BV2 Cells Research Articles

Effective treatment of traumatic brain injury by injection of a selenium-containing ointment

Traumatic brain injury (TBI) is an incurable and overwhelming disease accompanied with serve disability and huge financial burden, where the overproduced reactive oxygen species (ROS) can exacerbate the secondary injury, leading to massive apoptosis of neurons. In this study, β-cyclodextrin (CD)-capped hyperbranched polymers containing selenium element (HSE-CD) were crosslinked with CD-modified hyaluronic acid (HA-CD) and amantadine-modified hyaluronic acid (HA-AD) to obtain a ROS-responsive ointment (R-O). The structures of synthesized polymers were characterized with 1H nuclear magnetic resonance, and the properties of ointment were investigated with rheology and antioxidation. Compared to non-ROS-responsive ointment (N-O), the R-O ointment had stronger efficiency in decreasing the ROS level in BV2 cells in vitro. In a controlled rat cortical impact (CCI) model, the R-O ointment could relieve the DNA damage and decrease apoptosis in injured area via reducing the ROS level. Besides, after the R-O treatment, the rats showed significantly less activated astrocytes and microglia, a lower level of pro-inflammatory cytokines and a higher ratio of M2/M1 macrophage and microglia. Moreover, compared to the TBI group the R-O ointment promoted the doublecortin (DCX) expression and tissue structure integrity around the cavity, and promoted the recovery of nerve function post TBI. Statement of significanceTraumatic brain injury (TBI) is an incurable and overwhelming disease, leading to severe disability and huge social burden, where reactive oxygen species (ROS) are considered as one of the most significant factors in the secondary injury of TBI. A ROS responsive supramolecular ointment containing di-selenide bonds was injected in rats with controlled cortical impact. It relieved the DNA damage and decreased apoptosis in the injured area via reducing the ROS levels, downregulated neuroinflammation, and improved neurological recovery of TBI in vivo. This designed self-adaptive biomaterial effectively regulated the pathological microenvironment in injured tissue, and achieved better therapeutic effect.

Unraveling the Protective Role of Oleocanthal and Its Oxidation Product, Oleocanthalic Acid, against Neuroinflammation.

Neuroinflammation is a critical aspect of various neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. This study investigates the anti-neuroinflammatory properties of oleocanthal and its oxidation product, oleocanthalic acid, using the BV-2 cell line activated with lipopolysaccharide. Our findings revealed that oleocanthal significantly inhibited the production of pro-inflammatory cytokines and reduced the expression of inflammatory genes, counteracted oxidative stress induced by lipopolysaccharide, and increased cell phagocytic activity. Conversely, oleocanthalic acid was not able to counteract lipopolysaccharide-induced activation. The docking analysis revealed a plausible interaction of oleocanthal, with both CD14 and MD-2 leading to a potential interference with TLR4 signaling. Since our data show that oleocanthal only partially reduces the lipopolysaccharide-induced activation of NF-kB, its action as a TLR4 antagonist alone cannot explain its remarkable effect against neuroinflammation. Proteomic analysis revealed that oleocanthal counteracts the LPS modulation of 31 proteins, including significant targets such as gelsolin, clathrin, ACOD1, and four different isoforms of 14-3-3 protein, indicating new potential molecular targets of the compound. In conclusion, oleocanthal, but not oleocanthalic acid, mitigates neuroinflammation through multiple mechanisms, highlighting a pleiotropic action that is particularly important in the context of neurodegeneration.

RIPK1 inhibition mitigates neuroinflammation and rescues depressive-like behaviors in a mouse model of LPS-induced depression

BackgroundDepression is often linked to inflammation in the brain. Researchers have been exploring ways to reduce this inflammation to improve depression symptoms. One potential target is a protein called RIPK1, which is known to contribute to brain inflammation. However, it’s unclear how RIPK1 influences depression. Our study aims to determine whether RIPK1 inhibition could alleviate neuroinflammation-associated depression and elucidate its underlying mechanisms.MethodsTo investigate our research objectives, we established a neuroinflammation mouse model by administering LPS. Behavioral and biochemical assessments were conducted on these mice. The findings were subsequently validated through in vitro experiments.ResultsUsing LPS-induced depression models, we investigated RIPK1’s role, observing depressive-like behaviors accompanied by elevated cytokines, IBA-1, GFAP levels, and increased inflammatory signaling molecules and NO/H2O2. Remarkably, Necrostatin (Nec-1 S), a RIPK1 inhibitor, mitigated these changes. We further found altered expression and phosphorylation of eIF4E, PI3K/AKT/mTOR, and synaptic proteins in hippocampal tissues, BV2, and N2a cells post-LPS treatment, which Nec-1 S also ameliorated. Importantly, eIF4E inhibition reversed some of the beneficial effects of Nec-1 S, suggesting a complex interaction between RIPK1 and eIF4E in LPS-induced neuroinflammation. Moreover, citronellol, a RIPK1 agonist, significantly altered eIF4E phosphorylation, indicating RIPK1’s potential upstream regulatory role in eIF4E and its contribution to neuroinflammation-associated depression.ConclusionThese findings propose RIPK1 as a pivotal mediator in regulating neuroinflammation and neural plasticity, highlighting its significance as a potential therapeutic target for depression.

Sesquiterpenes from the seeds of Cichorium glandulosum and their anti- neuroinflammation activities

Three previously undescribed sesquiterpenes, along with three known ones were isolated from the seeds of Cichorium glandulosum. The structures of them were elucidated by the analysis of spectroscopic data. The isolated compounds were tested for their neuroprotective effects against LPS-induced neuroinflammation in BV-2 cells. Santamarine (5) exhibited inhibitory activity on LPS-induced NO production in BV-2 cells with IC50 of 0.89 ± 0.12 μM. The mechanism of the compound 5 was related to activating the NF-κB, MAPK and cGAS/STING pathways.

Nrf2 Deficiency Exacerbates Parkinson's Disease by Aggravating NLRP3 Inflammasome Activation in MPTP-Induced Mouse Models and LPS-Induced BV2 Cells.

Parkinson's disease (PD) is a movement disorder characterized by the progressive loss of dopamine neurons. Microglia-mediated neuroinflammation drives disease progression and becomes a critical factor in neuronal degeneration. Recent studies have found that nuclear factor-erythroid 2-related-2 (Nrf2) expression levels are reduced during aging and neurodegenerative diseases, but its regulatory mechanism on microglia-induced neuroinflammation has not been fully elucidated. In vivo, we used the intraperitoneal injection of the neurotoxic drug neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish an animal model of PD and, at the same time, administered Nrf2 inhibitors ML385 and dimethyl fumarate to regulate Nrf2 protein levels. In vitro, we used si-RNA to knock out the Nrf2 gene to intervene in BV2 cells and used lipopolysaccharide (LPS) to stimulate and induce the cell model. The study found that inhibition of Nrf2 expression aggravated the motor defects of PD mice, accompanied by a significant loss of dopaminergic neurons in the substantia nigra and striatum of the brain. In addition, after inhibition of Nrf2, the malondialdehyde (MDA) level in the substantia nigra of the midbrain of mice increased, and the levels of superoxide dismutase (SOD) and heme oxygenase-1 (HO-1) decreased, accompanied by the proliferation of microglia and astrocytes. In addition, the activation of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome, the assembly of apoptosis-associated speck-like protein containing a CARD (ASC) protein in microglia, and the release of downstream inflammatory factors caspase-1 and interleukin (IL)-1β, were aggravated. At the cellular level, it was found that knocking out the expression of Nrf2 would aggravate the activation of NLRP3 inflammasomes and the assembly of ASC in LPS-induced BV2 cells. Inhibited Nrf2 activity can reduce the downstream antioxidant enzyme HO-1 and antioxidant levels, induce NLRP3 inflammasome activation and ASC protein assembly in microglia, and ultimately aggravate PD inflammatory response and dopamine neuron degeneration.

P18-03 Ferroptosis mediated by mtROS is involved in the Ag2Se QDs induced NLRP3 inflammasome activation in BV2 cells

P18-03 Ferroptosis mediated by mtROS is involved in the Ag2Se QDs induced NLRP3 inflammasome activation in BV2 cells

Morphine induces inflammatory responses via both TLR4 and cGAS-STING signaling pathways

BackgroundOpioid activation of the microglia or macrophage Toll-like receptor 4 (TLR4) and associated inflammatory cytokine release are implicated in opioid-induced hyperalgesia and tolerance. The cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS-STING) signaling pathway, activated by double-stranded DNA including mitochondrial DNA (mtDNA), has emerged as another key mediator of inflammatory responses. This study tested the hypothesis that morphine induces immune inflammatory responses in microglia and macrophages involving TLR4 and cGAS-STING pathway. MethodsBV2 microglia and Raw 264.7 (Raw) macrophage cells were exposed to morphine with and without a STING inhibitor (C176) for 6 h or TLR 4 inhibitor (TAK242) for 24 h. Western blotting and RT-qPCR analyses assessed TLR4, cGAS, STING, nuclear factor-kappa B (NF-κB), and pro-inflammatory cytokine expression. Morphine-induced mitochondria dysfunction was quantified by reactive oxygen species (ROS) release using MitoSOX, mtDNA release by immunofluorescence, and RT-qPCR. Polarization of BV2 and Raw cells was assessed by inducible nitric oxide (iNOS) and CD86 expression. The role of mtDNA on morphine-related inflammation was investigated by mtDNA depletion of the cells with ethidium bromide (EtBr) or cell transfection of mtDNA extracted from morphine-treated cells. ResultsMorphine significantly increased the expression of TLR4, cGAS, STING, p65 NF-κB, and cytokines (IL-6 and TNF-α) in BV2 and Raw cells. Morphine-induced mitochondrial dysfunction by increased ROS and mtDNA release; the increased iNOS and CD86 evidenced inflammatory M1-like phenotype polarization. TLR4 and STING inhibitors reduced morphine-induced cytokine release in both cell types. The transfection of mtDNA activated inflammatory signaling proteins, cytokine release, and polarization. Conversely, mtDNA depletion led to the reversal of these effects. ConclusionMorphine activates the cGAS-STING pathway in macrophage cell types. Inhibition of the STING pathway can be an additional method to overcome immune cell inflammation-related morphine tolerance and opioid-induced hyperalgesia.

Inhibition of cGAS attenuates neonatal hypoxic-ischemic encephalopathy via regulating microglia polarization and pyroptosis.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a condition causing brain injury in newborns with unclear pathogenesis. Cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) signaling pathway and NOD-like receptor protein 3 (NLRP3) mediated pyroptosis are thought to be involved in the pathological process of HIE, but whether these two mechanisms act independently is still unknown. Therefore, we aim to clarify whether there is any interaction between these two pathways and thus synergistically affects the progression of HIE. The HIE model of neonatal rats was established using the Rice-Vannucci method. The potential therapeutic effect of RU.521 targeting cGAS on HIE was explored through rescue experiment. Twenty-four hours after modeling was selected as observation point, sham + vehicle group, HIE + vehicle group and HIE + RU.521 group were established. A complete medium of BV2 cells was adjusted to a glucose-free medium, and the oxygen-glucose deprivation model was established after continuous hypoxia for 4 hours and reoxygenation for 12 to 24 hours. 2,3,5-triphenyl tetrazolium chloride staining was employed to detect ischemic cerebral infarction in rat brain tissue, and hematoxylin and eosin staining was used to observe tissue injury. Immunofluorescence was applied to monitor the expression of cGAS. Real-time quantitative polymerase chain reaction and western blot were utilized to detect the expression of messenger RNA and protein. cGAS expression was increased in brain tissues of neonatal rats with HIE, and mainly localized in microglia. RU.521 administration reduced infarct size and pathological damage in rat HIE. Moreover, blocking cGAS with RU.521 significantly reduced inflammatory conditions in the brain by down-regulating STING expression, decreasing NLRP3 inflammasome activation and reducing microglial pyroptosis both in vivo and in vitro. Besides, RU.521 promoted the switching of BV2 cells towards the M2 phenotype. This study revealed a link between the cGAS/STING pathway and the NLRP3/GSDMD/pyroptosis pathway in neonatal HIE. Furthermore, the small molecule compound RU.521 can negatively regulate cGAS/STING/NLRP3/pyroptosis axis and promote M2 polarization in microglia, which provides a potential therapeutic strategy for the treatment of neuroinflammation in HIE.

Formononetin inhibits neuroinflammation in BV2 microglia induced by glucose and oxygen deprivation reperfusion through TLR4/NF-κB signaling pathway

Ischemic stroke, caused by diminished or interrupted cerebral blood flow, triggers the activation of microglial cells and subsequent inflammatory responses. Formononetin (FMN) has been observed to inhibit BV2 microglial cell activation and alleviate ensuing neuroinflammatory reactions. Despite extensive research, the precise underlying mechanism remains unclear. To investigate the neuroinflammatory response following FMN-mediated inhibition of BV2 microglial activation, we employed an in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model. BV2 microglial cells were categorized into four groups: control, FMN, OGD/R, and OGD/R+FMN. Cell viability was assessed using the CCK-8 assay, while flow cytometry assessed M1 and M2 cell populations within BV2 cells. Immunofluorescence was utilized to detect the expression levels of apoptosis-inducing factor (AIF), p53, Toll-like receptor 4 (TLR4), and NF-κB p65. Western blotting (WB) was conducted to quantify p65/p-p65, IκB-α/p-IκB-α, and TLR4 protein levels in each group. Additionally, ELISA was employed to measure IL-1β and TNF-α levels in cell supernatants from each group. The results revealed a significant increase in the proportion of iNOS/CD206-positive M1/M2 cells in the OGD/R group compared to the control group (p < 0.05). There was also a notable increase in nuclear translocation of NF-κB p65 and elevated expression of inflammatory factors IL-1β and TNF-α in cell supernatants. Moreover, levels of p-p65, p-IκB-α, and TLR4 proteins were significantly elevated in the OGD/R group (p < 0.05). However, the addition of FMN reversed these effects. Specifically, FMN administration notably attenuated cell death and inflammation in BV2 microglia induced by OGD/R through modulation of the TLR4/NF-κB signaling pathway.These findings suggest that FMN may serve as a potential therapeutic agent against neuroinflammation associated with ischemic stroke by targeting microglial activation pathways.

Design, synthesis, and biological evaluation of imidazolylacetophenone oxime derivatives as novel brain-penetrant agents for Alzheimer's disease treatment

Alzheimer's disease (AD, also known as dementia) has become a serious global health problem along with population aging, and neuroinflammation is the underlying cause of cognitive impairment in the brain. Nowadays, the development of multitarget anti-AD drugs is considered to be one effective approach. Imidazolylacetophenone oxime ethers or esters (IOEs) were multifunctional agents with neuroinflammation inhibition, metal chelation, antioxidant and neuroprotection properties against Alzheimer's disease. In this study, IOEs derivatives 1−8 were obtained by structural modifications of the oxime and imidazole groups, and the SARs showed that (Z)-oxime ether (derivative 2) had stronger anti-neuroinflammatory and neuroprotective ability than (E)-congener. Then, IOEs derivatives 9−30 were synthesized based on target-directed ligands and activity-based groups hybridization strategy. In vitro anti-AD activity screening revealed that some derivatives exhibited potentially multifunctional effects, among which derivative 28 exhibited the strongest inhibitory activity on NO production with EC50 value of 0.49 μM, and had neuroprotective effects on 6-OHDA-induced cell damage and RSL3-induced ferroptosis. The anti-neuroinflammatory mechanism showed that 28 could inhibit the release of pro-inflammatory factors PGE2 and TNF-α, down-regulate the expression of iNOS and COX-2 proteins, and promote the polarization of BV-2 cells from pro-inflammatory M1 phenotype to anti-inflammatory M2 phenotype. In addition, 28 can dose-dependently inhibit acetylcholinesterase (AChE) and Aβ42 aggregation. Moreover, the selected nuclide [18F]-labeled 28 was synthesized to explore its biodistribution by micro-PET/CT, of which 28 can penetrate the blood-brain barrier (BBB). These results shed light on the potential of 28 as a new multifunctional candidate for AD treatment.

Anti-Neuroinflammatory Potential of Areca Nut Extract and Its Bioactive Compounds in Anthracene-Induced BV-2 Microglial Cell Activation.

Particulate matter (PM2.5) containing polycyclic aromatic hydrocarbons (PAHs) is of considerable environmental importance worldwide due to its adverse effects on human health, which are associated with neurodegenerative diseases (NDDs). Areca catechu L. (AC) fruit is known to possess various pharmacological properties; however, the anti-neuroinflammatory roles of AC on the suppression of PAH-induced neuroinflammation are still limited. Thus, we focused on the effects and related signaling cascades of AC and its active compounds against anthracene-induced toxicity and inflammation in mouse microglial BV-2 cells. Phytochemicals in the ethanolic extract of AC (ACEE) were identified using LC-MS, and molecular docking was conducted to screen the interaction between compounds and target proteins. Significant bioactive compounds in ACEE such as arecoline, (-)-epicatechin, and syringic acid were evinced through the LC-MS spectrum. The docking study revealed that (-)-epicatechin showed the highest binding affinities against NF-κB. For cell-based approaches, anthracene induced intracellular ROS, mRNA levels of TNF-α, IL-1β, and IL-6, and the release of TNF-α through enhancing JNK, p38, and NF-κB signaling pathways. However, the co-treatment of cells with ACEE or (-)-epicatechin could reverse those anthracene-induced changes. The overall study suggested that ACEE-derived bioactive compounds such as (-)-epicatechin may be developed as a potential anti-neuroinflammatory agent by preventing inflammation-mediated NDDs.

Sufentanil-induced Nrf2 protein ameliorates cerebral ischemia-reperfusion injury through suppressing neural ferroptosis

As an oxidative stress and inflammation-related disease, cerebral ischemia-reperfusion injury (CIRI) is a prevalent pathogenic factor of ischemic stroke (IS) and seriously degrades the life quality of human beings. As an opioid analgesic for anesthesia, Sufentanil (SUF) can activate the Nrf2 protein-induced anti-oxidant effects, which indicate that SUF may be used as alternative drug for CIRI therapy, but little is known regarding to its molecular mechanisms. Thus, this research aimed to examine whether SUF pre-treatment alleviated CIRI through the modulation of Nrf2 protein-mediated antioxidant activity. Our research revealed that middle cerebral artery occlusion/reperfusion (MCAO/R)-treated rats exhibited apparent CIRI-related symptoms and induced damages in rats' brain, which were all notably mitigated in the MCAO/R rats. The subsequent in vitro cellular experiments verified that oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity were apparently reversed by SUF co-treatment in HT22 and BV2 cells, and it was also validated that SUF was capable of suppressing inflammation and ferroptosis in CIRI models by inhibiting oxidative stress-related damages. Mechanistically, the Akt/GSK-3β pathway was excessively activated by SUF to promote Nrf2 protein expressions and enhance Nrf2-meidated anti-oxidant effects, and it was found that SUF-induced protective effects during CIRI progression were all abrogated by co-treating cells with MK2206 (Akt inhibitor), NP-12 (GSK-3β inhibitor), or ML385 (Nrf2 inhibitor). In conclusion, SUF activated the Akt/GSK-3β pathway to initiate Nrf2 protein-mediated antioxidant effects, which further suppressed oxidative stress-related inflammation and ferroptosis to ameliorate CIRI progression, and SUF could potentially be used as novel therapeutic agent for CIRI treatment in clinic.

Systematic Characterization of Sesquiterpenes from Dendrobium nobile through Offline Two-Dimensional Chromatography Tandem Mass Spectrometry and Target Isolation.

Dendrobium nobile is a species of the genus Dendrobium that can be used as both a medicinal herb and healthy food. The sesquiterpenes in D. nobile have attracted extensive attention in recent years. In this study, Amide × RP offline two-dimensional chromatography separation tandem high-resolution mass spectrometry combined with GNPS (Global Natural Product Social Molecular Networking) was developed for the characterization of sesquiterpenes in D. nobile. After first-dimensional amide separation, the 70% ethanol extract of D. nobile was divided into 40 fractions, which were analyzed by second-dimensional reverse-phase system separation and LTQ-Orbitrap detection. The raw data was imported into the GNPS, resulting in the efficient clustering of similar substances. Finally, 594 sesquiterpene compounds were characterized, and 25 compounds were isolated based on molecular network analysis, including six new compounds. In vitro bioassays, the isolated compounds decreased NO production in the LPS-induced microglial BV-2 cell model and the content of MDA in PC12 cells, demonstrating neuroprotective activity. These findings unraveled the underlying material and provided valuable insights into the quality control of D. nobile.

Dynamic changes in pyroptosis following spinal cord injury and the identification of crucial molecular signatures through machine learning and single-cell sequencing

The pathological cascade of spinal cord injury (SCI) is highly intricate. The onset of neuroinflammation can exacerbate the extent of damage. Pyroptosis is a form of inflammation-linked programmed cell death (PCD), the inhibition of pyroptosis can partially mitigate neuroinflammation. It is imperative to delineate the principal cell types susceptible to pyroptosis and concomitantly identify key genes associated with this process. We initially defined the pyroptosis-related genes (PRGs) and analyzed their expression at different time points post SCI. The results demonstrate a substantial upregulation of differentially expressed genes (DEGs) related to pyroptosis on the 7 days post-injury (dpi), these DEGs in the 7 dpi are closely related to the inflammatory response. Subsequently, immune infiltration analysis revealed a predominant presence of inflammatory microglia. Through correlation analysis, we postulated that pyroptosis primarily manifested within the inflammatory microglia. Employing machine learning algorithms, we identified four pyroptosis-related molecular signatures, which were experimentally validated using BV2 cells and spinal cord tissue samples. The robustness of the identified molecular signatures was further confirmed through single-cell sequencing data analysis. Overall, our study elucidates the temporal dynamics of pyroptosis and identifies key molecular signatures following SCI. These findings can provide novel evidence for therapeutic interventions in SCI.

Rabies Virus Regulates Inflammatory Response in BV-2 Cells through Activation of Myd88 and NF-κB Signaling Pathways via TLR7.

Rabies is a fatal neurological infectious disease caused by rabies virus (RABV), which invades the central nervous system (CNS). RABV with varying virulence regulates chemokine expression, and the mechanisms of signaling pathway activation remains to be elucidated. The relationship between Toll-like receptors (TLRs) and immune response induced by RABV has not been fully clarified. Here, we investigated the role of TLR7 in the immune response induced by RABV, and one-way analysis of variance (ANOVA) was employed to evaluate the data. We found that different RABV strains (SC16, HN10, CVS-11) significantly increased CCL2, CXCL10 and IL-6 production. Blocking assays indicated that the TLR7 inhibitor reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). The activation of the Myd88 pathway in BV-2 cells stimulated by RABV was TLR7-dependent, whereas the inhibition of Myd88 activity reduced the expression of CCL2, CXCL10 and IL-6 (p < 0.01). Meanwhile, the RABV stimulation of BV-2 cells resulted in TRL7-mediated activation of NF-κB and induced the nuclear translocation of NF-κB p65. CCL2, CXCL10 and IL-6 release was attenuated by the specific NF-κB inhibitor used (p < 0.01). The findings above demonstrate that RABV-induced expression of CCL2, CXCL10 and IL-6 involves Myd88 and NF-κB pathways via the TLR7 signal.

Caspase-11 signaling promotes damage to hippocampal CA3 to enhance cognitive dysfunction in infection

BackgroundCognitive dysfunction caused by infection frequently emerges as a complication in sepsis survivor patients. However, a comprehensive understanding of its pathogenesis remains elusive.MethodsIn our in vivo experiments, an animal model of endotoxemia was employed, utilizing the Novel Object Recognition Test and Morris Water Maze Test to assess cognitive function. Various techniques, including immunofluorescent staining, Western blotting, blood‒brain barrier permeability assessment, Limulus Amebocyte Lysate (LAL) assay, and Proximity-ligation assay, were employed to identify brain pathological injury and neuroinflammation. To discern the role of Caspase-11 (Casp11) in hematopoietic or non-hematopoietic cells in endotoxemia-induced cognitive decline, bone marrow chimeras were generated through bone marrow transplantation (BMT) using wild-type (WT) and Casp11-deficient mice. In vitro studies involved treating BV2 cells with E. coli-derived outer membrane vesicles to mimic in vivo conditions.ResultsOur findings indicate that the deficiency of Casp11-GSDMD signaling pathways reverses infection-induced cognitive dysfunction. Moreover, cognitive dysfunction can be ameliorated by blocking the IL-1 effect. Mechanistically, the absence of Casp11 signaling significantly mitigated blood‒brain barrier leakage, microglial activation, and synaptic damage in the hippocampal CA3 region, ultimately leading to improved cognitive function.ConclusionThis study unveils the crucial contribution of Casp11 and GSDMD to cognitive impairments and spatial memory loss in a murine sepsis model. Targeting Casp11 signaling emerges as a promising strategy for preventing or treating cognitive dysfunction in patients with severe infections.

Exploring the Biomarkers and Potential Mechanisms of Botulinum Toxin Type A in the Treatment of Microglial Inflammatory Activation through P2X7 Receptors based on Transcriptome Sequencing.

This study aims to explore the potential mechanism by which Botulinum toxin type A (BoNT/ A) inhibits microglial inflammatory activation through P2X7 receptors (P2X7R). BoNT/A is a promising analgesic drug, and previous studies have established that it alleviates Neuropathic Pain (NP) by inhibiting microglial inflammatory activation. This study examined the biomarkers and potential mechanisms by which BoNT/A relieves neuropathic pain by mediating microglial P2X7R and analyzing transcriptome sequencing data from mouse BV-2 microglial cells. The P2X7R agonist Bz-ATP was used to induce microglial inflammatory activation, whilst RNAseq technology was used to explore the biomarkers and potential mechanisms through which BoNT/A suppresses microglial inflammation. RNA sequencing was performed on three BV-2 cell samples treated with a P2X7R specific activator (Bz-ATP) and three BV-2 cell samples pre-treated with BoNT/A. Only data that successfully passed quality control measures were included in subsequent analysis. Initially, Differentially Expressed Genes (DEGs) were identified from BoNT/A and control samples, followed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Biomarkers were then identified by constructing a Protein- Protein Interaction (PPI) network and utilizing the CytoHubba plug-in in Cytoscape software. Lastly, enrichment analysis and regulatory network analysis were performed to elucidate the potential mechanism of BoNT/A in the treatment of NP. 93 DEGs related to the "cell component size regulation" GO term and enriched in the "axon guidance" KEGG pathway were identified. Subsequently, 6 biomarkers were identified, namely PTPRF, CHDH, CKM, Ky, Sema3b, and Sema3f, which were enriched in pathways related to biosynthesis and metabolism, disease progression, signal transduction, and organelle function, including the "ribosome" and "Wnt signaling pathway." Finally, a competing endogenous RNA (ceRNAs) network was constructed from 6 mRNAs, 66 miRNAs, and 31 lncRNAs, forming a complex relationship network. Six genes (PTPRF, Sema3b, Sema3f, CHDH, CKM, and Ky) were identified as biomarkers of microglial inflammatory activation following BoNT/A treatment. This finding may provide a valuable reference for the relief and treatment of neuropathic pain.

Npc1 deficiency impairs microglia function via TREM2-mTOR signaling in Niemann-Pick disease type C

Niemann-Pick disease Type C (NPC) is a neurodegenerative disease mainly caused by the mutation in NPC1 gene, leading to massive accumulation of unesterified cholesterol in the late endosome/lysosome of cells. Impaired phenotype of microglia is a hallmark in Npc1 mutant mice (Npc1−/− mice). However, the mechanism of Npc1 in regulating microglial function is still unclear. Here, we showed that the reactive microglia in the neonatal Npc1−/− mice indicated by the increased lysosome protein CD68 and phagocytic activity were associated with disrupted TREM2-mTOR signaling in microglia. Furthermore, in Npc1-deficient BV2 cells, genetic deletion of Trem2 partially restored microglial function, probably via restored mTOR signaling. Taken together, our findings indicated that loss of Npc1 in microglia caused changes of their morphologies and the impairment of lysosomal function, which were linked to the TREM2-mTOR signaling pathway.

Prolonged extracellular low sodium concentrations and subsequent their rapid correction modulate nitric oxide production dependent on NFAT5 in microglia

Hyponatremia is the most common clinical electrolyte disorder. Chronic hyponatremia has been recently reported to be associated with falls, fracture, osteoporosis, neurocognitive impairment, and mental manifestations. In the treatment of chronic hyponatremia, overly rapid correction of hyponatremia can cause osmotic demyelination syndrome (ODS), a central demyelinating disease that is also associated with neurological morbidity and mortality. Using a rat model, we have previously shown that microglia play a critical role in the pathogenesis of ODS. However, the direct effect of rapid correction of hyponatremia on microglia is unknown. Furthermore, the effect of chronic hyponatremia on microglia remains elusive. Using microglial cell lines BV-2 and 6-3, we show here that low extracellular sodium concentrations (36 mmol/L decrease; LS) suppress Nos2 mRNA expression and nitric oxide (NO) production of microglia. On rapid correction of low sodium concentrations, NO production was significantly increased in both cells, suggesting that acute correction of hyponatremia partly directly contributes to increased Nos2 mRNA expression and NO release in ODS pathophysiology. LS also suppressed expression and nuclear translocation of nuclear factor of activated T cells-5 (NFAT5), a transcription factor that regulates the expression of genes involved in osmotic stress. Furthermore, overexpression of NFAT5 significantly increased Nos2 mRNA expression and NO production in BV-2 cells. Expressions of Nos2 and Nfat5 mRNA were also modulated in microglia isolated from cerebral cortex in chronic hyponatremia model mice. These data indicate that LS modulates microglial NO production dependent on NFAT5 and suggest that microglia contribute to hyponatremia-induced neuronal dysfunctions.

Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway

To investigate the mechanism underlying the neuroprotective effect of linarin (LIN) against microglia activation-mediated inflammation and neuronal apoptosis following spinal cord injury (SCI). Fifty C57BL/6J mice (8- 10 weeks old) were randomized to receive sham operation, SCI and linarin treatment at 12.5, 25, and 50 mg/kg following SCI (n=10). Locomotor function recovery of the SCI mice was assessed using the Basso Mouse Scale, inclined plane test, and footprint analysis, and spinal cord tissue damage and myelination were evaluated using HE and LFB staining. Nissl staining, immunofluorescence assay and Western blotting were used to observe surviving anterior horn motor neurons in injured spinal cord tissue. In cultured BV2 cells, the effects of linarin against lipopolysaccharide (LPS)‑induced microglia activation, inflammatory factor release and signaling pathway changes were assessed with immunofluorescence staining, Western blotting, RT-qPCR, and ELISA. In a BV2 and HT22 cell co-culture system, Western blotting was performed to examine the effect of linarin against HT22 cell apoptosis mediated by LPS-induced microglia activation. Linarin treatment significantly improved locomotor function (P < 0.05), reduced spinal cord damage area, increased spinal cord myelination, and increased the number of motor neurons in the anterior horn of the SCI mice (P < 0.05). In both SCI mice and cultured BV2 cells, linarin effectively inhibited glial cell activation and suppressed the release of iNOS, COX-2, TNF-α, IL-6, and IL-1β, resulting also in reduced neuronal apoptosis in SCI mice (P < 0.05). Western blotting suggested that linarin-induced microglial activation inhibition was mediated by inhibition of the TLR4/NF- κB signaling pathway. In the cell co-culture experiments, linarin treatment significantly decreased inflammation-mediated apoptosis of HT22 cells (P < 0.05). The neuroprotective effect of linarin is medicated by inhibition of microglia activation via suppressing the TLR4/NF‑κB signaling pathway, which mitigates neural inflammation and reduce neuronal apoptosis to enhance motor function of the SCI mice.