The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

Apigenin exhibits anti-inflammatory effects in LPS-stimulated BV2 microglia through activating GSK3β/Nrf2 signaling pathway

Abstract

Objective: Apigenin is a natural flavonoid compound extracted from Matricaria chamomilla. We evaluated the anti-inflammatory effects of apigenin in this study using the Lipopolysaccharide (LPS)-stimulated BV2 microglia.

Methods: BV2 cells were treated with apigenin for 1?h and then treated with LPS. The inflammatory cytokine productions were tested by qRT-PCR and ELISA. The expression of GSK3β, Nrf2, and NF-κB signaling pathways were measured by western blot analysis.

Results: Apigenin significantly attenuated LPS-induced TNF-α, IL-1β, and IL-6 production. Apigenin suppressed LPS-induced NF-κB activation. Furthermore, GSK3β, Nrf2, and HO-1 were concentration-dependently increased by apigenin. The suppression of apigenin on LPS-induced inflammatory response and NF-κB activation were prevented when Nrf2 was knocked out or by GSK3β inhibitor.

Conclusions: Collectively, apigenin suppressed LPS-induced microglia activation via activating GSK3β/Nrf2 signaling pathway.     

Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage through inhibition of microglia activation and proinflammatory factors generation

Activation of microglia and consequent release of proinflammatory factors, are believed to contribute to neurodegeneration in Parkinson's disease (PD). Hence, identification of compounds that prevent microglial activation is highly desirable in the search for therapeutic agents for inflammation-mediated neurodegenerative diseases. In this study, we reported that biochanin A, one of the predominant isoflavones in Trifolium pratense, attenuated lipopolysaccharide (LPS)-induced decrease in dopamine uptake and the number of dopaminergic neurons in a dose-dependent manner in rat mesencephalic neuron-glia cultures. Moreover, biochanin A also significantly inhibited LPS-induced activation of microglia and production of tumor necrosis factor-alpha, nitric oxide and superoxide in mesencephalic neuron-glia cultures and microglia-enriched cultures. This study suggested for the first time that biochanin A protected dopaminergic neurons against LPS-induced damage through inhibition of microglia activation and proinflammatory factors generation.     

Effect of sevoflurane treatment on microglia activation,NF-kB and MAPK activities

Microglia activation has been implicated in neurodegenerative disease. Sevoflurane is fluorinated methyl isopropyl ether with anti-inflammatory activity. In this study, we evaluated the potential effects of sevoflurane on lipopolysaccharides (LPS)-induced microglia activation. We treated primary microglia cells with sevoflurane prior to LPS treatment and tested the microglia migration, the productions of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-6 and interleukin-8. We also explored the effects of sevoflurane on NF-κB and p38 MAPK activation. Finally, we examined the effect of sevoflurane on cytokines production in rat brain. Sevoflurane significantly reduced LPS-induced microglial migration. Sevoflurane significantly decreased the production of pro-inflammatory cytokines both in vitro and in vivo. Sevoflurane attenuated activations of NF-κB and MAPK signaling pathways. Sevoflurane treatment decreased microglia activation by suppressing NF-kB and MAPK signaling pathways.     

The PI3K/Akt pathway is required for LPS activation of microglial cells

Upregulation of inflammatory responses in the brain is associated with a number of neurodegenerative diseases. Microglia are activated in neurodegenerative diseases, producing pro-inflammatory mediators. Critically, lipopolysaccharide (LPS)-induced microglial activation causes dopaminergic neurodegeneration in vitro and in vivo. The signaling mechanisms triggered by LPS to stimulate the release of pro-inflammatory mediators in microglial cells are still incompletely understood. To further explore the mechanisms of LPS-mediated inflammatory response of microglial cells, we studied the role of phosphatidylinositol 3-kinase (PI3K)/Akt signal transduction pathways known to be activated by toll-like receptor-4 signaling through LPS. In the current study, we report that the activation profile of LPS-induced pAkt activation preceded those of LPS-induced NF-κB activation, suggesting a role for PI3K/Akt in the pathway activation of NF-κB-dependent inflammatory responses of activated microglia. These results, providing the first evidence that PI3K dependent signaling is involved in the inflammatory responses of microglial cells following LPS stimulation, may be useful in preventing inflammatory based neurodegenerative processes.     

Walnut Extract Inhibits LPS-induced Activation of Bv-2 Microglia via Internalization of TLR4: Possible Involvement of Phospholipase D2

Walnuts are a rich source of essential fatty acids, including the polyunsaturated fatty acids alpha-linolenic acid and linoleic acid. Essential fatty acids have been shown to modulate a number of cellular processes in the brain, including the activation state of microglia. Microglial activation can result in the generation of cytotoxic intermediates and is associated with a variety of age-related and neurodegenerative conditions. In vitro, microglial activation can be induced with the bacterial cell wall component lipopolysaccharide (LPS). In the present study, we generated a methanolic extract of English walnuts (Juglans regia) and examined the effects of walnut extract exposure on LPS-induced activation in BV-2 microglial cells. When cells were treated with walnut extract prior to LPS stimulation, production of nitric oxide and expression of inducible nitric oxide synthase were attenuated. Walnut extract also induced a decrease in tumor necrosis-alpha (TNFα) production. We further found that walnut extract induced internalization of the LPS receptor, toll-like receptor 4, and that the anti-inflammatory effects of walnut were dependent on functional activation of phospholipase D2. These studies represent the first to describe the anti-inflammatory effects of walnuts in microglia, which could lead to nutritional interventions in the prevention and treatment of neurodegeneration.     

Minocycline attenuates lipopolysaccharide-induced white matter injury in the neonatal rat brain

Our previous studies have shown that intracerebral administration of endotoxin, lipopolysaccharide (LPS), induces selective white matter injury and hypomyelination in the neonatal rat brain and that the LPS-induced brain injury is associated with activation of microglia. To test the hypothesis that inhibition of microglial activation may protect against LPS-induced white matter injury, we examined roles of minocycline, a putative suppressor of microglial activation, on LPS-induced brain injury in the neonatal rat. A stereotactic intracerebral injection of LPS (1 mg/kg) was performed in postnatal day 5 Sprague-Dawley rats and control rats were injected with sterile saline. Minocycline (45 mg/kg) was administered intraperitoneally 12 h before and immediately after LPS injection and then every 24 h for 3 days. Inflammatory responses, activation of microglia and brain injury were examined 1 and 3 days after LPS injection. LPS injection resulted in brain injury in selective brain areas, including bilateral ventricular enlargement, cell death at the sub- and periventricular areas, loss of O4+ and O1+ oligodendrocyte (OL) immunoreactivity and hypomyelination, as indicated by decreased myelin basic protein immunostaining, in the neonatal rat brain. Minocycline administration significantly attenuated LPS-induced brain injury in these rat brains. The protective effect of minocycline was associated with suppressed microglial activation as indicated by the decreased number of activated microglial cells following LPS stimulation and with consequently decreased elevation of interleukin 1beta and tumor necrosis factor-alpha concentrations induced by LPS and a reduced number of inducible nitric oxide synthase expressing cells. Protection of minocycline was also linked with the reduction in LPS-induced oxidative stress, as indicated by 4-hydroxynonenal positive OLs. The overall results suggest that reduction in microglial activation may protect the neonatal brain from LPS-induced white matter injury and inhibition of microglial activation might be an effective approach for the therapeutic treatment of infection-induced white matter injury.     

成纤维细胞生长因子10抑制脂多糖刺激下BV2细胞的活化

目的:探索成纤维细胞生长因子10(fibroblast growth factor 10,FGF10)对脂多糖(lipopolysaccharide,LPS)刺激下小胶质细胞BV2活化的影响。方法:小鼠BV2小胶质细胞用细胞培养基DMEM培养,置于37℃、5%CO_2、饱和湿度的培养箱中培养,1~2 d换液,4~5 d传代。实验分为对照组、LPS组和FGF10组,FGF10组的BV2细胞预先给予FGF10 1 mg/L 30 min后,在LPS组和FGF10组中加入500 mg/L的LPS,在不同时点进行检测。用倒置显微镜观察小胶质细胞的形态学改变,RT-qPCR和ELISA分别检测肿瘤坏死因子α(TNF-α)转录和蛋白表达水平的改变来观测BV2细胞的活化情况。结果:静息状态下BV2细胞形态呈圆形或椭圆形,经过24 h LPS刺激后,BV2细胞形状向多极或纺锤样改变,活化细胞数量比值明显高于对照组;预先给予FGF10能抑制LPS刺激下的BV2细胞向活化形态改变,活化的BV2细胞明显减少。给予LPS刺激6 h后,LPS组TNF-α的mRNA水平相比于对照组显著升高,然而预先给予FGF10会显著抑制TNF-α的转录。LPS作用24 h后,细胞培养上清液内TNF-α的表达水平与对照组相比显著上升,而预先给予FGF10在蛋白水平显著抑制TNF-α的表达。结论:FGF10能够成功抑制LPS刺激下BV2细胞的活化,有望成为治疗经胶质细胞介导的神经系统炎症性疾病的一种有效药物。     

A novel fibrinogenase from Agkistrodon acutus venom protects against LPS-induced endotoxemia via regulating NF-κB pathway

Abstract

Context: Endotoxins including lipopolysaccharide (LPS) could cause endotoxemia which often results in excessive inflammation, organ dysfunction, sepsis, disseminated intravascular coagulation (DIC) or even death. Previously, a novel fibrinogenase (FII) showed protective effects on LPS-induced DIC via activating protein C and suppressing inflammatory cytokines.

Objective: To evaluate whether FII has protective effect on LPS-induced endotoxemia in mice and learn about the role of NF-κB pathway in TNF-α producing process.

Methods: BALB/C mice were intraperitoneally injected (i.p.) with (a) 30?mg/kg LPS, (b) LPS?+?0.3?mg/kg FII, (c) LPS?+?1.0?mg/kg FII, (d) LPS?+?3.0?mg/kg FII or (e) saline. Both survival rate and organ function were tested, including alanine aminotransferase (ALT), blood urine nitrogen (BUN) and tissue section, and TNF-α was examined by ELISA. RAW 264.7 macrophage was administered with (a) LPS, (b) LPS?+?FII, (c) FII alone or (d) saline, and TNF-α and phosphorylation (P)-NF-κB (P65) were determined by Western blot.

Results: The administration of LPS led to 65% mortality rate, a rise of serum TNF-α, BUN and ALT levels, and both liver and renal tissue damage were observed. While FII treatment significantly increased the survival rate of LPS-induced endotoxemia mice model, histopathology and protein analysis results also revealed that FII remarkably protected liver and renal from LPS damage as well as decreasing TNF-α level. In vitro, FII significantly decreased LPS-induced TNF-α production and the expression of P-NF-κB (P65).

Conclusions: Our findings suggested that FII had protective effect on LPS-induced endotoxemia and organ injuries by suppressing the activation of NF-κB which decreased TNF-α level.     

MCP-1-deficient mice show reduced neuroinflammatory responses and increased peripheral inflammatory responses to peripheral endotoxin insult

Background

Inflammation plays an important role in the pathogenesis of Parkinson's disease (PD) through over-activation of microglia, which consequently causes the excessive production of proinflammatory and neurotoxic factors, and impacts surrounding neurons and eventually induces neurodegeneration. Hence, prevention of microglial over-activation has been shown to be a prime target for the development of therapeutic agents for inflammation-mediated neurodegenerative diseases.

Methods

For in vitro studies, mesencephalic neuron-glia cultures and reconstituted cultures were used to investigate the molecular mechanism by which FLZ, a squamosamide derivative, mediates anti-inflammatory and neuroprotective effects in both lipopolysaccharide-(LPS)- and 1-methyl-4-phenylpyridinium-(MPP⁺)-mediated models of PD. For in vivo studies, a 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine-(MPTP-) induced PD mouse model was used.

Results

FLZ showed potent efficacy in protecting dopaminergic (DA) neurons against LPS-induced neurotoxicity, as shown in rat and mouse primary mesencephalic neuronal-glial cultures by DA uptake and tyrosine hydroxylase (TH) immunohistochemical results. The neuroprotective effect of FLZ was attributed to a reduction in LPS-induced microglial production of proinflammatory factors such as superoxide, tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E₂ (PGE₂). Mechanistic studies revealed that the anti-inflammatory properties of FLZ were mediated through inhibition of NADPH oxidase (PHOX), the key microglial superoxide-producing enzyme. A critical role for PHOX in FLZ-elicited neuroprotection was further supported by the findings that 1) FLZ's protective effect was reduced in cultures from PHOX^-/- mice, and 2) FLZ inhibited LPS-induced translocation of the cytosolic subunit of p47^PHOX to the membrane and thus inhibited the activation of PHOX. The neuroprotective effect of FLZ demonstrated in primary neuronal-glial cultures was further substantiated by an in vivo study, which showed that FLZ significantly protected against MPTP-induced DA neuronal loss, microglial activation and behavioral changes.

Conclusion

Taken together, our results clearly demonstrate that FLZ is effective in protecting against LPS- and MPTP-induced neurotoxicity, and the mechanism of this protection appears to be due, at least in part, to inhibition of PHOX activity and to prevention of microglial activation.     

Transcriptional regulation of iNOS and COX-2 by a novel compound from Curcuma comosa in lipopolysaccharide-induced microglial activation

Overproduction of pro-inflammatory mediators resulting from chronic activation of microglia has been implicated in many neurodegenerative disorders, such as Parkinson's disease and Alzheimer's disease. In this study, we investigated the effects of (3R) 1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol, or compound 049 on the production of pro-inflammatory mediators in lipopolysaccharide (LPS)-treated microglia. Compound 049 is a pure compound fractionated from the hexane extract of Curcuma comosa, an indigenous plant of Thailand traditionally used as an anti-inflammatory agent for the treatment of uterine inflammation. It was found that pretreatment of the highly aggressively proliferating immortalized (HAPI), rat microglial cell line, with compound 049, at the concentrations of 0.1, 0.5 and 1 μM significantly decreased LPS-induced NO and PGE₂ production in a concentration-dependent manner. Parallel to the decreases in NO and PGE₂ production was a reduction in the expression of inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) as measured by mRNA and protein levels. These results indicate that compound 049 possesses an anti-inflammatory activity and may have a therapeutic potential for the treatment of neurodegenerative diseases related to microglial activation.     

Parkinson's disease: connecting mitochondria to inflammasomes

Activated microglia foster a neurotoxic, inflammatory environment in the mammalian central nervous system (CNS) that drives the pathology of neurodegenerative diseases including Parkinson's disease (PD). Moreover, mitochondrial fission promotes microglial inflammatory responses in vitro. Given that the NLRP3 inflammasome and mitochondria are central regulators of both inflammation and PD, we explore potential functions for the NLRP3 inflammasome and mitochondrial dynamics in PD. Specifically, we propose that inducible microglial mitochondrial fission can promote NLRP3-dependent neuroinflammation in hereditary and idiopathic PD. Further in-depth exploration of this topic can prompt valuable discoveries of the underlying molecular mechanisms of PD neuroinflammation, identify novel candidate anti-inflammatory therapeutics for PD, and ideally provide better outcomes for PD patients.     

Sphingosine kinase 1 regulates the expression of proinflammatory cytokines and nitric oxide in activated microglia

Microglial activation has been implicated as one of the causative factors for neuroinflammation in various neurodegenerative diseases. The sphingolipid metabolic pathway plays an important role in inflammation, cell proliferation, survival, chemotaxis, and immunity in peripheral macrophages. In this study, we demonstrate that sphingosine kinase1 (SphK1), a key enzyme of the sphingolipid metabolic pathway, and its receptors are expressed in the mouse BV2 microglial cells and SphK1 alters the expression and production of proinflammatory cytokines and nitric oxide in microglia treated with lipopolysaccharide (LPS). LPS treatment increased the SphK1 mRNA and protein expression in microglia as revealed by the RT–PCR, Western blot and immunofluorescence. Suppression of SphK1 by its inhibitor, N, N Dimethylsphingosine (DMS), or siRNA resulted in decreased mRNA expression of TNF-α, IL-1β, and iNOS and release of TNF-α and nitric oxide (NO) in LPS-activated microglia. Moreover, addition of sphingosine 1 phosphate (S1P), a breakdown product of sphingolipid metabolism, increased the expression levels of TNF-α, IL-1β and iNOS and production of TNF-α and NO in activated microglia. Hence to summarize, suppression of SphK1 in activated microglia inhibits the production of proinflammatory cytokines and NO and the addition of exogenous S1P to activated microglia enhances their inflammatory responses. Since the chronic proinflammatory cytokine production by microglia has been implicated in neuroinflammation, modulation of SphK1 and S1P in microglia could be looked upon as a future potential therapeutic method in the control of neuroinflammation in neurodegenerative diseases.     

Inhibition of LPS-Induced Retinal Microglia Activation by Naloxone Does Not Prevent Photoreceptor Death

Microglia-associated inflammation is closely related to the pathogenesis of retinal degenerative disorders. We have previously shown in vivo that naloxone protected photoreceptors from light-induced apoptosis possibly through inhibiting microglial activation. In this study, we attempted to explore the effect of lipopolysaccharide (LPS)-activated microglia on photoreceptor death and the influence of naloxone treatment using an in vitro retinal microglia and 661 W photoreceptor co-culture system. Immunofluorescent staining and ELISA measurements demonstrated that LPS activated microglia by changing the morphology and increasing the production of proinflammatory factors interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha. Flow cytometry analysis of annexin V/propidium iodide staining showed that LPS-activated microglia promoted the apoptosis of co-cultured 661 W photoreceptor cells. Naloxone inhibited microglial activation and decreased the release of IL-1beta and TNF-alpha but could not prevent photoreceptors from undergoing apoptosis. Considering the dual role of microglia-associated inflammation in both neurotoxicity and neuroprotection, modulating the function, rather than simply inhibiting their activation, might be a new therapeutic method for preventing photoreceptor degeneration.     

Regulation of IL-18 production by IFN gamma and PGE2 in mouse microglial cells: involvement of NF-kB pathway in the regulatory processes

Interleukin (IL)-18, a recently identified proinflammatory cytokine, has been implicated in a variety of pathological conditions such as rheumatoid arthritis, insulin-dependent diabetes mellitus, and inflammatory liver injury. Microglial cells are the primary cellular source of IL-18 in the brain. Along with other inflammatory mediators in the central nervous system (CNS), IL-18 may play an important role in the pathogenesis of various neurodegenerative diseases. To understand how lymphokines and lipid mediators participate in the regulation of microglial IL-18 production, we assessed the effects of interferon (IFN)gamma, one of the major macrophage-activating lymphokines, and prostaglandin (PG)E(2), a lipid mediator produced in the brain, on IL-18 production and the expression of the IL-18 processing enzyme, caspase-1, in mouse microglial cells. IFNgamma increased lipopolysaccharide (LPS)-induced IL-18 production and caspase-1 expression, while PGE(2) inhibited LPS-induced IL-18 production. A similar pattern of IL-18 regulation by IFNgamma and PGE(2) was observed at the mRNA level. The regulation of microglial activation by IFNgamma and PGE(2) was accompanied by differential modulation of LPS-induced NF-kB activation. While IFNgamma enhanced LPS-induced NF-kB activation, PGE(2) suppressed its activation. These results indicate that IFNgamma and PGE(2) are the important regulators of proinflammatory microglial activation in CNS, and suggest the involvement of NF-kB pathway in these regulatory processes.     

CD45 isoform RB as a molecular target to oppose lipopolysaccharide-induced microglial activation in mice

CD45 is a membrane-bound protein tyrosine phosphatase expressed on all hemopoietic cells with multiple splice variants, including RA, RB, RC and RO. Our previous studies have shown that cross-linking of CD45 with an anti-CD45 antibody markedly inhibits LPS-induced microglia activation. In order to determine which of the CD45 isoforms may be responsible for these effects, we have investigated the expression of CD45 isoforms on cultured microglial cells using flow cytometric analysis. Data reveal that CD45RB is the predominant isoform expressed in murine primary cultured microglial cells. Furthermore, incubation of these cultured cells with anti-CD45RB antibody results in a reduction of microglial activation induced by LPS as evidenced by TNF-alpha production. As a validation of these findings in vivo, brain homogenates from anti-CD45RB antibody (MG23G2)-injected animals that had been treated with LPS demonstrate a significant decrease in TNF-alpha levels compared to control mice treated with LPS plus vehicle. Taken together, these findings suggest that therapeutic agents that specifically stimulate the microglial CD45RB signaling pathway may be effective in suppressing microglial activation associated with several neurodegenerative disorders.     

栀子苷下调Toll样受体4表达并拮抗脂多糖诱导的小胶质细胞炎性反应

目的:观察栀子昔对细菌脂多糖(LPS)诱导的BV2小胶质细胞炎性反应的影响并探讨其作用机制。方法:LPS诱导BV2小胶质细胞活化,CCK-8方法检测细胞存活率,Griess法测定NO释放量,ELISA测定肿瘤坏死因子-α(TNF-α)和白介素-1β(IL-1β)含量,免疫印迹检测Toll样受体4(TLR4)蛋白表达。结果:栀子苷在10～100μg/ml浓度范围内对小胶质细胞活力影响不显著,此浓度范围内,栀子苷剂量依赖性的减少LPS诱导的NO、TNF-α和IL-1β释放。此外,栀子苷还可抑制LPS诱导的BV2细胞形态活化改变,并降低LPS诱导的TLR4蛋白表达。结论:栀子苷可以拮抗LPS诱导的BV2小胶质细胞炎性反应,其机制可能与下调TLR4信号通路有关。