Migration Of BV-2 Cells Research Articles

Netrin-1 Is an Important Mediator in Microglia Migration.

Microglia migrate to the cerebral cortex during early embryonic stages. However, the precise mechanisms underlying microglia migration remain incompletely understood. As an extracellular matrix protein, Netrin-1 is involved in modulating the motility of diverse cells. In this paper, we found that Netrin-1 promoted microglial BV2 cell migration in vitro. Mechanism studies indicated that the activation of GSK3β activity contributed to Netrin-1-mediated microglia migration. Furthermore, Integrin α6/β1 might be the relevant receptor. Single-cell data analysis revealed the higher expression of Integrin α6 subunit and β1 subunit in microglia in comparison with classical receptors, including Dcc, Neo1, Unc5a, Unc5b, Unc5c, Unc5d, and Dscam. Microscale thermophoresis (MST) measurement confirmed the high binding affinity between Integrin α6/β1 and Netrin-1. Importantly, activation of Integrin α6/β1 with IKVAV peptides mirrored the microglia migration and GSK3 activation induced by Netrin-1. Finally, conditional knockout (CKO) of Netrin-1 in radial glial cells and their progeny led to a reduction in microglia population in the cerebral cortex at early developmental stages. Together, our findings highlight the role of Netrin-1 in microglia migration and underscore its therapeutic potential in microglia-related brain diseases.

Effect and mechanism of chlorogenic acid on cognitive dysfunction in mice by lipopolysaccharide-induced neuroinflammation.

Neuroinflammation is an important factor causing numerous neurodegenerative pathologies. Inflammation can lead to abnormal neuronal structure and function and even death, followed by cognitive dysfunction. There is growing evidence that chlorogenic acid has anti-inflammatory effects and immunomodulatory activity. The aim of this study was to elucidate the potential targets and molecular mechanisms of chlorogenic acid in the treatment of neuroinflammation. We used the lipopolysaccharide-induced neuroinflammation mouse model and the lipopolysaccharide-stimulated BV-2 cells in vitro model. Behavioral scores and experiments were used to assess cognitive dysfunction in mice. HE staining and immunohistochemistry were used to assess neuronal damage in the mouse brain. Immunofluorescence detected microglia polarization in mouse brain. Western blot and flow cytometry detected the polarization of BV-2 cells. The migration of BV-2 cells was detected by wound healing assay and transwell assay. Potential targets for chlorogenic acid to exert protective effects were predicted by network pharmacology. These targets were then validated using molecular docking and experiments. The results of in vivo experiments showed that chlorogenic acid had an obvious ameliorating effect on neuroinflammation-induced cognitive dysfunction. We found that chlorogenic acid was able to inhibit BV-2 cells M1 polarization and promote BV-2 cells M2 polarization in vitro while also inhibiting the abnormal migration of BV-2 cells. Based on the network pharmacology results, we identified the TNF signaling pathway as a key signaling pathway in which chlorogenic acid exerts anti-neuroinflammatory effects. Among them, Akt1, TNF, MMP9, PTGS2, MAPK1, MAPK14, and RELA are the core targets for chlorogenic acid to function. Chlorogenic acid can inhibit microglial polarization toward the M1 phenotype and improve neuroinflammation-induced cognitive dysfunction in mice by modulating these key targets in the TNF signaling pathway.

Microfluidic Wound-Healing Assay for ECM and Microenvironment Properties on Microglia BV2 Cells Migration.

Microglia cells, as the resident immune cells of the central nervous system (CNS), are highly motile and migratory in development and pathophysiological conditions. During their migration, microglia cells interact with their surroundings based on the various physical and chemical properties in the brain. Herein, a microfluidic wound-healing chip is developed to investigate microglial BV2 cell migration on the substrates coated with extracellular matrixes (ECMs) and substrates usually used for bio-applications on cell migration. In order to generate the cell-free space (wound), gravity was utilized as a driving force to flow the trypsin with the device. It was shown that, despite the scratch assay, the cell-free area was created without removing the extracellular matrix coating (fibronectin) using the microfluidic assay. It was found that the substrates coated with Poly-L-Lysine (PLL) and gelatin stimulated microglial BV2 migration, while collagen and fibronectin coatings had an inhibitory effect compared to the control conditions (uncoated glass substrate). In addition, the results showed that the polystyrene substrate induced higher cell migration than the PDMS and glass substrates. The microfluidic migration assay provides an in vitro microenvironment closer to in vivo conditions for further understanding the microglia migration mechanism in the brain, where the environment properties change under homeostatic and pathological conditions.

DOCK8 interference alleviates Aβ‑induced damage of BV2 cells by inhibiting STAT3/NLRP3/NF‑κB signaling.

Dementia is defined as memory loss and other cognitive decline and it severely influences daily life. Alzheimer's disease (AD) is the most common cause of dementia. Dedicator of cytokinesis 8 (DOCK8) is reported to be involved in neurological diseases. The present study focused on investigating the role that DOCK8 serves in AD and addressing its hidden regulatory mechanism. Initially, Aβ1-42 (Aβ) was applied for the administration of BV2 cells. Subsequently, the mRNA and protein expression levels of DOCK8 were evaluated utilizing reverse transcription-quantitative PCR (RT-qPCR) and western blotting. After the DOCK8 silencing, immunofluorescence staining (IF), ELISA, wound healing and Transwell assays were applied to assess ionized calcium binding adapter molecule-1 (IBA-1) expression, release of inflammatory factors, migration and invasion in Aβ-induced BV2 cells. IF was used to evaluate cluster of differentiation (CD)11b expression. RT-qPCR and western blotting were to analyze the levels of M1 cell markers inducible nitric oxide synthase (iNOS) and CD86. The expression of STAT3/NLR family pyrin domain containing 3 (NLRP3)/NF-κB signaling-related proteins were determined by western blotting. Finally, the viability and apoptosis in hippocampal HT22 cells with DOCK8 depletion were estimated. Results revealed that Aβ induction greatly stimulated the expression levels of IBA-1 and DOCK8. DOCK8 silencing suppressed Aβ-induced inflammation, migration and invasion of BV2 cells. Additionally, DOCK8 deficiency conspicuously decreased the expression levels of CD11b, iNOS and CD86. The expression of phosphorylated (p-)STAT3, NLRP3, ASC, caspase1 and p-p65 was downregulated in Aβ-induced BV2 cells after DOCK8 depletion. STAT3 activator Colivelin reversed the effects of DOCK8 knockdown on IBA-1 expression, inflammation, migration, invasion and M1 cell polarization. In addition, the viability and apoptosis in hippocampal HT22 cells stimulated by neuroinflammatory release of BV2 cells were repressed following DOCK8 deletion. Collectively, DOCK8 interference alleviated Aβ-induced damage of BV2 cells by inhibiting STAT3/NLRP3/NF-κB signaling.

The Role of Aldose Reductase in Beta-Amyloid-Induced Microglia Activation.

The occurrence of Alzheimer's disease has been associated with the accumulation of beta-amyloid (β-amyloid) plaques. These plaques activate microglia to secrete inflammatory molecules, which damage neurons in the brain. Thus, understanding the underlying mechanism of microglia activation can provide a therapeutic strategy for alleviating microglia-induced neuroinflammation. The aldose reductase (AR) enzyme catalyzes the reduction of glucose to sorbitol in the polyol pathway. In addition to mediating diabetic complications in hyperglycemic environments, AR also helps regulate inflammation in microglia. However, little is known about the role of AR in β-amyloid-induced inflammation in microglia and subsequent neuronal death. In this study, we confirmed that AR inhibition attenuates increased β-amyloid-induced reactive oxygen species and tumor necrosis factor α secretion by suppressing ERK signaling in BV2 cells. In addition, we are the first to report that AR inhibition reduced the phagocytotic capability and cell migration of BV2 cells in response to β-amyloid. To further investigate the protective role of the AR inhibitor sorbinil in neurons, we co-cultured β-amyloid-induced microglia with stem cell-induced neurons. sorbinil ameliorated neuronal damage in both cells in the co-culture system. In summary, our findings reveal AR regulation of microglia activation as a novel therapeutic target for Alzheimer's disease.

Activin A is a novel chemoattractant for migration of microglial BV2 cells

BackgroundMicroglia are involved in many neurodegenerative diseases and repairment of traumatic injury to the CNS. Activin A is a neurotrophic and neuroprotective factor that can regulate the activities of macrophages/microglia. However, the effects of activin A on the migration of microglia are still unclear. In this study, the role of activin A in regulation of the microglia migration was investigated with the murine microglial BV2 cell. MethodsThe levels of cytokines were detected by enzyme-linked immunosorbent assay (ELISA). The protein expression was examined by Western blotting. The adhesion of BV2 cells was assayed by real-time cell analysis (RTCA). The migration of BV2 cells was determined by transwell chamber and microfluidics device. Smad3 was overexpressed or knocked down in BV2 cells by transfection of Smad3 or Smad3 shRNA-expressing plasmids. ResultsActivin A inhibited the release of nitric oxide (NO) and inflammatory cytokines of TNF-α and IL-6 and the expression of TNF-α and IL-6 mRNA by BV2 cells. In contrast, activin A promoted the production of TGF-β1. Activin A inhibited adhesion, promoted wound healing and migration which is related to the expression of N-cadherin and E-cadherin expression. Additionally, Smad3 overexpression in BV2 cells decreased the levels of TNF-α and IL-6, and promoted the wound healing, whereas Smad3 knockdown showed the opposite effects. ConclusionsThese findings revealed that activin A regulated the biological behavior of BV2 cells via Smad3 signaling, suggesting that activin A may serve as a potential treatment target for neuroinflammation and glia scar formation in nervous system.

Naringin Regulates Microglia BV-2 Activation and Inflammation via the JAK/STAT3 Pathway.

Objective Microglial BV-2 cells are activated in the brain following insomnia. Naringin (NAR) is a polymethoxylated flavonoid that is also commonly found in citrus fruits and is known for its antioxidant potential. However, the effect of NAR on microglial cells has rarely been studied in the brain of an organism after insomnia. This study aimed to investigate the effects and potential mechanisms of action of NAR on microglial cell activation and inflammation. Methods BV-2 cells were obtained from the China Center for Type Culture Collection and randomly divided into five treatment groups: control, model, NAR (10 μM), WP1066 (5 μM), and NAR + WP1066. With the exception of the control group, all groups were stimulated with LPS (1 μg/mL) for 6 h. CCK8 was used to quantify cell viability and a scratch test was performed to detect cell migration. The expression levels of interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-α), interleukin 1 beta (IL-1β), nterleukin 10 (IL-10), and insulin like growth factor (1IGF-1) were measured by ELISA. Western blotting was performed to determine the levels of p-STAT3 and p-JAK. The Focalcheck™ Thin-Ring Fluorescent Microspheres kit was used to detect cell phagocytosis. Immunofluorescence was used to observe the expression of iNOS and arginase1 in BV-2 cells. Results Compared with the control group, cell migration, cell viability, and the expression of IL-1β, IL-6, TNF-α, and iNOS were significantly increased in the model group, whereas the expression levels of IL-10, IGF-1, and arginase 1, as well as cell phagocytosis were reduced. With the increase in NAR concentration, cell migration, cell viability, the expression levels of IL-1β, IL-6, TNF-α, and iNOS decreased, while the expression of IL-10, IGF-1, and arginase 1 increased. Compared with the control group, p-STAT3, and p-JAK expression in the model group were significantly increased (P<0.05). Compared with the model group, the expression of p-STAT3 and p-JAK in the NAR, NAR + WP1066, and WP1066 groups was significantly decreased (P < 0.05). Conclusion NAR treatment inhibited the proliferation, migration, and inflammation of BV-2 cells as well as the activation of microglia to the M1 phenotype. Conversely, NAR treatment promoted the activation of microglia to the M2 phenotype and enhanced the phagocytic function of BV-2 cells by regulating the activity of the JAK/STAT3 pathway.

Morphine promotes microglial activation by upregulating the EGFR/ERK signaling pathway.

Although they represent the cornerstone of analgesic therapy, opioids, such as morphine, are limited in efficacy by drug tolerance, hyperalgesia and other side effects. Activation of microglia and the consequent production of proinflammatory cytokines play a key pathogenic role in morphine tolerance, but the exact mechanisms are not well understood. This study aimed to investigate the regulatory mechanism of epidermal growth factor receptor (EGFR) on microglial activation induced by morphine in mouse microglial BV-2 cells. In this research, BV-2 cells were stimulated with morphine or pretreated with AG1478 (an inhibitor of EGFR). Expression levels of cluster of differentiation molecule 11b (CD11b), EGFR, and phospho-EGFR were detected by immunofluorescence staining. Cell signaling was assayed by Western blot. The migration ability of BV-2 cells was tested by Transwell assay. The production of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) in the cell supernatant was determined by ELISA. We observed that the expression of CD11b induced by morphine was increased in a dose- and time- dependent manner in BV-2 cells. Phosphorylation levels of EGFR and ERK1/2, migration of BV-2 cells, and production of IL-1β and TNFα were markedly enhanced by morphine treatment. The activation, migration, and production of proinflammatory cytokines in BV-2 cells were inhibited by blocking the EGFR signaling pathway with AG1478. The present study demonstrated that the EGFR/ERK signaling pathway may represent a novel pharmacological strategy to suppress morphine tolerance through attenuation of microglial activation.

Suppression of LPS-Induced Inflammation and Cell Migration by Azelastine through Inhibition of JNK/NF-κB Pathway in BV2 Microglial Cells.

The c-Jun N-terminal kinases (JNKs) are implicated in many neuropathological conditions, including neurodegenerative diseases. To explore potential JNK3 inhibitors from the U.S. Food and Drug Administration-approved drug library, we performed structure-based virtual screening and identified azelastine (Aze) as one of the candidates. NMR spectroscopy indicated its direct binding to the ATP-binding site of JNK3, validating our observations. Although the antihistamine effect of Aze is well documented, the involvement of the JNK pathway in its action remains to be elucidated. This study investigated the effects of Aze on lipopolysaccharide (LPS)-induced JNK phosphorylation, pro-inflammatory mediators, and cell migration in BV2 microglial cells. Aze was found to inhibit the LPS-induced phosphorylation of JNK and c-Jun. It also inhibited the LPS-induced production of pro-inflammatory mediators, including interleukin-6, tumor necrosis factor-α, and nitric oxide. Wound healing and transwell migration assays indicated that Aze attenuated LPS-induced BV2 cell migration. Furthermore, Aze inhibited LPS-induced IκB phosphorylation, thereby suppressing nuclear translocation of NF-κB. Collectively, our data demonstrate that Aze exerts anti-inflammatory and anti-migratory effects through inhibition of the JNK/NF-κB pathway in BV2 cells. Based on our findings, Aze may be a potential candidate for drug repurposing to mitigate neuroinflammation in various neurodegenerative disorders, including Alzheimer’s and Parkinson’s diseases.

Fatty Acid Amide Hydrolase (FAAH) Inhibition Modulates Amyloid-Beta-Induced Microglia Polarization.

The ability of endocannabinoid (eCB) to change functional microglial phenotype can be explored as a possible target for therapeutic intervention. Since the inhibition of fatty acid amide hydrolase (FAAH), the main catabolic enzyme of anandamide (AEA), may provide beneficial effects in mice model of Alzheimer’s disease (AD)-like pathology, we aimed at determining whether the FAAH inhibitor URB597 might target microglia polarization and alter the cytoskeleton reorganization induced by the amyloid-β peptide (Aβ). The morphological evaluation showed that Aβ treatment increased the surface area of BV-2 cells, which acquired a flat and polygonal morphology. URB597 treatment partially rescued the control phenotype of BV-2 cells when co-incubated with Aβ. Moreover, URB597 reduced both the increase of Rho protein activation in Aβ-treated BV-2 cells and the Aβ-induced migration of BV-2 cells, while an increase of Cdc42 protein activation was observed in all samples. URB597 also increased the number of BV-2 cells involved in phagocytosis. URB597 treatment induced the polarization of microglial cells towards an anti-inflammatory phenotype, as demonstrated by the decreased expression of iNOS and pro-inflammatory cytokines along with the parallel increase of Arg-1 and anti-inflammatory cytokines. Taken together, these data suggest that FAAH inhibition promotes cytoskeleton reorganization, regulates phagocytosis and cell migration processes, thus driving microglial polarization towards an anti-inflammatory phenotype.

Exosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-\u03baB pathway

ObjectiveNeuroinflammation plays a critical role in central nervous system diseases. Exosomal miRNAs released from various cells are implicated in cell-to-cell communication. Prior studies have placed substantial emphasis on the role of cytokines in mast cell-microglia interactions during neuroinflammation. However, it has never been clearly determined whether exosomal miRNAs participate in the interaction between mast cells and microglia and thus mediate neuroinflammation.MethodsThe characteristics of exosomes isolated from cell culture supernatants were confirmed by transmission electron microscopy (TEM), nanoparticle-tracking analysis (NTA) and Western blot. The transfer of PKH67-labelled exosomes and Cy3-labelled miR-409-3p was observed by fluorescence microscopy. Migration and activation of murine BV-2 microglial cells were evaluated through Transwell assays and immunofluorescence staining for Iba1 and CD68. CD86, IL-1β, IL-6 and TNF-α were assessed via qRT-PCR and ELISA. MiR-409-3p was detected by qRT-PCR. Nr4a2 and NF-κB levels were measured by western blot. Regulatory effects were identified by luciferase reporter assays.ResultsLipopolysaccharide (LPS)-stimulated murine P815 mast cells secreted exosomes that were efficiently taken up by murine BV-2 cells, which promoted murine BV-2 cell migration and activation. LPS-P815 exosomes increased the CD86, IL-1β, IL-6 and TNF-α levels in murine BV-2 microglia. Furthermore, activated mast cells delivered exosomal miR-409-3p to murine BV-2 microglia. Upregulated miR-409-3p promoted murine BV-2 microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway.ConclusionExosomal miR-409-3p secreted from activated mast cells promotes microglial migration, activation and neuroinflammation by targeting Nr4a2 to activate the NF-κB pathway, which provides evidence that not only cytokines but also exosomal miRNAs participate in neuroinflammation. In the future, targeting exosomal miRNAs may provide new insights into neuroinflammation.

Potassium Channels Kv1.3 and Kir2.1 But Not Kv1.5 Contribute to BV2 Cell Line and Primary Microglial Migration.

(1) Background: As membrane channels contribute to different cell functions, understanding the underlying mechanisms becomes extremely important. A large number of neuronal channels have been investigated, however, less studied are the channels expressed in the glia population, particularly in microglia. In the present study, we focused on the function of the Kv1.3, Kv1.5 and Kir2.1 potassium channels expressed in both BV2 cells and primary microglia cultures, which may impact the cellular migration process. (2) Methods: Using an immunocytochemical approach, we were able to show the presence of the investigated channels in BV2 microglial cells, record their currents using a patch clamp and their role in cell migration using the scratch assay. The migration of the primary microglial cells in culture was assessed using cell culture inserts. (3) Results: By blocking each potassium channel, we showed that Kv1.3 and Kir2.1 but not Kv1.5 are essential for BV2 cell migration. Further, primary microglial cultures were obtained from a line of transgenic CX3CR1-eGFP mice that express fluorescent labeled microglia. The mice were subjected to a spared nerve injury model of pain and we found that microglia motility in an 8 µm insert was reduced 2 days after spared nerve injury (SNI) compared with sham conditions. Additional investigations showed a further impact on cell motility by specifically blocking Kv1.3 and Kir2.1 but not Kv1.5; (4) Conclusions: Our study highlights the importance of the Kv1.3 and Kir2.1 but not Kv1.5 potassium channels on microglia migration both in BV2 and primary cell cultures.

Sphingosine-1-phosphate induces migration of microglial cells via activation of volume-sensitive anion channels, ATP secretion and activation of purinergic receptors

Microglia cells are versatile players coordinating inflammatory and regenerative processes in the central nervous system in which sphingosine-1-phosphate (S1P)-mediated migration is essential. We investigated the involved signaling cascade by means of voltage clamp, measurement of ATP secretion, and wound healing assay in murine microglial BV-2 cells. S1P and extracellular hypoosmolar solution evoked an anion conductance of the cell membrane. The corresponding ion currents were inhibited by intracellular hypoosmolar solution and by the anion channel antagonists NPPB, tamoxifen, and carbenoxolone, pointing to the activation of volume-regulated anion channels (VRAC). The knockdown by siRNA indicates the involvement of LRRC8A subunits. The S1PR1-antagonist W123 and pertussis-toxin prevented the S1P-induced currents, showing the involvement of the Gi-protein-coupled S1P receptor 1 (S1PR1). Furthermore, S1P and hypoosmolar extracellular solution induced an increase of ATP levels in the supernatants of BV-2 cells, which was inhibited by NPPB, tamoxifen, and W123. S1P, ATP, and ADP stimulated cell migration into the scratch area. The inhibition of S1PR1 and the downstream Gi proteins hampered cell migration. Antagonists of VRAC were also able to diminish the migration of BV-2 cells. Furthermore, direct inhibition of ATP-gated P2X4 or P2X7 receptors or ADP-stimulated P2Y12 receptors blocked the stimulating effects of S1P on BV-2 cell migration. We conclude that there is an interaction between S1P receptors and purinergic receptors mediated by an S1P-induced ATP release via VRAC and that the amount of released ATP is capable of stimulating cell migration of BV-2 microglia cells via activation of P2X4, P2X7, and P2Y12 receptors.

Repressor Element 1 Silencing Transcription Factor (REST) Governs Microglia-Like BV2 Cell Migration via Progranulin (PGRN)

Microglia activation contributes to Alzheimer's disease (AD) etiology, and microglia migration is a fundamental function during microglia activation. The repressor element-1 silencing transcription factor (REST), a powerful transcriptional factor, was found to play a neuroprotective role in AD. Despite its possible role in disease progression, little is known about whether REST participates in microglia migration. In this study, we aimed to explore the function of REST and its molecular basis during microglia migration under Aβ1-42-treated pathological conditions. When treated by Aβ1-42 REST was upregulated through JAK2/STAT3 signal pathway in BV2 cells. And transwell coculture system was used to evaluate cell migration function of microglia-like BV2. Small interfering RNA (siRNA) targeting progranulin (PGRN) were delivered into BV2 cells, and results showed that PGRN functions to promote BV2 migration. REST expression was inhibited by sh-RNA, which induced BV2 cell migration obviously. On the contrary, REST was overexpressed by REST recombinant plasmid transfection, which repressed BV2 cell migration, indicating that REST may act as a repressor of cell migration. To more comprehensively examine the molecular basis, we analyzed the promoter sequence of PGRN and found that it has the potential binding site of REST. Moreover, knocking-down of REST can increase the expression of PGRN, which confirms the inhibiting effect of REST on PGRN expression. Further detection of double luciferase reporter gene also confirmed the inhibition of REST on the activity of PGRN promoter, indicating that REST may be an inhibitory transcription factor of PGRN which governs microglia-like BV2 cell migration. In conclusion, the present study demonstrates that transcription factor REST may act as a repressor of microglia migration through PGRN.

Lipopolysaccharide induces BV2 microglial cell migration via a decrease in SET8 expression.

Excessively activated microglia exhibit increased migration, resulting in tissue damage and chronic inflammation. Src was confirmed to play an important role in regulation of cell motility following lipopolysaccharide (LPS) treatment. SET8 plays an important part in multiple cellular signal pathways. In this study, we speculated that SET8 is involved in LPS-induced microglial migration via regulation of Src expression. Our study showed that LPS promoted cell migration via augmentation of Src expression in BV2 cells. Moreover, LPS treatment decreased SET8 expression and upregulated the expression of the transcription factor ETS proto-oncogene 1 (ETS1). Overexpression of both SET8 and small interfering ETS1 reversed LPS-induced Src expression and cell migration. The effects of short hairpin SET8 (shSET8) and ETS1 overexpression are the same as the effects of LPS treatment. Decrease of Src expression reversed the shSET8-induced and ETS1 overexpression-induced migration of BV2 cells. Furthermore, SET8 was observed to associate with ETS1. Chromatin immunoprecipitation assay indicated H4K20me1, a downstream target of SET8, in addition to ETS1, was enriched at the Src promoter region. Furthermore, shSET8 increased Src promoter activity and also increased the positive effect of ETS1 overexpression on Src promoter activity. This study shows that SET8 associates with ETS1 to regulate Src expression, which is involved in LPS-induced BV2 cell migration.

Aβ-Induced Repressor Element 1-Silencing Transcription Factor (REST) Gene Delivery Suppresses Activation of Microglia-Like BV-2 Cells.

Compelling evidence from basic molecular biology has demonstrated the crucial role of microglia in the pathogenesis of Alzheimer's disease (AD). Microglia were believed to play a dual role in both promoting and inhibiting Alzheimer's disease progression. It is of great significance to regulate the function of microglia and make them develop in a favorable way. In the present study, we investigated the function of repressor element 1-silencing transcription factor (REST) in Aβ1-42-induced BV-2 cell dysfunction. We concluded that Aβ1-42 could promote type I activation of BV-2 cells and induce cell proliferation, migration, and proinflammation cytokine TNF-α, IL-1β, and IL-6 expression. Meanwhile, REST was upregulated, and nuclear translocalization took place due to Aβ1-42 stimulation. When REST was knocked down by a specific short hairpin RNA (sh-RNA), BV-2 cell proliferation, migration, and proinflammation cytokine expression and secretion induced by Aβ1-42 were increased, demonstrating that REST may act as a repressor of microglia-like BV-2 cell activation.

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

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

Osteopontin heptamer peptide containing the RGD motif enhances the phagocytic function of microglia

Osteopontin (OPN) is a phosphorylated glycoprotein expressed in various tissues, including brain, and mediates a wide range of cellular activities. In our previous studies, we reported recombinant OPN and RGD and SLAY-containing OPN-peptide icosamer (OPNpt20) exhibited robust neuroprotective activities in an animal model of transient focal ischemia, and attributed these effects to the anti-inflammatory, pro-angiogenic, and phagocytic functions of OPNpt20. In the present study, we truncated OPNpt20 to 13 or 7 amino acid peptides containing RGD (R) and/or SLAY (S) motif (OPNpt13RS, OPNpt7R, OPNpt7RS, and OPNpt7S) and their cell motility and migration inducing activities were examined in BV2 cells (a microglia cell line). All four peptides significantly enhanced BV2 cell motility and migration, but OPNpt7R, an RGD-containing 7-amino-acid OPN peptide (VPNGRGD), was found to be most potent and its potency was comparable to OPNpt20. Phagocytic activity and F-actin polymerization were also significantly enhanced in OPNpt7R-treated BV2 cells. Importantly, studies using two mutant peptides (OPNpt7R-RAA and OPNpt7R-RAD, wherein RGD in OPNpt7R was replaced with RAA or RAD, respectively) revealed that all these effects of OPNpt7R, motility, migration, F-actin polymerization, and phagocytosis induction, were RGD-dependent. Furthermore, the Erk, Fak, and Akt signaling pathways appeared to be involved in the induction of phagocytic activity by OPNpt7R. Co-treating cells with OPNpt7R and D98059 or wortmannin (pharmacological inhibitors of Erk and Akt, respectively) significantly suppressed OPNpt7R-mediated phagocytosis induction. These results indicate the RGD-containing OPN heptamer OPNpt7R triggers microglial motility, migration, and phagocytic activity and that the RGD motif plays a critical role in these activities.

Anti-inflammatory Effect of AZD6244 on Acrolein-Induced Neuroinflammation.

Clinically, high levels of acrolein (a highly reactive α, β-unsaturated aldehyde) and acrolein adducts are detected in the brain of patients with CNS neurodegenerative diseases, including Alzheimer's disease and spinal cord injury. Our previous study supports this notion by showing acrolein as a neurotoxin in a Parkinsonian animal model. In the present study, the effect of AZD6244 (an ATP non-competitive MEK1/2 inhibitor) on acrolein-induced neuroinflammation was investigated using BV-2 cells and primary cultured microglia. Our immunostaining study showed that lipopolysaccharide (LPS, an inflammation inducer as a positive control) increased co-localized immunoreactivities of phosphorylated ERK and ED-1 (a biomarker of activated microglia) in the treated BV-2 cells. Similar elevation in co-localized immunoreactivities of phosphorylated ERK and ED-1 was detected in the acrolein-treated BV-2 cells. Furthermore, Western blot assay showed increases in phosphorylated ERK in BV-2 cells subjected to LPS (1μg/mL) or acrolein (30μM); these increases were blocked by AZD6244 (10μM). At the same time, AZD6244 attenuated LPS-induced TNF-α (a pro-inflammatory cytokine) and cyclooxygenase-II (COX II, a pro-inflammatory enzyme). Consistently, AZD6244 reduced acrolein-induced elevations in COX-II mRNA and COX-II protein expression. In addition, AZD6244 inhibited acrolein-induced increases in activated caspase 1 (a biomarker of inflammasome activation) and heme oxygenase-1 (a redox-regulated chaperone protein) in BV-2 cells. Using a transwell migration assay, AZD6244 attenuated acrolein (5μM)-induced migration of BV-2 cells and primary cultured microglia. In conclusion, our study shows that acrolein is capable of inducing neuroinflammation which involved ERK activation in microglia. Furthermore, AZD6244 is capable of inhibiting acrolein-induced neuroinflammation. Our study suggests that ERK inhibition may be a neuroprotective target against acrolein-induced neuroinflammation in the CNS neurodegenerative diseases.

Inhibition of calcium-calmodulin-dependent phosphodiesterase (PDE1) suppresses inflammatory responses

A novel, potent, and highly specific inhibitor of calcium-calmodulin-dependent phosphodiesterases (PDE) of the PDE1 family, ITI-214, was used to investigate the role of PDE1 in inflammatory responses. ITI-214 dose-dependently suppressed lipopolysaccharide (LPS)-induced gene expression of pro-inflammatory cytokines in an immortalized murine microglial cell line, BV2 cells. RNA profiling (RNA-Seq) was used to analyze the impact of ITI-214 on the BV2 cell transcriptome in the absence and the presence of LPS. ITI-214 was found to regulate classes of genes that are involved in inflammation and cell migration responses to LPS exposure. The gene expression changes seen with ITI-214 treatment were distinct from those elicited by inhibitors of other PDEs with anti-inflammatory activity (e.g., a PDE4 inhibitor), indicating a distinct mechanism of action for PDE1. Functionally, ITI-214 inhibited ADP-induced migration of BV2 cells through a P2Y12-receptor-dependent pathway, possibly due to increases in the extent of cAMP and VASP phosphorylation downstream of receptor activation. Importantly, this effect was recapitulated in P2 rat microglial cells in vitro, indicating that these pathways are active in native microglial cells. These studies are the first to demonstrate that inhibition of PDE1 exerts anti-inflammatory effects through effects on microglia signaling pathways. The ability of PDE1 inhibitors to prevent or dampen excessive inflammatory responses of BV2 cells and microglia provides a basis for exploring their therapeutic utility in the treatment of neurodegenerative diseases associated with increased inflammation and microglia proliferation such as Parkinson's disease and Alzheimer's disease.