Cell Line BV-2 Research Articles

Lipofuscin-dependent stimulation of microglial cells.

To examine the reaction of microglial cells (MG) when incubated with lipofuscin (LP) in vitro with emphasis on the immunological reaction of the MG toward LP and the suppression of this reaction by immunomodulatory agents. MG are involved in the pathogenesis of degenerative eye disorders such as age-related macular degeneration (AMD). LP is a heterogeneous waste material that accumulates in the retinal pigment epithelium (RPE) cells with advancing age. LP is known to have toxic effects on RPE cells and therefore an elevated LP-derived fundus autofluorescence is a risk factor for AMD development. MG in the subretinal space have been reported in eyes affected by AMD. Moreover, in senescent mice, subretinal MG were found, which display an autofluorescence that may be derived from LP uptake. In this study, we incubated MG (BV-2 cell line and primary cells from murine brain) in vitro with LP isolated from the human RPE. We observed phagocytosis, studied cell morphologies, and analyzed the cell culture supernatants. We also investigated the effect of the immunomodulatory agents hydrocortisone (HC), minocycline, and the tripeptide TKP. The MG phagocytosed the LP quickly and completely. We detected highly elevated levels of pro-inflammatory cytokines (especially of IL-6, IL-23p19, TNF-α, KC, RANTES, and IL-1α) in the cell culture supernatants. Furthermore, levels of vascular endothelial growth factor (VEGF) were raised in BV-2 cells. Anti-inflammatory agents added to the cell cultures inhibited the inflammatory reaction, in particular hydrocortisone (HC). Minocycline and TKP had less impact on the cytokine release. The interaction of MG and LP could play a role in the development of retinal degeneration by triggering an inflammatory reaction and angiogenesis.

MicroRNA-146a protects against cognitive decline induced by surgical trauma by suppressing hippocampal neuroinflammation in mice

Postoperative cognitive dysfunction (POCD) is a common postoperative complication that is associated with increased morbidity and mortality. However, the neuropathogenesis of this complication remains largely unknown. Neuroinflammation, in particular hippocampal inflammation, contributes to POCD. Recently, increasing evidence has supported the involvement of microRNAs (miRNAs) in the regulation of neuroinflammation in human neurological disorders. In the present study, we investigated the role of miR-146a, a key regulator of the innate immune response, in surgery-induced hippocampal inflammation and cognitive impairment.The expression of miR-146a was measured in BV-2 microglial cells stimulated with lipopolysaccharide (LPS) and hippocampal tissues of mice with POCD. Loss of function and overexpression studies were performed via transfection with miR-146a mimic/inhibitor in cultured BV-2 cell lines and intrahippocampal injection of miR-146a agomir/antagomir before surgery/anesthesia to identify the role of miR-146a in neuroinflammation and cognitive impairment. QPCR, Western blot and ELISA were used to determine the expression levels of downstream adaptor proteins and proinflammatory cytokines. Immunofluorescence staining was applied to evaluate the activation of microglia.Increased expression of miR-146a was observed in BV-2 microglial cells stimulated with LPS and hippocampal tissues of mice with POCD. Modulation of miR-146a expression via transfection of microglia with miR-146a mimic or inhibitor regulated the mRNA and protein expression levels of downstream targets of miR-146a (IRAK1 and TRAF6) as well as the release of proinflammatory cytokines (TNF-α, IL-1β and IL-6). In addition, overexpression of miR-146a attenuated hippocampus-dependent learning and memory impairment in mice with POCD, which was accompanied by decreased expression of the IRAK1/TRAF6/nuclear factor (NF)-κB pathway and downregulation of microglial activation in the hippocampus. Conversely, knockdown of miR-146a expression may exacerbate hippocampus-dependent learning and memory deficiency and hippocampal inflammation in mice with POCD.Collectively, our findings demonstrate the important role of miR-146a in the neuropathogenesis of POCD and suggest that miR-146a may be a potential therapeutic target for POCD.

Triggering microglia through toll-like receptor 2 pathway induced interferon β expression in cell and animal model of Alzheimer's disease.

Synaptic function and memory performance are disrupted by soluble form of β-amyloid (Aβ). In the previous study, we found that early activation of microglia by toll-like receptor 2 (TLR2) attenuated Alzheimer's disease-associated cognitive deficit. This study was designed to investigate whether pretreatment with the TLR2 receptor ligand can regulate microglia to produce interferon β (INFβ) in a rat model of Alzheimer's disease. For this purpose, the BV-2 cell line was cultured in a 24-well plate, treated with Pam3Cys (1 μg/ml), and then incubated with oligomeric Aβ for 24 h. The expression of TRIF, IRF3, and INFβ was measured by western blot technique. For in-vivo study, bilateral guide cannulas were streotaxically implanted in the right and left lateral ventricles. Pam3Cys/vehicle was microinjected into the right ventricle every 3 days. Two weeks later, an osmotic pump was inserted into the left ventricle to microinfuse oligomeric Aβ/placebo over 14 days. In the meanwhile, treatment with Pam3Cys was continued every 3 days. Then, expression of TRIF, IRF3, and INFβ was measured in the hippocampus. The results showed that although oligomeric Aβ could not alter the expression of these proteins at the cell and tissue level, treatment with Pam3Cys resulted in enhanced expression of them at both cell culture and hippocampal tissue following treatment with oligomeric Aβ. It is concluded that stimulation of microglia through TLR2 pathway induces INFβ expression, which may in part mediate neuroprotection against oligomeric Aβ.

DNA damage in BV-2 cells: An important supplement to the neurotoxicity of CdTe quantum dots.

Microglial cells are resident immune cells in the central nervous system. Activation of microglia as induced by CdTe quantum dots (QDs) can trigger damage to neurons. To quantify the intracellular QDs, we monitored the intracellular Cd concentration in the QD-exposed mouse microglial cells (BV-2 cell line). The extent of cell injury at different times correlated with the Cd concentration in cells at that time. In addition to Cd ion detection, we also monitored the intracellular fluorescence of the QDs. More QDs accumulated in the nucleus than in the cytoplasm. Comet assays confirmed that QDs induce DNA damage. However, DNA cannot interact with QDs, so the DNA damage was not caused by CdTe QDs adducts to DNA but by the increase of the Cd ion concentration and the secondary oxidative damage. In addition to DNA damage, biofilm injury and endogenous reduced glutathione depletion were also apparent in QD-exposed BV-2 cells. These changes can be prevented or even reversed by exogenous reduced glutathione administration.

Palmitate Induces an Anti-Inflammatory Response in Immortalized Microglial BV-2 and IMG Cell Lines that Decreases TNFα Levels in mHypoE-46 Hypothalamic Neurons in Co-Culture

Background and Objectives: Elevated levels of saturated fatty acids (SFA) induce a state of neuroinflammation in the hypothalamus. It has been suggested that microglia sense palmitate, a prevalent circulating SFA, and act as mediators of this inflammatory process by communicating with neurons, particularly those involved in appetite regulation. In this study, we examined the inflammatory response to palmitate in immortalized microglial cell lines, BV-2 and IMG, and the subsequent effects on inflammatory gene expression in a model of NPY/AgRP neurons, mHypoE-46. Methods: The BV-2 cells were treated with 50 µM palmitate for 4 and 24 h, and the transcriptional regulation of markers for inflammation and cellular stress was assessed using an RT² Profiler PCR Array. Select genes were verified with qRT-PCR. The BV-2 and IMG cells were then co-cultured using 1.0-µm cell culture inserts with an immortalized hypothalamic cell line, mHypoE-46, to investigate potential intercellular communication between microglia and neurons. Results: We found that palmitate increased the mRNA levels of specific inflammatory genes, and a general anti-inflammatory profile was revealed in the microglia cells. The mRNA changes in TNFα at 4 and 24 h in BV-2 cells were abrogated with the toll-like receptor 4 (TLR4) inhibitor, TAK-242, indicating the involvement of TLR4. Co-culture of mHypoE-46 neurons with microglia pre-treated with palmitate resulted in repression of TNFα expression in the hypothalamic neurons. As palmitate significantly increased IL-13 expression in microglia, the effect of this cytokine was tested in mHypoE-46 neurons. The addition of IL-13 to neuronal cultures normalized the palmitate-mediated increase in IL-6 and AgRP expression, suggesting that microglia may protect surrounding neurons, at least in part, through the release of IL-13. Conclusions: These results suggest a potential anti-inflammatory role of microglia towards the palmitate-induced neuroinflammation, and potentially energy homeostasis, in hypothalamic neurons.

A microglial cell model for acyl-CoA oxidase 1 deficiency

Acyl-CoA oxidase 1 (ACOX1) deficiency is a rare and severe peroxisomal leukodystrophy associated with a very long-chain fatty acid (VLCFA) β–oxidation defect. This neurodegenerative disease lacks relevant cell models to further decipher the pathomechanisms in order to identify novel therapeutic targets. Since peroxisomal defects in microglia appear to be a key component of peroxisomal leukodystrophies, we targeted the Acox1 gene in the murine microglial BV-2 cell line. Using CRISPR/Cas9 gene editing, we generated an Acox1-deficient cell line and validated the allelic mutations, which lead to the absence of ACOX1 protein and enzymatic activity. The activity of catalase, the enzyme degrading H2O2, was increased, likely in response to the alteration of redox homeostasis. The mutant cell line grew more slowly than control cells without obvious morphological changes. However, ultrastructural analysis revealed an increased number of peroxisomes and mitochondria associated with size reduction of mitochondria. Changes in the distribution of lipid droplets containing neutral lipids have been observed in mutant cells; lipid analysis revealed the accumulation of saturated and monounsaturated VLCFA. Besides, expression levels of genes encoding interleukin-1 beta and 6 (IL-1β and IL-6), as well as triggering receptor expressed on myeloid cells 2 (Trem2) were found modified in the mutant cells suggesting modification of microglial polarization and phagocytosis ability. In summary, this Acox1-deficient cell line presents the main biochemical characteristics of the human disease and will serve as a promising model to further investigate the consequences of a specific microglial peroxisomal β–oxidation defect on oxidative stress, inflammation and cellular functions.

Ethanol extract of Glycyrrhizae Radix modulates the responses of antigen-specific splenocytes in experimental autoimmune encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disorder resulting in paralysis, and the responses of reactive T cells against self-antigens are hallmarks. Glycyrrhizae Radix (GR) has been used for detoxification and reducing inflammation. However, very few reports have described the effects of GR on MS. PurposeThe immunomodulatory effects of GR extract on autoimmune responses were evaluated through in vitro, ex vivo, and in vivo assays using primary mouse splenocytes (SPLC), mouse microglia BV2 cell line, and a mouse model of experimental autoimmune encephalomyelitis (EAE). Study designEthanol extract of GR was used in vitro with primary SPLC in the condition of anti-CD3/CD28 stimulation and interferon (IFN)-γ-producing CD4+ (TH1)/CD8+ (TC1) polarization as well as IFN-γ-stimulated BV2 cells. For EAE induction, female C57BL/6 mice were immunized with 200 μg of myelin oligodendrocyte glycoprotein (MOG)35–55 without pertussis toxin. EAE SPLC (ex vivo) and EAE mice (in vivo) were treated with GR extract to evaluate the changes in antigen-specific responses. SPLC media containing antigen-specific responses were used to stimulate BV2 cells. ResultsGR extract effectively modulated the responses of reactive splenic T cells through the reduction in IFN-γ+ T cell populations, the expressions of cell adhesion molecules (CAMs), and secretions of cytokines containing IFN-γ and a chemokine IFN-γ-induced protein 10 (IP-10) in vitro. In addition, GR extract significantly decreased nitric oxide production and secretion of tumor necrosis factor (TNF)-α and IP-10 in IFN-γ-stimulated BV2 cells. The antigen-specific TH1 and TC1 populations were decreased following administration of 100 mg/kg of GR extract, whereas CD8+IL-17A+ (TC17) population was increased on day 36 after EAE induction. Moreover, IFN-γ, which showed the highest secretion among examined cytokines, and IP-10 decreased on day 36. SPLC media derived from 100 mg/kg GR extract-administered EAE mice revealed the ameliorative effects on BV2 cell stimulation. ConclusionThis is the first report on the immunomodulatory effects of GR extract on antigen-specific SPLC responses in EAE. These results could be helpful for the discovery of drug candidates for MS by focusing on IFN-γ-related autoimmune responses.

Crocin, a natural product attenuates lipopolysaccharide-induced anxiety and depressive-like behaviors through suppressing NF-kB and NLRP3 signaling pathway

Depression is one of the foremost psychological illness which is closely leagued with inflammation. Crocin is a natural product that exhibits both anti-inflammatory and anti-oxidant activities. However, little is known about anti-inflammatory mechanisms of crocin on LPS-induced anxiety and depressive-like behaviors. The objective of this study is emphasized on neuroprotective role of crocin against LPS-induced anxiety and depressive-like behaviors in mice. It is observed that crocin inhibited LPS-induced production of NO, TNF-α, IL-1β and ROS in BV-2 microglial cells. Moreover, crocin significantly declined the expression of iNOS, NF-κB p65 and CD16/32 (M1 marker), as well as elevated the expression of CD206 (M2 marker) in BV-2 cell line with decreased LPS-induced anxiety and depressive-like behaviors by improved locomotor activity, reduced sucrose intake, and decreased immobility time in forced swim and tail suspension test in Kunming mice. Expression of NLRP3, ASC and caspase-1 by i.p administration of LPS found to be neutralized with reduction in level of IL-1β, IL-18 and TNF-α in mouse hippocampus. In conclusion, these results suggested that crocin as a potential therapeutic candidate for neuro-inflammation and depressive-like behaviors induced by LPS. The effect was found to be due to inhibition of NLRP3 inflammasome and NF-κB and its promoted M1 to M2 phenotypic conversion of microglia.

Carrageenan polysaccharides and oligosaccharides with distinct immunomodulatory activities in murine microglia BV-2 cells

This study was to investigate the anti-inflammatory activities in vitro of various carrageenans (Car) fractions (κ-, ι-, and λ-types) with well characterized molecular properties, using murine microglia BV-2 cell line treated with lipopolysaccharide (LPS) as model. It is indicated that pretreatments with the oligosaccharide fractions from κ- or ι-carrageenan acid hydrolysates (κ- and ι-CarAOS, respectively) at 125–500 μg/mL significantly and dose-dependently decreased the levels of tumor necrosis factor α (TNF-α) secreted from LPS-treated BV-2 cells, showing promisingly anti-inflammatory effects. Differently, pretreatments of most of polymeric carrageenans at 250–500 μg/mL significantly increased the TNF-α level, implying the co-inflammatory effects with LPS. The co-inflammatory effectiveness of pure carrageenans at 125 μg/mL was notable for λ-Car, followed by ι-Car, and insignificantly for κ-Car. Generally, cytokine TNF-α was a more sensitive biomarker to the presence of carrageenans than was the IL-6. The TNF-α level varied greatly at a low carrageenan concentration (125 μg/mL) and high polymer percentage (e.g. purified κ- and ι-Car). Conclusively, the anti-inflammatory effects on LPS-treated BV-2 cells could be attenuated by pretreatments with κ- and ι-CarAOS at 125–500 μg/mL.

Telmisartan Protects a Microglia Cell Line from LPS Injury Beyond AT1 Receptor Blockade or PPARγ Activation.

The Angiotensin II Receptor Blocker (ARB) Telmisartan reduces inflammation through Angiotensin II AT1 receptor blockade and peroxisome proliferator-activated receptor gamma (PPARγ) activation. However, in a mouse microglia-like BV2 cell line, imitating primary microglia responses with high fidelity and devoid of AT1 receptor gene expression or PPARγ activation, Telmisartan reduced gene expression of pro-injury factors, enhanced that of anti-inflammatory genes, and prevented LPS-induced increase in inflammatory markers. Using global gene expression profiling and pathways analysis, we revealed that Telmisartan normalized the expression of hundreds of genes upregulated by LPS and linked with inflammation, apoptosis and neurodegenerative disorders, while downregulating the expression of genes associated with oncological, neurodegenerative and viral diseases. The PPARγ full agonist Pioglitazone had no neuroprotective effects. Surprisingly, the PPARγ antagonists GW9662 and T0070907 were neuroprotective and enhanced Telmisartan effects. GW9226 alone significantly reduced LPS toxic effects and enhanced Telmisartan neuroprotection, including downregulation of pro-inflammatory TLR2 gene expression. Telmisartan and GW9662 effects on LPS injury negatively correlated with pro-inflammatory factors and upstream regulators, including TLR2, and positively with known neuroprotective factors and upstream regulators. Gene Set Enrichment Analysis (GSEA) of the Telmisartan and GW9662 data revealed negative correlations with sets of genes associated with neurodegenerative and metabolic disorders and toxic treatments in cultured systems, while demonstrating positive correlations with gene sets associated with neuroprotection and kinase inhibition. Our results strongly suggest that novel neuroprotective effects of Telmisartan and GW9662, beyond AT1 receptor blockade or PPARγ activation, include downregulation of the TLR2 signaling pathway, findings that may have translational relevance.

Impact of steroid hormones E2 and P on the NLRP3/ASC/Casp1 axis in primary mouse astroglia and BV-2 cells after in vitro hypoxia

Clinical and animal model studies have demonstrated the neuroprotective and anti-inflammatory effects of 17beta-estradiol (E2) and progesterone (P) in different disease models of the central nervous system (CNS) including ischemic stroke. Inflammasomes are involved in the interleukin-1 beta (IL1beta) maturation, in particular, NLRP3, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and the active caspase-1 (Casp1) form. Recently, we showed that administration of E2 or P selectively regulated these components after experimental ischemic stroke in rats. Therefore, we investigated the impact of E2 and P on the NLRP3/ASC/Casp1 axis in the murine microglia-like cell line BV-2 cells and primary astrocytes after short-term in vitro hypoxia.The inflammatory cytokine IL1beta but not IL18 was increased after short-term hypoxia in astroglia and BV-2 cells. The same applied to NLPR3 and ASC. Casp1 activity was also elevated in astroglia and BV-2 cells after hypoxia. The administration of E2 or P selectively dampened IL1beta, ASC and NLRP3 expression mainly in BV-2 cells. Both steroid hormones failed to reduce Casp1 activity after hypoxia.We conclude that E2- and P-mediated anti-inflammatory mechanisms occur upstream of Casp1 through the regulation of NLRP3 and its adaptor ASC.

Mitochonic acid 5 activates the MAPK\u2013ERK\u2013yap signaling pathways to protect mouse microglial BV-2 cells against TNF\u03b1-induced apoptosis via increased Bnip3-related mitophagy

BackgroundThe regulation of microglial function via mitochondrial homeostasis is important in the development of neuroinflammation. The underlying mechanism for this regulatory function remains unclear. In this study, we investigated the protective role of mitochonic acid 5 (MA-5) in microglial mitochondrial apoptosis following TNFα-induced inflammatory injury.MethodsTNFα was used to induce inflammatory injury in mouse microglial BV-2 cells with and without prior MA-5 treatment. Cellular apoptosis was assessed using the MTT and TUNEL assays. Mitochondrial functions were evaluated via mitochondrial membrane potential JC-1 staining, ROS flow cytometry analysis, mPTP opening assessment, and immunofluorescence of cyt-c. Mitophagy was examined using western blots and immunofluorescence. The pathways analysis was carried out using western blots and immunofluorescence with a pathway blocker.ResultsOur results demonstrated that TNFα induced apoptosis in the microglial BV-2 cell line by activating the caspase-9-dependent mitochondrial apoptotic pathway. Mechanistically, inflammation reduced mitochondrial potential, induced ROS production, and contributed to the leakage of mitochondrial pro-apoptotic factors into the cytoplasm. The inflammatory response reduced cellular energy metabolism and increased oxidative stress. By contrast, treatment with MA-5 reduced mitochondrial apoptosis via upregulation of mitophagy. Increased mitophagy degraded damaged mitochondria, disrupting mitochondrial apoptosis, neutralizing ROS overproduction, and improving cellular energy production. We also identified that MA-5 regulated mitophagy via Bnip3 through the MAPK–ERK–Yap signaling pathway. Inhibiting this signaling pathway or knocking down Bnip3 expression prevented MA-5 from having beneficial effects on mitochondrial homeostasis and increased microglial apoptosis.ConclusionsAfter TNFα-induced inflammatory injury, MA-5 affects microglial mitochondrial homeostasis in a manner mediated via the amplification of protective, Bnip3-related mitophagy, which is mediated via the MAPK–ERK–Yap signaling pathway.

Paraquat-induced inflammatory response of microglia through HSP60/TLR4 signaling.

Previous studies showed that paraquat (PQ) caused the apoptosis of dopaminergic neurons by inducing the generation of oxygen radical. The purpose of this study is to explore PQ-induced microglial inflammatory response and its underlying molecular mechanisms. The murine microglia BV2 cell line was used. After stimulation with PQ and lipopolysaccharides (positive control), the concentrations of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6) in the culture supernatant and mRNA expression of TNF-α and IL-1β were determined by ELISA and quantitative real-time Polymerase Chain Reaction (PCR), respectively. The protein expression of heat shock protein 60 (HSP60) and toll-like receptor 4 (TLR4), along with the mRNA expression of transcription factors of nuclear factor κB-p65 (NF-κB-p65) and activated protein 1 (AP1, c-fos, and c-jun dimer) were evaluated with western blot and quantitative real-time PCR, respectively. The results showed that PQ activated microglia, which was characterized by increasing the generation and upregulated mRNA expression of pro-inflammatory cytokines, TNF-α, IL-1β, and IL-6. In addition, PQ significantly enhanced the expressions of HSP60 and TLR4 proteins in BV2 cells, as well as NF-κB-p65, c-fos, and c-jun mRNA. These findings suggest that PQ can activate microglia and enhance the expression and secretion of pro-inflammatory cytokines in a HSP60/TLR4 signaling, leading to the inflammatory response.

Elevated Pressure Changes the Purinergic System of Microglial Cells.

Glaucoma is the second cause of blindness worldwide and is characterized by the degeneration of retinal ganglion cells (RGCs) and optic nerve atrophy. Increased microglia reactivity is an early event in glaucoma that may precede the loss of RGCs, suggesting that microglia and neuroinflammation are involved in the pathophysiology of this disease. Although global changes of the purinergic system have been reported in experimental and human glaucoma, it is not known if this is due to alterations of the purinergic system of microglial cells, the resident immune cells of the central nervous system. We now studied if elevated hydrostatic pressure (EHP), mimicking ocular hypertension, changed the extracellular levels of ATP and adenosine and the expression, density and activity of enzymes, transporters and receptors defining the purinergic system. The exposure of the murine microglial BV-2 cell line to EHP increased the extracellular levels of ATP and adenosine, increased the density of ecto-nucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1, CD39) and decreased the density of the equilibrative nucleotide transporter 2 as well as the activity of adenosine deaminase. The expression of adenosine A1 receptor also decreased, but the adenosine A3 receptor was not affected. Notably, ATP and adenosine selectively control migration rather than phagocytosis, both bolstered by EHP. The results show that the purinergic system is altered in microglia in conditions of elevated pressure. Understanding the impact of elevated pressure on the purinergic system will help to unravel the mechanisms underlying inflammation and neurodegeneration associated with glaucoma.

Inhibition of cyclin-dependent kinase 5 affects early neuroinflammatory signalling in murine model of amyloid beta toxicity

BackgroundCyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinases and plays a critical role in neuronal differentiation, migration, synaptogenesis, plasticity, neurotransmission and apoptosis. The deregulation of Cdk5 activity was observed in post mortem analysis of brain tissue of Alzheimer’s disease (AD) patients, suggesting the involvement of Cdk5 in the pathomechanism of this neurodegenerative disease. However, our recent study demonstrated the important function of Cdk5 in regulating inflammatory reaction.MethodsSince the role of Cdk5 in regulation of inflammatory signalling in AD is unknown, we investigated the involvement of Cdk5 in neuroinflammation induced by single intracerebroventricular (icv) injection of amyloid beta protein (Aβ) oligomers in mouse. The brain tissue was analysed up to 35 days post injection. Roscovitine (intraperitoneal administration) was used as a potent Cdk5 inhibitor. The experiments were also performed on human neuroblastoma SH-SY5Y as well as mouse BV2 cell lines treated with exogenous oligomeric Aβ.ResultsOur results demonstrated that single injection of Aβ oligomers induces long-lasting activation of microglia and astrocytes in the hippocampus. We observed also profound, early inflammatory response in the mice hippocampus, leading to the significant elevation of pro-inflammatory cytokines expression (e.g. TNF-α, IL-1β, IL-6). Moreover, Aβ oligomers elevated the formation of truncated protein p25 in mouse hippocampus and induced overactivation of Cdk5 in neuronal cells. Importantly, administration of roscovitine reduced the inflammatory processes evoked by Aβ in the hippocampus, leading to the significant decrease of cytokines level.ConclusionsThese studies clearly show the involvement of Cdk5 in modulation of brain inflammatory response induced by Aβ and may indicate this kinase as a novel target for pharmacological intervention in AD.

Asiatic acid attenuates methamphetamine-induced neuroinflammation and neurotoxicity through blocking of NF-kB/STAT3/ERK and mitochondria-mediated apoptosis pathway

BackgroundMethamphetamine (METH) is a commonly abused drug that may result in neurotoxic effects. Recent studies have suggested that involvement of neuroinflammatory processes in brain dysfunction is induced by misuse of this drug. However, the mechanism underlying METH-induced inflammation and neurotoxicity in neurons is still unclear. In this study, we investigated whether asiatic acid (AA) effected METH-mediated neuroinflammation and neurotoxicity in dopaminergic neuronal cells. And we further determined whether the effect involved in the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and signal transducer and activator of transcription (STAT)3 and extracellular signal-regulated kinase (ERK) pathway.MethodsWe used the human dopaminergic neuroblastoma SH-SY5Y cell line, murine microglial BV2 cell line, and primary culture of rat embryo mesencephalic neurons. Pro-inflammatory cytokine production was monitored by ELISA and RT/real-time PCR. The cell cycle distribution and mitochondrial membrane integrity was analyzed by flow cytometry. We used immunoblotting, DNA-binding activity, and immunofluorescence staining to analyze the effect of AA on activation of the NF-κB, STAT3, MAPK-ERK, and apoptosis signaling pathways.ResultsMETH induced TNF receptor (TNFR) expression and led to morphological changes of cells. Additionally, this drug increased pro-inflammatory cytokine (TNFα and IL-6) expression. AA significantly suppressed METH-induced TNFR expression in concentration dependent. Increased secretion of TNFα and IL-6 was inhibited in METH-stimulated neuronal cells by AA administration. AA showed significant protection against METH-induced translocation of NF-κB/STAT3 and ERK phosphorylation. AA inhibited METH-induced proteolytic fragmentation of caspase-3 and PARP. The pro-apoptotic protein Bax was significantly decreased, while the anti-apoptotic protein Bcl-xL was increased by AA treatment in METH-stimulated cells. A similar protective effect of AA on mitochondrial membrane integrity was also confirmed by flow cytometry and immunofluorescence staining.ConclusionsBased on the literatures and our findings, AA is a promising candidate for an anti-neurotoxic agent, and it can potentially be used for the prevention and treatment of various neurological disorders.

Blocking ATP-sensitive potassium channel alleviates morphine tolerance by inhibiting HSP70-TLR4-NLRP3-mediated neuroinflammation

BackgroundLong-term use of morphine induces analgesic tolerance, which limits its clinical efficacy. Evidence indicated morphine-evoked neuroinflammation mediated by toll-like receptor 4 (TLR4) - NOD-like receptor protein 3 (NLRP3) inflammasome was important for morphine tolerance. In our study, we investigated whether other existing alternative pathways caused morphine-induced activation of TLR4 in microglia. We focused on heat shock protein 70 (HSP70), a damage-associated molecular pattern (DAMP), which was released from various cells upon stimulations under the control of KATP channel and bound with TLR4-inducing inflammation. Glibenclamide, a classic KATP channel blocker, can improve neuroinflammation by inhibiting the activation of NLRP3 inflammasome. Our present study investigated the effect and possible mechanism of glibenclamide in improving morphine tolerance via its specific inhibition on the release of HSP70 and activation of NLRP3 inflammasome induced by morphine.MethodsCD-1 mice were used for tail-flick test to evaluate morphine tolerance. The microglial cell line BV-2 and neural cell line SH-SY5Y were used to investigate the pharmacological effects and the mechanism of glibenclamide on morphine-induced neuroinflammation. The activation of microglia was accessed by immunofluorescence staining. Neuroinflammation-related cytokines were measured by western blot and real-time PCR. The level of HSP70 and related signaling pathway were evaluated by western blot and immunofluorescence staining.ResultsMorphine induced the release of HSP70 from neurons. The released HSP70 activated microglia and triggered TLR4-mediated inflammatory response, leading to the phosphorylation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) p65 and the activation of NLRP3 inflammasome. Moreover, anti-HSP70 neutralizing antibody partly attenuated chronic morphine tolerance. The secretion of HSP70 was under the control of MOR/AKT/KATP/ERK signal pathway. Glibenclamide as a classic KATP channel blocker markedly inhibited the release of HSP70 induced by morphine and suppressed HSP70-TLR4-NLRP3 inflammasome-mediated neuroinflammation, which consequently attenuated morphine tolerance.ConclusionsOur study indicated that morphine-induced extracellular HSP70 was an alternative way for the activation of TLR4-NLRP3 in analgesic tolerance. The release of HSP70 was regulated by MOR/AKT/KATP/ERK pathway. Our study suggested a promising target, KATP channel and a new leading compound, glibenclamide, for treating morphine tolerance.

Investigation of chemical constituents from Spiraea prunifolia var. simpliciflora and their biological activities

Quantum mechanics (QM)-based calculations for elucidating full structures of natural compounds are growing in importance and reliability. Two new phenolic glycosides (1 and 2) and 11 known compounds were isolated from the twigs of Spiraea prunifolia var. simpliciflora. The chemical structures of the new compounds (1 and 2) were initially established through different NMR techniques (1H and 13C NMR, COSY, HSQC, and HMBC), HRMS data analysis, and chemical hydrolysis. These structure assignments were further verified by QM-based NMR chemical shift calculations. All of the purified compounds (1–13) were evaluated for their cytotoxicity against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and BT549). Those phytochemicals were also evaluated for both anti-inflammatory activity through the measurement of nitric oxide (NO) production levels in lipopolysaccharide (LPS)-stimulated murine microglia BV-2 cell lines and neuroprotective effects via induction of nerve growth factor (NGF) in C6 glioma cells.

Inhibition of rabies virus replication by interferon-stimulated gene 15 and its activating enzyme UBA7

It was reported that ISG15 and its activating enzyme UBA7 have antiviral functions. However, there is no study to demonstrate whether ISG15 and UBA7 have anti-rabies virus function. In the current study, In vivo and in vitro anti-rabies virus function of ISG15 and UBA7 were investigated using RNAi technology. The results showed that shRNA knock-down of expression of ISG15 and UBA7 increased the titers of RABV in neuroblastoma cell line NA and microglial cell line BV-2 cells and shRNA knockdown of ISG15 conjugation alleviates the IFN-induced inhibition of RABV gene expression in vitro. Lentiviral vector mediated-shRNA knock-down of expression of ISG15 and UBA7 increased the titers of RABV in mouse brains and decreased the survivorship of mice. The study showed that ISG15 and UBA7 inhibit RABV replication in vitro and in vivo. To our knowledge, we for the first time documented the anti-RABV function of ISG15 and UBA7, which may provide a means of understanding the pathogenesis of rabies and improving therapeutic methods.

Magnetofection is superior to other chemical transfection methods in a microglial cell line

BackgroundMicroglia, the resident phagocytic cells of the brain, have recently been the subject of intense investigation given their role in pathology and normal brain physiology. In general, phagocytic cells are hard to transfect with plasmid DNA. The BV2 cell line is a murine cell line of microglial origin which is often used to study this cell type in vitro. Unfortunately, this microglial cell line is, like other phagocytic cells, resistant to transfection. New methodMagnetofection is a well-established transfection method that combines DNA with magnetic particles which, under the influence of a magnetic field, ensures a high concentration of particles in proximity of cultured cells. Only recently, Glial-Mag was specifically developed for efficient transfection of microglia and microglial cell lines. ResultsMagnetofection with Glial-Mag yielded a transfection efficiency of 34.95% in BV2 cells, 24h after transfection with an eGFP-expressing plasmid. Efficient gene delivery caused a modest and short-lived cell activation (as measured by IL6 secretion) that ceased by 24h after transfection. Comparison with existing methodsHere we show that Glial-Mag magnetofection of BV2 cells yielded a significantly higher transfection efficiency (34.95%) compared to other chemical transfection methods including calcium-phoshate precipication (0.34%), X-tremeGENE (3.30%) and Lipofectamine 2000 (12.51%). ConclusionTransfection of BV2 cells using Glial-Mag magnetofection is superior compared to other chemical transfection methods and could be considered as the method of choice to chemically transfect microglial cell lines.