M2 Microglia Research Articles

The role of JAK/STAT signaling pathway in cerebral ischemia-reperfusion injury and the therapeutic effect of traditional Chinese medicine: A narrative review.

Cerebral ischemia is a cerebrovascular disease with symptoms caused by insufficient blood or oxygen supply to the brain. When blood supplied is restored after cerebral ischemia, secondary brain injury may occur, which is called cerebral ischemia-reperfusion injury (CIRI). In this process, the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway plays an important role. It mediates neuroinflammation and participates in the regulation of physiological activities, such as cell proliferation, differentiation, and apoptosis. After CIRI, M1 microglia is activated and recruited by the damaged tissue. The inflammatory factors are produced by M1 microglia through the JAK/STAT pathway, eventually leading to cell apoptosis. Meanwhile, the JAK2/STAT3 signaling pathway and the expression of lipocalin-2 and caspase-3 could increase. In the pathway, phosphorylated JAK2 and phosphorylated STAT3 function of 2 ways. They not only promote the proliferation of neurons, but also affect the differentiation direction of neural stem cells by further acting on the Notch signaling pathway. Recently, traditional Chinese medicine (TCM) is a key player in CIRI, through JAK2, STAT3, STAT1 and their phosphorylation. Therefore, the review focuses on the JAK/STAT signaling pathway and its relationship with CIRI as well as the influence of the TCM on this pathway. It is aimed at providing the basis for future clinical research on the molecular mechanism of TCM in the treatment of CIRI.

PPAR-γ promotes the polarization of rat retinal microglia to M2 phenotype by regulating the expression of CD200-CD200R1 under hypoxia.

Recent reports suggest that peroxisome proliferator-activated receptor-γ (PPAR-γ) could promote microglial M2 polarization to inhibit inflammation. However, the specific molecular mechanisms that trigger PPAR-γ's anti-inflammatory ability in microglia are yet to be expounded. Thus, in this study, we aimed to explore the molecular mechanisms behind the anti-inflammatory effects of PPAR-γ in hypoxia-stimulated rat retinal microglial cells. We used shRNA expressing lentivirus to knock down PPAR-γ and CD200 genes, and we assessed hypoxia-induced polarization markers release - M1 (iNOS, IL-1β, IL-6, and TNF-α) and M2 (Arg-1, YM1, IL-4, and IL-10) by RT-PCR. We also monitored PPAR-γ-related signals (PPAR-γ, PPAR-γ in cytoplasm or nucleus, CD200, and CD200Rs) by Western blot and RT-PCR. Our results showed that hypoxia enhanced PPAR-γ and CD200 expressions in microglial cells. Moreover, PPAR-γ agonist 15d-PGJ2 elevated CD200 and CD200R1 expressions, whereas sh-PPAR-γ had the opposite effect. Following hypoxia, expressions of M1 markers increased significantly, while those of M2 markers decreased, and the above effects were attenuated by 15d-PGJ2. Conversely, knocking down PPAR-γ or CD200 inhibited the polarization of microglial cells to M2 phenotype. Our findings demonstrated that PPAR-γ performed an anti-inflammatory function in hypoxia-stimulated microglial cells by promoting their polarization to M2 phenotype via the CD200-CD200R1 pathway.

Pseudoginsenoside GQ mitigates chronic intermittent hypoxia-induced cognitive damage by modulating microglia polarization

Obstructive sleep apnea (OSA), a state of sleep disruption, is characterized by recurrent apnea, chronic intermittent hypoxia (CIH) and hypercapnia. Previous studies have showed that CIH-induced neuroinflammatory plays a crucial role in cognitive deficits. Pseudoginsenoside GQ (PGQ) is a new oxytetracycline-type saponin formed by the oxidation and cyclization of the 20(S) Rg3 side chain. Rg3 has been found to afford anti-inflammatory effects, while whether PGQ plays a role of anti-neuroinflammatory remains unclear. The purpose of this study was to investigate whether PGQ attenuates CIH-induced neuroinflammatory and cognitive impairment and the possible mechanism it involves. We found that PGQ significantly ameliorated CIH-induced spatial learning deficits, and inhibited microglial activation, pro-inflammatory cytokine release, and neuronal apoptosis in the hippocampus of CIH mice. In addition, PGQ pretreatment promoted microglial M1 to M2 phenotypic transition in IH-induced BV-2 microglial, as well as indirectly inhibited IH-induced neuronal injury via modulation of microglia polarization. Furthermore, we noted that activation of HMGB1/TLR4/NF-κB signaling pathway induced by IH was inhibited by PGQ. Molecular docking results revealed that PGQ could bind to the active sites of HMGB1 and TLR4. Taken together, this work supports that PGQ inhibits M1 microglial polarization via the HMGB1/TLR4/NF-κB signaling pathway, and indirectly exerts neuroprotective effects, suggesting that PGQ may be a potential therapeutic strategy for cognitive impairment accompanied OSA.

Takinib inhibits microglial M1 polarization and oxidative damage after subarachnoid hemorrhage by targeting TAK1-dependent NLRP3 inflammasome signaling pathway.

Transforming growth factor-β-activated kinase 1 (TAK1) positively regulates oxidative stress and inflammation in different diseases. Takinib, a novel and specific TAK1 inhibitor, has beneficial effects in a variety of disorders. However, the effects of takinib on early brain injury (EBI) after subarachnoid hemorrhage (SAH) and the underlying molecular mechanisms remain unknown. Our study showed that takinib administration significantly inhibited phosphorylated TAK1 expression after SAH. In addition, takinib suppressed M1 microglial polarization and promoted M2 microglial polarization. Furthermore, blockade of TAK1 by takinib reduced neuroinflammation, oxidative damage, brain edema, and neuronal apoptosis, and improved neurological behavior after SAH. Mechanistically, we revealed that TAK1 inhibition by takinib mitigated reactive oxygen species (ROS) production and ROS-mediated nod-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome activation. In contrast, NLRP3 activation by nigericin abated the neuroprotective effects of takinib against EBI after SAH. In general, our study demonstrated that takinib could protect against EBI by targeting TAK1-ROS-NLRP3 inflammasome signaling. Inhibition of TAK1 might be a promising option in the management of SAH.

CNSC-19. HYPOXIA-INDUCED NEURONAL ACTIVITY IN GLIOMA PATIENTS POLARIZES MICROGLIA BY POTENTIATING RNA M6A DEMETHYLATION

Abstract Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. In this study, by applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. Here, we demonstrate that hypoxia induces glioma stem cells (GSCs) to produce glutamate, which activates local neurons. The hypoxia-induced neuronal activity promotes GBM progression by facilitating microglial M2 polarization. We found that when microglia were co-cultured with activated neurons, the level of RNA m6A methylation in microglia was impaired, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted regional microglial M2 polarization while cortical neuronal inhibition decreased regional microglial M2 polarization in GBM xenografts. Together, these findings indicate that hypoxic GSC-induced neuronal activation promotes GBM progression by polarizing microglia via decreasing their m6A methylation levels.

The ratio of M1 to M2 microglia in the striatum determines the severity of L-Dopa-induced dyskinesias.

L-Dopa, while treating motor symptoms of Parkinson's disease, can lead to debilitating L-Dopa-induced dyskinesias, limiting its use. To investigate the causative relationship between neuro-inflammation and dyskinesias, we assessed if striatal M1 and M2 microglia numbers correlated with dyskinesia severity and whether the anti-inflammatories, minocycline and indomethacin, reverse these numbers and mitigate against dyskinesia. In 6-OHDA lesioned mice, we used stereology to assess numbers of striatal M1 and M2 microglia populations in non-lesioned (naïve) and lesioned mice that either received no L-Dopa (PD), remained non-dyskinetic even after L-Dopa (non-LID) or became dyskinetic after L-Dopa treatment (LID). We also assessed the effect of minocycline/indomethacin treatment on striatal M1 and M2 microglia and its anti-dyskinetic potential via AIMs scoring. We report that L-Dopa treatment leading to LIDs exacerbates activated microglia numbers beyond that associated with the PD state; the severity of LIDs is strongly correlated to the ratio of the striatal M1 to M2 microglial numbers; in non-dyskinetic mice, there is no M1/M2 microglia ratio increase above that seen in PD mice; and reducing M1/M2 microglia ratio using anti-inflammatories is anti-dyskinetic. Parkinson's disease is associated with increased inflammation, but this is insufficient to underpin dyskinesia. Given that L-Dopa-treated non-LID mice show the same ratio of M1/M2 microglia as PD mice that received no L-Dopa, and, given minocycline/indomethacin reduces both the ratio of M1/M2 microglia and dyskinesia severity, our data suggest the increased microglial M1/M2 ratio that occurs following L-Dopa treatment is a contributing cause of dyskinesias.

Electroacupuncture Alleviates Neuroinflammation by Regulating Microglia Polarization via STAT6/PPARγ in Ischemic Stroke Rats.

Previous study showed that electroacupuncture (EA) produced a protective effect on cerebral ischemia-reperfusion injury (CIRI) in rats and may correlate with the anti-inflammatory effects of microglia. This study aimed to investigate further whether EA could modulate neuroinflammation by targeting the Signal Transducer and Activator of Transcription 6 (STAT6) and Peroxisome Proliferator-Activated Receptor γ (PPARγ) pathway, the key regulator of microglia. Middle cerebral artery occlusion (MCAO) rats were used, and 6h after reperfusion, EA interventions were performed in Chize (LU 5), Hegu (LI 4), Sanyinjiao (SP 6), and Zusanli (ST 36) on the affected side of the rats, the group that received EA+STAT6 phosphorylation inhibitor AS1517499 was used as a parallel control. The degree of neurological impairment, infarct volume, microglia polarization, inflammation levels and activity of STAT6/PPARγ pathway were then assessed by neurological deficit score, triphenyl tetrazolium chloride (TTC) staining, immunofluorescence, western blotting (WB), quantitative real-time PCR (qPCR) and Enzyme linked immunosorbent assay (ELISA). The data showed that EA significantly alleviated nerve injury, reduced infarct volume, enhanced the expression and activity of STAT6/PPARγ pathway, inhibited NF-κB activity, increased M2 microglia numbers and anti-inflammatory factor release, and inhibited microglia M1-type polarization and pro-inflammatory factor expression. In contrast, inhibition of STAT6 phosphorylation exacerbated neural damage, inhibited STAT6/PPARγ pathway activity, promoted microglia M1-type polarization and exacerbated neuroinflammation, resulting in an attenuated positive effect of EA intervention. Therefore, we concluded that EA intervention could attenuate microglia-associated neuroinflammation by enhancing the expression and activity of STAT6/PPARγ pathway, thereby reducing CIRI in MCAO rats.

Baicalein promotes the microglia M2 polarization and suppresses apoptosis by targeting HMOX1/PDE4D to alleviate Alzheimer’s disease

Alzheimer's disease (AD) is a neurodegenerative disorder that has quickly becoming one of the most expensive, lethal, and burdening diseases of this century. In the past twenty years, hundreds of drugs have been tested while only several have been authorized by FDA for AD treatment, hence, searching for candidate agent with therapeutic potential for AD is imminent. Controlling polarization direction of microglia is crucial in AD therapy. Recent research suggests that baicalein has potential to reduce neuroinflammation and prevent neurodegenerative diseases by affecting microglia, while the specific molecular mechanism of baicalein in regulating microglia in the treatment of AD is still unclear. In this study, we investigated how baicalein affected microglial polarization in AD and potential biological mechanisms. In cell experiments, it was verified that baicalein significantly shifted the BV-2 microglia phenotype from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype, inhibited the microglial apoptosis and pro-inflammatory factors, promoted the microglial Aβ uptake and anti-inflammatory factors after LPS stimulated. In APP/PS1 mice, it was found that baicalein decreased the Aβ plaque deposition in brain, attenuated NLRP3 inflammasome activation and neuronal apoptosis in APP/PS1 mice. Furthermore, bioinformatics analysis and experiment validated that HMOX1 is a target of baicalein, and we elucidated that baicalein modulated the microglial polarization to inhibit neuroinflammation and neural injury through targeting on the HMOX1/PDE4D axis in AD. In conclusion, our findings indicate the therapeutic effect of baicalein for AD, and baicalein might serve a potential agent for AD treatment.

Multi-parametric MRI assessment of melatonin regulating the polarization of microglia in rats after cerebral ischemia/reperfusion injury

ObjectivesMulti-parametric magnetic resonance imaging (MRI) can provide comprehensive and valuable information for precise diagnosis and treatment evaluation of a number of diseases. In this study, the neuroprotective effects of melatonin (Mel) on a rat model of cerebral ischemia/reperfusion injury (CIRI) were assessed by multi-parametric MRI combined with histopathological techniques for longitudinal monitoring of the lesion microenvironment. MethodsSixty Sprague Dawley (SD) rats were randomly divided into three groups: the Sham, CIRI and CIRI + Mel groups. At multiple time points after ischemia, MRI scanning was performed on a 7.0 Tesla MRI scanner. Multi-parametric MRI includes T2-weighted imaging (T2WI), diffusion weighted imaging (DWI), and chemical exchange saturation transfer (CEST)-MRI. CEST effects were calculated by the Lorentzian difference method, 3.5 ppm indicates amide protons of mobile proteins/peptide (Amide-CEST) and 2.0 ppm indicates amine protons (Guan-CEST). Multiple histopathological techniques were used to examine the histopathological changes and explore the therapeutic effects of Mel. ResultsT2WI and DWI-MRI could localize the infarct foci and areas in CIRI rats, which was further validated by staining, 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin and eosin (H&E) staining, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) staining. After Mel treatment, T2WI and DWI-MRI showed smaller infarct volume, and neurons displayed improved morphology with less apoptosis rates. Notably, Amide-CEST and Guan-CEST signal decreased as early as 2 h after CIRI (all P <0.001), reflecting the change of pH after ischemia. After Mel treatment, both Amide-CEST and Guan-CEST signal increased in ischemic cortex and striatum compared with control group (all P < 0.001). The immunofluorescence staining and western blotting analysis suggested the expression of M2 microglia increased after Mel treatment; While，after Mel treatment the inflammatory factor interleukin-1β (IL-1β) decreased compared with control CIRI rats. ConclusionsMulti-parametric MRI was shown to be an effective method to monitor the brain damage in a rat model of CIRI and assess the therapeutic effects of Mel treatment. Amide-CEST and Guan-CEST were especially sensitive to the changes in brain microenvironment during the early stage after CIRI. Furthermore, the neuroprotective effect of Mel treatment is associated with its promotion of the microglia polarized to M2 type in CIRI rats.

Glioma-derived S100A9 polarizes M2 microglia to inhibit CD8+T lymphocytes for immunosuppression via αvβ3 integrin/AKT1/TGFβ1

Our previous studies showed that S100A9 was overexpressed in glioma and promoted tumor growth. However, S100A9 can also be secreted by tumor cells to regulate the tumor microenvironment (TME). In this study, we aimed to explore the functions of glioma derived-S100A9 in microglial M2 polarization, resulting in inhibition of CD8+ T lymphocytes and promotion of immunosuppression. We first showed that glioma exhibited higher expression and secretion of S100A9 than astrocytes. After knocking down S100A9 in two glioma cell lines, the secretion of S100A9 was repressed. Then, the medium was collected and considered as conditioned medium (CM), which was incubated with microglia. We found that glioma-derived S100A9 drove microglial M2 polarization and increased TGFβ1 secretion. These molecular mechanisms were related to the interaction of S100A9 with αvβ3 integrin and the subsequent activation of AKT1 in microglia. Furthermore, we demonstrated that S100A9-induced M2 microglia negatively affected cell viability, IL-2 and IFN-γ secretion, together with increased early apoptosis in CD8+T lymphocytes via TGFβ1. Additionally, glioma cells were implanted into mouse brains, and we confirmed that S100A9 stimulated microglial M2 polarization, enhanced TGFβ1 levels and repressed CD8+ T lymphocytes in orthotopically transplanted tumors. In human glioma samples, S100A9 expression was positively associated with CD206 expression, but negatively correlated with CD8+T lymphocyte accumulation in the TME. Our data indicated that glioma-derived S100A9 has a promising ability to manipulate non-malignant cells and promote immune evasion in the TME, providing valuable insight into the mechanism by which S100A9 participates in the progression of glioma.

Interleukin-4 Modulates Neuroinflammation by Inducing Phenotypic Transformation of Microglia Following Subarachnoid Hemorrhage

Neuroinflammation, a key pathological feature following subarachnoid hemorrhage (SAH), can be therapeutically targeted by inhibiting microglia M1 polarization and promoting phenotypic transformation to M2 microglia. Interleukin-4 (IL-4) is a pleiotropic cytokine known to its regulation of physiological functions of the central nervous system (CNS) and mediate neuroinflammatory processes. However, its specific role in neuroinflammation and microglia responses following SAH remains unexplored. In this investigation, we established both in vivo and in vitro SAH models and employed a comprehensive array of assessments, including ELISA, neurofunctional profiling, immunofluorescence staining, qRT-PCR, determination of phagocytic capacity, and RNA-Seq analyses. The findings demonstrate an elevated expression of IL-4 within cerebrospinal fluid (CSF) subsequent to SAH. Furthermore, exogenous administration of IL-4 ameliorates post-SAH neurofunctional deficits, attenuates cellular apoptosis, fosters M2 microglia phenotype conversion, and mitigates neuroinflammatory responses. The RNA-Seq analysis signifies that IL-4 governs the modulation of neuroinflammation in microglia within an in vitro SAH model through intricate cascades of signaling pathways, encompassing interactions between cytokines and cytokine receptors. These discoveries not only augment comprehension of the neuropathogenesis associated with post-SAH neuroinflammation but also present novel therapeutic targets for the management thereof.Graphical

LncRNA-AC020978 Promotes Metabolic Reprogramming in M1 Microglial Cells in Postoperative Cognitive Disorder via PKM2.

The present work aimed to explore the role of long non-coding RNA (lncRNA)-AC020978 in postoperative cognitive disorder (POCD) and the underlying mechanism. The POCD mouse model was constructed through isoflurane anesthesia + abbreviated laparotomy. The AC020978 expression in brain tissue was silenced after lentivirus injection, then Morris water maze test was conducted to detect the cognitive disorder level, flow cytometry was performed to analyze M1 macrophage level, ELISA was carried out to measure inflammatory factor levels, H&E, Nissl and immunohistochemical staining was performed to detect the pathological changes in brain tissue, and Western blotting assay was adopted to detect protein expression. In addition, microglial cells were cultured in vitro, after lentivirus infection, the effect of AC020978 on the M1 polarization of microglial cells and glycolysis was observed. AC020978 overexpression promoted POCD progression and aggravated cognitive disorder in mice; in addition, the proportion of peripheral and central M1 cells increased, the inflammatory factor levels were upregulated, and microglial cells were activated. By contrast, AC020978 silencing led to cognitive disorder in mice and suppressed microglial cell activation and M1 polarization. In vitro experimental results indicated that AC020978 promoted the expression and phosphorylation of PKM2, which promoted inflammatory response through enhancing microglial cell glycolysis and M1 polarization. AC020978 interacts with PKM2 to promote the glycolysis and M1 polarization of microglial cells, thus regulating cognitive disorder and central inflammation in POCD.

Salvianolic acid B exerts protective effects against Aβ-induced neuroinflammation through the inhibition of NLRP3 inflammasome activation and switching of M1/M2 polarization

BackgroundSalvianolic acid B (SalB) is a bioactive extract of Salvia miltiorrhiza with the ability to ameliorate amyloid beta (Aβ)-induced neuronal degeneration and neuroinflammation in Alzheimer’s disease (AD). However, the underlying mechanisms of this action have not been elucidated. Herein, we aimed to investigate whether the neuroprotective effect of SalB is attributable to the modulation of microglial polarization and NLRP3 inflammasome-mediated neuroinflammation. MethodsBased on the TMT-labeled proteomics analysis, immunofluorescence, western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to investigate the effects of SalB on neuroinflammation in Aβ1–42-stimulated BV2 microglia cells. ResultsAt the proteomic level, a total of 6631 proteins were quantified, and of these, 104 were significantly influenced under Aβ1–42 treatment. The expression of 36 Aβ1–42-induced differentially expressed proteins were significantly recovered by SalB treatment (13 upregulated and 23 downregulated). NLRP3 was significantly recovered and was identified as one of the hub proteins. Consistent with the result of the proteomic analysis, western blot and qRT-PCR demonstrated that SalB reduced Aβ1–42-induced NLRP3 upregulation at both the protein and mRNA levels. In addition, SalB significantly blocked M1 microglia polarization, enhanced M2 microglial polarization, and inhibited the production of caspase-1 and interleukin-1β in BV2 microglia cells. Conclusionour study demonstrated, for the first time, that the anti-inflammatory effects of SalB were mediated by the regulation of NLRP3 activation and promotion of microglial M2 polarization, indicating the potential of SalB as a novel therapeutic candidate for AD.

Circular RNA PTP4A2 regulates microglial polarization through STAT3 to promote neuroinflammation in ischemic stroke.

Microglial polarization plays a critical role in neuroinflammation and may be a potential therapeutic target for ischemic stroke. This study was to explore the role and underlying molecular mechanism of Circular RNA PTP4A2 (circPTP4A2) in microglial polarization after ischemic stroke. C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO), while primary mouse microglia and BV2 microglial cells experienced oxygen glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions. CircPTP4A2 shRNA lentivirus and Colivelin were used to knock down circPTP4A2 and upregulate signal transducer and activator of transcription 3 (STAT3) phosphorylation, respectively. Microglial polarization was assessed using immunofluorescence staining and Western blot. RNA pull-down and RNA binding protein immunoprecipitation (RIP) were applied to detect the binding between circPTP4A2 and STAT3. The levels of circPTP4A2 were significantly increased in plasma and peri-infarct cortex in tMCAO mice. CircPTP4A2 knockdown reduced infarct volume, increased cortical cerebral blood flow (CBF), and attenuated neurological deficits. It also decreased pro-inflammatory factors levels in peri-infarct cortex and plasma, and increased anti-inflammatory factors concentrations 24 h post-stroke. In addition, circPTP4A2 knockdown suppressed M1 microglial polarization and promoted M2 microglial polarization in both tMCAO mice and OGD/R-induced BV2 microglial cells. Moreover, circPTP4A2 knockdown inhibited the phosphorylation of STAT3 induced by oxygen-glucose deprivation. In contrast, increased phosphorylation of STAT3 partly counteracted the effects of circPTP4A2 knockdown. RNA pull-down and RIP assays further certified the binding between circPTP4A2 and STAT3. These results revealed regulatory mechanisms of circPTP4A2 that stimulated neuroinflammation by driving STAT3-dependent microglial polarization in ischemic brain injury. CircPTP4A2 knockdown reduced cerebral ischemic injury and promoted microglial M2 polarization, which could be a novel therapeutic target for ischemic stroke.

Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate neuropathic pain.

(Switching from the microglial M1 phenotype to the M2 phenotype is a promising therapeutic strategy for neuropathic pain (NP). This study aimed to investigate the potential use of stigmasterol for treating NP. In animal experiments, 32 male Sprague-Dawley rats were randomly divided into the sham operation group, chronic constriction injury (CCI) group, CCI + ibuprofen group, and CCI + stigmasterol group. We performed behavioral tests, enzyme-linked immunosorbent assay, hematoxylin-esoin staining (H&E) staining and immunohistochemistry, immunofluorescence, and Western blotting. In cell experiments, we performed flow cytometry, immunofluorescence, Western blotting, and qRT-PCR. Stigmasterol reduced thermal and mechanical hyperalgesia and serum IL-1β and IL-8 levels and increased serum IL-4 and TGF-β levels in CCI rats. Stigmasterol reduced IL-1β, COX-2, and TLR4 expression in the right sciatic nerve and IL-1β expression in the spinal cord. Stigmasterol reduced the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, COX-2, IL-1β, and CD32 in the spinal cord of CCI rats while increasing the expression of IL-10 and CD206. Stigmasterol decreased M1 polarization markers and increased M2 polarization markers in lipopolysaccharide (LPS)-induced microglia and decreased the expression of Iba-1, TLR4, MyD88, pNF-κB, pP38 MAPK, pJNK, pERK, iNOS, COX-2, and IL-1β in LPS-treated microglia while increasing the expression of Arg-1 and IL-10. Stigmasterol regulates microglial M1/M2 polarization via the TLR4/NF-κB pathway to alleviate NP.

Hypoxia-Induced Neuronal Activity in Glioma Patients Polarizes Microglia by Potentiating RNA m6A Demethylation.

Neuronal activity in the brain has been reported to promote the malignant progression of glioma cells via nonsynaptic paracrine and electrical synaptic integration mechanisms. However, the interaction between neuronal activity and the immune microenvironment in glioblastoma (GBM) remains largely unclear. By applying chemogenetic techniques, we enhanced and inhibited neuronal activity in vitro and in a mouse model to study how neuronal activity regulates microglial polarization and affects GBM progression. We demonstrate that hypoxia drove glioma stem cells (GSC) to produce higher levels of glutamate, which activated local neurons. Neuronal activity promoted GBM progression by facilitating microglial M2 polarization through enriching miR-200c-3p in neuron-derived exosomes, which decreased the expression of the m6A writer zinc finger CCCH-type containing 13 (ZC3H13) in microglia, impairing methylation of dual specificity phosphatase 9 (DUSP9) mRNA. Downregulation of DUSP9 promoted ERK pathway activation, which subsequently induced microglial M2 polarization. In the mouse model, cortical neuronal activation promoted microglial M2 polarization whereas cortical neuronal inhibition decreased microglial M2 polarization in GBM xenografts. miR-200c-3p knockdown in cortical neurons impaired microglial M2 polarization and GBM xenograft growth, even when cortical neurons were activated. Treatment with the anti-seizure medication levetiracetam impaired neuronal activation and subsequently reduced neuron-mediated microglial M2 polarization. These findings indicated that hypoxic GSC-induced neuron activation promotes GBM progression by polarizing microglia via the exosomal miR-200c-3p/ZC3H13/DUSP9/p-ERK pathway. Levetiracetam, an antiepileptic drug, blocks the abnormal activation of neurons in GBM and impairs activity-dependent GBM progression. See related commentary by Cui et al., p. 1073.

Long Non-Coding RNA MALAT1 Protects Against Spinal Cord Injury via Suppressing microRNA-125b-5p Mediated Microglial M1 Polarization, Neuroinflammation, and Neural Apoptosis.

Our previous studies have discovered that long non-coding RNA (lncRNA) MALAT1 and its target microRNA-125b-5p (miR-125b-5p) are implicated in neurological diseases via regulating neuroinflammation and neuronal injury. This study aimed to further explore the relationship between lncRNA MALAT1 and miR-125b-5p, as well as their effect on microglial activation, neuroinflammation, and neural apoptosis in spinal cord injury (SCI).Primary microglia from Sprague Dawley rats were stimulated with lipopolysaccharide (LPS). Then, microglia were transfected with lncRNA MALAT1 overexpression or knock-down adenovirus-associated virus with or without miR-125b-5p mimic. The culture medium of microglia was incubated with primary neurons. SCI rats were established for in vivo validation.LncRNA MALAT1 expression was reduced by LPS treatment in a dose-dependent manner. LncRNA MALAT1 overexpression suppressed the microglial M1 polarization (decreased iNOS but increased ARG1), neuroinflammation (declined PTGS2, TNF-α, IL-1β, and IL-6), and microglia-induced neural apoptosis (lower TUNEL positive cells and C-caspase3 but higher BCL2) under LPS treatment; its knock-down displayed the opposite trend. Moreover, lncRNA MALAT1 directly bound to and negatively regulated miR-125b-5p. MiR-125b-5p mimic promoted microglial M1 polarization, neuroinflammation, and microglia-induced neural apoptosis following LPS treatment; also, it could attenuate the effect of lncRNA MALAT1. Further in vivo study displayed that lncRNA MALAT1 overexpression elevated the Basso-Beattie-Bresnahan motor function score and improved neural injury. Also, in vivo validation indicated a similar effect of lncRNA MALAT1 on microglial polarization and neuroinflammation as in vitro.LncRNA MALAT1 improves SCI recovery via miR-125b-5p mediated microglial M1 polarization, neuroinflammation, and neural apoptosis.

Inhibition of the TLR4/NF-κB pathway promotes the polarization of LPS-induced BV2 microglia toward the M2 phenotype.

This study aimed to investigate whether the inhibition of the TLR4/NF-κB pathway can promote lipopolysaccharide (LPS)-induced microglial polarization from the M1 to M2 phenotype, and thus exert neuroprotection. LPS-induced microglia were used as a model for inflammation in vitro. TLR4-specific inhibitor resatorvid (TAK-242) and NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) were used to verify the effect of the TLR4/NF-κB pathway on microglia activation and polarization. Cell proliferation was measured by cell counting, and nitric oxide (NO) and reactive oxygen species (ROS) release was measured using the Griess reagent and ROS kit, respectively. Immunofluorescence and RT-qPCR analyses were used to detect the expression of microglial activation markers, phenotypic markers, related pathway molecules, and inflammatory factors. TLR4 specific inhibitor TAK-242 and NF-κB inhibitor PDTC alleviated LPS-induced microglia over-activation by inhibiting the TLR4/NF-κB pathway, and reduced LPS-stimulated cell proliferation and the release of NO, ROS, TNF-a, and IL-6 and IL-1β. Meanwhile, TAK-242 and PDTC promoted LPS-induced polarization of microglia from M1 to M2 phenotype, decreased the expression of microglial activation marker Iba1 and M1 phenotypic markers (TNF-a and CD86), and increased the expression of M2 phenotypic markers (Arg-1 and CD206). The mechanism may be related to inhibiting the TLR4/NF-κB pathway. The inhibition of the TLR4/NF-κB pathway can promote LPS-induced polarization of BV2 microglia from M1 phenotype to M2 phenotype.

Complement receptor 3-mediated neurotoxic glial activation contributes to rotenone-induced cognitive decline in mice

Microglia-mediated chronic neuroinflammation has been associated with cognitive decline induced by rotenone, a well-known neurotoxic pesticide used in agriculture. However, the mechanisms remain unclear. This work aimed to elucidate the role of complement receptor 3 (CR3), a highly expressed receptor in microglia, in cognitive deficits induced by rotenone. Rotenone up-regulated the expression of CR3 in the hippocampus and cortex area of mice. CR3 deficiency markedly ameliorated rotenone-induced cognitive impairments, neurodegeneration and phosphorylation (Ser129) of α-synuclein in mice. CR3 deficiency also attenuated rotenone-stimulated microglial M1 activation. In microglial cells, siRNA-mediated knockdown of CR3 impeded, while CR3 activation induced by LL-37 exacerbated, rotenone-induced microglial M1 activation. Mechanistically, CR3 deficiency blocked rotenone-induced activation of nuclear factor κB (NF-κB), signal transducer and activator of transcription 1 (STAT1) and STAT3 signaling pathways. Pharmacological inhibition of NF-κB or STAT3 but not STAT1 was confirmed to suppress microglial M1 activation elicited by rotenone. Further study revealed that CR3 deficiency or knockdown also reduced rotenone-induced expression of C3, an A1 astrocyte marker, and production of microglial C1q, TNFα and IL-1α, a cocktail for activated microglia to induce neurotoxic A1 astrocytes, via NF-κB and STAT3 pathways. Finally, a small molecule modulator of CR3 efficiently mitigated rotenone-elicited cognitive deficits in mice even administered after the establishment of cognitive dysfunction. Taken together, our findings demonstrated that CR3 is a key factor in mediating neurotoxic glial activation and subsequent cognitive impairments in rotenone-treated mice, giving novel insights into the immunopathogenesis of cognitive impairments in pesticide-related Parkinsonism.

Koumine ameliorates neuroinflammation by regulating microglia polarization via activation of Nrf2/HO-1 pathway

BackgroundGelsemium elegans (Gardner & Chapm.) Benth (G. elegans) has been widely used as a traditional folk medicine in China and Southeast Asia. As the most abundant alkaloid in G. elegans, Koumine (KM) has been revealed the effect of inflammatory attenuation modulating by macrophage activation and polarization. PurposeThis study aimed to explore the effect of KM on modulation of microglia polarization that led to the suppression of neuroinflammation and further improved neurodegenerative behavior. MethodsInflammatory mediators, microglia M1 and M2 phenotype markers and Nrf2/HO-1 pathway related protein were assessed in LPS-induced BV2 cells and LPS-treated mice by RT-PCR, immunohistochemistry, immunofluorescence and Western blotting. Moreover, the learning and memory abilities of mice were evaluated by Morris water maze test, and the neuronal damage was evaluated by the Nissl staining. ResultsKM attenuated LPS-induced viability and morphological changes in BV2 microglial cells. Our findings showed that KM activated the Nrf2/HO-1 signaling pathway to promote phenotypic switch from M1 to M2 phenotypes. This switch suppresses the release of inflammatory mediators in LPS-induced BV2 cells. Meanwhile, KM attenuated neuroinflammation through modulating microglia polarization and subsequently reversed the behavioral alterations in LPS-induced mice model of neuroinflammation. ConclusionsKM may alleviate neuroinflammation by regulating microglia polarization with the involvement of Nrf2/HO-1 pathway, resulting of the neuroprotective effect.