LPS-activated Microglial Cells Research Articles

Influence of a pro-inflammatory stimulus on the miRNA and lipid content of human dental stem cell-derived extracellular vesicles and their impact on microglial activation

Neuro-inflammation occurs in numerous disorders such as multiple sclerosis, Alzheimer's disease and Parkinson's disease. However, anti-inflammatory drugs for the central nervous system have failed to show significant improvement when compared to a placebo in clinical trials. Our previous work demonstrated that stem cells from the apical papilla (SCAP) can decrease neuro-inflammation and stimulate oligodendrocyte progenitor cell differentiation. One hypothesis is that the therapeutic effect of SCAP could be mediated by their secretome, including extracellular vesicles (EV). Here, our objectives were to characterize SCAP-EV and to study their effect on microglial cells. We isolated EV from non-activated SCAP and from SCAP activated with TNFα and IFN-γ and characterized them according to their size, EV markers, miRNA and lipid content. Their ability to decrease pro-inflammatory cytokine expression in vitro and ex vivo was also assessed. We showed that the miRNA content was impacted by a pro-inflammatory environment but not their lipid composition. SCAP-EV reduced the expression of pro-inflammatory markers in LPS-activated microglial cells while their effect was limited on mouse spinal cord sections. In conclusion, we were able to isolate EV from SCAP, to show that their miRNA content was impacted by a pro-inflammatory stimulus, and to describe that SCAP-EV and not the protein fraction of conditioned medium could reduce pro-inflammatory marker expression in LPS-activated BV2 cells.

Agarperoxinols A and B: Two Unprecedented Tricyclic 6/6/7 Rearranged Humulene-Type Sesquiterpenoids That Attenuated the Neuroinflammation in LPS-Stimulated Microglial Models.

Agarperoxinols A and B (1-2), two naturally occurring humulene-type sesquiterpenoids with an unprecedented tricyclic 6/6/7 ring, were discovered from the agarwood of Aquilaria malaccensis. Their structures were unambiguously determined by various spectroscopic data, experimental ECD calculations, and single-crystal X-ray diffraction analysis. Agarperoxinol B showed significant and dose-dependent neuroinflammatory inhibitory effects on various proinflammatory mediators, including NO, TNF-α, IL-6, and IL-1β, and suppressed iNOS and COX-2 enzymes in LPS-activated microglial cells. A mechanistic study demonstrated that agarperoxinol B remarkably inhibited the phosphorylation of the Akt and JNK signaling pathways. Agarperoxinol B also significantly reduced the expression of the microglial markers Iba-1, COX-2, and TNF-α in the mouse cerebral cortex. Our findings introduce a bioactive compound from natural products that decreases proinflammatory factor production and has application for the treatment of neurodegenerative diseases.

The nuclear receptor Nurr1 regulates the expression and activity of PPARγ in human pro-inflammatory macrophages

Abstract Nurr1 is a member of the orphan nuclear receptor family NR4A that modulates inflammation in several cell lineages through inhibition of the NF-κB signaling pathway. Nurr1 was found to be able to interact with the peroxisome proliferator-activated receptor (PPAR)γ, as well as to be recruited to the PPARG promoter in LPS-activated microglial cells; however, the functional relationship between Nurr1 and PPARγ is not clearly established. Here, we aimed to investigate the role of Nurr1 on PPARγ activity in human macrophages. Blood monocytes were cultured with GM-CSF or M-CSF to generate pro- (GM-MDMs) and anti-inflammatory (M-MDMs) macrophages, respectively. GM-MDMs showed enhanced expression of both Nurr1 and PPARγ compared to M-MDMs, and they were preferentially located at the cytoplasmic (Nurr1) and nuclear (PPARγ) compartments. PPARγ activation in GM-MDMs with the agonist rosiglitazone did not modify Nurr1 expression. Conversely, Nurr1 activation with the agonist C-DIM12 increased PPARγ expression. This was concomitant with enhanced expression of two PPARγ target genes, CD36 and PLIN2, while silencing Nurr1 expression led to a decrease in the amounts of CD36 transcripts. Co-treatment of GM-MDMs with C-DIM12 and rosiglitazone synergistically enhanced the transcriptional activity of PPARγ, whereas C-DIM12 alone had no effect. PPARγ and Nurr1 agonists alone exerted anti-inflammatory effects on GM-MDMs, and also showed synergistic activity in combined form. In sum, our data suggests that Nurr1 positively regulates PPARγ activity at least partially through induction of PPARγ expression, which may represent a novel mechanism of Nurr1 targeting metabolic pathways and inflammation. Supported by grants from the Secretaría de Educación Pública-Consejo Nacional de Ciencia y Tecnología (SEP-CONACYT, #A1-S-9430)

Two Argan Oil Phytosterols, Schottenol and Spinasterol, Attenuate Oxidative Stress and Restore LPS-Dysregulated Peroxisomal Functions in Acox1-/- and Wild-Type BV-2 Microglial Cells.

Oxidative stress and inflammation are the key players in neuroinflammation, in which microglia dysfunction plays a central role. Previous studies suggest that argan oil attenuates oxidative stress, inflammation, and peroxisome dysfunction in mouse brains. In this study, we explored the effects of two major argan oil (AO) phytosterols, Schottenol (Schot) and Spinasterol (Spina), on oxidative stress, inflammation, and peroxisomal dysfunction in two murine microglial BV-2 cell lines, wild-ype (Wt) and Acyl-CoA oxidase 1 (Acox1)-deficient cells challenged with LPS treatment. Herein, we used an MTT test to reveal no cytotoxicity for both phytosterols with concentrations up to 5 µM. In the LPS-activated microglial cells, cotreatment with each of these phytosterols caused a significant decrease in intracellular ROS production and the NO level released in the culture medium. Additionally, Schot and Spina were able to attenuate the LPS-dependent strong induction of Il-1β and Tnf-α mRNA levels, as well as the iNos gene and protein expression in both Wt and Acox1-/- microglial cells. On the other hand, LPS treatment impacted both the peroxisomal antioxidant capacity and the fatty acid oxidation pathway. However, both Schot and Spina treatments enhanced ACOX1 activity in the Wt BV-2 cells and normalized the catalase activity in both Wt and Acox1-/- microglial cells. These data suggest that Schot and Spina can protect cells from oxidative stress and inflammation and their harmful consequences for peroxisomal functions and the homeostasis of microglial cells. Collectively, our work provides a compelling argument for the protective mechanisms of two major argan oil phytosterols against LPS-induced brain neuroinflammation.

Activated microglia promote invasion and barrier dysfunction of brain endothelial cells via regulating the CXCL13/CXCR5 axis

The blood brain barrier (BBB) is a protective border that prevents noxious substances from gaining access to the central nervous system (CNS). CXCL13 is a chemokine from the CXC chemokine family, which has been shown to destroy the barrier function of umbilical vein endothelial cells with its receptor CXCR5. Here, we aimed to investigate the role of CXCL13/CXCR5 signaling axis in BBB. The invasive ability of bEnd.3 cells was determined by the Transwell invasion assay. The barrier integrity of bEnd.3 cells was assessed by detecting trans-endothelial electrical resistance, the permeability to fluorescein isothiocyanate-dextran, and the expression levels of the tight junction protein E-cadherin. Lipopolysaccharide (LPS)-activated microglia promoted invasion and barrier dysfunction, and upregulated CXCR5 and p-p38 expression levels in cocultured bEnd.3 cells. However, the effects of activated microglia were alleviated by knocking down CXCR5 in cocultured bEnd.3 cells. Furthermore, recombinant CXCL13 promoted invasion and barrier dysfunction, and upregulated the expression levels of p-p38 in bEnd.3 cells; however, its effects were abolished by treating bEnd.3 cells with the p38 inhibitor SB203580. Our data tentatively demonstrated that LPS-activated microglial cells may promote invasion and barrier dysfunction in bEnd.3 cells by regulating the CXCL13/CXCR5 axis and p38 signaling.

Antineuroinflammatory Effect of Amburana cearensis and Its Molecules Coumarin and Amburoside A by Inhibiting the MAPK Signaling Pathway in LPS-Activated BV-2 Microglial Cells.

Microglia plays an important role in the neuroinflammatory response, identified as one of the major factors in the development and progression of neurodegenerative diseases. Amburana cearensis and its bioactive compounds, including coumarin (CM), vanillic acid (VA), and amburoside A (AMB), exert antioxidant, anti-inflammatory, and neuroprotective activities, on 6-OHDA-induced neurotoxicity in rat mesencephalic cells determined by our group. The present study investigated the anti-inflammatory effect of the dry extract from A. cearensis (DEAC), CM, AMB, and VA on lipopolysaccharide- (LPS-) stimulated microglial cells and elucidated the possible molecular mechanism of action. The DEAC was characterized by HPLC-PDA (chemical markers: CM, AMB, and VA). The BV-2 microglial cell line was pretreated with increasing concentrations of DEAC, CM, AMB, or VA in the presence or absence of LPS to evaluate the toxicity and anti-inflammatory activity. The cytotoxicity of DEAC, CM, AMB, or VA on BV-2 cells was evaluated by the MTT test, the free radical scavenging activity of test drugs was investigated, and the nitric oxide (NO) production was determined using the Griess reagent, while cytokine levels were measured by ELISA. The expressions of toll-like receptor 4 (TLR-4), nuclear factor kappa B (NF-κB), MAPK members (JNK and ERK1/2), and iNOS were determined through Western blot analysis. DEAC, CM, AMB, or VA (5-100 μg/mL) did not induce any detectable cytotoxicity in BV-2 cells. All test drugs (100 μg/mL) showed free radical scavenging activity (hydroxyl and superoxide radicals); however, only DEAC, CM, and AMB (5-100 μg/mL) significantly reduced NO production. DEAC (100 μg/mL), as well as CM (50 and 100 μg/mL) and AMB (25 μg/mL), reduced at least 50% of NO produced and markedly decrease the production of TNF-α and IL-6 but they did not significantly affect IL-10 levels. Only DEAC (100 μg/mL) and AMB (25 μg/mL) reduced the expression of iNOS, and they did not affect arginase activity. DEAC (100 μg/mL) suppressed the activation of the MAPKs JNK and ERK1/2 in LPS-activated BV-2 cells but it did not suppress the expression of TLR-4 nor the phosphorylation of NF-κB. In conclusion, DEAC, CM, and AMB exerted anti-inflammatory activity in LPS-activated microglial cells as observed by the reduction in the production of inflammatory mediators and the expression of iNOS. We identified the MAPK signaling pathway as a probable mechanism of action to the anti-inflammatory effects observed.

3­‘,4‘­,7-Trihydroxyflavone inhibits NO production in LPS-activated MG6 microglial cells by suppressing the JNK–STAT1 pathway

3­‘,4‘­,7-Trihydroxyflavone inhibits NO production in LPS-activated MG6 microglial cells by suppressing the JNK–STAT1 pathway

Ergosta-7,9(11),22-trien-3β-ol Alleviates Intracerebral Hemorrhage-Induced Brain Injury and BV-2 Microglial Activation.

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by an exacerbation of neuroinflammation and neuronal injury, for which few effective therapies are available at present. Inhibition of excessive neuroglial activation has been reported to alleviate ICH-related brain injuries. In the present study, the anti-ICH activity and microglial mechanism of ergosta-7,9(11),22-trien-3β-ol (EK100), a bioactive ingredient from Asian medicinal herb Antrodia camphorate, were evaluated. Post-treatment of EK100 significantly attenuated neurobehavioral deficit and MRI-related brain lesion in the mice model of collagenase-induced ICH. Additionally, EK100 alleviated the inducible expression of cyclooxygenase (COX)-2 and the activity of matrix metalloproteinase (MMP)-9 in the ipsilateral brain regions. Consistently, it was shown that EK100 concentration-dependently inhibited the expression of COX-2 protein in Toll-like receptor (TLR)-4 activator lipopolysaccharide (LPS)-activated microglial BV-2 and primary microglial cells. Furthermore, the production of microglial prostaglandin E2 and reactive oxygen species were attenuated by EK100. EK100 also attenuated the induction of astrocytic MMP-9 activation. Among several signaling pathways, EK100 significantly and concentration-dependently inhibited activation of c-Jun N-terminal kinase (JNK) MAPK in LPS-activated microglial BV-2 cells. Consistently, ipsilateral JNK activation was markedly inhibited by post-ICH-treated EK100 in vivo. In conclusion, EK100 exerted the inhibitory actions on microglial JNK activation, and attenuated brain COX-2 expression, MMP-9 activation, and brain injuries in the mice ICH model. Thus, EK100 may be proposed and employed as a potential therapeutic agent for ICH.

Effects of a Novel GPR55 Antagonist on the Arachidonic Acid Cascade in LPS-Activated Primary Microglial Cells.

Neuroinflammation is a crucial process to maintain homeostasis in the central nervous system (CNS). However, chronic neuroinflammation is detrimental, and it is described in the pathogenesis of CNS disorders, including Alzheimer’s disease (AD) and depression. This process is characterized by the activation of immune cells, mainly microglia. The role of the orphan G-protein-coupled receptor 55 (GPR55) in inflammation has been reported in different models. However, its role in neuroinflammation in respect to the arachidonic acid (AA) cascade in activated microglia is still lacking of comprehension. Therefore, we synthesized a novel GPR55 antagonist (KIT 10, 0.1–25 µM) and tested its effects on the AA cascade in lipopolysaccharide (LPS, 10 ng / mL)-treated primary rat microglia using Western blot and EIAs. We show here that KIT 10 potently prevented the release of prostaglandin E2 (PGE2), reduced microsomal PGE2 synthase (mPGES-1) and cyclooxygenase-2 (COX-2) synthesis, and inhibited the phosphorylation of Ikappa B-alpha (IκB-α), a crucial upstream step of the inflammation-related nuclear factor-kappaB (NF-κB) signaling pathway. However, no effects were observed on COX-1 and -2 activities and mitogen-activated kinases (MAPK). In summary, the novel GPR55 receptor antagonist KIT 10 reduces neuroinflammatory parameters in microglia by inhibiting the COX-2/PGE2 pathway. Further experiments are necessary to better elucidate its effects and mechanisms. Nevertheless, the modulation of inflammation by GPR55 might be a new therapeutic option to treat CNS disorders with a neuroinflammatory background such as AD or depression.

Hp-s1 Ganglioside Suppresses Proinflammatory Responses by Inhibiting MyD88-Dependent NF-κB and JNK/p38 MAPK Pathways in Lipopolysaccharide-Stimulated Microglial Cells.

Hp-s1 ganglioside is isolated from the sperm of sea urchin (Hemicentrotus pulcherrimus). In addition to neuritogenic activity, the biological function of Hp-s1 in neuroinflammation is unknown. In this study, we investigated the anti-neuroinflammatory effect of Hp-s1 on lipopolysaccharide (LPS)-stimulated microglial cells. MG6 microglial cells were stimulated with LPS in the presence or absence of different Hp-s1 concentrations. The anti-inflammatory effect and underlying mechanism of Hp-s1 in LPS-activated microglia cells were assessed through a Cell Counting kit-8 assay, Western blot analysis, and immunofluorescence. We found that Hp-s1 suppressed not only the expression of inducible nitric oxide synthase and cyclooxygenase-2 but also the expression of proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6. Hp-s1 inhibited the LPS-induced NF-κB signaling pathway by attenuating the phosphorylation and translocation of NF-κB p65 and by disrupting the degradation and phosphorylation of inhibitor κB-α (IκBα). Moreover, Hp-s1 inhibited the LPS-induced phosphorylation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK). Hp-s1 also reduced the expression of myeloid differentiation factor 88 (MyD88) and TNF receptor-associated factors 6 (TRAF6), which are prerequisites for NF-κB and MAPKs activation. These findings indicated that Hp-s1 alleviated LPS-induced proinflammatory responses in microglial cells by downregulating MyD88-mediated NF-κB and JNK/p38 MAPK signaling pathways, suggesting further evaluation as a new anti-neuroinflammatory drug.

Betanin purification from red beetroots and evaluation of its anti-oxidant and anti-inflammatory activity on LPS-activated microglial cells.

Microglial activation can release free radicals and various pro-inflammatory cytokines, which implicates the progress of a neurodegenerative disease. Therefore suppression of microglial activation can be an appropriate strategy for combating neurodegenerative diseases. Betanin is a red food dye that acts as free radical scavenger and can be a promising candidate for this purpose. In this study, purification of betanin from red beetroots was carried out by normal phase colum chromatography, yielding 500 mg of betanin from 100 g of red beetroot. The purified betanin was evaluated by TLC, UV-visible, HPLC, ESI-MASS, FT-IR spectroscopy. Investigation on the inhibitory effect of betanin on activated microglia was performed using primary microglial culture. The results showed that betanin significantly inhibited lipopolysaccharide induced microglial function including the production of nitric oxide free radicals, reactive oxygen species, tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-1 beta (IL-1β). Moreover, betanin modulated mitochondrial membrane potential, lysosomal membrane permeabilization and adenosine triphosphate. We further investigated the interaction of betanin with TNF-α, IL-6 and Nitric oxide synthase (iNOS or NOS2) using in silico molecular docking analysis. The docking results demonstrated that betanin have significant negative binding energy against active sites of TNF-α, IL-6 and iNOS.

Mesenchymal stem cells inhibited the inflammation and oxidative stress in LPS-activated microglial cells through AMPK pathway.

Microglia are the resident mononuclear immune cells of the central nervous system (CNS) and the activation of microglia contributes to the production of excessive neurotoxic factors. In particular, the overproduction of neurotoxic factors has critical effects on the development of brain injuries and neurodegenerative diseases. The human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have blossomed into an effective approach with great potential for the treatment of neurodegenerative diseases and gliomas. The present study aimed to investigate the mechanism behind the therapeutic effect of hBM-MSCs on the activation of microglia in vitro. Specifically, the hBM-MSCs significantly inhibited the proliferation of lipopolysaccharide-activated microglial cells (LPS)-activated microglial cells. Additionally, we investigated whether the adenosine-monophosphate-activated protein kinase signaling (AMPK) pathway was involved in this process. Our data demonstrated that hBM-MSCs significantly increased the phosphorylated AMPK in LPS-activated microglial cells. In addition, our study indicated the inhibitory effect of hBM-MSCs on the pro-inflammatory mediators and oxidative stress by the AMPK pathway in LPS-activated microglial cells. These results could shed light on the understanding of the molecular basis for the inhibition of hBM-MSCs on LPS-activated microglial cells and provide a molecular mechanism for the hBM-MSCs implication in brain injuries and neurodegenerative diseases.

Abstract # 3250 Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells

Abstract # 3250 Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells

Cryptotanshinone Attenuates Inflammatory Response of Microglial Cells via the Nrf2/HO-1 Pathway.

Cryptotanshinone (CTN), a monomer compound extracted from the dried roots and rhizomes of Salvia miltiorrhiza Bge, has a variety of pharmacological effects. However, little research has been done on the mechanism of CTN in attenuating neuroinflammation. The present study aimed to investigate whether CTN can ameliorate neuroinflammation induced by lipopolysaccharide (LPS) through the Nrf2/heme-oxygenase 1 (HO-1) signaling pathway in BV-2 microglial cells. We found that CTN attenuated the upregulated expression of inducible nitric oxide synthase, cyclooxygenase 2, NOD-like receptor pyrin domains-3, and nitric oxide induced by LPS in microglial cells. In addition, it curtailed the increased release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α in LPS-activated microglial cells. Furthermore, CTN significantly increased the levels of NF-κB, Nrf2, HO-1, and Akt proteins. We demonstrated that the anti-inflammatory action of CTN in BV-2 microglial cells was partially through the activation of the Nrf2/HO-1 pathway, which was regulated by the PI3K/Akt signaling pathway. Taken together, our results indicated that CTN downregulated the production and release of proinflammatory mediators in BV-2 microglial cells through activating the Nrf2/HO-1 pathway and subsequently protected neurons from inflammatory injury.

Mechanism underlying β2-AR agonist-mediated phenotypic conversion of LPS-activated microglial cells

Fundamentally, microglia have two activation states, a pro-inflammatory neurotoxic (M1) and an anti-inflammatory neuroprotective (M2) phenotype, and their conversion from M1-like to M2-like microglia may provide therapeutic benefits to prevent neuronal loss in neurodegenerative diseases such as Parkinson's disease (PD). Previously, we showed that Salmeterol, a long-acting β2-adrenergic receptor (β2-AR) agonist, has neuroprotective effects in PD models in vitro and in vivo through the β-arrestin2-dependent inhibition of pro-inflammatory M1-type mediator production. In the present study, we explored whether Salmeterol can mediate phenotypic conversion in LPS-activated murine microglial BV2 cells from the neurotoxic M1-like to a neuroprotective M2-like phenotype. Salmeterol inhibited the production of LPS-induced mediators of the pro-inflammatory M1 phenotype such as tumor necrosis factor-α (TNF-α), IL-(interleukin) 18, IL-6, chemokines (CCL2, CCL3, CCL4) and reactive oxygen species from BV2 cells. Conversely, treatment with Salmeterol and other β2-AR agonists robustly enhances the production of the M2 cytokine IL-10 from LPS-activated microglia. In addition, Salmeterol upregulates the expression of arginase-1 and CXCL14. Furthermore, using siRNA approach we found that silencing of the transcription factor Creb abrogates the Salmeterol-mediated production of IL-10 in LPS-activated BV2 cells, but silencing of β-arrestin2 with Arrb2 siRNA did not. In addition, our data shows conversion from an M1- to M2-like phenotype in LPS-activated microglia by β2-AR agonists involves activation of the classical cAMP/PKA/CREB as well as the PI3K and p38 MAPK signaling pathways, and provides a novel therapeutic approach targeting microglial cell activation and inducing their phenotypic conversion in the treatment of neuroinflammatory diseases such as PD.

HADC8 Inhibitor WK2-16 Therapeutically Targets Lipopolysaccharide-Induced Mouse Model of Neuroinflammation and Microglial Activation.

Glial activation and neuroinflammatory processes play important roles in the pathogenesis of brain abscess and neurodegenerative diseases. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which contribute to the exacerbation of neuronal cell death. The inhibition of glial activation has been shown to alleviate neurodegenerative conditions. The present study was to investigate the specific HDAC8 inhibitor WK2-16, especially its effects on the neuroinflammatory responses through glial inactivation. WK2-16 significantly reduced the gelatinolytic activity of MMP-9, and expression of COX-2/iNOS proteins in striatal lipopolysaccharide (LPS)-induced neuroinflammation in C57BL/6 mice. The treatment of WK2-16 markedly improved neurobehavioral deficits. Immunofluorescent staining revealed that WK2-16 reduced LPS-stimulated astrogliosis and microglial activation in situ. Consistently, cellular studies revealed that WK2-16 significantly suppressed LPS-induced mouse microglia BV-2 cell proliferation. WK2-16 was proven to concentration-dependently induce the levels of acetylated SMC3 in microglial BV-2 cells. It also reduced the expression of COX-2/iNOS proteins and TNF-α production in LPS-activated microglial BV-2 cells. The signaling studies demonstrated that WK2-16 markedly inhibited LPS-activated STAT-1/-3 and Akt activation, but not NF-κB or MAPK signaling. In summary, the HDAC8 inhibitor WK2-16 exhibited neuroprotective effects through its anti-neuroinflammation and glial inactivation properties, especially in microglia in vitro and in vivo.

Anti-neuroinflammatory effects of GPR55 antagonists in LPS-activated primary microglial cells

BackgroundNeuroinflammation plays a vital role in Alzheimer’s disease and other neurodegenerative conditions. Microglia are the resident mononuclear immune cells of the central nervous system, and they play essential roles in the maintenance of homeostasis and responses to neuroinflammation. The orphan G-protein-coupled receptor 55 (GPR55) has been reported to modulate inflammation and is expressed in immune cells such as monocytes and microglia. However, its effects on neuroinflammation, mainly on the production of members of the arachidonic acid pathway in activated microglia, have not been elucidated in detail.MethodsIn this present study, a series of coumarin derivatives, that exhibit GPR55 antagonism properties, were designed. The effects of these compounds on members of the arachidonic acid cascade were studied in lipopolysaccharide (LPS)-treated primary rat microglia using Western blot, qPCR, and ELISA.ResultsWe demonstrate here that the various compounds with GPR55 antagonistic activities significantly inhibited the release of PGE2 in primary microglia. The inhibition of LPS-induced PGE2 release by the most potent candidate KIT 17 was partially dependent on reduced protein synthesis of mPGES-1 and COX-2. KIT 17 did not affect any key enzyme involved on the endocannabinoid system. We furthermore show that microglia expressed GPR55 and that a synthetic antagonist of the GPR receptor (ML193) demonstrated the same effect of the KIT 17 on the inhibition of PGE2.ConclusionsOur results suggest that KIT 17 is acting as an inverse agonist on GPR55 independent of the endocannabinoid system. Targeting GPR55 might be a new therapeutic option to treat neurodegenerative diseases with a neuroinflammatory background such as Alzheimer’s disease, Parkinson, and multiple sclerosis (MS).

IDH2 Deficiency in Microglia Decreases the Pro-inflammatory Response via the ERK and NF-κB Pathways.

In various neuronal diseases, the activation of microglia contributes to the production of excessive neurotoxic factors, such as pro-inflammatory mediators. In particular, the overproduction of pro-inflammatory cytokines and nitric oxide (NO) has critical effects on the development of neurodegenerative diseases and gliomas in the brain. Recent studies have suggested that isocitrate dehydrogenase 2 (IDH2) plays a key role in inducing gliomas and neurodegeneration. IDH2 dysfunction has been linked to various cancers and neurodegenerative diseases associated with uncontrolled inflammatory responses, such as the excessive generation of pro-inflammatory cytokines. In this study, we demonstrate that IDH2 contributes to the regulation of pro-inflammatory mediators in microglia. The downregulation of IDH2 decreased the lipopolysaccharide (LPS)-induced pro-inflammatory response in BV-2 and primary microglial cells. Furthermore, IDH2 deficiency downregulated pro-inflammatory mediators via modulation of the ERK and NF-κB pathways. These results indicate that IDH2 is a potential target for the regulation of pro-inflammatory responses in LPS-activated microglial cells. Our findings also provide a basis for the development of new therapies for pro-inflammatory responses in dysfunction-associated neuronal diseases.

Methamphetamine modulates the production of interleukin-6 and tumor necrosis factor-alpha via the cAMP/PKA/CREB signaling pathway in lipopolysaccharide-activated microglia

Methamphetamine (METH) elicits neuroinflammatory effects that may implicate its regulatory role on the microglial immune response. However, the mechanism underlying this remains unclear. In the present study, the effects of METH on lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) productions were tested in BV-2 cells and primary microglial cells. Additionally, western blot analysis was used to examine the phosphorylation of mitogenactivated protein kinases (MAPKs). Next, we detected the alterations in cAMP content and the phosphorylation levels of CREB in microglial cells to determine the involvement of the cAMP/CREB signaling pathway. We also used an adenylyl cyclase (AC) agonist (forskolin) and antagonist (MDL-12330A) and a PKA antagonist (H89) to confirm their participation. We observed that METH alone did not affect the production of IL-6 or TNF-α. In contrast, METH augmented the IL-6 production and inhibited the TNF-α production induced by LPS. A similar effect of forskolin was also observed in BV-2 cells. While MAPK activation was not influenced by METH alone, the LPS-induced phosphorylation of p38, JNK and ERK1/2 were all reduced by METH. Both the concentration of cAMP and the phosphorylation of CREB were increased by METH in LPS-activated microglial cells. The effects of METH were altered by MDL-12330A and H89. Moreover, the inhibition of the phosphorylation of ERK1/2 by METH was also reversed. These results suggest that the differential regulation of IL-6 and TNF-α by METH in LPS-activated microglial cells may be attributable to the cAMP/PKA/CREB signaling pathway.

Effect of simvastatin on neuroinflammation in microglial cells via mitogen-activated protein kinase and nuclear factor κB pathways

Background: Activated microglial cells are found in different sorts of the neurodegenerative process including Parkinson and Alzheimer. Suppressing the activated microglial cells developed as a novel procedure for the treatment of neuroinflammation-based neurodegeneration. Materials and Methods: We have investigated the effects of simvastatin on memory impairment and inflammatory cytokines expression induced by transient cerebral ischemia in cultured microglial cells. Results: The lipopolysaccharide (LPS)-activated microglial cells treated with simvastatin 3 μmol has decreased the inflammation which was indicated by the reduced levels of the nitric oxide (NO), tumor necrosis factor-α, interleukin-1 β, cyclooxygenase-2, and inducible NO synthase. Simvastatin also delayed the activation of atomic component nuclear factor-κB, p38 mitogen-activated protein kinase, and the reactive oxygen species in LPS-activated microglial cells. Moreover, simvastatin has provoked the outflow of heme oxygenase-1 in BV-2 microglial cells. Conclusions: The present study showed that the simvastatin antagonizes neuroinflammation and can be a potential restorative operator for treating neuroinflammatory ailments. Abbreviations used: LPS: Lipopolysaccharide, TNF-α: Tumor necrosis factor, NO: Nitric oxide, IL-1β: Interleukin, COX-2: Cyclooxygenase, iNOS: Inducible nitric oxide synthase, MAPK: Mitogen-activated protein kinase, HO-1: Heme oxygenase