Activated BV-2 Cells Research Articles

D-beta-hydroxybutyrate protects against microglial activation in lipopolysaccharide-treated mice and BV-2 cells.

Microglial activation is a key event in neuroinflammation, which, in turn, is a central process in neurological disorders. In this study, we investigated the protective effects of D-beta-hydroxybutyrate (BHB) against microglial activation in lipopolysaccharide (LPS)-treated mice and BV-2 cells. The effects of BHB in mice were assessed using behavioral testing, morphological analysis and immunofluorescence labeling for the microglial marker ionizing calcium-binding adaptor molecule 1 (IBA-1) and the inflammatory cytokine interleukin-6 (IL-6) in the hippocampus. Moreover, we examined the levels of the inflammatory IL-6 and tumor necrosis factor-α (TNF-α), as well as those of the neuroprotective brain-derived neurotrophic factor (BDNF) and transforming growth factor-β (TGF-β) in the brain. In addition, we examined the effects of BHB on IL-6, TNF-α, BDNF, TGF-β, reactive oxygen species (ROS) level and cell viability in LPS-stimulated BV-2 cells. BHB treatments attenuated behavioral abnormalities, reduced the number of IBA-1-positive cells and the intensity of IL-6 fluorescence in the hippocampus, with amelioration of microglia morphological changes in the LPS-treated mice. Furthermore, BHB inhibited IL-6 and TNF-α generation, but promoted BDNF and TGF-β production in the brain of LPS-treated mice. In vitro, BHB inhibited IL-6 and TNF-α generation, increased BDNF and TGF-β production, reduced ROS level, ameliorated morphological changes and elevated cell viability of LPS-stimulated BV-2 cells. Together, our findings suggest that BHB exerts protective effects against microglial activation in vitro and in vivo, thereby reducing neuroinflammation.

Pazopanib alleviates neuroinflammation and protects dopaminergic neurons in LPS-stimulated mouse model by inhibiting MEK4-JNK-AP-1 pathway.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons and the accumulation of Lewy bodies (LB) in the substantia nigra (SN). Evidence shows that microglia-mediated neuroinflammation plays a key role in PD pathogenesis. Using TNF-α as an indicator for microglial activation, we established a cellular model to screen compounds that could inhibit neuroinflammation. From 2471 compounds in a small molecular compound library composed of FDA-approved drugs, we found 77 candidates with a significant anti-inflammatory effect. In this study, we further characterized pazopanib, a pan-VEGF receptor tyrosine kinase inhibitor (that was approved by the FDA for the treatment of advanced renal cell carcinoma and advanced soft tissue sarcoma). We showed that pretreatment with pazopanib (1, 5, 10 μM) dose-dependently suppressed LPS-induced BV2 cell activation evidenced by inhibiting the transcription of proinflammatory factors iNOS, COX2, Il-1β, and Il-6 through the MEK4-JNK-AP-1 pathway. The conditioned medium from LPS-treated microglia caused mouse DA neuronal MES23.5 cell damage, which was greatly attenuated by pretreatment of the microglia with pazopanib. We established an LPS-stimulated mouse model by stereotactic injection of LPS into mouse substantia nigra. Administration of pazopanib (10 mg·kg-1·d-1, i.p., for 10 days) exerted significant anti-inflammatory and neuronal protective effects, and improved motor abilities impaired by LPS in the mice. Together, we discover a promising candidate compound for anti-neuroinflammation and provide a potential repositioning of pazopanib in the treatment of PD.

TSAb Modulates Microglia M1 Polarization via Activation of the TSHR-Mediated NF-κB Signaling Pathway

Introduction: Thyrotropin receptor-stimulating antibody (TSAb) is a pathogenic antibody in the serum of patients with Graves’ disease. The binding of TSAb to thyroid-stimulating hormone receptor (TSHR) in non-thyroid tissue may be associated with the occurrence and development of Graves’ disease-related complications. However, only few studies have been conducted on the effects of TSAb on the brain, and the pathogenesis of acute hyperthyroidism myopathy (ATM) is unclear. Therefore, this study aimed to explore the effect of TSAb on the polarization of BV-2 cells in the brain and its possible mechanism and provide a basic experimental basis for ATM. Methods: BV-2 cells were treated with different concentrations of TSAb. The relative survival rate of BV-2 cells was determined using the CCK-8 assay; the migration ability of BV-2 cells was detected using the Transwell migration assay; and the expression levels of M1/M2 polarization markers (CD86, inducible nitric oxide synthase [iNOS], CD206, and arginase 1 [Arg-1]), TSHR, tumor necrosis factor-alpha (TNF-α), and nuclear factor-kappa B (NF-κB) protein in BV-2 cells were measured using WB. Results: Compared with the negative control group, the proliferative activity of BV-2 cells was significantly increased in the 20, 50, and 100 ng/mL TSAb groups, and the migration ability of BV-2 cells was significantly enhanced in the 50 and 100 ng/mL TSAb groups. The expression levels of M1 polarization markers (CD86 and iNOS), TSHR, TNF-α, and NF-κB protein in BV-2 cells treated with 50 and 100 ng/mL TSAb for 24 h were significantly upregulated, whereas those of M2 polarization markers (CD206 and Arg-1) significantly decreased. Conclusions: TSAb can induce abnormal activation of microglia, polarize to the M1 phenotype, and promote the inflammatory cascade reaction, in which TSHR plays a key role in NF-κB activation and proinflammatory cytokine release.

BAG3 promotes autophagy and suppresses NLRP3 inflammasome activation in Parkinson’s disease

Neuroinflammation mediated by microglia plays a key role in the pathogenesis of Parkinson's disease (PD), and our previous studies showed this was significantly inhibited by enhanced autophagy. In the autophagy pathway, Bcl2-associated athanogene (BAG)3 is a prominent co-chaperone, and we have shown BAG3 can regulate autophagy to clear the PD pathogenic protein α-synuclein. However, the connection between BAG3 and microglia mediated neuroinflammation is not clear. In this study, we explored whether BAG3 regulated related neuroinflammation and its original mechanism in PD. An inflammatory model of PD was established by injecting adeno-associated virus (AAV)-BAG3 into the bilateral striatum of C57BL/6 male mice to induce overexpression of BAG3, followed by injection of lipopolysaccharide (LPS). The striatum was extracted at 3 days after injection of LPS for Western blotting and reverse transcription quantitative polymerase chain reaction (RT-qPCR), and immunohistochemical staining was performed at 21 days after injection. At the same time, LPS was used to induce activation of BV2 cells to verify the effect of BAG3 in vitro. Overexpression of BAG3 reduced LPS-induced pyroptosis by reducing activation of caspase-1, the NOD-like receptor family, and the pyrin domain-containing 3 (NLRP3) inflammasome, and by release of interleukin (IL)-1β and tumor necrosis factor (TNF)-α. The LPS-induced inflammatory environment inhibits autophagy, and overexpression of BAG3 can restore autophagy, which may be the mechanism by which BAG3 reduces neuronal inflammation in PD. Our results demonstrate BAG3 promotes autophagy and suppresses NLRP3 inflammasome formation in PD.

Discovery of a NADPH oxidase inhibitor, (E)-3-cyclohexyl-5-(4-((2-hydroxyethyl)(methyl)amino)benzylidene)-1-methyl-2-thioxoimidazolidin-4-oneone, as a novel therapeutic for Parkinson's disease

Several lines of evidence indicated that generation of NADPH oxidase (Nox)-mediated reactive oxygen species are associated with neuronal inflammation, leading to Parkinson's disease (PD). Novel benzylidene-1-methyl-2-thioxoimidazolidin-one derivatives as Nox inhibitors were designed and synthesized in order to increase blood-brain barrier (BBB) permeability to target Nox in brain cells. In lucigenin chemiluminescence assay, eight compounds showed excellent inhibition activity against NADPH oxidases and parallel artificial membrane permeability assay (PAMPA) identified compound 11 with high passive permeability. To validate the effect of compound 11 on neuronal inflammation, we tested the regulatory activity of compound 11 in lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines in BV-2 microglial cells and LPS-mediated microglial migration. Treatment of BV2 cells with compound 11 resulted in suppressed production of pro-inflammatory cytokines and migration activity of BV2 cells in response to LPS. To evaluate the therapeutic efficacy of compound 11 in PD animal model, compound 11 was applied to MPTP-induced PD mouse model. Oral administration of compound 11 (30 mg/kg/daily, 4 weeks) into the mice resulted in suppression of dopaminergic neuronal death in substantia nigra (SN) and in striatum as well as inhibition of microglial migration into SN. These results implicate compound 11 as a novel therapeutic agent for the treatment of PD.

Copper induces microglia-mediated neuroinflammation through ROS/NF-κB pathway and mitophagy disorder.

The epidemiological correlation between copper exposure and higher risk of Parkinson disease (PD) has been recognized for a long time, and microglia-mediated neuroinflammation has reported to be an important component of the pathogenesis of PD. The present study aimed to investigate the role of microglial activation and neuroinflammation in copper neurotoxicity and the underlying mechanism of copper-induced activation of microglia. Based on the inflammatory changes in mouse brain tissues, the activation of microglia, the loss of dopaminergic neurons and the aggregation of α-syn were found in the substantia nigra. In this study we found that copper significantly caused inflammatory activation of BV2 cells. Importantly, copper increased the level of reactive oxygen species (ROS) in BV2 cells, and then activated the NF-κB pathway which acted as an early survival signal. Further study indicated that sustained copper accumulation in BV2 cells led to the decrease of mitochondrial membrane potential, reduction of Parkin and PINK1 expression, increase of P62 expression and LC3BⅡ/I ratio, as well as upregulation of NLRP3/caspase1/GSDMD axis proteins. In addition, the increased release of inflammatory factors was rescued by redox agent, NF-κB pathway inhibitor and mitophagy inducer. This work illustrated that copper exposure activates microglia to secrete inflammatory products, resulting in the pyroptosis of dopaminergic neurons, which was related to the early activation of ROS/NF-κB pathway and subsequent mitophagy disorder in BV2 cells.

Angiotensin II receptor type 1 blocker candesartan improves morphine tolerance by reducing morphine‑induced inflammatory response and cellular activation of BV2 cells via the PPARγ/AMPK signaling pathway.

Morphine is the most common drug of choice in clinical pain management; however, morphine tolerance presents a significant clinical challenge. The pathogenesis of morphine tolerance is known to be closely associated with angiotensin II receptor type 1 (AT1R) in microglia. As an AT1R antagonist, candesartan may serve an important role in regulating morphine tolerance. Therefore, the present study aimed to investigate the role of candesartan in morphine tolerance, and to explore the underlying mechanism. To meet this aim, BV2 microglial cells were treated with morphine or candesartan alone, or as a combination, and the expression levels of AT1R in BV2 cells were detected by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The levels of the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were subsequently detected by ELISA and western blotting. In addition, immunofluorescence analysis, western blotting and RT-qPCR were used to detect the expression levels of the BV2 cell activation marker, ionized calcium-binding adaptor molecule 1 (IBA-1). Western blotting was also used to detect the expression levels of peroxisome proliferator-activated receptor-γ/AMP-activated protein kinase (PPARγ/AMPK) signaling pathway-associated proteins. Finally, the cells were treated with the PPARγ antagonist GW9662 and the AMPK inhibitor compound C to further explore the mechanism underlying the effects of candesartan on improving morphine tolerance. The expression levels of AT1R were revealed to be significantly increased following morphine induction; however, candesartan treatment inhibited the expression levels of AT1R, the levels of inflammatory cytokines and the protein expression levels of IBA-1 in morphine-induced BV2 cells in a dose-dependent manner. These processes may be associated with activation of the PPARγ/AMPK signaling pathway. Taken together, the present study revealed that treatment with candesartan reduced morphine-induced inflammatory response and cellular activation of BV2 cells via PPARγ/AMPK signaling.

Neuroprotection of NSC Therapy is Superior to Glibenclamide in Cardiac Arrest-Induced Brain Injury via Neuroinflammation Regulation.

Cardiac arrest (CA) is common and devastating, and neuroprotective therapies for brain injury after CA remain limited. Neuroinflammation has been a target for two promising butunderdevelopedpost-CA therapies: neural stem cell (NSC) engrafting and glibenclamide (GBC). It is critical to understand whether one therapy has superior efficacy over the other and to further understand their immunomodulatory mechanisms. In this study, we aimed to evaluate and compare the therapeutic effects of NSC and GBC therapies post-CA. Inin vitro studies,BV2 cells underwent oxygen-glucose deprivation (OGD) for three hours and were then treated with GBC or co-cultured with human NSCs (hNSCs). Microglial polarization phenotype and TLR4/NLRP3 inflammatory pathway proteins were detected by immunofluorescence staining. Twenty-four Wistar rats were randomly assigned to three groups (control, GBC, and hNSCs, N = 8/group). After 8min of asphyxial CA, GBC was injected intraperitoneally or hNSCs were administered intranasally in the treatment groups. Neurological-deficit scores (NDSs) were assessed at 24, 48, and 72h after return of spontaneous circulation (ROSC). Immunofluorescence was used to track hNSCs and quantitatively evaluate microglial activation subtype and polarization. The expression of TLR4/NLRP3 pathway-related proteins was quantified via Western blot. The in vitro studies showedthe highest proportion of activated BV2 cells with an increased expression of TLR4/NLRP3 signaling proteins were found in the OGD group compared to OGD + GBC and OGD + hNSCsgroups. NDS showed significant improvement after CAin hNSC and GBC groups compared to controls, and hNSC treatment was superior to GBCtreatment. The hNSC group had more inactive morphology and anti-inflammatory phenotype of microglia. The quantified expression of TLR4/NLRP3 pathway-related proteins was significantly suppressed by both treatments, and the suppression was more significant in the hNSC group compared to the GBC group. hNSC and GBC therapy regulate microglial activation and the neuroinflammatory response in the brain after CA through TLR4/NLRP3 signaling and exert multiple neuroprotective effects, including improved neurological function and shortened time of severe neurological deficit. In addition, hNSCs displayed superior inflammatory regulation over GBC.

Taurine inhibits KDM3a production and microglia activation in lipopolysaccharide-treated mice and BV-2 cells

Microglia activation has been suggested as the key factor in neuro-inflammation and thus participates in neurological diseases. Although taurine exhibits anti-inflammatory and neuro-protective effects, its underlying epigenetic mechanism is unknown. In this study, taurine was administered to lipopolysaccharide (LPS)-treated mice and BV-2 cells. Behavioral test, morphological analyze, detection of microglia activation, and lysine demethylase 3a (KDM3a) measurements were performed to investigate the mechanism by which taurine regulates KDM3a and subsequently antagonizes microglia activation. Taurine improved the sociability of LPS-treated mice, inhibited microglia activation in the hippocampus, and reduced generation of brain inflammatory factors, such as interleukin-6, tumor necrosis factor-α, inducible nitric oxide synthase, and cyclooxygenase-2. Meanwhile, taurine suppressed the LPS-induced increase in microglial KDM3a, and increased the level of mono-, di- or tri-methylation of lysine 9 on histone H3 (H3K9me1/2/3). Furthermore, taurine inhibited the LPS-induced increase in KDM3a, elevated the H3K9me1/2/3 level, and reduced inflammatory factors and reactive oxygen species in a concentration-dependent manner in LPS-stimulated BV-2 cells. In conclusion, taurine inhibited KDM3a and microglia activation, thereby playing an anti-inflammatory role in LPS-treated mice and BV-2 cells.

Dim Blue Light at Night Induces Spatial Memory Impairment in Mice by Hippocampal Neuroinflammation and Oxidative Stress.

Light pollution is one of the most serious public problems, especially the night light. However, the effect of dim blue light at night (dLAN-BL) on cognitive function is unclear. In this study, we evaluated the effects of exposure to dLAN-BL in C57BL/6J mice for 4 consecutive weeks. Our results showed dLAN-BL significantly impaired spatial learning and memory and increased plasma corticosterone level in mice. Consistent with these changes, we observed dLAN-BL significantly increased the numbers and activation of microglia and the levels of oxidative stress product MDA in the hippocampus, decreased the levels of antioxidant enzymes Glutathione peroxidase (GSH-Px), Superoxide dismutase (SOD), Gluathione reductase (Gsr), total antioxidants (T-AOC) and the number of neurons in the hippocampus, up-regulated the mRNA expression levels of IL6, TNF-α and the protein expression levels of iNOS, COX2, TLR4, p-p65, Cleaved-Caspase3 and BAX, and down-regulated the mRNA expression levels of IL4, IL10, Psd95, Snap25, Sirt1, Dcx and the protein expression level of BCL2. In vitro results further showed corticosterone (10uM)-induced BV2 cell activation and up-regulated content of IL6, TNF-α in the cell supernatant and the protein expression levels of iNOS, COX2, p-p65 in BV2 cells. Our findings suggested dLAN-BL up-regulated plasma corticosterone level and hippocampal microglia activation, which in turn caused oxidative stress and neuroinflammation, leading to neuronal loss and synaptic dysfunction, ultimately leading to spatial learning and memory dysfunction in mice.

Particulate Matter Exacerbates the Death of Dopaminergic Neurons in Parkinson's Disease through an Inflammatory Response.

Particulate matter (PM), a component of air pollution, has been epidemiologically associated with a variety of diseases. Recent reports reveal that PM has detrimental effects on the brain. In this study, we aimed to investigate the biological effects of ambient particles on the neurodegenerative disease Parkinson’s disease (PD). We exposed mice to coarse particles (PM10: 2.5–10 μm) for short (5 days) and long (8 weeks) durations via intratracheal instillation. Long-term PM10 exposure exacerbated motor impairment and dopaminergic neuron death in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse models. Short-term PM10 exposure resulted in both pulmonary and systemic inflammatory responses in mice. We further investigated the mechanism underlying PM10-induced neurotoxicity in cocultures of lung LA-4 epithelial cells and RAW264.7 macrophages. PM10 treatment elicited a dramatic increase in proinflammatory mediators in LA-4/RAW264.7 coculture. Treating BV2 microglial cells with PM10-treated conditioned medium induced microglial activation. Furthermore, 1-methyl-4-phenylpyridinium (MPP+) treatment caused notable cell death in N2A neurons cocultured with activated BV2 cells in PM10-conditioned medium. Altogether, our results demonstrated that PM10 plays a role in the neurodegeneration associated with PD. Thus, the impact of PM10 on neurodegeneration could be related to detrimental air pollution-induced systemic effects on the brain.

Circ-Ncam2 (mmu_circ_0006413) Participates in LPS-Induced Microglia Activation and Neuronal Apoptosis via the TLR4/NF-κB Pathway.

Spinal cord injury (SCI) can cause permanent neurological deficits. Circular RNA Ncam2 (circ-Ncam2 also termed mmu_circ_0006413) has been reported to be overexpressed in SCI mouse models. However, the function of circ-Ncam2 in SCI has not been validated. Lipopolysaccharide (LPS) was used to activate mouse microglia (BV2 cells). Expression levels of circ-Ncam2 were determined by RT-qPCR. Relative protein levels were evaluated by western blotting. Cytokines were determined by ELISA. The regulatory mechanism of circ-Ncam2 was validated by dual-luciferase reporter and RNA pull-down assays. Effects of LPS-induced BV2 cells on mouse neuronal (HT22 cells) viability, proliferation, and apoptosis were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry assays. LPS stimulation promoted circ-Ncam2 expression in BV2 cells. Inhibition of circ-Ncam2 mitigated LPS-induced BV2 cell activation and inflammation. Mechanically, circ-Ncam2 adsorbed miR-544-3p to regulate TLR4 expression. Also, either miR-544-3p inhibition or TLR4 overexpression weakened circ-Ncam2 silencing-mediated effects on LPS-induced BV2 cell activation and inflammation. Furthermore, LPS-induced BV2 cells suppressed HT22 cell proliferation and promoted HT22 cell apoptosis through the circ-Ncam2/miR-544-3p axis. Importantly, circ-Ncam2 activated the NF-κB signaling via the miR-544-3p/TLR4axis. circ-Ncam2 silencing lowered LPS-induced microglia activation and neuronal apoptosis via blocking the TLR4/NF-κB pathway through acting as a miR-544-3p sponge, suggesting that circ-Ncam2 may be involved in secondary SCI.

A Critical Role of δ-Opioid Receptor in Anti-microglial Activation Under Stress

Microglia are involved in the regulation of cerebral homeostasis and pathogen confrontation. There is, however, evidence showing that excessive microglia activation is implicated in various age-related cerebral diseases. On the other hand, microglia may experience complex changes of polarization in pathological insults, i.e., from a proinflammatory M1 to an anti-inflammatory M2 phenotype, which differentially contribute to the exacerbation or alleviation of cellular injury. Remolding the phenotype of microglia or inhibiting the excessive activation of microglia seems to be a promising approach against neurodegenerative pathologies. Since δ-opioid receptor (DOR) activation exhibits a strong protective capacity against various neuronal injuries, especially the hypoxic/ischemic injury, we asked if the DOR-induced neuroprotection is associated with its effect on microglia. We explored this fundamental issue by using pharmacological and genetic approaches in the BV2 cell line, a general type of microglial cells. The results showed that DOR expression significantly increased in the activated microglial M2 phenotype, but slightly decreased in the microglial M1 phenotype. Hypoxia induced dual polarizations of BV2 cells with an increase in DOR expression. Administration of a specific DOR agonist, UFP-512, largely inhibited lipopolysaccharide (LPS) or hypoxia-induced microglial M1 activation and inflammatory activity with high concentrations of UFP-512 being effective to reverse the interleukin-4 (IL4)-induced microglial activation. Consistent with these observations, inhibiting DOR or knocking-down DOR promoted the excessive activation of BV2 cells in both M1 and M2 directions, while DOR overexpression did the opposite. Furthermore, the PC12 cells exposed to the conditioned medium of BV2 cells treated by UFP-512 grew better than those treated directly with UFP-512 under LPS or hypoxic insults. DOR inhibitor naltrindole could block all the effects of DOR activation. The medium from the BV2 cells with DOR knock-down decreased the viability of PC12 cell, while the medium from the BV2 cells with DOR overexpression largely attenuated LPS or hypoxic injury in the PC12 cells. These first data suggest a close linkage between DOR expression/function and microglial polarization and a critical role of DOR in negative controlling microglial activation. Our work provides a novel clue for new protective strategies against neurodegenerative pathophysiology through DOR-mediated regulation of microglia.

Terpenoids from Glechoma hederacea var. longituba and their biological activities.

Glechoma hederacea var. longituba (common name: ground ivy) has been used for the treatment of asthma, bronchitis, cholelithiasis, colds, and inflammation. In the present study, three new sesquiterpene glycosides (1–3), two new diterpene glycosides (4 and 5), and four known compounds (6–9) were isolated from its MeOH extract. A structure elucidation was performed for the five new compounds (1–5) using 1D and 2D NMR, HRESIMS, DP4+ and ECD calculations, and chemical methods. All the isolates (1–9) were assessed for their antineuroinflammatory activities on nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV-2 cells, nerve growth factor (NGF) secretion stimulation activities in C6 glioma cells, and cytotoxic activities against four human cancer cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15). Compounds 2 and 5–7 exhibited inhibitory effects on the NO production with IC50 values of 52.21, 47.90, 61.61, and 25.35 μM, respectively. Compound 5 also exhibited a significant stimulating effect on NGF secretion (122.77 ± 8.10%). Compound 9 showed potent cytotoxic activity against SK-OV-3 (IC50 = 3.76 μM) and SK-MEL-2 (IC50 = 1.48 μM) cell lines, while 7 displayed a strong cytotoxic activity against the SK-MEL-2 (IC50 = 9.81 μM) cell line.

Remifentanil inhibits the inflammatory response of BV2 microglia and protects PC12 cells from damage caused by microglia activation

ABSTRACT Microglia acts as a critical player in neuroinflammation and neuronal injury. Remifentanil (Rem) has been reported to exert anti-inflammatory activity in several types of diseases. However, the role of Rem in microglia-mediated neuroinflammation is unclear. The present study was designed to investigate the effects of Rem against lipopolysaccharide (LPS)-activated BV2 microglial and PC12 cell induced by activated BV2 microglia. Cell proliferative ability was assessed with cell counting kit-8 assay and cellular morphology was observed. ELISA assay was used to measure the expressions of PGE2 and inflammatory factors. The contents of p-NF-KB p65, p-IKKα/β, and COX2 were evaluated with the aid of western blot. The levels of NO and iNOS were assessed with Griess assay, qRT-PCR, and western blot. In addition, Tunel assay and western blot were performed to assess cell apoptosis. The data revealed that Rem alleviated BV2 microglial morphological injury induced by LPS. Furthermore, Rem suppressed inflammatory releases, iNOS, NO and PGE2 stimulated by LPS in activated BV2 cells. Moreover, Rem suppressed PC12 cell injury, the generations of inflammatory factors and cell apoptosis triggered by inflammatory mediators secreted from activated BV2 cells. These results suggest that Rem exhibited anti-neuroinflammatory activity in protecting PC12 cells against injury derived from LPS-stimulated BV2 microglia.

Zileuton, a 5-Lipoxygenase Inhibitor, Attenuates Haemolysate-Induced BV-2 Cell Activation by Suppressing the MyD88/NF-κB Pathway.

M1 microglia induce neuroinflammation-related neuronal death in animal models of spontaneous subarachnoid haemorrhage. Zileuton is a 5-lipoxygenase inhibitor that reduces the levels of downstream pro-inflammatory cytokines. This study aimed to investigate whether zileuton inhibits microglial activation and describe its underlying mechanisms. BV-2 cells were exposed to 1 mg/mL haemolysate for 30 min, followed by treatment with different concentrations (5, 10, 15, or 20 μM) of zileuton for 24 h. The cells were then assessed for viability, polarisation, and protein expression levels. Haemolysate increases the viability of BV-2 cells and induces M1 polarisation. Subsequent exposure to high concentrations of zileuton decreased the viability of BV-2 cells, shifted the polarisation to the M2 phenotype, suppressed the expression of 5-lipoxygenase, decreased tumour necrosis factor α levels, and increased interleukin-10 levels. Furthermore, high concentrations of zileuton suppressed the expression of myeloid differentiation primary response protein 88 and reduced the phosphorylated-nuclear factor-kappa B (NF-kB)/NF-kB ratio. Therefore, phenotype reversal from M1 to M2 is a possible mechanism by which zileuton attenuates haemolysate-induced neuroinflammation after spontaneous subarachnoid haemorrhage.

Polystyrene nanoplastics penetrate across the blood-brain barrier and induce activation of microglia in the brain of mice

Microplastics (MPs) have been well demonstrated as potential threats to the ecosystem, whereas the neurotoxicity of MPs in mammals remains to be elucidated. The current study was designed to investigate whether 50 nm polystyrene nanoplastics (PS-NPs) could pass through the blood-brain barrier (BBB), and to elucidate the underlying mechanisms and the following neurotoxic manifestation. In vivo study showed that PS-NPs (0.5–50 mg/kg. bw PS-NPs for 7 days) significantly induced the increase of permeability of BBB, and dose-dependently accumulated in the brain of mice. In addition, PS-NPs were found to be present in microglia, and induced microglia activation and neuron damage in the mouse brain. In vitro studies using the immortalized human cerebral microvascular endothelial cell (hCMEC/D3), the most commonly used cell model for BBB-related studies, revealed that PS-NPs could be internalized into cells, and caused reactive oxygen species (ROS) production, nuclear factor kappa-B (NF-κB) activation, tumor necrosis factors α (TNF-α) secretion, and necroptosis of hCMEC/D3 cells. Furthermore, PS-NPs exposure led to disturbance of the tight junction (TJ) formed by hCMEC/D3, as demonstrated by the decline of transendothelial electrical resistance (TEER) and decreased expression of occludin. Lastly, PS-NPs exposure resulted in the activation of murine microglia BV2 cells, and the cell medium of PS-NPs-exposed BV2 induced obvious damage to murine neuron HT-22 cells. Collectively, these results suggest that PS-NPs could pass through BBB and induce neurotoxicity in mammals probably by inducing activation of microglia.

Corticotropin-Releasing Factor Aggravates Ischemic Stroke Injury by the Inflammatory Activation of Microglia.

Ischemic stroke is the second leading cause of death worldwide. Therefore, exploring effective and emerging molecular targets for ischemic stroke is a primary task of basic and clinical research. The aim of the present study was to investigate the function of corticotropin-releasing factor (CRF) in ischemic stroke and its related mechanisms, to provide a reference for the treatment of ischemic stroke. CRF, antalarmin, or astressin-2B were used to activate or block the CRF1 (CRF receptor 1) or CRF2 (CRF receptor 2) in BV2 cells and adult male mice, thus constructing a distal middle cerebral artery occlusion (dMCAO) model. CRF not only accelerated microglial activity by promoting transcription and production of inflammatory factors, but also promoted the transformation of activated BV2 cells from a neuroprotective phenotype (M2) to cytotoxic phenotype (M1), and these effects were mediated by the TLR4/NF-κB signaling pathway. These effects can be blocked by antalarmin but not by astressin-2B. CRF significantly aggravated the neurological deficit, increased infarction volume, and exacerbated neuronal injuries. Additionally, CRF significantly improved the levels of TNF-α and phospho-NF-κB in the ischemia penumbra. Finally, CRF significantly increased the number of CD16/Iba-1-positive cells and decreased the number of CD206/Iba-1-positive cells in the ischemia penumbra. These results provide evidence of the proinflammatory role of CRF in an ischemic stroke model and a possible underlying mechanism, which may facilitate the elucidation of potential treatment approaches for ischemic stroke.

Dexmedetomidine alleviates microglial activation of neuropathic pain by modulating miR-23a/PDE10A axis in streptozotocin-induced diabetic mice


 
 
 
 Purpose: To elaborate the functional role of dexmedetomidine (DEX) in alleviating microglial activation of diabetic neuropathic pain (DNP) and explore the involved signaling pathways.
 Methods: The viability of BV-2 cells was measured using a commercial kit. Levels of interleukin 1β (IL- 1β) and tumor necrosis factor-α (TNF-α) were measured using commercial ELISA kit. The mRNA target was predicted and confirmed using TargetScan and luciferase assay. Protein expression levels were determined by western blotting. Diabetes was indiced in C57BL/6J mice using streptozotocin (STZ) and antidiabetic parameters evaluated in vivo.
 Results: DEX suppressed HG-induced microglial activation in BV-2 cells. The levels of IL-1β and TNF-α increased in HG-treated cells, but this was counteracted following DEX treatment. Phosphorylation of p65 (p-p65) was upregulated in cells treated with HG, while DEX repressed this upregulation. MiR-23a was downregulated in BV-2 cells treated with HG, but upregulated by addition of DEX. MiR-23a mimics repressed the induction of IL-1β and TNF-α levels and expression of p-p65. Results from TargetScan and luciferase assays showed that the 3-untranslated region (UTR) of PDE10A was directly targeted by miR-23a. The in vivo studies showed that miR-23a agomir relieved neuropathic pain and reduced the expressions of PDE10A and p-p65 in STZ-induced diabetic mice, but these effects were aggravated by DEX.
 Conclusion: The results show that upregulation of miR-23a, DEX alleviates microglial activation of neuropathic pain and reduces levels of inflammatory factors in STZ-induced diabetic C57BL/6J mice. The underlying mechanism was at least partially mediated by PDE10A.
 
 
 

Phoenixin-14 Promotes the Recovery of Neurological Dysfunction After Spinal Cord Injury by Regulating Microglial Polarization via PTEN/Akt Signaling Pathway.

Spinal cord injury (SCI) is a destructive event in central nervous system (CNS) with the hallmark of deficits in neuronal function. Phoenixin-14 (PNX-14) is a reproductive peptide that also has neuroprotective effects. However, the role of PNX-14 in SCI has not yet been studied. In this study, we firstly investigated the effects of PNX-14 on the recovery of neurological dysfunction and microglial polarization in a SCI mice model. We demonstrated that PNX-14 improved the recovery of neurological dysfunction with increased Basso Mouse Scale (BMS) scores, reduced lesion area volume and Evans blue (EB) dye extravasation. PNX-14 alleviated neuronal apoptosis and neuroinflammation in mice underwent SCI. In vitro co-culture assay proved that PNX-14 protected neurons injury in response to LPS- activated BV-2 cells. PNX-14 suppressed the LPS- induced microglia M1 phenotype polarization with decreased expression of M1-associated markers (CD16 and iNOS) and increased expression of M2-associated markers (CD206 and Arg1). PNX-14 also suppressed LPS- caused decrease in anti-inflammatory cytokines TGF-β, IL-10, and IL-13, as well increase in pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 in BV2 cells. PNX-14 treatment caused increased PTEN expression and decreased p-Akt expression in BV2 cells against LPS induction. While inhibition of PTEN by SF1670 reversed the effects of PNX-14 on LPS- induced phenotypic transition of BV2 cells. Taken together, we found that PNX-14 exerted protective effects on neurological dysfunction and inflammation in SCI mice through modulating microglial polarization via PTEN/Akt signaling pathway.