which may initiate or amplify neurodegenerative diseases. Bromodomain and extraterminal domain (BET) proteins are a group of epigenetic regulators that associate with acetylated histones and facilitate the transcription of target genes. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities by inhibiting the expression of IL-6 and Tnf-α in macrophages. However, a genome-wide search for JQ1 molecular targets is largely unexplored in microglia.MethodsThe present study was aimed at evaluating the anti-inflammatory function and underlying genes targeted by JQ1 in lipopolysaccharide (LPS)-stimulated BV-2 microglial cells using two transcriptomic techniques: global transcriptomic biological duplicate RNA sequencing and quantitative real-time PCR. Associated biological pathways and functional gene ontology were also evaluated.ResultsWith a cutoff value of P ≤ 0.01 and fold change ≥1.5 log2, the expression level of 214 and 301 genes, including pro-inflammatory cytokine, chemokine, and transcription factors, was found to be upregulated in BV-2 cells stimulated with LPS for 2 and 4 h, respectively. Among these annotated genes, we found that JQ1 selectively reduced the expression of 78 and 118 genes (P ≤ 0.01, and fold change ≥ 1.5, respectively). Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced the expression of key inflammation- and immunity-related genes as well as cytokines/chemokines in the supernatants of LPS-treated primary microglial cells isolated from 3-day-old ICR mice. Utilizing functional group analysis, the genes affected by JQ1 were classified into four categories related to biological regulation, immune system processes, and response to stimuli. Moreover, the biological pathways and functional genomics obtained in this study may facilitate the suppression of different key inflammatory genes through JQ1-treated BV-2 microglial cells.ConclusionsThese unprecedented results suggest the BET inhibitor JQ1 as a candidate for the prevention or therapeutic treatment of inflammation-mediated neurodegenerative diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0260-5) contains supplementary material, which is available to authorized users.

Hypoxia potentiates cytotoxicity of LPS-activated microglial BV2 cells in vitro by synergistic effects on cytokine and nitric oxide secretion

Results Activation of BV2 microglial cells by LPS exposure stimulated significant and persistent production of NO, IL-1b, IL-6, and TNF-a. Even after LPS removal, ongoing NO and cytokine secretion could be observed. While hypoxia alone mediated exclusively a significant, shortterm increase of IL-1b, oxygen deprivation enhanced LPS-induced secretion of NO, IL-1b, IL-6, and TNF-a significantly. Surprisingly, pre-stimulation of BV2 cells by hypoxia prior LPS exposure abolished microglial activation suppressing LPS-induced NO production. Hereby, cell-free supernatants derived from LPS-activated microglial cells exhibited a stronger cytotoxic effect in glial and neuronal cells than LPS exposition per se (P < 0.001). Again, hypoxia potentiated LPS-induced cytotoxicity. Conclusion Present data prove that i) the outcome of hypoxia is determined by the microglial activation status and that ii) LPS-induced soluble factors rather than LPS are mediators of microglial neurotoxicity under conditions of hypoxia in vitro. Activation of pro-inflammatory pathways may sensitize microglial cells to promote hypoxia-induced injury of the developing brain. Consequently, our findings may promote neuroprotective therapeutic strategies in the field of perinatal brain injury.

Biological evaluation of synthetic chalcone and flavone derivatives as anti-inflammatory agents.

Flavonoids and chalcones are natural plant derived compounds with inherent therapeutic value for a range of human pathologies. In this study, a series of 24 substituted chalcones and flavones were synthesized and subsequently screened for anti-inflammatory effects on lipopolysaccharide (1 µg/ml)-activated BV-2 microglial cells by assessing initial production/release of nitric oxide (NO). The data obtained eliminate the majority of compounds as weak or non-effective, whereas 2'-hydroxy-3,4,5,3',4'-pentamethoxychalcone (1) and 2'-hydroxy-3,4,5-trimethoxychalcone (2) were potent, having an IC50 of 1.10 and 2.26 µM, respectively; with greater potency than L-N6-(1-iminoethyl)lysine selective iNOS inhibitor (IC50 = 3.1 µM) but less than steroidal dexamethasone (IC50 < 200 nM). The most potent compound (chalcone 1) attenuated NO parallel to reducing iNOS protein expression, events also corresponding to reduction of IL-1α, IL-10 and IL-6 pro-inflammatory cytokines. These findings suggest that the presence of electron donating groups OH and OCH3 on both A and B rings of synthetic compounds correlate to stronger anti-inflammatory potency.

Mesenchymal stem cells inhibit lipopolysaccharide-induced inflammatory responses of BV2 microglial cells through TSG-6.

Microglia are the primary immunocompetent cells in brain tissue and microglia-mediated inflammation is associated with the pathogenesis of various neuronal disorders. Recently, many studies have shown that mesenchymal stem cells (MSCs) display a remarkable ability to modulate inflammatory and immune responses through the release of a variety of bioactive molecules, thereby protecting the central nervous system. Previously, we reported that MSCs have the ability to modulate inflammatory responses in a traumatic brain injury model and that the potential mechanisms may be partially attributed to upregulated TNF-α stimulated gene/protein 6 (TSG-6) expression. However, whether TSG-6 exerts an anti-inflammatory effect by affecting microglia is not fully understood. In this study, we investigated the anti-inflammatory effects of MSCs and TSG-6 in an in vitro lipopolysaccharide (LPS)-induced BV2 microglial activation model. We found that MSCs and TSG-6 significantly inhibited the expression of pro-inflammatory mediators in activated microglia. However, MSC effects on microglia were attenuated when TSG-6 expression was silenced. In addition, we found that the activation of nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) pathways in LPS-stimulated BV2 microglial cells was significantly inhibited by TSG-6. Furthermore, we found that the presence of CD44 in BV2 microglial cells was essential for MSC- and TSG-6-mediated inhibition of pro-inflammatory gene expression and of NF-κB and MAPK activation in BV2 microglial cells. The results of this study suggest that MSCs can modulate microglia activation through TSG-6 and that TSG-6 attenuates the inflammatory cascade in activated microglia. Our study indicates that novel mechanisms are responsible for the immunomodulatory effect of MSCs on microglia and that MSCs, as well as TSG-6, might be promising therapeutic agents for the treatment of neurotraumatic injuries or neuroinflammatory diseases associated with microglial activation.

β-Asarone (cis-2,4,5-trimethoxy-1-allyl phenyl), attenuates pro-inflammatory mediators by inhibiting NF-κB signaling and the JNK pathway in LPS activated BV-2 microglia cells

Acorus species contains diverse pharmacologically active phytochemicals including α-asarone, β-asarone, and eugenol. We determined if β-asarone isolated from Acorus gramineus (AG) Solander would be efficacious in protecting BV-2 microglia cells from lipopolysaccharide (LPS)-induced stress signaling. BV-2 microglial cells were pretreated with an AG ethanol extract (1, 10, and 100μg/mL) or β-asarone (10, 50, and 100μM) prior to exposure to LPS (100ng/mL). AG and β-asarone inhibited LPS-induced production of nitric oxide in a dose-dependent manner. The mRNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 also decreased dose dependently following AG and β-asarone treatments. Immunostaining and immunoblot studies revealed that β-asarone also suppressed nuclear factor (NF)-κB activation by blocking IkB degradation. Further mechanistic studies revealed that β-asarone acted through the JNK/MAPK pathway. Taken together, our findings demonstrate that β-asarone exhibits anti-inflammatory effects by suppressing the production of pro-inflammatory mediators through NF-κB signaling and the JNK pathways in activated microglial cells and might be developed as a promising candidate to treat various neuroinflammatory diseases.

Tumor necrosis factor-α-stimulated brain pericytes possess a unique cytokine and chemokine release profile and enhance microglial activation

Brain pericytes are involved in neurovascular dysfunction, neurodegeneration and/or neuroinflammation. In the present study, we focused on the proinflammatory properties of brain pericytes to understand their participation in the induction of inflammation at the neurovascular unit (NVU). The NVU comprises different cell types, namely, brain microvascular endothelial cells, pericytes, astrocytes and microglia. Among these, we found pericytes to be the most sensitive to tumor necrosis factor (TNF)-α, possessing a unique cytokine and chemokine release profile. This was characterized by marked release of interleukin (IL)-6 and macrophage inflammatory protein-1α. Furthermore, TNF-α-stimulated pericytes induced expression of inducible nitric oxide synthase and IL-1β mRNAs, as an index of BV-2 microglial cell activation state, to the highest levels. Based on these findings, the possibility that brain pericytes act specifically as TNF-α-sensitive cells and as effectors of TNF-α through the release of proinflammatory factors, and that, as such, they have a role in inducing brain inflammation, should be considered.

Resolvin D1 promotes the interleukin-4-induced alternative activation in BV-2 microglial cells

BackgroundMicroglia play key roles in innate immunity, homeostasis, and neurotropic support in the central nervous system. Similar to macrophages, microglia adopt two different activation phenotypes, the classical and alternative activation. Resolvin D1 (RvD1) is considered to display potent anti-inflammatory and pro-resolving actions in inflammatory models. In this present study, we investigate the effect of RvD1 on IL-4-induced alternative activation in murine BV-2 microglial cells.MethodsBV-2 cells were incubated with RvD1 alone, IL-4 alone, or the combination of RvD1 and IL-4. Western blot and immunofluorescence were performed to detect protein levels of alternative activation markers arginase 1 (Arg1), chitinase 3-like 3 (Ym1). Moreover, we investigated the effects of RvD1 on IL-4-induced activation of signal transducer and activators of transcription 6 (STAT6) and peroxisome proliferator-activated receptor gamma (PPARγ).ResultsRvD1 promoted IL-4-induced microglia alternative activation by increasing the expression of Arg1 and Ym1. RvD1 also enhanced phosphorylation of STAT6, nuclear translocation of PPARγ and the DNA binding activity of STAT6 and PPARγ. These effects were reversed by butyloxycarbonyl-Phe-Leu-Phe-Leu-Phe (a formyl peptide receptor 2 antagonist). Further, the effects of RvD1 and IL-4 on Arg1 and Ym1 were blocked by the application of leflunomide (a STAT6 inhibitor) or GW9662 (a PPARγ antagonist).ConclusionsOur studies demonstrate that RvD1 promotes IL-4-induced alternative activation via STAT6 and PPARγ signaling pathways in microglia. These findings suggest that RvD1 may have therapeutic potential for neuroinflammatory diseases.