BV2 Cells Research Articles

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.

Anti-neuroinflammatory effects of the KIOM -patented Polygonum multiflorum maximized root tuber against LPS-stimulated BV2 Cells

The main pathological mechanism of neurodegeneration is neuroinflammation. It is known that the persistent neuroinflammatory response is harmful by causing secondary nerve tissue damage. Meanwhile, P. multiflorum is a traditional oriental medicinal herb. It has been used as a hematopoietic agent and is used to treat a variety of diseases and conditions. The aim of the present study was to compare the anti-inflammatory efficacy between the commonly available P. multiflorum (C1) and the KIOM-patented in vitro-propagated P. multiflorum (K1), which had higher content of active ingredients and biomass, using culture and cultivation conditions of LPS-induced neuroinflammation. After stimulation with LPS and treatment with C1 and K1 in mouse microglial BV-2 cells, nitric oxide (NO) production, pro-inflammatory cytokine secretion, inducible NO synthase (iNOS) expression, MAPK phosphorylation and transcription factor activity were assessed. We examined the antioxidant effect using DPPH and production of nitric oxide (NO). C1 and K1 suppressed the expression of iNOS and COX-2 and the production of pro-inflammatory cytokines. Furthermore, we determined the levels of inflammatory mediators, such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and mitogen-activated protein kinases and IκBα via Western blotting to understand the regulating mechanisms. Additionally, C1 and K1 also inhibited the activation of p38 and nuclear factor-kappa B (NF-κB) in LPS-stimulated BV2 cells. In all experimental results, excellent anti-neuroinflammatory effects were confirmed at a lower dose in K1 than in C1, which is believed to be due to the increased biomass. Therefore, K1 is expected to be more effective than C1 and can be applied more broadly in the development of prevention and treatment of various inflammatory-mediated neurodegenerative diseases.

ELK4 ameliorates cognitive impairment and neuroinflammation induced by obstructive sleep apnea

Intermittent hypoxia (IH) in patients with obstructive sleep apnea (OSA) syndrome elicited neuron injury (especially in the hippocampus and cortex), contributing to cognitive dysfunction. This study investigated the effects and clarified the mechanisms of ETS domain-containing protein Elk-4 (ELK4) on the cognitive function and neuroinflammation of mice with IH. Mouse microglia BV2 cells were induced with IH by exposure to fluctuating O2 concentrations (alternating from 5 % to 21 % every 30 min), and mice with OSA were developed and subjected to lentivirus-mediated gene intervention. ELK4 expression was significantly reduced in IH-induced microglia and brain tissues of mice with OSA. Overexpression of ELK4 attenuated oxidative stress, decreased the pro-inflammatory factors IL-1β, IL-6, and TNF-α, and increased the level of the anti-inflammatory factors IL-10 and TGF-β1, as well as the neuroprotective factor BDNF. ELK4 promoted the transcription of fibronectin type III domain-containing protein 5 (FNDC5) by binding to the promoter of FNDC5. Knockdown of FNDC5 in IH-induced microglia and animals reversed the protective effects of ELK4 on OSA-associated neuroinflammation and cognitive dysfunction. Overall, the results demonstrated that ELK4 overexpression repressed microglial activation by inducing the transcription of FNDC5, thus attenuating neuroinflammation and cognitive dysfunction induced by OSA.

Exploring Novel GSK-3β Inhibitors for Anti-Neuroinflammatory and Neuroprotective Effects: Synthesis, Crystallography, Computational Analysis, and Biological Evaluation.

In the pathogenesis of Alzheimer's disease, the overexpression of glycogen synthase kinase-3β (GSK-3β) stands out due to its multifaced nature, as it contributes to the promotion of amyloid β and tau protein accumulation, as well as neuroinflammatory processes. Therefore, in the present study, we have designed, synthesized, and evaluated a new series of GSK-3β inhibitors based on the N-(pyridin-2-yl)cyclopropanecarboxamide scaffold. We identified compound 36, demonstrating an IC50 of 70 nM against GSK-3β. Subsequently, through crystallography studies and quantum mechanical analysis, we elucidated its binding mode and identified the structural features crucial for interactions with the active site of GSK-3β, thereby understanding its inhibitory potency. Compound 36 was effective in the cellular model of hyperphosphorylated tau-induced neurodegeneration, where it restored cell viability after okadaic acid treatment and showed anti-inflammatory activity in the LPS model, significantly reducing NO, IL-6, and TNF-α release. In ADME-tox in vitro studies, we confirmed the beneficial profile of 36, including high permeability in PAMPA (Pe equals 9.4) and high metabolic stability in HLMs as well as lack of significant interactions with isoforms of the CYP enzymes and lack of considerable cytotoxicity on selected cell lines (IC50 > 100 μM on HT-22 cells and 89.3 μM on BV-2 cells). Based on promising pharmacological activities and favorable ADME-tox properties, compound 36 may be considered a promising candidate for in vivo research as well as constitute a reliable starting point for further studies.

Heat-Processed Soybean Germ Extract and Lactobacillus gasseri NK109 Supplementation Reduce LPS-Induced Cognitive Impairment and Colitis in Mice.

Soybean alleviates cognitive impairment. In our preparatory experiment, we found that dry-heat (90 °C for 30 min)-processed soybean embryo ethanol extract (hSE) most potently suppressed lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-α expression in BV2 cells among dry-heat-, steaming-, and oil exclusion-processed soybean embryo ethanol extracts (SEs). Heat processing increased the absorbable soyasaponin Bb content of SE. Therefore, we investigated whether hSE and its supplement could mitigate LPS-impaired cognitive function in mice. Among dry-heat-, steaming-, and oil exclusion-processed SEs, hSE mitigated LPS-impaired cognitive function more than parental SE. hSE potently upregulated LPS-suppressed brain-derived neurotropic factor (BDNF) expression in the hippocampus, while LPS-induced TNF-α and IL-1β expression in the hippocampus and colon were downregulated. Lactobacillus gasseri NK109 additively increased the cognitive function-enhancing activity of hSE in mice with LPS-induced cognitive impairment as follows: the hSE and NK109 mix potently increased cognitive function and hippocampal BDNF expression and BDNF-positive neuron cell numbers and decreased TNF-α expression and NF-κB-positive cell numbers in the hippocampus and colon. These findings suggest that hSE and its supplement may decrease colitis and neuroinflammation by suppressing NF-κB activation and inducing BDNF expression, resulting in the attenuation of cognitive impairment.

Jiao-tai-wan and its effective component-coptisine alleviate cognitive impairment in db/db mice through the JAK2/STAT3 signaling pathway

BackgroundCognitive impairment (CI) is now well-accepted as a complication and comorbidity of diabetes mellitus (DM), becoming a serious medical and social problem. Jiao-tai-wan (JTW), one of noted traditional Chinese medicine (TCM), showed dual therapeutic effects on DM and CI. Nevertheless, the potential mechanism is unclear. PurposeThis study sought to investigate the mechanism how JTW protected against DM and CI and screen the active component in JTW. MethodsDb/db mice were used as mouse models. Mice were treated by gavage with 0.9 % saline (0.1 mL/10g/d), low dose of JTW (2.4 g/kg/d) or high dose of JTW (4.8 g/kg/d) for 8 weeks separately. To access the effects of JTW, the levels of OGTT, HOMA-IR, blood lipids, inflammatory cytokines in serum and hippocampus were measured, behavioral tests were conducted, and histopathological changes were observed. The mechanism exploration was performed via network pharmacology, RT-qPCR, western blot, and immunofluorescence staining (IF). The impact and mechanism of coptisine in vitro were investigated using BV2 cells induced by LPS as cellular models. In vitro experiments were conducted in two parts. The first part comprised four groups: Control group, LPS group, LPS+LCOP group and LPS+HCOP group. The second part consisted of four groups: Control group, LPS group, LPS+HCOP group, and LPS+ Fed group. The western blot and RT-qPCR methods were used to examine the changes in biomarkers of the JAK2/STAT3 signaling pathways in BV2 cells. ResultsThe results demonstrated that JTW could improve OGTT and HOMA-IR, reduce the serum levels of LDL-C, HDL-C, TG, and TC, restore neuronal dysfunction and synaptic plasticity, and decrease the deposition of Aβ in the hippocampus. The findings from ELISA, IF, and RT-qPCR revealed that JTW could alleviate microglial activation and inflammatory status in vivo and coptisine could play the same role in vitro. Moreover, the changes of the JAK2/STAT3 signaling pathway in LPS-induced BV2 cells or hippocampus of db/db mice were distinctly reversed by coptisine or JTW, respectively. ConclusionOur study suggested that JTW and its effective component coptisine could alleviate diabetes mellitus-related cognitive impairment, closely linked to the JAK2/STAT3 signaling pathway.

BV2-derived extracellular vesicles modulate microglia inflammatory profile, neuronal plasticity, and behavioural performances in late adult mice

BackgroundDuring aging, both the brain and the immune system undergo a progressive impairment of physiological functions. Microglia, the immunocompetent cells of the central nervous system, shift towards a chronic mild inflammatory state that impacts brain homeostasis. Extracellular vesicles (EVs) released by microglia transport packages of molecular information that mirror the inflammatory status of donor cells and modulate the inflammatory phenotype of recipient microglia and other cell types. ResultsWe demonstrated that intranasal administration of EVs derived from microglial-like BV2 cells to late adult mice (16–20 months of age) shifts microglia toward a “juvenile” morphology affecting their inflammatory profile. Mice treated with BV2-derived EVs have a reduction of anxiety-like behavior and an increased spatial learning, with sex-dependent differences. Further, BV2-derived EVs increased neuronal plasticity both in male and female mice. These findings suggest the involvement of microglial cells in vesicles-mediated anti-aging effect. ConclusionsOur data indicate that BV2-derived EVs could represent a resource to slow down age-dependent inflammation in the mouse brain.

Noralashinols D-F, New Norlignans from Syringa pinnatifolia and its Anti-Inflammation in BV2 Cells.

Four new norlignans, noralashinols D-F (1a/b-3), and two known analogues (4 and 5) were isolated from the peeled stems of Syringa pinnatifolia Hemsl. The structures were elucidated by analysis of spectroscopic data, such as IR, HR-ESI-MS, 1D and 2D NMR, and ECD. All compounds were evaluated for anti-inflammatory activities against NO production induced by LPS in BV2 microglia cells. Compounds 1b and 2 exhibited moderate activities with IC50 values of 32.39±9.1 and 47.83±10.44 μM, respectively, compared with positive control indomethacin (IC50=21.62 μM). It is worth to note that 1, 3, and 4 have a distinctive woody fragrance.

Role of Microglial Mitophagy in Alleviating Postoperative Cognitive Dysfunction: a Mechanistic Study.

Postoperative cognitive dysfunction (POCD), a common complication following anesthesia and surgery, is influenced by hippocampal neuroinflammation and microglial activation. Mitophagy, a process regulating inflammatory responses by limiting the accumulation of damaged mitochondria, plays a significant role. This study aimed to determine whether regulating microglial mitophagy and the cGAS-STING pathway could alleviate cognitive decline after surgery. Exploratory laparotomy was performed to establish a POCD model using mice. Western blotting, immunofluorescence staining, transmission electron microscopy, and mt-Keima assays were used to examine microglial mitophagy and the cGAS-STING pathway. Quantitative polymerase chain reaction (qPCR) was used to detect inflammatory mediators and cytosolic mitochondrial DNA (mtDNA) levels in BV2 cells. Exploratory laparotomy triggered mitophagy and enhanced the cGAS-STING pathway in mice hippocampi. Pharmacological treatment reduced microglial activation, neuroinflammation, and cognitive impairment after surgery. Mitophagy suppressed the cGAS-STING pathway in mice hippocampi. In vitro, microglia-induced inflammation was mediated by mitophagy and the cGAS-STING pathway. Small interfering RNA (siRNA) of PINK1 hindered mitophagy activation and facilitated the cytosolic release of mtDNA, resulting in the initiation of the cGAS-STING pathway and innate immune response. Microglial mitophagy inhibited inflammatory responses via the mtDNA-cGAS-STING pathway inducing microglial mitophagy and inhibiting the mtDNA-cGAS-STING pathway may be an effective therapeutic approach for patients with POCD.

Minocycline alleviates microglia ferroptosis by inhibiting HO-1 during cerebral ischemia-reperfusion injury.

Ischemic stroke is a leading cause of death and disability in individuals worldwide. Cerebral ischemia-reperfusion injury (CIRI) typically results in severe secondary injury and complications following reperfusion therapy. Microglia play critical roles in the inflammatory reaction of CIRI. However, less attention has been given to microglial death in this process. Our study aims to explore microglial death in CIRI and the effects and mechanism of minocycline treatment on microglia. A middle cerebral artery occlusion (MCAO) model was applied to induce CIRI in rats. At 0h, 24h and 48h post-operation, rats were intraperitoneally injected with 45mg/kg minocycline. Neurological deficit scoring, 2,3,5-triphenyltetrazolium chloride (TTC) staining, assessment of activated microglia and examination of mitochondrial structure were conducted and checked at 72h after reperfusion. Additionally, an in vitro model of oxygen-glucose deprivation/reperfusion (OGD/R) model was established. BV-2 cells were treated with various pharmacological inhibitors of cell death or minocycline. Cell viability, lipid peroxidation, mitochondrial structure and function, and labile Fe2+ and ferroptosis-associated gene/protein levels were measured. Hemin was used for further validation after transcriptome analysis. In the MCAO and OGD/R models, ferroptosis was identified as a major form of microglial death. Minocycline inhibited microglia ferroptosis by reducing HO-1 expression. In addition, minocycline improved mitochondrial membrane potential, mitochondrial structures and microglial survival in vivo. Minocycline also decreased labile Fe2+ levels, lipid peroxidation, and expression of ferritin heavy chain (FTH) and it improved mitochondrial structure and function in vitro. Upregulation of HO-1 counteracted the protective effect of minocycline. Ferroptosis is a major form of microglial death in CIRI. The protective mechanism of minocycline in CIRI partially hinges on its ability to effectively ameliorate microglia ferroptosis by downregulating HO-1 expression. Consequently, targeting microglia ferroptosis is a promising treatment for CIRI.

Platycodon grandiflorum exhibits anti-neuroinflammatory potential against beta-amyloid-induced toxicity in microglia cells.

Platycodon grandiflorum (PG) is used in traditional oriental medicine to treat several ailments. The study investigated the anti-inflammatory and neuroprotective effects of PGW (P. grandiflorum) extract in Aβ25-35-induced inflammation in BV2 microglia cells. PGW demonstrated significant inhibition of nitric oxide (NO) production, with reductions of 30.4, 36.7, and 61.2% at concentrations of 50, 100, and 200 μg/mL, respectively. Moreover, PGW effectively suppressed the production of pro-inflammatory cytokines IL-1β and IL-6 and exhibited significant inhibitory activity against TNF-α at 200 μg/mL. Furthermore, PGW treatment mitigated apoptosis in Aβ-induced BV2 cells by modulating the mitochondrial apoptosis pathway, regulating Bcl-2 family protein synthesis, and inhibiting caspase activation. Mechanistically, PGW attenuated the activation of the MAPK (JNK, ERK, p38) pathway induced by Aβ, showing a concentration-dependent decrease in phosphorylation levels of these proteins. Additionally, PGW inhibited the NF-κB pathway activation by reducing the phosphorylation levels of p65 and IκBα in a concentration-dependent manner. PGW demonstrated anti-inflammatory and neuroprotective effects in Aβ-induced neuronal cells, suggesting its potential as a therapeutic agent for neuroinflammatory associated with neurodegenerative diseases.

Highly BBB-permeable nanomedicine reverses neuroapoptosis and neuroinflammation to treat Alzheimer's disease

The prevalence of Alzheimer's disease (AD) is increasing globally due to population aging. However, effective clinical treatment strategies for AD still remain elusive. The mechanisms underlying AD onset and the interplay between its pathological factors have so far been unclear. Evidence indicates that AD progression is ultimately driven by neuronal loss, which in turn is caused by neuroapoptosis and neuroinflammation. Therefore, the inhibition of neuroapoptosis and neuroinflammation could be a useful anti-AD strategy. Nonetheless, the delivery of active drug agents into the brain parenchyma is hindered by the blood-brain barrier (BBB). To address this challenge, we fabricated a black phosphorus nanosheet (BP)-based methylene blue (MB) delivery system (BP-MB) for AD therapy. After confirming the successful preparation of BP-MB, we proved that its BBB-crossing ability was enhanced under near-infrared light irradiation. In vitro pharmacodynamics analysis revealed that BP and MB could synergistically scavenge excessive reactive oxygen species (ROS) in okadaic acid (OA)-treated PC12 cells and lipopolysaccharide (LPS)-treated BV2 cells, thus efficiently reversing neuroapoptosis and neuroinflammation. To study in vivo pharmacodynamics, we established a mouse model of AD mice, and behavioral tests confirmed that BP-MB treatment could successfully improve cognitive function in these animals. Notably, the results of pathological evaluation were consistent with those of the in vitro assays. The findings demonstrated that BP-MB could scavenge excessive ROS and inhibit Tau hyperphosphorylation, thereby alleviating downstream neuroapoptosis and regulating the polarization of microglia from the pro-inflammatory M1 phenotype to the anti-inflammatory M2 phenotype. Overall, this study highlights the therapeutic potential of a smart nanomedicine with the capability of reversing neuroapoptosis and neuroinflammation for AD treatment.

Fisetin Promotes Functional Recovery after Spinal Cord Injury by Inhibiting Microglia/Macrophage M1 Polarization and JAK2/STAT3 Signaling Pathway.

Spinal cord injury (SCI) is one of the most serious health problems, with no effective therapy. Recent studies indicate that Fisetin, a natural polyphenolic flavonoid, exhibits multiple functions, such as life-prolonging, antioxidant, antitumor, and neuroprotection. However, the restorative effects of Fisetin on SCI and the underlying mechanism are still unclear. In the present study, we found that Fisetin reduced LPS-induced apoptosis and oxidative damage in PC12 cells and reversed LPS-induced M1 polarization in BV2 cells. Additionally, Fisetin safely and effectively promoted the motor function recovery of SCI mice by attenuating neurological damage and promoting neurogenesis at the lesion. Moreover, Fisetin administration inhibited glial scar formation, modulated microglia/macrophage polarization, and reduced neuroinflammation. Network pharmacology, RNA-seq, and molecular biology revealed that Fisetin inhibited the activation of the JAK2/STAT3 signaling pathway. Notably, Colivelin TFA, an activator of JAK2/STAT3 signaling, attenuated Fis-mediated neuroinflammation inhibition and therapeutic effects on SCI mice. Collectively, Fisetin promotes functional recovery after SCI by inhibiting microglia/macrophage M1 polarization and the JAK2/STAT3 signaling pathway. Thus, Fisetin may be a promising therapeutic drug for the treatment of SCI.

BACE1 inhibitors from the fruits of Alpinia oxyphylla have efficacy to treat T2DM-related cognitive disorder

The fruits of Alpinia oxyphylla (Alpiniae Oxyphyllae Fructus, AOF) are one of the “Four Famous South Medicines” in China. In this study, beta-site amyloid protein precursor cleaving enzyme 1 (BACE1) was applied to explore the active components in AOF responsible for type 2 diabetes mellitus (T2DM)-related cognitive disorder. As a result, 24 compounds including three unreported ones (1, 3, 4) were isolated from AOF. Compound 1 is an unusual carbon‑carbon linked diarylheptanoid dimer, and compound 4 is the first case of 3,4-seco-eudesmane sesquiterpenoid with a 5/6-bicyclic skeleton. Four diarylheptanoids (3, 5–7), one flavonoid (9) and two sesquiterpenoids (14 and 20) showed BACE1 inhibitory activity, of which the most active 6 was revealed to be a non-competitive and anti-competitive mixed inhibitor. Docking simulation suggested that OH-4′ of 6 played important roles in maintaining activity by forming hydrogen bonds with Ser36 and Ile126 residues. Compounds 3, 5, 9 and 20 displayed neuroprotective effects against amyloid β (Aβ)-induced damage in BV2 cells. Mechanism study revealed that compounds 5 and 20 downregulated the expression of BACE1 and upregulated the expression of Lamp2 to exert effects. Thus, the characteristic diarylheptanoids and sesquiterpenoids in AOF had the efficacy to alleviate T2DM-related cognitive disorder by inhibiting BACE1 activity and reversing Aβ-induced neuronal damage.

IL-33 relieves nerve injury by mediating microglial polarization in neuromyelitis optica spectrum disorders via the IL-33/ST2 pathway

Interleukin-33 (IL-33) is a member of the interleukin-1 cytokine family. Its function in regulating microglial M1/M2 polarization in neuromyelitis optica spectrum disorder (NMOSD) is still unelucidated. To evaluate the role of IL-33 in NMOSD, we constructed NMOSD mice model by injecting purified serum IgG from AQP4-IgG seropositive NMOSD patients into experimental autoimmune encephalomyelitis (EAE) mice, and IL-33 was intraperitoneally injected into NMOSD mice 3 d before the model induction. We found that pretreatment of the NMOSD mice with IL-33 relieved brain neuron loss, and demyelination and improved the structure of axons, astrocytes, and mitochondria. In the neuronal and microglial coculture system, pretreatment with IL-33 in microglia alleviated NMOSD serum-induced inflammation and damaged morphology in cultured neurons. IL-33 transformed microglia to the M2 phenotype, and NMOSD serum promoted microglia to the M1 phenotype in cultured BV2 cells. Moreover, IL-33 influenced microglial polarity via the IL-33/ST2 pathway. IL-33 may be a novel insight useful for further developing NMOSD-targeted therapy and drug development.

Curcumin loaded hydrogel with double ROS-scavenging effect regulates microglia polarization to promote poststroke rehabilitation

Cyclodextrins are used to include curcumin to form complex, which is subsequently loaded into a reactive oxygen species (ROS) responsive hydrogel (Cur gel). This gel exhibits a dual ROS scavenging effect. The gel can neutralize extracellular ROS to lead to a ROS-sensitive curcumin release. The released curcumin complex can eliminate intracellular ROS. Furthermore, the Cur gel effectively downregulates the expression of CD16 and IL-1β while upregulating CD206 and TGF-β in oxygen and glucose-deprived (OGD) BV2 cells. Additionally, it restores the expression of synaptophysin and PSD95 in OGD N2a cells. Upon injection into the stroke cavity, the Cur gel reduces CD16 expression and increases CD206 expression in the peri-infarct area of stroke mice, indicating an in vivo anti-inflammatory polarization of microglia. Colocalization studies using PSD95 and VGlut-1 stains, along with Golgi staining, reveal enhanced neuroplasticity. As a result, stroke mice treated with the Cur gel exhibit the most significant motor function recovery. Mechanistic investigations demonstrate that the released curcumin complex scavenges ROS and suppresses the activation of the ROS-NF-κB signaling pathway by inhibiting the translocation of p47-phox and p67-phox to lead to anti-inflammatory microglia polarization. Consequently, the Cur gel exhibits promising potential for promoting post-stroke rehabilitation in clinics.

Microfluidic Wound-Healing Assay for Comparative Study on Fluid Dynamic, Chemical and Mechanical Wounding on Microglia BV2 Migration.

Microglial cells, or brain immune cells, are highly dynamic and continuously migrate in pathophysiological conditions. Their adhesion, as a physical characteristic, plays a key role in migration. In this study, we presented a microfluidic chip combination of two assays: a microglial BV2 adhesion assay and a wound-healing migration assay. The chip could create the cell-free area (wound) under chemical stimuli with trypsin (chemical assay) and also mechanical stimuli with the PBS flow (mechanical assay). The microfluidic chip functioned as the cell adhesion assay during wounding, when the cell adhesion of microglia BV2 cells was characterized by the cell removal time under various shear stress ranges. The cell detachment pattern on the glass substrate was found under physiological conditions. After wounding, the chip operated as a migration assay; it was shown that cell migration in the cell-free area generated chemically with trypsin was highly improved compared to mechanical cell-free area creations with PBS flow and the scratch assay. Our findings indicated that the increase in inlet flow rate in the mechanical assay led to a reduced experiment time and mechanical force on the cells, which could improve cell migration. Furthermore, the study on the effect of the device geometry showed that the increased channel width had an inhibitory effect on cell migration. The bi-functional chip offers an opportunity for the development of new models for a better understanding of cellular adhesion and migration in in vitro microenvironments.