LPS-induced Microglial Activation Research Articles

FKBP51 is involved in LPS-induced microglial activation via NF-κB signaling to mediate neuroinflammation

AimsFKBP5 encodes FKBP51, which has been implicated in stress-related psychiatric disorders, and its expression is often increased under chronic stress, contributing to mental dysfunctions. However, the precise role of FKBP51 in brain inflammation remains unclear. This study aimed to investigate the role of FKBP51 in microglia-mediated inflammatory responses in the central nervous system. Main methodsWe employed a peripheral lipopolysaccharide (LPS) administration model to compare microglial activation and cytokine gene expression between Fkbp5 knockout (Fkbp5-KO) and wild-type (WT) male mice. Additionally, we used both BV2 and primary microglia in vitro to examine how Fkbp5 deletion influenced inflammation-related pathways and microglial functions. Key findingsThis study revealed that systemic LPS-induced microglial activation was significantly attenuated in Fkbp5-KO mice compared with WT mice. In Fkbp5-KO mice following the LPS challenge, there was a notable decrease in the expression of pro-inflammatory genes, coupled with an increase in the anti-inflammatory gene Arg1. Furthermore, Fkbp5 knockdown in BV2 microglial cells led to reduced expression of LPS-induced inflammatory markers, and targeted inhibition of NF-κB activation, while Akt signaling remained unaffected. Similar results were observed in Fkbp5-KO primary microglia, which exhibited not only decreased microglial activation but also a significant reduction in phagocytic activity in response to LPS stimulation. SignificanceThis study highlights the critical role of FKBP51 in LPS-induced microglial activation and neuroinflammation. It shows that reducing FKBP51 levels attenuates inflammation through NF-κB signaling in microglia. This suggests that FKBP51 is a potential target for alleviating neuroinflammation-induced stress responses.

GHS-R1a deficiency protects against lipopolysaccharide-induced spatial memory impairment in mice

Neuroinflammation has been implicated in cognitive deficits of neurological and neurodegenerative diseases. There is abundant evidence that the application of ghrelin, an orexigenic hormone regulating appetite and energy balance, abrogates neuroinflammation and rescues associated memory impairment. However, the underlying mechanism is uncertain. In this study, we find that both intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) administration of lipopolysaccharide (LPS) impairs spatial memory in mice. LPS treatment causes neuroinflammation and microglial activation in the hippocampus. Ghsr1a deletion suppresses LPS-induced microglial activation and neuroinflammation, and rescued LPS-induced memory impairment. Our findings thus suggest that GHS-R1a signaling may promote microglial immunoactivation and contribute to LPS-induced neuroinflammation. GHS-R1a may be a new therapeutic target for cognitive dysfunction associated with inflammatory conditions.

Microglial forkhead box O3a deficiency attenuates LPS-induced neuro-inflammation and depressive-like behaviour through regulating the expression of peroxisome proliferator-activated receptor-γ.

Depression is closely linked with microglial activation and neuro-inflammation. Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in M2 activation of microglia. Forkhead box (FOX) O3a has been implicated in the regulation of mood-relevant behaviour. However, little is known about the inflammatory mechanisms of in the microglia of the brain. Here, we have investigated the role of microglial FOXO3a/PPAR-γ in the development of depression. The effect of FOXO3a on microglia inflammation was analysed in vitro and in lipopolysaccharide (LPS)-induced depression-like behaviours in vivo. ChIP-seq and Dual-luciferase reporter assays were used to confirm the interaction between FOXO3a and PPAR-γ. Behavioural changes were measured, while inflammatory cytokines, microglial phenotype and morphological properties were determined by ELISA, qRT-PCR, western blotting and immunostaining. Overexpression of FOXO3a significantly attenuated expression of PPAR-γ and enhanced the microglial polarization towards the M1 phenotype, while knockdown of FOXO3a had the opposite effect. FOXO3a binds to the promoters of PPAR-γ and decreases its transcription activity. Importantly, deacetylation and activation of FOXO3a regulate LPS-induced neuro-inflammation by inhibiting the expression of PPAR-γ in microglia cells, supporting the antidepressant potential of histone deacetylase inhibitors. Microglial FOXO3a deficiency in mice alleviated LPS-induced neuro-inflammation and depression-like behaviours but failed to reduce anxiety behaviour, whereas pharmacological inhibition of PPAR-γ by GW9662 restored LPS-induced microglial activation and depressive-like behaviours in microglial FOXO3a-deficient mice. FOXO3a/PPAR-γ axis plays an important role in microglial activation and depression, identifying a new therapeutic avenue for the treatment of major depression.

Microglial activation induces nitric oxide signalling and alters protein S-nitrosylation patterns in extracellular vesicles.

Neuroinflammation is an underlying feature of neurodegenerative conditions, often appearing early in the aetiology of a disease. Microglial activation, a prominent initiator of neuroinflammation, can be induced through lipopolysaccharide (LPS) treatment resulting in expression of the inducible form of nitric oxide synthase (iNOS), which produces nitric oxide (NO). NO post-translationally modifies cysteine thiols through S-nitrosylation, which can alter function of the target protein. Furthermore, packaging of these NO-modified proteins into extracellular vesicles (EVs) allows for the exertion of NO signalling in distant locations, resulting in further propagation of the neuroinflammatory phenotype. Despite this, the NO-modified proteome of activated microglial EVs has not been investigated. This study aimed to identify the protein post-translational modifications NO signalling induces in neuroinflammation. EVs isolated from LPS-treated microglia underwent mass spectral surface imaging using time of flight-secondary ion mass spectrometry (ToF-SIMS), in addition to iodolabelling and comparative proteomic analysis to identify post-translation S-nitrosylation modifications. ToF-SIMS imaging successfully identified cysteine thiol side chains modified through NO signalling in the LPS treated microglial-derived EV proteins. In addition, the iodolabelling proteomic analysis revealed that the EVs from LPS-treated microglia carried S-nitrosylated proteins indicative of neuroinflammation. These included known NO-modified proteins and those associated with LPS-induced microglial activation that may play an essential role in neuroinflammatory communication. Together, these results show activated microglia can exert broad NO signalling changes through the selective packaging of EVs during neuroinflammation.

Gibberellic acid targeting ZBTB16 reduces NF-κB dependent inflammatory stress in sepsis-induced neuroinflammation

ObjectiveSepsis is frequently complicated by neuroinflammation. Gibberellic acid (GA3) is recognized for its anti-inflammatory properties. In this study, our objective was to investigate whether GA3 could alleviate Nuclear factor-kappa B (NF-κB) -dependent inflammatory stress in sepsis-induced neuroinflammation. MethodsC57BL/6 J mice were administered 10 mg/kg lipopolysaccharide (LPS) to induce sepsis. BV2 cells were pre-incubated with GA3 and subjected lipopolysaccharide stimulation to replicate the inflammatory microglia during sepsis. Subsequently, we assessed the release of IL-6, TNF-α, and IL-1β, along with the expression of Zbtb16, NF-κB, and IκB. To investigate whether any observed anti-inflammatory effects of GA3 were mediated through a Zbtb16-dependent mechanism, Zbtb16 was silenced using siRNA. ResultsGA3 improved the survival of sepsis mice and alleviated post-sepsis cognitive impairment. Additionally, GA3 attenuated microglial M1 activation (pro-inflammatory phenotype), inflammation, and neuronal damage in the brain. Moreover, GA3 inhibited the release of TNF-α, IL-6, and IL-1β in microglia stimulated with LPS. The NF-κB signaling pathway emerged as one of the key molecular pathways associated with the impact of GA3 on LPS-stimulated microglia. Lastly, GA3 upregulated Zbtb16 expression in microglia that had been downregulated by LPS. The inhibitory effects of GA3 on microglial M1 activation were partially reversed through siRNA knockdown of Zbtb16. ConclusionsPre-incubation of microglia with GA3 led to the upregulation of the NF-κB regulator, Zbtb16. This process counteracted LPS-induced microglial M1 activation, resulting in an anti-inflammatory effect upon subsequent LPS stimulation.

Positive Effect of 6-Gingerol on Functional Plasticity of Microglia in a rat Model of LPS-induced Depression.

Neuroinflammation has emerged as a crucial factor in the development of depression. Despite the well-known anti-inflammatory properties of 6-gingerol, its potential impact on depression remains poorly understood. This study aimed to investigate the antidepressant effects of 6-gingerol by suppressing microglial activation. In vivo experiments were conducted to evaluate the effect of 6-gingerol on lipopolysaccharide (LPS)-induced behavioral changes and neuroinflammation in rat models. In vitro studies were performed to examine the neuroprotective properties of 6-gingerol against LPS-induced microglial activation. Furthermore, a co-culture system of microglia and neurons was established to assess the influence of 6-gingerol on the expression of synaptic-related proteins, namely synaptophysin (SYP) and postsynaptic density protein 95 (PSD95), which are influenced by microglial activation. In the in vivo experiments, administration of 6-gingerol effectively alleviated LPS-induced depressive behavior in rats. Moreover, it markedly suppressed the activation of rat prefrontal cortex (PFC) microglia induced by LPS and the activation of the NF-κB/NLRP3 inflammatory pathway, while also reducing the levels of inflammatory cytokines IL-1β and IL-18. In the in vitro experiments, 6-gingerol mitigated nuclear translocation of NF-κB p65, NLRP3 activation, and maturation of IL-1β and IL-18, all of which were induced by LPS. Furthermore, in the co-culture system of microglia and neurons, 6-gingerol effectively restored the decreased expression of SYP and PSD95. The findings of this study demonstrate the neuroprotective effects of 6-gingerol in the context of LPS-induced depression-like behavior. These effects are attributed to the inhibition of microglial hyperactivation through the suppression of the NF-κB/NLRP3 inflammatory pathway.

Temporin-GHaR Peptide Alleviates LPS-Induced Cognitive Impairment and Microglial Activation by Modulating Endoplasmic Reticulum Stress.

Neuroinflammation is considered an important factor that leads to cognitive impairment. Microglia play a crucial role in neuroinflammation, which leads to cognitive impairment. This study aimed at determining whether temporin-GHaR peptide (GHaR) could improve cognitive function and at uncovering the underlying mechanisms. We found that GHaR treatment alleviated LPS-induced cognitive impairment and inhibited activation of microglia in LPS-induced mice. Furthermore, GHaR inhibited activation of endoplasmic reticulum stress (ERS) and the NF-κB signaling pathway in LPS-induced mice. In vitro, GHaR inhibited M1 polarization of BV2 cells and suppressed TNF-α and IL-6 secretion. Additionally, GHaR neuronal cell viability and apoptosis were induced by LPS-activated microglia-conditioned medium. Moreover, in LPS-induced BV2 cells, GHaR inhibited activation of ERS and the NF-κB signaling pathway. In summary, GHaR improved LPS-induced cognitive and attenuated inflammatory responses via microglial activation reversal. In conclusion, the neuroprotective effects of GHaR were mediated via the ERS signaling pathway.

The role of ZC3H12D-regulated TLR4-NF-κB pathway in LPS-induced pro-inflammatory microglial activation

Lipopolysaccharide (LPS) is an important neurotoxin that can cause inflammatory activation of microglia. ZC3H12D is a novel immunomodulator, which plays a remarkable role in neurological pathologies. It has not been characterized whether ZC3H12D is involved in the regulation of microglial activation. The aim of this study was to investigate the role of ZC3H12D in LPS-induced pro-inflammatory microglial activation and its potential mechanism. To elucidate this, we established animal models of inflammatory injury by intraperitoneal injection of LPS (10 mg/kg). The results of the open-field test showed that LPS caused impaired motor function in mice. Meanwhile, LPS caused pro-inflammatory activation of microglia in the mice cerebral cortex and inhibited the expression of ZC3H12D. We also constructed in vitro inflammatory injury models by treating BV-2 microglia with LPS (0.5 μg/mL). The results showed that down-regulated ZC3H12D expression was associated with LPS-induced pro-inflammatory microglial activation, and further intervention of ZC3H12D expression could inhibited LPS-induced pro-inflammatory activation of microglia. In addition, LPS activated the TLR4-NF-κB signaling pathway, and this process can also be reversed by promoting ZC3H12D expression. At the same time, the addition of resveratrol, a nutrient previously proven to inhibit pro-inflammatory microglial activation, can also reverse this process by increasing the expression of ZC3H12D. Summarized, our data elucidated that ZC3H12D in LPS-induced pro-inflammatory activation of brain microglia via restraining the TLR4-NF-κB pathway. This study may provide a valuable clue for potential therapeutic targets for neuroinflammation-related injuries.

Decreased hippocampal microglial cell activation by methanolic extract from the leaves of Mallotus oppositifolius (Geiseler) Müll. Arg contributes to its antidepressant-like effect

Background Natural remedies with neuroprotective effect are useful in neuroinflammation-associated depression. Although Mallotus oppositifolius extract (MOE) has previously demonstrated antidepressant and anti-inflammatory properties, its neuroprotective effect remains unknown. Thus, the study evaluated the effect of MOE on lipopolysaccharide (LPS)-induced neuroinflammation-associated depression in mice. Methods Antidepressant-like effect of MOE (10 – 100 mg/kg), fluoxetine (20 mg/kg) and minocycline (50 mg/kg) was established in naïve Institute of Cancer Research (ICR) mice using the forced swim (FST), tail suspension (TST) and open-space swim (OSST) tests. In a separate experiment, FST and TST were used to assess the effect of an 11-day pre-treatment with MOE (10 – 100 mg/kg) or minocycline (50 mg/kg) on LPS (1 mg/kg) neuroinflammation at 6 and 24 hours post LPS. Following these tests, mice were sacrificed and their hippocampi isolated to evaluate their resting and activated microglial cells using Golgi-Cox staining technique. Open-field test was used to assess locomotor activity. Results MOE, fluoxetine and minocycline significantly reduced immobility in FST, TST and OSST compared to vehicle (p < 0.05), confirming their antidepressant-like effect. Interestingly, MOE’s antidepressant-like effect was faster than fluoxetine and minocycline. Conversely, LPS treatment increased immobility behavior at 6 and 24 hours, suggestive of neuroinflammation-induced depression. Compared to vehicle group, pre-treatment with MOE and minocycline ameliorated LPS-induced hippocampal microglial activation and reversed increased immobility behavior without affecting locomotor activity (p < 0.05). Resting microglial cell count was significantly increased by MOE pre-treatment in the OSST-challenged mice compared to vehicle group (p < 0.01). Similarly, MOE pre-treatment reversed LPS-induced reduction in resting microglial count, and restored resting microglial count to normal levels compared to LPS naive vehicle group. Conclusions Collectively, the results suggest that MOE exerts neuroprotective effect against LPS-induced neuroinflammation by decreasing the activation of microglia and increasing resting microglial count. This contributes to its antidepressant-like effect.

Human adipose tissue-derived stem cell extracellular vesicles attenuate ocular hypertension-induced retinal ganglion cell damage by inhibiting microglia- TLR4/MAPK/NF-κB proinflammatory cascade signaling

Microglia-mediated neuroinflammatory responses are recognized as a predominant factor during high intraocular pressure (IOP)-induced retinal and optic nerve injury along with potential therapeutic targets for the disease. Our previous research indicated that mesenchymal stem cell (MSC) treatment could reduce high IOP-induced neuroinflammatory responses through the TLR4 pathway in a rat model without apparent cell replacement and differentiation, suggesting that the anti-neuroinflammatory properties of MSCs are potentially mediated by paracrine signaling. This study aimed to evaluate the anti-neuroinflammatory effect of human adipose tissue-derived extracellular vesicles (ADSC-EVs) in microbead-induced ocular hypertension (OHT) animals and to explore the underlying mechanism since extracellular vesicles (EVs) are the primary transporters for cell secretory action. The anti-neuroinflammatory effect of ADSC-EVs on LPS-stimulated BV-2 cells in vitro and OHT-induced retinal and optic nerve injury in vivo was investigated. According to the in vitro research, ADSC-EV treatment reduced LPS-induced microglial activation and the TLR4/NF-κB proinflammatory cascade response axis in BV-2 cells, such as CD68, iNOS, TNF-α, IL-6, and IL-1β, TLR4, p-38 MAPK, NF-κB. According to the in vivo data, intravitreal injection of ADSC-EVs promoted RGC survival and function, reduced microglial activation, microglial-derived neuroinflammatory responses, and TLR4/MAPK/NF-κB proinflammatory cascade response axis in the OHT mice. Our findings provide preliminary evidence for the RGC protective and microglia-associated neuroinflammatory reduction effects of ADSC-EVs by inhibiting the TLR4/MAPK/NF-κB proinflammatory cascade response in OHT mice, indicating the therapeutic potential ADSC-EVs or adjunctive therapy for glaucoma.

Amlexanox attenuates LPS-induced neuroinflammatory responses in microglial cells via inhibition of NF–κB and STAT3 signaling pathways

Amlexanox is an anti-inflammatory and anti-allergic agent used clinically for the treatment of aphthous ulcers, allergic rhinitis, and asthma. Recent studies have demonstrated that amlexanox, a selective inhibitor of IkB kinase epsilon (IKKε) and TANK-binding kinase 1 (TBK1), suppresses a range of diseases or inflammatory conditions, such as obesity-related metabolic dysfunction and type 2 diabetes. However, the effects of amlexanox on neuroinflammatory responses to amlexanox have not yet been comprehensively studied. In this study, we investigated the novel therapeutic effect of amlexanox on LPS-induced neuroinflammation in vivo, and intraperitoneal injection of amlexanox markedly reduced LPS-induced IKKε levels, proinflammatory cytokines, and microglial activation, as evidenced by ionized calcium-binding adapter molecule 1 (Iba1) immunostaining. Furthermore, amlexanox significantly reduced proinflammatory cytokines and chemokines in LPS-induced bone marrow-derived macrophages (BMDM), murine BV2, and human HMC3 microglial cells. This data provided considerable evidence that amlexanox can be used as a preventive and curative therapy for neuroinflammatory and neurodegenerative diseases. In terms of mechanism aspects, our results demonstrated that the anti-inflammatory action of amlexanox in BV2 microglial cells was through the downregulation of NF-κB and STAT3 signaling pathways. In addition, the combination of amlexanox and SPI (a STAT3 selective inhibitor) showed high efficiency in inhibiting the production of neurotoxic and pro-inflammatory mediators. Overall, our data provide rational insights into the mechanisms of amlexanox as a potential therapeutic strategy for neuroinflammation-related diseases.

Lumbricus Extract Prevents LPS-Induced Inflammatory Activation of BV2 Microglia and Glutamate-Induced Hippocampal HT22 Cell Death by Suppressing MAPK/NF-κB/NLRP3 Signaling and Oxidative Stress.

Microglia-induced inflammatory signaling and neuronal oxidative stress are mutually reinforcing processes central to the pathogenesis of neurodegenerative diseases. Recent studies have shown that extracts of dried Pheretima aspergillum (Lumbricus) can inhibit tissue fibrosis, mitochondrial damage, and asthma. However, the effects of Lumbricus extracts on neuroinflammation and neuronal damage have not been previously studied. Therefore, to evaluate the therapeutic potential of Lumbricus extract for neurodegenerative diseases, the current study assessed the extract's anti-inflammatory and antioxidant activities in BV2 microglial cultures stimulated with lipopolysaccharide (LPS) along with its neuroprotective efficacy in mouse hippocampal HT22 cell cultures treated with excess glutamate. Lumbricus extract dose-dependently inhibited the LPS-induced production of multiple proinflammatory cytokines (tumor necrosis factor-α, interleukin (IL)-6, and IL-1β) and reversed the upregulation of proinflammatory enzymes (inducible nitric oxide synthase and cyclooxygenase-2). Lumbricus also activated the antioxidative nuclear factor erythroid 2-relayed factor 2/heme oxygenase-1 pathway and inhibited LPS-induced activation of the nuclear factor-κB/mitogen-activated protein kinases/NOD-like receptor family pyrin domain containing 3 inflammatory pathway. In addition, Lumbricus extract suppressed the glutamate-induced necrotic and apoptotic death of HT22 cells, effects associated with upregulated expression of antiapoptotic proteins, downregulation of pro-apoptotic proteins, and reduced accumulation of reactive oxygen species. Chromatography revealed that the Lumbricus extract contained uracil, hypoxanthine, uridine, xanthine, adenosine, inosine, and guanosine. Its effects against microglial activation and excitotoxic neuronal death reported herein support the therapeutic potential of Lumbricus for neurodegenerative diseases.

Direct inhibition of microglial activation by a μ receptor selective agonist alleviates inflammatory-induced pain hypersensitivity

Opioids are widely used in the treatment of moderate and severe pain. Nociceptive stimulation has been reported to potentially promote microglial activation and neuroinflammation, which also causes chronic pain sensitization. The aim of this study was to demonstrate whether the novel μ receptor agonist MEL-0614 could inhibit activated microglia directly and the associated signaling pathway. Mice were administered lipopolysaccharide and formalin to induce allodynia. Von Frey test was used to detect the anti-allodynia effect of MEL-0614 before and after LPS and formalin injection. In the spinal cord, the levels of proinflammatory cytokines and microglial activation were determined after MEL-0614 administration. BV2 and primary microglia were cultured to further explore the effect of MEL-0614 on LPS-induced microglial activation and key signaling pathways involved. MEL-0614 partially prevented and reversed allodynia induced by LPS and formalin in vivo, which was not inhibited by the μ receptor antagonist CTAP. Minocycline was effective in reversing the established allodynia. MEL-0614 also downregulated the activation of microglia and related proinflammatory cytokines in the spinal cord. Additionally, in BV2 and primary microglia, MEL-0614 inhibited the LPS-induced upregulation of proinflammatory factors, which was unaffected by CTAP. The NLR family pyrin domain containing 3 (NLRP3) related signaling pathway may be involved in the interaction between MEL-0614 and microglia. The opioid agonist MEL-0614 inhibited the activation of microglia and the subsequent upregulation of proinflammatory factors both in vivo and in vitro. Notably, this effect is partially mediated by the μ receptor.

Deficient chaperone-mediated autophagy facilitates LPS-induced microglial activation via regulation of the p300/NF-κB/NLRP3 pathway.

Neuroinflammation is a pathological change that is involved in the progression of Parkinson's disease. Dysfunction of chaperone-mediated autophagy (CMA) has proinflammatory effects. However, the mechanism by which CMA mediates inflammation and whether CMA affects microglia and microglia-mediated neuronal damage remain to be elucidated. In the present study, we found that LAMP2A, a limiting protein for CMA, was decreased in lipopolysaccharide (LPS)-treated primary microglia. Activation of CMA by the activator CA significantly repressed LPS-induced microglial activation, whereas CMA dysfunction exacerbated microglial activation. We further identified that the protein p300 was a substrate of CMA. Degradation of p300 by CMA reduced p65 acetylation, thereby inhibiting the transcription of proinflammatory factors and the activation of the NLRP3 inflammasome. Furthermore, CA pretreatment inhibited microglia-mediated inflammation and, in turn, attenuated neuronal death in vitro and in vivo. Our findings suggest repressive effects of CMA on microglial activation through the p300-associated NF-κB signaling pathway, thus uncovering a mechanistic link between CMA and neuroinflammation.

NLRC5 Deficiency Reduces LPS-Induced Microglial Activation via Inhibition of NF-κB Signaling and Ameliorates Mice's Depressive-like Behavior.

Microglia are believed to be the key immune effectors of the central immune microenvironment, and their dysregulation is associated with neuroinflammation and mood disorders. Nucleotide-binding oligomerization domain-like receptor family caspase recruitment domain-containing five (NLRC5) is a new member of the Nod-like receptor family. Recently, NLRC5 has been reported to be expressed by microglia. Nonetheless, the exact roles of NLRC5 in microglial activation and its function in depression have not been investigated yet. Herein, we found that reducing NLRC5 decreased lipopolysaccharide (LPS)-induced secretion of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) in primary cultured microglia and microglial cell lines but not in bone marrow-derived macrophages (BMDMs). In more detail, reducing NLRC5 diminished the secretion of LPS-induced cytokines by attenuating IKKα/β phosphorylation and inhibiting NF-κB signaling. Moreover, the expression of Nlrc5 in the hippocampus of LPS- or chronic unpredictable mild stress (CUMS)-induced depressive mice was increased. In line with the in vitro findings, Nlrc5 deficiency inhibited microglial activation in the mouse hippocampus and improved LPS- or CUMS-induced depressive-like behaviors. In summary, we demonstrated the critical role of NLRC5 in LPS-induced microglial activation and LPS- or CUMS-induced depressive mouse models.

GKLF, a transcriptional activator of Txnip, drives microglia activation in kainic acid-induced murine models of epileptic seizures.

Neuroinflammation is a major component of epilepsy. Gut-enriched Kruppel-like factor (GKLF), a transcription factor of Kruppel-like factor family, has been reported to promote microglia activation and mediate neuroinflammation. However, the role of GKLF in epilepsy remains poorly characterized. This study focused on the function of GKLF in neuron loss and neuroinflammation in epilepsy and the molecular mechanism underlying microglia activation induced by GKLF upon lipopolysaccharides (LPS) treatment. An experimental epileptic model was induced by an intraperitoneal injection of 25mg/kg kainic acid (KA). Lentivirus vectors (Lv) carrying Gklf CDS or short hairpin RNA targeting Gklf (shGKLF) was injected into the hippocampus, resulting in Gklf overexpression or knockdown in the hippocampus. BV-2 cells were co-infected with Lv-shGKLF or/and Lv carrying thioredoxin interacting protein (Txnip) CDS for 48h and treated with 1μg/mL LPS for 24h. Results showed that GKLF enhanced KA-induced neuronal loss, pro-inflammatory cytokine secretion, activation of NOD-like receptor protein-3 (NLRP3) inflammasomes and microglia, and TXNIP expression in the hippocampus. GKLF inhibition showed negative effects on LPS-induced microglia activation, as evidenced by reduced pro-inflammatory cytokine secretion and activation of NLRP3 inflammasomes. GKLF bound to Txnip promoter and increased TXNIP expression in LPS-activated microglia. Interestingly, Txnip overexpression reversed the inhibitory effect of Gklf knockdown on microglia activation. These findings indicated that GKLF was involved in microglia activation via TXNIP. This study demonstrates the underlying mechanism of GKLF in the pathogenesis of epilepsy and uncovers that GKLF inhibition may be a therapeutic strategy for epilepsy treatment.

Lithium Chloride Exerts Anti-Inflammatory and Neuroprotective Effects by Inhibiting Microglial Activation in LPS-Induced Retinal Injury.

To explore the anti-inflammatory and neuroprotective effects of lithium chloride (LiCl) in LPS-induced retinal injury. In vitro, primary retinal microglia were pretreated with LiCl and stimulated with lipopolysaccharide (LPS). Pro-inflammatory cytokine production, microglial morphological changes, and inflammation-associated signaling pathways were measured by real-time PCR (RT-PCR), western blotting, and immunofluorescence. Primary retinal neurons were cultured with microglial-derived conditioned medium in the absence or presence of LiCl. Neurotoxicity was evaluated by Cell Counting Kit-8 (CCK-8), terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, and γ-H2AX detection. In vivo, an endotoxin-induced uveitis mice model was established, and each animal was given intraperitoneal injection of LiCl or vehicle. The retinal inflammatory response was measured by hematoxylin and eosin and fluorescent staining, RT-PCR, western blotting, and TUNEL assay. Retinal thickness and function were evaluated by spectral-domain optical coherence tomography and electroretinography. In vitro, LiCl exerted no obvious toxic effects on microglia and significantly decreased proinflammatory factor (inducible nitric oxide synthase, tumor necrosis factor α, interleukin 6) production, inhibited microglial activation in morphology, and suppressed nuclear factor kappa B (NF-κB), Akt, and phosphatidylinositol 3-kinase (PI3K) phosphorylation. Moreover, LiCl promoted retinal neuron survival and reduced cell apoptosis and the expression of γ-H2AX. In vivo, LiCl reduced inflammatory infiltrating cells in the vitreous cavity and decreased proinflammatory cytokine expression in retinas. LiCl suppressed LPS-induced microglial activation, proliferation, and migration. Additionally, LiCl reduced LPS-induced apoptosis of ganglion cells and retinal edema and rescued retinal functional damage. This study demonstrates that LiCl exerts anti-inflammatory and neuroprotective effects by inhibiting microglial activation via the PI3K/Akt/NF-κB pathway in LPS-induced retinal injury. LiCl provides a novel and promising option to treat retinal inflammatory diseases.

Decreased hippocampal microglial cell activation by methanolic extract from the leaves of Mallotus oppositifolius (Geiseler) Müll. Arg contributes to its antidepressant-like effect

Background: Natural remedies with neuroprotective effect are useful in neuroinflammation-associated depression. Although Mallotus oppositifolius extract (MOE) has previously demonstrated antidepressant and anti-inflammatory properties, its neuroprotective effect remains unknown. Thus, the study evaluated the effect of MOE on lipopolysaccharide (LPS)-induced neuroinflammation-associated depression in mice. Methods: Antidepressant-like effect of MOE (10 - 100 mg/kg), fluoxetine (20 mg/kg) and minocycline (50 mg/kg) was established in naïve Institute of Cancer Research (ICR) mice using the forced swim (FST), tail suspension (TST) and open-space swim (OSST) tests. In a separate experiment, FST and TST were used to assess the effect of an 11-day pre-treatment with MOE (10 - 100 mg/kg) or minocycline (50 mg/kg) on LPS (1 mg/kg) neuroinflammation at 6 and 24 hours post LPS. Following these tests, mice were sacrificed and their hippocampi isolated to evaluate their resting and activated microglial cells using Golgi-Cox staining technique. Open-field test was used to assess locomotor activity. Results: MOE, fluoxetine and minocycline significantly reduced immobility in FST, TST and OSST compared to vehicle (p < 0.05), confirming their antidepressant-like effect. Interestingly, MOE’s antidepressant-like effect was faster than fluoxetine and minocycline. Conversely, LPS treatment increased immobility behavior at 6 and 24 hours, suggestive of neuroinflammation-induced depression. Compared to vehicle group, pre-treatment with MOE and minocycline ameliorated LPS-induced hippocampal microglial activation and reversed increased immobility behavior without affecting locomotor activity (p < 0.05). Resting microglial cell count was significantly increased by MOE pre-treatment in the OSST-challenged mice compared to vehicle group (p < 0.01). Similarly, MOE pre-treatment reversed LPS-induced reduction in resting microglial count, and restored resting microglial count to normal levels compared to LPS naive vehicle group. Conclusions: Collectively, the results suggest that MOE exerts neuroprotective effect against LPS-induced neuroinflammation by decreasing the activation of microglia and increasing resting microglial count. This contributes to its antidepressant-like effect.

Ergosterol Isolated from Antrodia camphorata Suppresses LPS-Induced Neuroinflammatory Responses in Microglia Cells and ICR Mice.

Inflammation caused by microglial activation is important in neurodegenerative diseases. In this research, we tried to identify safe and effective anti-neuroinflammatory agents by screening a natural compounds library and found that Ergosterol can inhibit the nuclear factor kappa-light-chain enhancer of the activated B cells (NF-κB) pathway induced by lipopolysaccharide (LPS) in microglia cells. Ergosterol has been reported to be an effective anti-inflammatory agent. Nevertheless, the potential regulatory role of Ergosterol in neuroinflammatory responses has not been fully investigated. We further investigated the mechanism of Ergosterol that regulates LPS-induced microglial activation and neuroinflammatory reactions both in vitro and in vivo. The results showed that Ergosterol can significantly decrease the pro-inflammatory cytokines induced by LPS in BV2 and HMC3 microglial cells, possibly by inhibiting the NF-κB, protein kinase B (AKT), and mitogen-activated protein kinase (MAPK) signaling pathways. In addition, we treated Institute of Cancer Research (ICR) mice with a safe concentration of Ergosterol following LPS injection. Ergosterol treatment significantly decreased microglial activation-associated ionized calcium-binding adapter molecule-1 (IBA-1), NF-κB phosphorylation, and pro-inflammatory cytokine levels. Moreover, Ergosterol pretreatment clearly reduced LPS-induced neuron damage by restoring the expression of synaptic proteins. Our data may provide insight into possible therapeutic strategies for neuroinflammatory disorders.

Endothelial Toll-like receptor 4 is required for microglia activation in the murine retina after systemic lipopolysaccharide exposure

BackgroundClustering of microglia around the vasculature has been reported in the retina and the brain after systemic administration of lipopolysaccharides (LPS) in mice. LPS acts via activation of Toll-like receptor 4 (TRL4), which is expressed in several cell types including microglia, monocytes and vascular endothelial cells. The purpose of this study was to investigate the effect of systemic LPS in the pigmented mouse retina and the involvement of endothelial TLR4 in LPS-induced retinal microglia activation.MethodsC57BL/6J, conditional knockout mice that lack Tlr4 expression selectively on endothelial cells (TekCre−posTlr4loxP/loxP) and TekCre−negTlr4loxP/loxP mice were used. The mice were injected with 1 mg/kg LPS via the tail vein once per day for a total of 4 days. Prior to initiation of LPS injections and approximately 5 h after the last injection, in vivo imaging using fluorescein angiography and spectral-domain optical coherence tomography was performed. Immunohistochemistry, flow cytometry, electroretinography and transmission electron microscopy were utilized to investigate the role of endothelial TLR4 in LPS-induced microglia activation and retinal function.ResultsActivation of microglia, infiltration of monocyte-derived macrophages, impaired ribbon synapse organization and retinal dysfunction were observed after the LPS exposure in C57BL/6J and TekCre−negTlr4loxP/loxP mice. None of these effects were observed in the retinas of conditional Tlr4 knockout mice after the LPS challenge.ConclusionsThe findings of the present study suggest that systemic LPS exposure can have detrimental effects in the healthy retina and that TLR4 expressed on endothelial cells is essential for retinal microglia activation and retinal dysfunction upon systemic LPS challenge. This important finding provides new insights into the role of microglia–endothelial cell interaction in inflammatory retinal disease.