M2 Microglia Research Articles

Spinal voltage-gated potassium channel Kv1.3 contributes to neuropathic pain via the promotion of microglial M1 polarization and activation of the NLRP3 inflammasome.

Studies have shown that the activation of microglia is the main mechanism of neuropathic pain. Kv1.3 channel is a novel therapeutic target for treating neuroinflammatory disorders due to its crucial role in subsets of microglial cells. As such, it may be involved in the processes of neuropathic pain, however, whether Kv1.3 plays a role in neuroinflammation following peripheral nerve injury is unclear. The spared nerve injury model (SNI) was used to establish neuropathic pain. Western blot and immunofluorescence were used to examine the effect of Kv1.3 in the SNI rats. PAP-1, a Kv1.3 specific blocker was administered to alleviate neuropathic pain in the SNI rats. Neuropathic pain and allodynia occurred after SNI, the levels of M1 (CD68, iNos) and M2 (CD206, Arg-1) phenotypes were up-regulated in the spinal cord, and the protein levels of NLRP3, caspase-1 and IL-1β were also increased. Pharmacological blocking of Kv1.3 with PAP-1 alleviated hyperpathia induced by SNI. Meanwhile, intrathecal injection of PAP-1 reduced M1 polarization and decreased NLRP3, caspase-1 and IL-1β expressions of protein levels. Our research indicates that the Kv1.3 channel in the spinal cord contributes to neuropathic pain by promoting microglial M1 polarization and activating the NLRP3 inflammasome.

Milmed Yeast Alters the LPS-Induced M1 Microglia Cells to Form M2 Anti-Inflammatory Phenotype.

Microglial cells polarized towards a proinflammatory phenotype are considered the main cellular players of neuroinflammation, underlying several neurodegenerative diseases. Many studies have suggested that imbalance of the gut microbial composition is associated with an increase in the pro-inflammatory cytokines and oxidative stress that underlie chronic neuroinflammatory diseases, and perturbations to the gut microbiota were detected in neurodegenerative conditions such as Parkinson's disease and Alzheimer's disease. The importance of gut-brain axis has been uncovered and the relevance of an appropriate microbiota balance has been highlighted. Probiotic treatment, rebalancing the gut microbioma, may reduce inflammation. We show that Milmed yeast, obtained from S. cerevisiae after exposure to electromagnetic millimeter wavelengths, induces a reversal of LPS-M1 polarized microglia towards an anti-inflammatory phenotype, as demonstrated morphologically by the recovery of resting phenotype by microglia, by the decrease in the mRNAs of IL-1β, IL-6, TNF-α and in the expression of iNOS. Moreover, Milmed stimulated the secretion of IL-10 and the expression of Arginase-1, cell markers of M2 anti-inflammatory polarized cells. The present findings data suggest that Milmed may be considered to be a probiotic with diversified anti-inflammatory activity, capable of directing the polarization of microglial cells.

M2 microglia activation and proNGF inhibition : Possible Mechanisms Behind Cognitive Improvements by Low‐Dose Ionizing Radiation on Alzheimer's Disease; A Literature Review

AbstractBackgroundRecent studies showed that Low‐Dose Ionizing Radiation (LDIR) may induce significant cognitive improvements on Alzheimer's disease (AD). The mechanism behind it is still remain unclear.MethodIn this review, numerous research papers and clinical studies results from the last 10 years related to LDIR exposure on AD as a treatment, immunochemistry in AD, and neurogenesis are collected. The results of the studies are then comprehensively analyzed to find any associations that could explain or give clues on how LDIR exposure may induce significant cognitive improvements in AD.ResultThere is evidence that LDIR modulates microglia phenotypes by switching phenotype M1 to M2. The anti‐inflammatory M2 microglia may associated in amyloid plaques and tau tangles reduction that showed in many studies on LDIR for AD treatment. Further study shows that LDIR also promotes proNGF inhibition that may triggers neurogenesis.ConclusionLDIR shows that it may be a promising novel treatment for AD retrospectively via immunomodulation and triggers neurogenesis.

Bone Marrow-derived Mesenchymal Stem Cells Promote Microglia/Macrophage M2 Polarization and Enhance Neurogenesis in the Acute and Chronic Stages after Ischemic Stroke

• BMSCs promote microglia/macrophage M2 polarization in acute and chronic stages after stroke. • BMSCs enhance neurogenesis after cerebral ischemia/reperfusion. • The enhancement of neurogenesis by BMSCs could be associated with microglia M2 polarization. : Ischemic stroke has been regarded as a major cause of disability and death around the world due to limited effective therapies. Accumulating evidence have shown that although microglia are polarized to an anti-inflammatory M2 phenotype in the early stage of ischemia, they transform progressively into a proinflammatory M1 phenotype. Bone marrow-derived mesenchymal stem cells (BMSCs) may be used to treat ischemic injury through regulating the poststroke inflammatory response. However, the mechanism by which BMSCs can treat ischemic stroke remains unclarified. : This study aimed to investigate whether BMSCs shift M1-to-M2 phenotype transformation of microglia/macrophages and enhance neurogenesis in a rat transient middle cerebral artery occlusion (tMCAO) model. : Ninety-minute tMCAO was applied to the rats, followed by reperfusion. BMSCs were transplanted into the rats via intravenous injection at 24 h after tMCAO. After being randomly divided into the sham group, the MCAO group, and the BMSCs group, the rats’ behavior was assessed at 1, 3, 7, and 14 days following tMCAO. qRT-PCR, double-immunofluorescence staining, and Western blot were performed at 3 and 14 days after tMCAO to determine M1/M2 polarization of microglia/macrophages. Neurogenesis was examined by double-immunofluorescence staining at 14 days after tMCAO. Expression of brain-derived neurotrophic factor (BDNF) was measured on the protein level by immunofluorescence staining at 3 and 14 days after tMCAO. : We found that BMSCs treatment promoted the recovery of neurological function after tMCAO, inhibited the expression of TNF-α, iNOS and CD16/32, which are markers of M1 microglia/macrophage, and enhanced the expression of IL-10, TGF-β and CD206 that are markers of M2 microglia/macrophage. Moreover, BMSCs treatment promoted neurogenesis and M2-derived BDNF expression after tMCAO. : It is indicated by the results that BMSCs modulate neuroinflammation and enhance neurogenesis, which could be due to transforming microglia/macrophages from the M1 polarization state towards M2 in a rat tMCAO model.

M1 Microglia Induced Neuronal Injury on Ischemic Stroke via Mitochondrial Crosstalk between Microglia and Neurons.

Among the middle-aged and senile populations, ischemic stroke (IS) is a frequently occurring acute condition of the cerebrovascular system. Traditionally, it is recognized that when stroke occurs, microglia are activated into M1 phenotype and release cytotoxic cytokines, reactive oxygen species, proteases, and other factors, thus exacerbating the injury by further destroying or killing nearby neurons. In the latest research, the crucial role of the intercellular mitochondrial crosstalk on the stroke management has been demonstrated. Therefore, we tried to clarify mitochondrial crosstalk between microglia and neurons, and evaluated M1 microglial mitochondria-mediated neurological performance in transient middle cerebral artery occlusion (tMCAO) rats. We found that when microglia was activated into the proinflammatory M1 type after stroke, mitochondrial fission process was accelerated, and damaged mitochondria were released, further transferred to neurons and fused with neuronal mitochondria. As a result, the function of neuronal mitochondria was damaged by decreasing adenosine triphosphate (ATP), mitochondria membrane potential, and increasing excessive reactive oxygen species (ROS), thus inducing mitochondria-mediated neuronal death and finally aggravating ischemia injury. Taken together, it provides a novel neuroglial crosstalk mechanism at the mitochondrial level.

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.

Icaritin inhibits neuroinflammation in a rat cerebral ischemia model by regulating microglial polarization through the GPER–ERK–NF-κB signaling pathway

BackgroundActivated microglia play a key role in initiating the inflammatory cascade following ischemic stroke and exert proinflammatory or anti-inflammatory effects, depending on whether they are polarized toward the M1 or M2 phenotype. The present study investigated the regulatory effect of icaritin (ICT) on microglial polarization in rats after cerebral ischemia/reperfusion injury (CI/RI) and explored the possible anti-inflammatory mechanisms of ICT.MethodsA rat model of transient middle cerebral artery occlusion (tMCAO) was established. Following treatment with ICT, a G protein-coupled estrogen receptor (GPER) inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, the Garcia scale and rotarod test were used to assess neurological and locomotor function. 2,3,5-Triphenyltetrazolium chloride (TTC) and Fluoro-Jade C (FJC) staining were used to evaluate the infarct volume and neuronal death. The levels of inflammatory factors in the ischemic penumbra were evaluated using enzyme-linked immunosorbent assays (ELISAs). In addition, western blotting, immunofluorescence staining and quantitative PCR (qPCR) were performed to measure the expression levels of markers of different microglial phenotypes and proteins related to the GPER–ERK–nuclear factor kappa B (NF-κB) signaling pathway.ResultsICT treatment significantly decreased the cerebral infarct volume, brain water content and fluorescence intensity of FJC; improved the Garcia score; increased the latency to fall and rotation speed in the rotarod test; decreased the levels of interleukin-1 beta (IL-1β), tumor necrosis factor-alpha (TNF-α), Iba1, CD40, CD68 and p-P65-NF-κB; and increased the levels of CD206 and p-ERK. U0126 (an inhibitor of ERK) and G15 (a selective antagonist of GPER) antagonized these effects.ConclusionsThese findings indicate that ICT plays roles in inhibiting the inflammatory response and achieving neuroprotection by regulating GPER–ERK–NF-κB signaling and then inhibiting microglial activation and M1 polarization while promoting M2 polarization, which provides a new therapeutic for against cerebral ischemic stroke.Graphical

Aerobic Glycolysis Induced by mTOR/HIF-1α Promotes Early Brain Injury After Subarachnoid Hemorrhage via Activating M1 Microglia.

M1 microglial activation is crucial for the pathogenesis of early brain injury (EBI) following subarachnoid hemorrhage (SAH), and there is growing evidence that glucose metabolism is frequently involved in microglial activation. However, the molecular mechanism of glycolysis and its role in M1 microglial activation in the context of EBI are not yet fully understood. In this study, firstly, the relationship between aerobic glycolysis and M1 microglial activation as well as SAH-induced EBI was researched in vivo. Then, intervention on mammalian target of rapamycin (mTOR) was performed to investigate the effects on glycolysis-dependent M1 microglial activation and EBI and its relationship with hypoxia-inducible factor-1α (HIF-1α) in vivo. Next, Hif-1α was inhibited to analyze its role in aerobic glycolysis, M1 microglial activation, and EBI in vivo. Lastly, both in vivo and in vitro, mTOR inhibition and Hif-1α enhancement were administered simultaneously, and the combined effects were further confirmed again. The results showed that aerobic glycolysis and M1 microglial polarization were increased after SAH, and glycolytic inhibition could attenuate M1 microglial activation and EBI. Inhibition of mTOR reduced glycolysis-dependent M1 microglial polarization and EBI severity by down-regulating HIF-1α expression, while enhancement had the opposite effects. Blockading HIF-1α had the similar effects as suppressing mTOR, while HIF-1α agonist worked against mTOR antagonist when administered simultaneously. In conclusion, the present study showed new evidence that aerobic glycolysis induced by mTOR/HIF-1α might promote EBI after SAH by activating M1 microglia. This finding provided new insights for the treatment of EBI.

Fingolimod Alleviates Cognitive Deficit in Type 2 Diabetes by Promoting Microglial M2 Polarization via the pSTAT3-jmjd3 Axis.

Sphingosine receptors (S1PRs) are implicated in the progression of neurodegenerative diseases and metabolic disorders like obesity and type 2 diabetes (T2D). The link between S1PRs and cognition in type 2 diabetes, as well as the mechanisms that underpin it, are yet unknown. Neuroinflammation is the common pathology shared among T2D and cognitive impairment. However, the interplay between the M1 and M2 polarization state of microglia, a primary driver of neuroinflammation, could be the driving factor for impaired learning and memory in diabetes. In the present study, we investigated the effects of fingolimod (S1PR1 modulator) on cognition in high-fat diet and streptozotocin-induced diabetic mice. We further assessed the potential pathways linking microglial polarization and cognition in T2D. Fingolimod (0.5mg/kg and 1mg/kg) improved M2 polarization and synaptic plasticity while ameliorating cognitive decline and neuroinflammation. Sphingolipid dysregulation was mimicked in vitro using palmitate in BV2 cells, followed by conditioned media exposure to Neuro2A cells. Mechanistically, type 2 diabetes induced microglial activation, priming microglia towards the M1 phenotype. In the hippocampus and cortex of type 2 diabetic mice, there was a substantial drop in pSTAT3, which was reversed by fingolimod. This protective effect of fingolimod on microglial M2 polarization was primarily suppressed by selective jmjd3 blockade in vitro using GSK-J4, revealing that jmjd3 was involved downstream of STAT3 in the fingolimod-enabled shift of microglia from M1 to M2 polarization state. This study suggested that fingolimod might effectively improve cognition in type 2 diabetes by promoting M2 polarization.

Microglia-Neutrophil Interactions Drive Dry AMD-like Pathology in a Mouse Model.

In dry age-related macular degeneration (AMD), inflammation plays a key role in disease pathogenesis. Innate immune cells such as microglia and neutrophils infiltrate the sub-retinal space (SRS) to induce chronic inflammation and AMD progression. But a major gap in our understanding is how these cells interact with each other in AMD. Here, we report a novel concept of how dynamic interactions between microglia and neutrophils contribute to AMD pathology. Using well-characterized genetically engineered mouse models as tools, we show that in the diseased state, retinal pigmented epithelial (RPE) cells trigger pro-inflammatory (M1) transition in microglia with diminished expression of the homeostatic marker, CX3CR1. Activated microglia localize to the SRS and regulate local neutrophil function, triggering their activation and thereby inducing early RPE changes. Ligand receptor (LR)-loop analysis and cell culture studies revealed that M1 microglia also induce the expression of neutrophil adhesion mediators (integrin β1/α4) through their interaction with CD14 on microglia. Furthermore, microglia-induced neutrophil activation and subsequent neutrophil-mediated RPE alterations were mitigated by inhibiting Akt2 in microglia. These results suggest that the Akt2 pathway in microglia drives M1 microglia-mediated neutrophil activation, thereby triggering early RPE degeneration and is a novel therapeutic target for early AMD, a stage without treatment options.

Synergistic regulation of microglia differentiation by CD93 and integrin β1 in the rat pneumococcal meningitis model

Streptococcus pneumoniae is the main bacterial pathogen of meningitis worldwide, which has a high mortality rate and survivors are prone to central nervous system (CNS) sequelae. In this regard, microglia activation has been associated with injury to the CNS. The aim of this study was to investigate the relationship between CD93, integrin β1, and microglia activation. In the rat pneumococcal meningitis model, we found significant increases of CD93 and integrin β1 expression and differentiation of M1 phenotype microglia. Furthermore, we showed in vitro siRNA-mediated downregulation of CD93 and integrin β1 expression after infecting highly aggressive proliferating immortalized (HAPI) microglia cells with S. pneumoniae. We observed differentiation of S. pneumonia-infected HAPI microglia cells to the M1 phenotype and significant release of soluble CD93 (sCD93) and integrin β1 expression. Complement C1q and metalloproteinases promoted sCD93 release. We also showed that downregulation of CD93 significantly reduced differentiation to M1 microglia and increased differentiation to M2 microglia. However, addition of recombinant CD93 may regulate microglia differentiation to the M1 phenotype. Furthermore, the downregulation of integrin β1 resulted in downregulation of the CD93 protein. In conclusion, interaction between integrin β1 and CD93 promotes differentiation of microglia to the M1 phenotype, increases the release of pro-inflammatory factors, and leads to nervous system injury in pneumococcal meningitis.

Exploring the pharmacological mechanism of Naoxueshu oral liquid in the treatment of intracerebral hemorrhage through weighted gene co-expression network analysis, network pharmacological and experimental validation

BackgroundIntracerebral hemorrhage (ICH) is a life-threatening stroke subtype with high rates of disability and mortality. Naoxueshu oral liquid is a proprietary Chinese medicine that absorbs hematoma and exhibits neuroprotective effects in patients with ICH. However, the underlying mechanisms remain obscure. PurposeExploring and elucidating the pharmacological mechanism of Naoxueshu oral liquid in the treatment of ICH. Study design and methodsThe Gene Expression Omnibus (GEO) database was used to download the gene expression data on ICH. ICH-related hub modules were obtained by weighted gene co-expression network analysis (WGCNA) of differentially co-expressed genes (DEGs). The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the obtained key modules to identify the ICH-related signaling pathways. Network pharmacology technology was applied to forecast the targets of Naoxueshu oral liquid and to establish a protein-protein interaction (PPI) network of overlapping targets between Naoxueshu oral liquid and ICH. Functional annotation and enrichment pathway analyses of the intersectional targets were performed using the omicsbean database. Finally, we verified the therapeutic role and mechanism of Naoxueshu oral liquid in ICH through molecular docking and experiments. ResultsThrough the WGCNA analysis, combined with network pharmacology, it was found that immune inflammation was closely related to the early pathological mechanism of ICH. Naoxueshu oral liquid suppressed the inflammatory response; hence, it could be a potential drug for ICH treatment. Molecular docking further confirmed that the effective components of Naoxueshu oral liquid docked well with CD163. Finally, the experimental results showed that Naoxueshu oral liquid treatment in the ICH rat model attenuated neurological deficits and neuronal injury, decreased hematoma volume, and promoted hematoma absorption. In addition, Naoxueshu oral liquid treatment also significantly increased the levels of Arg-1, CD163, Nrf2, and HO-1 around hematoma after ICH. ConclusionThis study demonstrated that Naoxueshu oral liquid attenuated neurological deficits and accelerated hematoma absorption, possibly by suppressing inflammatory responses, which might be related to the regulation of Nrf2/CD163/HO-1 that interfered with the activation of M2 microglia, thus accelerating the clearance and decomposition of hemoglobin in the hematoma.

Schisandra chinensis Lignans Exert Antidepressant Effects by Promoting BV2 Microglia Polarization toward the M2 Phenotype through the Activation of the Cannabinoid Receptor Type-2-Signal Transducer and Activator of Transcription 6 Pathway.

Based on the current results, they showed that Schisandra chinensis lignans (SCL) ameliorated depressive-like behaviors in chronic unpredictable mild stress (CUMS) mice, alleviated neuroinflammation, and improved neuronal injury. This study aimed to explore whether SCL exerted antidepressant effects through inhibiting neuroinflammation, in turn improving neuronal injury. In vitro studies revealed that SCL blocked lipopolysaccharide-increased BV2 microglial M1 but promoted the M2 phenotype. The BV2-N2a interaction model suggested that increasing the M2 phenotype of BV2 played neuroprotective effects. The current studies demonstrated that SCL up-regulated the expression of CUMS- and LPS-decreased cannabinoid receptor type-2 (CB2R) mRNA. In vitro studies showed that the transfection of BV2 with siCrn2 blocked the SCL-increased M2 phenotype via the inactivating signal transducer and activator of transcription 6 (STAT6) pathway, further decreasing the viability of N2a cells. Finally, the possible pharmacodynamic compounds, γ-schisandrin and schisantherin A, were indicated by AutoDuck analysis. Overall, our study showed that SCL promoted microglia polarization toward the M2 phenotype, in turn exerting neuroprotective effects by activating CB2R-STAT6 signaling further to play antidepressant roles.

Palmitic acid induces nDNA release to cytosol and promotes microglial M1 polarization via cGAS-STING signaling pathway

Palmitic acid (PA), the most common statured fatty acid in diets, is involved in peripheral as well as central inflammation. The M1 polarization of microglia plays an important role in PA-induced neuroinflammation. However, it is still unclear on the key factor and molecule mechanism of microglial polarization among it. Thus, we investigated whether the release of self-DNA into the cytoplasm of microglia was a consequence of PA treatment, as in aortic endothelial cells and adipocytes. RT-qPCR and immunofluorescence were performed to detect the status of cytosolic DNA and microglial polarization after PA treatment. We found that the content of cytosolic nDNA rather than mtDNA increased after PA treatment and the M1 polarization of microglia was associated with this. Moreover, the knockdown of cGAS in BV2 microglial cells demonstrated that the cGAS-STING pathway is involved in polarization process. Our results revealed that nDNA and cGAS-STING pathway are critically involved in PA-induced microglial M1 polarization. This mechanism may pose a new insight on targeting microglia may be a promising way to mitigate diet-induced early neuroinflammation.

The protective effects of nesfatin-1 in neurological dysfunction after spinal cord injury by inhibiting neuroinflammation.

Spinal cord injury (SCI) is one of the most severe neurological diseases. However, there is still no effective treatment for it. Nesfatin, a precursor neuropeptide derived from nucleobindin 2 (NUCB2), has displayed a wide range of protective effects in different types of cells and tissue. However, the effects of nesfatin-1 in SCI have not been reported before. A SCI model was established. The behavior of mice was assessed using the Basso, Beattie, and Bresnahan (BBB) assessment. Here, we report that the administration of nesfatin-1 improved neurological recovery in SCI mice by increasing BBB scores, reducing lesion area volume and spinal cord water content. Also, nesfatin-1 ameliorated oxidative stress by reducing reactive oxygen species (ROS) levels and increasing superoxide dismutase (SOD) activity. We also found that nesfatin-1 prevented neuronal apoptosis in SCI mice by reducing caspase 3 activity and the expression of Bax, as well as increasing B-cell lymphoma-2 (Bcl-2). Additionally, nesfatin-1 reduced the levels of interleukin 6 (IL-6), interleukin-1β (IL-1β), and tumor necrosis factor-α (TNF-α). Nesfatin-1 also promoted microglia towards M2 polarization by increasing the marker CD206 but reducing CD16. Importantly, nesfatin-1 enhanced the phosphorylation of signal transducer and activator of transcription 1 (STAT1) but reduced the expression levels of toll-like receptor 4 (TLR4) and phosphorylated nuclear factor kappa-B p65 (p-NF-κB p65). Our findings imply that nesfatin-1 exerts neuroprotective actions in SCI by promoting the activation of M2 microglia, and its underlying mechanisms might be related to the activation of STAT1 and inhibition of the TLR4/NF-κB signaling pathway.

Cycloastragenol suppresses M1 and promotes M2 polarization in LPS-stimulated BV-2 cells and ischemic stroke mice

There are two distinct phenotypes of activated microglia, pro-inflammatory M1 and anti-inflammatory M2. Accumulating evidence indicates that shifting the microglial polarization from M1 to M2 is a potential strategy for the treatment of neuroinflammation-associated brain diseases, including ischemic stroke. Cycloastragenol (CAG) is a hydrolysis product of astragaloside IV, the major active component of Astragalus radix. We have previously demonstrated that CAG has anti-inflammatory effect in a mouse model of ischemic stroke. This study investigated the effect of CAG on the phenotype polarization of microglia in lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial cells and ischemic stroke mice. In LPS-treated BV-2 cells, we found that CAG significantly reduced the expression of M1 markers, including pro-inflammatory cytokines and enzymes. In contrast, CAG promoted the expression of M2 markers, including anti-inflammatory cytokines and growth factor. In addition, CAG inhibited the activation of nuclear factor-κB (NF-κB) and enhanced the activation of nuclear factor E2-related factor 2 (Nrf2) and the expression of its downstream heme oxygenase-1 (HO-1). Furthermore, CAG also inhibited levels of M1 markers, promoted those of M2 markers, and enhanced Nrf2 activation and HO-1 expression in ischemic mouse brain. Importantly, the effect of CAG on M2 markers, but not M1 markers, was reversed by Nrf2 siRNA in LPS-stimulated BV-2 cells. Together, our results suggested that CAG promoted microglial M2 and suppressed M1 polarization through activating Nrf2 and inhibiting NF-κB, respectively, in LPS-stimulated BV-2 cells and ischemic mouse brain. CAG is a promising candidate for the treatment of neuroinflammation-related diseases, including ischemic stroke.

TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway

BackgroundMicroglia, the innate immune cells in the central nervous system, play an essential role in brain homeostasis, neuroinflammation and brain infections. Dysregulated microglia, on the other hand, are associated with neurodegenerative diseases, yet the mechanisms underlying pro-inflammatory gene expression in microglia are incompletely understood.MethodsWe investigated the role of the actin-associated protein tropomyosin 1 (TPM1) in regulating pro-inflammatory phenotype of microglia in the retina by using a combination of cell culture, immunocytochemistry, Western blot, qPCR, TUNEL, RNA sequencing and electroretinogram analysis. TREM2−/− mice were used to investigate whether TPM1 regulated pro-inflammatory responses downstream of TREM2. To conditionally deplete microglia, we backcrossed CX3CR1CreER mice with Rosa26iDTR mice to generate CX3CR1CreER:Rosa26iDTR mice.ResultsWe revealed a vital role for TPM1 in regulating pro-inflammatory phenotype of microglia. We found that TPM1 drove LPS-induced inflammation and neuronal death in the retina via the PKA/CREB pathway. TPM1 knockdown ameliorated LPS-induced inflammation in WT retinas yet exaggerated the inflammation in TREM2−/− retinas. RNA sequencing revealed that genes associated with M1 microglia and A1 astrocytes were significantly downregulated in LPS-treated WT retinas but upregulated in LPS-treated TREM2−/− retinas after TPM1 knockdown. Mechanistically, we demonstrated that CREB activated by TPM1 knockdown mediated anti-inflammatory genes in LPS-treated WT retinas but pro-inflammatory genes in LPS-treated TREM2−/− retinas, suggesting a novel role for TREM2 as a brake on TPM1-mediated inflammation. Furthermore, we identified that TPM1 regulated inflammation downstream of TREM2 and in a microglia-dependent manner.ConclusionsWe demonstrate that TPM1 mediates inflammation downstream of TREM2 via the PKA/CREB signaling pathway. Our findings suggest that TPM1 could be a potential target for therapeutic intervention in brain diseases.

Molecular Mechanism of the Protective Effects of M2 Microglia on Neurons: A Review Focused on Exosomes and Secretory Proteins.

Microglia, as innate immune cells in the brain, closely monitor changes in the internal environment and participate in the maintenance of homeostasis in the central nervous system (CNS). Microglia can be polarized to the M1 or M2 phenotype in response to various stimuli in vivo or in vitro, affecting the functions of peripheral neurons. M2 microglia have attracted increasing attention in recent years owing to their beneficial effects on various diseases and injuries of the CNS, such as traumatic brain injury, stroke, Alzheimer's disease and multiple sclerosis. They exert neuroprotective effects by various mechanisms, e.g., suppressing inflammation, promoting the degradation of misfolded and aggregated proteins, promoting neurite growth, enhancing neurogenesis, inhibiting autophagy and apoptosis, promoting myelination, maintaining blood-brain barrier integrity, and enhancing phagocytic activity.This review summarizes the molecular mechanisms by which M2 microglia exert protective effects on neurons and provides a reference for the selection of therapeutic targets for CNS diseases.

RNA interference-mediated silencing of DNA methyltransferase 1 attenuates neuropathic pain by accelerating microglia M2 polarization

BackgroundDNA methyltransferase 1 (DNMT1) exerts imperative functions in neuropathic pain (NP). This study explored the action of RNA interference-mediated DNMT1 silencing in NP by regulating microglial M2 polarization.MethodsNP rat models were established using chronic constriction injury (CCI) and highly aggressive proliferating immortalized (HAPI) microglia were treated with lipopolysaccharide (LPS) to induce microglia M1 polarization, followed by treatment of DNMT1 siRNA or si-DNMT1/oe-DNMT1, respectively. The pain threshold of CCI rats was assessed by determining mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL). Levels of inflammatory factors (TNF-α/IL-1β/IL-6/IL-10) and DNMT1 in rat L4-L6 spinal cord samples and HAPI cells were measured using ELISA, RT-qPCR, and Western blot. iNOS and Arg-1 mRNA levels were measured via RT-qPCR. DNMT1, M1 marker (iNOS), and M2 marker (Arg-1) levels in microglia of CCI rats were detected by immunofluorescence. Percentages of M1 microglia phenotype (CD16) and M2 microglia phenotype (CD206) were detected by flow cytometry. The phosphorylation of PI3K/Akt pathway-related proteins was determined by Western blot.ResultsCCI rats exhibited diminished MWT and TWL values, increased pro-inflammatory cytokines, and decreased anti-inflammatory cytokine IL-10. Additionally, DNMT1 was upregulated in CCI rat microglia. DNMT1 siRNA alleviated CCI-induced NP and facilitated M2 polarization of microglia in CCI rats. DNMT1 knockdown inhibited LPS-induced M1 polarization of HAPI cells and promoted M2 polarization by blocking the PI3K/Akt pathway, but DNMT1 overexpression inhibited the M1-to-M2 polarization of microglia.ConclusionRNA interference-mediated DNMT1 silencing accelerates microglia M2 polarization by impeding the PI3K/Akt pathway, thereby alleviating CCI-induced NP.

Ginsenoside Rb1 attenuates lipopolysaccharide-induced chronic neuroinflammation in mice by tuning glial cell polarization

To evaluate whether ginsenoside Rb1 (Rb1) can attenuate lipopolysaccharide (LPS)-induced chronic neuroinflammation in mice and to explore its relationship with glial cell polarization. Intraperitoneal injection with an escalating dose of LPS was used to establish a chronic neuroinflammation model in mice. Once LPS was initiated, 10 or 20 mg/kg Rb1, or sterile saline, was administered for 14 consecutive days. Open field test and beam walking test were used to monitor the changes in behavior. The concentration of cytokines in the serum and brain were used to monitor the systemic inflammation and neuroinflammation, respectively. Molecules specific to each glial cell phenotype were used to investigate glial cell polarization. Mice in the LPS group had reduced spontaneous activities and impaired beam walking performance. Rb1 obviously eased LPS-induced behavior disturbances. Regarding the levels of serum cytokines, both tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were significantly increased, while interleukin-10 (IL-10) and transforming growth factor β (TGF-β) remarkably decreased after LPS treatment (all P < .001). Rb1 treatment significantly attenuated LPS-induced serum cytokine changes (all P < .05). The results of quantitative polymerase chain reaction and western blotting showed that the mRNA and protein expression levels of TNF-α and complement component 3 (C3) in the brain were significantly increased after LPS treatment (all P < .01). Rb1 treatment significantly inhibited LPS-induced inflammation in the brain (all P < .05). Glial cell polarization analysis showed that M1 and M2 microglia, and A1 astrocytes increased following LPS treatment, while A2 astrocytes decreased. Rb1 treatment reduced M1 and M2 microglia, and A1 astrocytes, and significantly increased A2 astrocytes. Rb1 can attenuate chronic neuroinflammation induced by LPS in mice, which may be partially attributable to its fine tuning of microglia and astrocyte polarization. Rb1 has potential value for treating neurodegenerative diseases.