LPS-induced Microglial Activation Research Articles

Tert-butylhydroquinone prevents neuroinflammation and relieves depression via regulation of NLRP3 signaling in mice

Depression is a common psychiatric disorder, which seriously affects people’s health and quality of life. Current treatments, which mainly focus on neurotransmitter levels, are not effective in many patients. Recent studies have shown that neuroinflammation has certain correlation with the pathogenesis of depression. Tert-butylhydroquinone (TBHQ) is an antioxidant with an anti-inflammatory effect. The present study evaluated the effects of TBHQ on the improvement of depression-like behaviors induced by lipopolysaccharide (LPS) in mice and its possible mechanism. Behavioral test results showed that TBHQ treatment could significantly improve the depression-like behaviors of mice. Western blot results showed that TBHQ treatment inhibited the protein expression of NLRP3, Caspase-1, IL-1β, and IL-18, which induced by LPS. Immunofluorescence staining results showed that TBHQ treatment inhibited the activation of microglia induced by LPS. These results suggested that, by inhibiting LPS-induced neuroinflammation and microglia activation, TBHQ could effectively improve LPS-induced inflammation-related depression-like behavior through modulating the NLRP3 signaling pathway.

Natural bear bile powder suppresses neuroinflammation in lipopolysaccharide-treated mice via regulating TGR5/AKT/NF-κB signaling pathway

Ethnopharmacological relevanceAccording to the Tang Dynasty classics Dietetic Material Medica and the Ming Dynasty classics Compendium of Materia Medica records, bear bile powder (BBP) has been used to treat a variety of diseases, such as febrile seizures, the pathogenesis of which is associated to neuroinflammation. However, the mechanism of BBP on alleviating neuroinflammation remains unclear. Aims of the studyMicroglia can be activated by peripheral lipopolysaccharide (LPS) and play an important role in the pathogenesis of neuroinflammation. The purpose of this study is to investigate the effects and mechanism of BBP in inhibiting LPS-induced microglia inflammation in vitro and in vivo. Materials and methodsThe anti-microglia inflammatory effects and mechanism of BBP were assessed in LPS-treated BV2 microglial cells and in LPS-treated mice. The mRNA expression levels of the inflammatory factor and the protein expressions of cyclooxygenase-2 (COX2), inducible nitric oxide synthase (iNOS), takeda G-protein coupled receptor 5 (TGR5), nuclear factor-κB (NF-κB), inhibitor of NF-κB (IκBɑ), protein kinase B (AKT) in BV2 cells, mouse hippocampus and cortex were detected. The NF-κB transcription activity and NF-κB nuclear translocation were observed. ResultsOur findings showed that BBP reduces branched process retraction and NO in LPS-treated BV2 cells, inhibits the protein expression of ionized calcium binding adaptor molecule 1 in the hippocampus of LPS-treated mice. Moreover, we observed that BBP decreases tumor necrosis factor α, interleukin (IL)-6 and IL-1β mRNA levels, deceases iNOS and COX-2 protein levels, increases TGR5 protein levels, suppresses the phosphorylation of AKT, NF-κB and IκBɑ protein in microglia both in vitro and in vivo. Further, we found that triamterene, the inhibitor of TGR5, abolishes the effects of BBP in LPS- treated BV2 cells. ConclusionBBP inhibits LPS-induced microglia activation, and the mechanism of its action is partly through TGR5/AKT/NF-κB signaling pathway.

Blueberry treatment administered before and/or after lipopolysaccharide stimulation attenuates inflammation and oxidative stress in rat microglial cells

ABSTRACT Microglia are key regulators of inflammation and oxidative stress (OS) in the CNS. Microglia activation can lead to chronic inflammation, OS, and neurodegeneration. Blueberries (BB) reduce inflammation and OS when administered to microglia before stressors such as lipopolysaccharide (LPS), but the therapeutic value of BBs administered after activation by stressors has not been examined. Therefore, this study investigated the differential effects of pre-, post-, and pre-/post-BB on inflammation and OS in LPS-activated microglia. Rat microglia were pretreated with BB (0.5 mg/mL) or control media (C) for 24 hours, incubated overnight with LPS (0 or 200 ng/mL), and post-treated with BB or C for 24 hours. Biomarkers of inflammation (e.g. nitrite [NO2 −], tumor necrosis factor-ɑ [TNFɑ], inducible nitric oxide synthase [iNOS], cyclooxygenase-2 [COX-2], phosphorylated IκB-α [pIκB-ɑ]) and OS (e.g. NADPH oxidase [NOX2]) were assessed. LPS increased NO2 −, TNFɑ, COX-2, iNOS, pIκB-ɑ, and NOX2 compared to non-stressed conditions (P < 0.05), however BB before and/or after LPS significantly reduced these markers compared to no BB (P < 0.05). Pre-BB was more effective than post-BB at reducing LPS-induced NO2 −, TNFɑ, and COX-2 (P < 0.05). Pre-BB was also more effective than pre-/post-BB at attenuating LPS-induced NO2 − and TNFɑ (P < 0.05). All BB treatments were equally effective in reducing LPS-induced iNOS, pIκB-ɑ, and NOX2. Results suggest that BBs can target the downstream events of LPS-induced microglial activation and prevent stressor-induced neuroinflammation and OS. Furthermore, BBs may not need to be present prior to microglial activation for beneficial effects, suggesting that dietary interventions may be effective even after initiation of disease processes. Graphical . Cascade of inflammatory and OS-inducing events associated with self-propelling microglial activation by LPS and the effects of blueberry (0.5 mg/mL) administered before and/or after LPS on these processes (blue arrows). BB, blueberry; COX2, cyclooxygenase-2; IκB-ɑ, inhibitor kappa-B-ɑ; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; NF-κB, nuclear factor kappa-B; NO, nitric oxide; NOX2, NADPH oxidase; OS, oxidative stress; ROS, reactive oxygen species; TNFɑ, tumor necrosis factor-ɑ

Anti-Neuroinflammatory Effects of a Semi-Synthetic Isoorientin-Based Glycogen Synthase Kinase-3β Inhibitor in Lipopolysaccharide-Activated Microglial Cells.

Neuroinflammation contributes to the pathogenesis of several neurodegenerative disorders. Glycogen synthase kinase-3β (GSK-3β) regulates the release of proinflammatory cytokines and promotes inflammatory responses in immune cells. Microglia are the resident mononuclear immune cells of the central nervous system. Here, we investigated the anti-neuroinflammatory effects of (2S,3S,4R,5R,6S)-6-(2-(3,4-dimethoxyphenyl)-5,7-dimethoxy-4-oxo-4H-chromen-6-yl)-3,4,5-trihydroxy-N-((S)-1,1,1-trifluoropropan-2-yl)tetrahydro-2H-pyran-2-carboxamide (TFGF-18), a semisynthetic GSK-3β inhibitor, in lipopolysaccharide (LPS) activation of spontaneously immortalized SIM-A9 microglial cells and of mouse cortical microglia. TFGF-18 at 2.5 μM concentration inhibited LPS-induced production of nitric oxide by 56.3% and the proinflammatory cytokines TNF-α and IL-1β by 28.3 and 59.2% in SIM-A9 cells, respectively, relative to the LPS treatment control group. Pretreatment of mouse primary microglial cells with TFGF-18 at 2.5 μM concentration led to a reduction of 58.7% in TNF-α+ microglial cells at 24 h post-LPS stimulation. The migration of LPS-activated SIM-A9 cells was also reduced by 26.7% with pretreatment of TFGF-18 in a scratch assay. Analyses of signaling pathways demonstrated that TFGF-18 led to the suppression of LPS-induced GSK-3β activation and p65/NF-κB activity. Furthermore, the co-culture of SIM-A9 with SH-SY5Y neuroblastoma cells showed the suppression of TFGF-18 to microglia-mediated neurotoxicity in vitro. The findings indicate strong inhibitory effects of TFGF-18 on LPS-induced microglia activation via regulation of GSK-3β and downstream p65/NF-κB signaling. The results suggest a potential role of TFGF-18 in neuroprotection via its anti-neuroinflammatory effect.

Characterization of the Leucocyte Immunoglobulin-like Receptor B4 (Lilrb4) Expression in Microglia.

Simple SummaryIn the present study, we provide a detailed characterization of Lilrb4 expression in microglia and peripheral myeloid cells. Our data demonstrate that LILRB4 is a marker for microglia activation, as evidenced by upregulation after lipopolysaccharide treatment and inhibition of microglial TGFβ signaling. Moreover, we provide evidence that microglia express low levels of Lilrb4 in vivo and high levels in vitro, and we clearly demonstrate that LILRB4 is also expressed by bone marrow-derived monocytes and, to a greater extent, by peritoneal macrophages, defining LILRB4 as a surface marker of myeloid cells and not as a microglia-specific marker.As resident innate immune cells of the CNS, microglia play important essential roles during physiological and pathological situations. Recent reports have described the expression of Lilrb4 in disease-associated and aged microglia. Here, we characterized the expression of Lilrb4 in microglia in vitro and in vivo in comparison with bone marrow-derived monocytes and peritoneal macrophages in mice. Using BV2 cells, primary microglia cultures as well as ex vivo isolated microglia and myeloid cells in combination with qPCR and flow cytometry, we were able to provide a comprehensive characterization of Lilrb4 expression in distinct mouse myeloid cells. Whereas microglia in vivo display low expression of Lilrb4, primary microglia cultures present high levels of surface LILRB4. Among the analyzed peripheral myeloid cells, peritoneal macrophages showed the highest expression levels of Lilrb4. Moreover, LPS treatment and inhibition of microglial TGFβ signaling resulted in significant increases of LILRB4 cell surface levels. Taken together, our data indicate that LILRB4 is a reliable surface marker for activated microglia and further demonstrate that microglial TGFβ signaling is involved in the regulation of Lilrb4 expression during LPS-induced microglia activation.

Inhibition or Deletion of Hydroxylases-Prolyl-4-Hydroxyases 3 Alleviates Lipopolysaccharide-induced Neuroinflammation and Neurobehavioral Deficiency

It is well known that neuroinflammation plays a key role in neurodegenerative diseases. Hypoxia-inducible factor (HIF) and its hydroxylases-Prolyl-4-hydroxyases (PHDs) have been found to modulate the inflammatory processes. Here, the effects of PHDs enzyme onlipopolysaccharide-induced neuroinflammation and neurocognitive deficits were investigated. BV2 microglia cells were stimulated by LPS (1 μg/ml) as neuroinflammation model in vitro. Dimethyloxalylglycine (DMOG, 100 μM) and PHD3-siRNA were used to suppress the expression of PHD3. In vivo, mice received consecutive intraperitoneal injection of LPS (500 μg/kg) for 7 days, and intraperitoneal injection of DMOG (100 mg/kg) was applied 1 h before LPS at the same days. Several neurobehavioral tests (Open field, Novel object recognition and Morris water maze) were used to measure cognitive function. RT-qPCR and Western blotting were used to investigate the expression of inflammatory cytokines, HIF-PHDs protein. Metabolic reprogramming was measured by seahorse method. The results revealed that LPS induced neuroinflammation and PHD3 expression in vivo and vitro. DMOG and PHD3knockout decreased expression of inflammatory cytokines and improved the metabolic reprogramming caused by LPS treatment. Furthermore, pretreatment of DMOG reversed learning and memory deficits in systemic LPS-exposed mice through anti-neuroinflammation, which is independent of DMOG angiogenesis.These findings suggested that PHD3 may mediate LPS-induced microglial activation and neuroinflammation-associated neurobehavioral deficits.

Epimedii Folium and Curculiginis Rhizoma ameliorate lipopolysaccharides-induced cognitive impairment by regulating the TREM2 signaling pathway

Ethnopharmacological relevanceNeuroinflammation induced by microglia is closely related to a variety of neurodegenerative diseases including Alzheimer's disease (AD). Previous study has found that aqueous extract of Epimedii Folium and Curculiginis Rhizoma (EX) had anti-inflammatory effect on AD by activating the NLRP3 inflammasome and inhibiting NF-κB/MAPK pathway. However, whether the anti-neuroinflammatory effect of EX is related to microglia or not remains unclear. Aim of the studyThe present study aimed to investigate the protective effect of EX on cognitive impairment induced by LPS and explore the underlying mechanism of EX. Materials and methodsHigh performance liquid chromatography-tandem mass spectrometry (HPLC-MS) was performed to qualify the major components of EX, EX in the serum and cerebrospinal fluid. To evaluate the anti-inflammatory effects of EX in vivo, the mice were orally administrated with EX (2.34, 4.68 g kg−1•d−1) for 28 days before cotreatment with LPS (1 mg kg−1•d−1, i.p.). The leaning and memory abilities of mice were examined by Morris water maze test. The expression of inflammatory related proteins and the activation of microglia were detected by ELISA, immunofluorescence, real-time PCR and Western blotting. ResultsHPLC-MS analysis confirmed and quantified 9 components in EX, 5 components in the serum and 4 components in the cerebrospinal fluid. In a LPS-induced neuroinflammatory mouse model, EX was found to exert anti-inflammatory activity by reducing the levels of tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta (IL-1β), regulating the expression of different phenotypes of microglia, and increasing the expression of proteins related with TREM2 in the hippocampus tissue. Moreover, LPS-induced microglia activation was markedly attenuated in the hippocampus. ConclusionsThese findings demonstrate that EX exerts anti-neuroinflammatory effects via reducing the production of inflammatory mediators, regulating the conversion of microglia and activating the proteins related with TREM2. EX might become a novel herb pairs to treat neuroinflammatory diseases.

PACAP and VIP Modulate LPS-Induced Microglial Activation and Trigger Distinct Phenotypic Changes in Murine BV2 Microglial Cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP) are two structurally related immunosuppressive peptides. However, the underlying mechanisms through which these peptides regulate microglial activity are not fully understood. Using lipopolysaccharide (LPS) to induce an inflammatory challenge, we tested whether PACAP or VIP differentially affected microglial activation, morphology and cell migration. We found that both peptides attenuated LPS-induced expression of the microglial activation markers Iba1 and iNOS (### p < 0.001), as well as the pro-inflammatory mediators IL-1β, IL-6, Itgam and CD68 (### p < 0.001). In contrast, treatment with PACAP or VIP exerted distinct effects on microglial morphology and migration. PACAP reversed LPS-induced soma enlargement and increased the percentage of small-sized, rounded cells (54.09% vs. 12.05% in LPS-treated cells), whereas VIP promoted a phenotypic shift towards cell subpopulations with mid-sized, spindle-shaped somata (48.41% vs. 31.36% in LPS-treated cells). Additionally, PACAP was more efficient than VIP in restoring LPS-induced impairment of cell migration and the expression of urokinase plasminogen activator (uPA) in BV2 cells compared with VIP. These results suggest that whilst both PACAP and VIP exert similar immunosuppressive effects in activated BV2 microglia, each peptide triggers distinctive shifts towards phenotypes of differing morphologies and with differing migration capacities.

PDCD4 Simultaneously Promotes Microglia Activation via PDCD4-MAPK-NF-κB Positive Loop and Facilitates Neuron Apoptosis During Neuroinflammation.

Neuroinflammation and neuron injury are common features of the central nervous system (CNS) diseases. It is of great significance to identify their shared key regulatory molecules and thus explore the potential therapeutic targets. Programmed cell death factor 4 (PDCD4), an apoptosis-related molecule, extensively participates in tumorigenesis and inflammatory diseases, but its expression and biological function during CNS neuroinflammation remain unclear. In the present study, utilizing the lipopolysaccharide (LPS)-induced neuroinflammation model in mice, we reported an elevated expression of PDCD4 both in injured neurons and activated microglia of the inflamed brain. A similar change in PDCD4 expression was observed in vitro in the microglial activation model. Silencing PDCD4 by shRNA significantly inhibited the phosphorylation of MAPKs (p38, ERK, and JNK), prevented the phosphorylation and nuclear translocation of NF-κB p65, and thus attenuated the LPS-induced microglial inflammatory activation. Interestingly, LPS also required the MAPK/NF-κB signaling activation to boost PDCD4 expression in microglia, indicating the presence of a positive loop. Moreover, a persistent elevation of PDCD4 expression was detected in the H2O2-induced neuronal oxidative damage model. Knocking down PDCD4 significantly inhibited the expression of pro-apoptotic proteins BAX and Cleaved-PARP, suggesting the proapoptotic activity of PDCD4 in neurons. Taken together, our data indicated that PDCD4 may serve as a hub regulatory molecule that simultaneously promotes the microglial inflammatory activation and the oxidative stress-induced neuronal apoptosis within CNS. The microglial PDCD4-MAPK-NF-κB positive feedback loop may act as pivotal signaling for neuroinflammation which subsequently exaggerates neuronal injury, and thus may become a potential therapeutic target for neuroinflammatory diseases.

The inhibitory effects of Aster yomena extract on microglial activation-mediated inflammatory response and pain by modulation of the NF-κB and MAPK signaling pathways

• Aster yomena extract (AYE) inhibited LPS-induced microglial activation in BV2 cells. • LPS-induced NF-κB and MAPK signaling pathways were inhibited via treatment of AYE. • CFA-induced inflammatory pain were alleviated by oral administration of AYE in mouse. • Chlorogenic acid and t-Ferulic acid were identified as the major active components. This study investigated the inhibitory effect and underlying mechanisms of Aster yomena extract (AYE) on microglial activation-mediated inflammatory pain. Lipopolysaccharide (LPS)-induced microglial activation exhibited increased expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and pro-inflammatory cytokines. LPS-induced microglial activation was suppressed by AYE treatment via inhibition of the NF-κB and MAPK signaling pathways. To investigate the protective effects of AYE on inflammatory pain, complete Freund’s adjuvant (CFA) was injected into the right paw of mice. Pain hypersensitivity and microglial activation in the spinal cord were markedly alleviated by oral administration of AYE. HPLC was performed and identified chlorogenic acid (CGA) and t-Ferulic acid (FRA) as the major active components in AYE. Both components showed inhibitory effects on LPS-induced microglial activation in BV2 cells. Our study demonstrates that AYE inhibits microglial activation-mediated inflammatory pain and that AYE has potential application as a natural analgesic.

Achyranthes bidentata polypeptide alleviates neurotoxicity of lipopolysaccharide-activated microglia via PI3K/Akt dependent NOX2/ROS pathway.

BackgroundAchyranthes bidentata polypeptide fraction k (ABPPk) has been shown to protect ischemic stroke and Parkinson’s disease, and can inhibit neuroinflammation in lipopolysaccharide (LPS)-activated BV2 microglia. However, the effect of ABPPk responsible for alleviating microglial neurotoxicity remains unknown.MethodsPrimary microglia were cultured to investigate the effect of ABPPk on LPS-induced neuroinflammation. Microglia conditioned medium (MCM) was collected to stimulate primary cortical neurons and then the neuronal viability, lactate dehydrogenase (LDH) release, intracellular calcium influx, mitochondria membrane potential (MMP) were assessed, respectively. Postnatal day 5 Sprague-Dawley rat pups were intracerebral injected with LPS to establish an LPS-induced brain injury model. Double immunohistochemical staining for NeuN and Iba1 was performed to evaluate the effects of ABPPk on LPS-induced neuronal damage and microglial activation. TUNEL assay was conducted to detect cell apoptosis in LPS-injected brain. The effect of ABPPk on LPS-induced NADPH oxidase 2 (NOX2) expression and reactive oxygen species (ROS) production as well as the phosphorylation of protein kinase B (Akt) was detected. Moreover, LY294002 (a specific PI3K inhibitor) and SC79 (a specific Akt activator) were used to further reveal the underlying mechanism.ResultsABPPk pretreatment inhibited LPS-induced NLRP3 and cleaved caspase 1 expressions as well as the mRNA levels of IL-1β and IL-18. Moreover, ABPPk inhibited glutamate release from LPS-activated microglia in a concentration-dependent manner. MCM stimulation resulted in characteristic neuronal toxicity including neuronal viability decrease, LDH release increase, calcium overload, and MMP drop. However, ABPPk pretreatment on microglia reduced the neurotoxicity of MCM. LPS intracerebral injection led to neuronal damage, microglial activation and cell apoptosis in the brain, while ABPPk preadministration significantly inhibited LPS-induced microglial activation and alleviated the brain injury. ABPPk pretreatment inhibited NOX2 expression and ROS production in LPS-activated primary microglia. Signaling pathway analysis showed that ABPPk promoted the phosphorylation of Akt in microglia and inhibited LPS-upregulated NOX2 expression, ROS production, and glutamate release, which can be eliminated by pharmacological inhibition of PI3K. Specific Akt activator could inhibit LPS-induced NOX2 expression, ROS production and glutamate release.ConclusionsThe present results suggested that ABPPk could alleviate neurotoxicity of LPS-activated microglia via PI3K/Akt dependent NOX2/ROS pathway.

Donepezil Regulates LPS and Aβ-Stimulated Neuroinflammation through MAPK/NLRP3 Inflammasome/STAT3 Signaling.

The acetylcholinesterase inhibitors donepezil and rivastigmine have been used as therapeutic drugs for Alzheimer’s disease (AD), but their effects on LPS- and Aβ-induced neuroinflammatory responses and the underlying molecular pathways have not been studied in detail in vitro and in vivo. In the present study, we found that 10 or 50 μM donepezil significantly decreased the LPS-induced increases in the mRNA levels of a number of proinflammatory cytokines in BV2 microglial cells, whereas 50 μM rivastigmine significantly diminished only LPS-stimulated IL-6 mRNA levels. In subsequent experiments in primary astrocytes, donepezil suppressed only LPS-stimulated iNOS mRNA levels. To identify the molecular mechanisms by which donepezil regulates LPS-induced neuroinflammation, we examined whether donepezil alters LPS-stimulated proinflammatory responses by modulating LPS-induced downstream signaling and the NLRP3 inflammasome. Importantly, we found that donepezil suppressed LPS-induced AKT/MAPK signaling, the NLRP3 inflammasome, and transcription factor NF-kB/STAT3 phosphorylation to reduce neuroinflammatory responses. In LPS-treated wild-type mice, a model of neuroinflammatory disease, donepezil significantly attenuated LPS-induced microglial activation, microglial density/morphology, and proinflammatory cytokine COX-2 and IL-6 levels. In a mouse model of AD (5xFAD mice), donepezil significantly reduced Aβ-induced microglial and astrocytic activation, density, and morphology. Taken together, our findings indicate that donepezil significantly downregulates LPS- and Aβ-evoked neuroinflammatory responses in vitro and in vivo and may be a therapeutic agent for neuroinflammation-associated diseases such as AD.

Systemic LPS-induced microglial activation results in increased GABAergic tone: A mechanism of protection against neuroinflammation in the medial prefrontal cortex in mice

Neuroinflammation with excess microglial activation and synaptic dysfunction are early symptoms of most neurological diseases. However, how microglia-associated neuroinflammation regulates synaptic activity remains obscure. We report here that acute neuroinflammation induced by intraperitoneal injection of lipopolysaccharide (LPS) results in cell-type-specific increases in inhibitory postsynaptic currents in the glutamatergic, but not the GABAergic, neurons of medial prefrontal cortex (mPFC), coinciding with excessive microglial activation. LPS causes upregulation in levels of GABAAR subunits, glutamine synthetase and vesicular GABA transporter, and downregulation in brain-derived neurotrophic factor (BDNF) and its receptor, pTrkB. Blockage of microglial activation by minocycline ameliorates LPS-induced abnormal expression of GABA signaling-related proteins and activity of synaptic and network. Moreover, minocycline prevents the mice from LPS-induced aberrant behavior, such as a reduction in total distance and time spent in the centre in the open field test; decreases in entries into the open arm of elevated-plus maze and in consumption of sucrose; increased immobility in the tail suspension test. Furthermore, upregulation of GABA signaling by tiagabine also prevents LPS-induced microglial activation and aberrant behavior. This study illustrates a mode of bidirectional constitutive signaling between the neural and immune compartments of the brain, and suggests that the mPFC is an important area for brain–immune system communication. Moreover, the present study highlights GABAergic signaling as a key therapeutic target for mitigating neuroinflammation-induced abnormal synaptic activity in the mPFC, together with the associated behavioral abnormalities.

Gα13 Contributes to LPS-Induced Morphological Alterations and Affects Migration of Microglia.

Microglia are the resident immune cells of the CNS that are activated in response to a variety of stimuli. This phenotypical change is aimed to maintain the local homeostasis, also by containing the insults and repair the damages. All these processes are tightly regulated and coordinated and a failure in restoring homeostasis by microglia can result in the development of neuroinflammation that can facilitate the progression of pathological conditions. Indeed, chronic microglia activation is commonly recognized as a hallmark of many neurological disorders, especially at an early stage. Many complex pathways, including cytoskeletal remodeling, are involved in the control of the microglial phenotypical and morphological changes that occur during activation. In this work, we focused on the small GTPase Gα13 and its role at the crossroad between RhoA and Rac1 signaling when microglia is exposed to pro-inflammatory stimulation. We propose the direct involvement of Gα13 in the cytoskeletal rearrangements mediated by FAK, LIMK/cofilin, and Rac1 during microglia activation. In fact, we show that Gα13 knockdown significantly inhibited LPS-induced microglial cell activation, in terms of both changes in morphology and migration, through the modulation of FAK and one of its downstream effectors, Rac1. In conclusion, we propose Gα13 as a critical factor in the regulation of morphological and functional properties of microglia during activation, which might become a target of intervention for the control of microglia inflammation.

Long Noncoding RNA SNHG1 Promotes Lipopolysaccharide-Induced Activation and Inflammation in Microglia via Targeting miR-181b

Introduction: Long noncoding RNA small nuclear host gene 1 (SNHG1) was involved in neuroinflammation in microglial BV-2 cells; however, its interaction with microRNA (miR)-181b in lipopolysaccharide (LPS)-induced BV-2 cells remained poor. Methods: BV-2 cells were treated with LPS and then were subjected to observation on morphology and immunofluorescence staining. After transfection, levels of inflammatory cytokines interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were determined with enzyme-linked immunosorbent assay (ELISA). The potential binding sites between SNHG1 and miR-181b were confirmed using dual-luciferase reporter assay. Quantitative real-time polymerase chain reaction and Western blot were applied for detecting the mRNA and protein expressions of proinflammatory cytokines, ionized calcium-binding adapter molecule 1 (Iba1), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS). Results: LPS led to the morphological changes and activation of BV-2 cells. The transfection of SNHG1 overexpression vector further promoted LPS-induced SNHG1 upregulation, inflammatory cytokines (IL-1β, IL-6, and TNF-α) generation and Iba-1, COX-2, and iNOS expressions, whereas silencing SNHG1 did the opposite. miR-181b functions as a downstream miRNA of SNHG1. In LPS-treated cells, the inhibition of miR-181b induced by SNHG1 promoted inflammation response and the expressions of Iba-1, COX-2, and iNOS. Conclusion: SNHG1 was involved in LPS-induced microglial activation and inflammation response via targeting miR-181b, providing another evidence of the roles of SNHG1 implicated in neuroinflammation of microglia.

GPER and IGF-1R mediate the anti-inflammatory effect of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in rats

Neuroinflammation plays an important role in the pathogenesis of Parkinson's disease (PD). Genistein is an estrogen-like phytoestrogen that can exert biological effects via the crosstalk of estrogen receptor and insulin-like growth factor 1 receptor (IGF-1R). The present study aimed to evaluate the involvement of G protein-coupled estrogen receptor (GPER) and IGF-1R in the anti-inflammatory effects of genistein against lipopolysaccharide (LPS)-induced nigrostriatal injury in ovariectomized rats. Our results showed that genistein treatment could ameliorate the apomorphine-induced rotational behavior in LPS-induced inflammatory PD rat model. Genistein attenuated LPS-induced decrease of the contents of dopamine (DA) and its metabolites in striatum as well as the loss of tyrosine hydroxylase-immunoreactive (TH-IR) neurons in the substantia nigra (SN) of the lesioned side, which could be blocked by GPER antagonist G15 or IGF-1R antagonist JB1. Meanwhile, G15 or JB1 could attenuate the anti-inflammatory effects of genistein in LPS-induced microglial activation and production of tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, genistein could inhibit the LPS-induced phosphorylation of p38, JNK, ERK and IκB in the lesioned side of SN and these effects could also be blocked by G15 or JB1. Taken together, our data provide the first evidence that genistein can inhibit the increase of microglia and protect dopaminergic neurons at least in part via GPER and IGF-1R signaling pathways in ovariectomized PD rat model.

Propofol Suppresses Microglia Inflammation by Targeting TGM2/NF-κB Signaling.

Background Propofol is a known intravenous hypnotic drug used for induction and maintenance of sedation and general anesthesia. Emerging studies also reveal a neuroprotective effect of propofol in diverse diseases of neuronal injuries via modulating microglia activation. In this study, we aimed to uncover the downstream targets of propofol in this process. Methods RNA sequencing analysis to identify genes implicated in the propofol-mediated neuroprotective effect. Quantitative real-time PCR, enzyme-linked immunosorbent assay, and Western blotting analysis were performed to analyze inflammatory gene expression, cytokine levels, and TGM2. BV2 cells and primary microglia were used for functional verification and mechanism studies. Results The multifunctional enzyme transglutaminase 2 (TGM2) was identified as a putative functional mediator of propofol. TGM2 was significantly upregulated in lipopolysaccharide- (LPS-) primed BV2 cells. Genetic silencing of TGM2 abolished LPS-induced microglial activation. Notably, gain-of-function experiments showed that the proinflammatory effects of TGM2 were dependent on its GTP binding activity instead of transamidase activity. Then, TGM2 was revealed to activate the NF-κB signaling pathway to facilitate microglial activation. Propofol can inhibit TGM2 expression and NF-κB signaling in BV2 cells and primary microglia. Ectopic expression of TGM2 or constitutively active IKKβ (CA-IKKβ) can compromise propofol-induced anti-inflammatory effects. Conclusions Our findings suggest that TGM2-mediated activation of NF-κB signaling is an important mechanism in the propofol-induced neuroprotective effect that prevents microglial activation.

Naloxone Protects against Lipopolysaccharide-Induced Neuroinflammation and Microglial Activation via Inhibiting ATP-Sensitive Potassium Channel.

Aim The aim of this study was to evaluate the anti-inflammatory effects and underlying mechanism of naloxone on lipopolysaccharide- (LPS-) induced neuronal inflammation and microglial activation. Methods LPS-treated microglial BV-2 cells and mice were used to investigate the anti-inflammatory effects of naloxone. Results The results showed that naloxone dose-dependently promoted cell proliferation in LPS-induced BV-2 cells, downregulated the expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and proinflammatory enzymes iNOS and COX-2 as well as the expression of free radical molecule NO, and reduced the expression of Iba-1-positive microglia in LPS-stimulated BV-2 cells and mouse brain. Moreover, naloxone improved LPS-induced behavior degeneration in mice. Mechanically, naloxone inhibited LPS-induced activation in the ATP-sensitive potassium (KATP) channel. However, the presence of glibenclamide (Glib), an antagonist of KATP channel, ameliorated the suppressive effects of naloxone on inflammation and microglial activation. Conclusion Naloxone prevented LPS-induced neuroinflammation and microglial activation partially through the KATP channel. These findings might highlight the potential of naloxone in neuroinflammation therapy.

Malate-Aspartate Shuttle Plays an Important Role in LPS-Induced Neuroinflammation of Mice Due to its Effect on STAT3 Phosphorylation.

Neuroinflammation is a key pathological factor in numerous neurological disorders. Cumulating evidence has indicated critical roles of NAD+/NADH metabolism in multiple major diseases, while the role of malate-aspartate shuttle (MAS) - a major NADH shuttle - in inflammation has remained unclear. In this study we investigated the roles of MAS in LPS-induced neuroinflammation both in vivo and in vitro. Immunofluorescence staining, Western blot assay and Real-time PCR assays were conducted to determine the activation of Iba-1, the protein levels of iNOS and COX2 and the mRNA levels of IL-1β, IL-6, and TNF-α in vivo, showing that both pre-treatment and post-treatment of aminooxyacetic acid (AOAA) - an MAS inhibitor - profoundly decreased the LPS-induced neuroinflammation in mice. BV2 microglia was also used as a cellular model to investigate the mechanisms of this finding, in which such assays as Western blot assay and nitrite assay. Our study further indicated that AOAA produced its effects on LPS-induced microglial activation by its effects on MAS: Pyruvate treatment reversed the effects of AOAA on the cytosolic NAD+/NADH ratio, which also restored the LPS-induced activation of the AOAA-treated microglia. Moreover, the lactate dehydrogenase (LDH) inhibitor GSK2837808A blocked the effects of pyruvate on the AOAA-produced decreases in both the cytosolic NAD+/NADH ratio and LPS-induced microglial activation. Our study has further suggested that AOAA produced inhibition of LPS-induced microglial activation at least partially by decreasing STAT3 phosphorylation. Collectively, our findings have indicated AOAA as a new and effective drug for inhibiting LPS-induced neuroinflammation. Our study has also indicated that MAS is a novel mediator of LPS-induced neuroinflammation due to its capacity to modulate LPS-induced STAT3 phosphorylation, which has further highlighted a critical role of NAD+/NADH metabolism in inflammation.

Isoginkgetin treatment attenuated lipopolysaccharide-induced monoamine neurotransmitter deficiency and depression-like behaviors through downregulating p38/NF-κB signaling pathway and suppressing microglia-induced apoptosis.

Background:Microglia activation-induced neuroinflammation may contribute to the etiology of depression. Podocarpus nagi containing high concentration of isoginkgetin could effectively treat mental diseases in ancient times. However, the therapeutic role, peculiarly in the brain–immune modulation in depression is still unclear. This study aimed to determine effects of isoginkgetin on lipopolysaccharide (LPS)-induced depression-like changes. Furthermore, its modulation on the p38/nuclear factor-kappa B (NF-κB) pathway in LPS-activated microglia was evaluated.Methods:Adult Kunming mice were intraperitoneally injected vehicle or isoginkgetin (4 mg/kg) daily for 14 days before saline or LPS (0.83 mg/kg) administration. Depression-like behavior, neurotransmitter levels, and markers of neuroinflammation were determined. Isoginkgetin effect on LPS-induced microglial activation was then assessed in BV2 cells. Finally, conditioned medium (CM) derived from isoginkgetin-treated BV2 cells was co-cultured with SH-SY5Y cells for 24 h. Cell viability and apoptosis were evaluated.Results:LPS significantly induced helplessness and anxiety, which were associated with decreased 5-HT, noradrenaline, and dopamine concentrations. Meanwhile, LPS increased microglia M1 hallmark Iba1 expression and serum interleukin (IL)-1β concentration. These changes were attenuated by isoginkgetin treatment. In vitro, isoginkgetin markedly suppressed the production of IL-1β, IL-6, tumor necrosis factor-alpha, cyclooxygenase-2, inducible nitric oxide, and reactive oxygen species, which are released from LPS-stimulated BV2 cells. More interestingly, CM from isoginkgetin-treated BV2 cells significantly alleviated SH-SY5Y cell apoptosis and restored cell viability compared to LPS-treated group through the inhibition of p38/NF-κB signaling pathway.Conclusion:These data demonstrate that isoginkgetin is an effective therapeutic agent for depression-like behaviors and neuropathological changes via potent anti-inflammatory property.