The immunocytes microglia in the central nervous system (CNS) were reported to play a crucial role in neurodegeneration. As a member of P2 receptors family, purinoceptor P2Y6 has attracted much attention recently. Previous studies showed that purinoceptor P2Y6 mainly contributed to microglia activation and their later phagocytosis in CNS, while in immune system, it participated in the secretion of interleukin (IL)-8 from monocytes and macrocytes. So there raises a question: whether purinoceptor P2Y6 also takes part in neuroinflammation? Thus, this review mainly concerns about the properties and roles of purinoceptor P2Y6, including (1) structure of purinoceptor P2Y6; (2) distribution and properties of purinoceptor P2Y6; (3) relationships between purinoceptor P2Y6 and microglia; (4) relationships between purinoceptor P2Y6 and immunoinflammation. Itos proposed that purinoceptor P2Y6 may play a role in neuroinflammation in CNS, although further research is still required.
Animals ; Humans ; Inflammation ; immunology ; metabolism ; Microglia ; drug effects ; metabolism ; Monocytes ; metabolism ; Phagocytosis ; physiology ; Receptors, Purinergic P2 ; chemistry ; genetics ; metabolism

Present study demonstrated that fibrillar beta-amyloid peptide (fAbeta(1-42)) induced ATP release, which in turn activated NADPH oxidase via the P2X(7) receptor (P2X(7)R). Reactive oxygen species (ROS) production in fAbeta(1-42)-treated microglia appeared to require Ca2+ influx from extracellular sources, because ROS generation was abolished to control levels in the absence of extracellular Ca2+. Considering previous observation of superoxide generation by Ca2+ influx through P2X(7)R in microglia, we hypothesized that ROS production in fAbeta-stimulated microglia might be mediated by ATP released from the microglia. We therefore examined whether fAbeta(1-42)-induced Ca2+ influx was mediated through P2X(7)R activation. In serial experiments, we found that microglial pretreatment with the P2X(7)R antagonists Pyridoxal-phosphate-6-azophenyl-2',4'- disulfonate (100 micrometer) or oxidized ATP (100 micrometer) inhibited fAbeta-induced Ca2+ influx and reduced ROS generation to basal levels. Furthermore, ATP efflux from fAbeta(1-42)-stimulated microglia was observed, and apyrase treatment decreased the generation of ROS. These findings provide conclusive evidence that fAbeta-stimulated ROS generation in microglial cells is regulated by ATP released from the microglia in an autocrine manner.
Adenosine Triphosphate/*metabolism ; Amyloid beta-Protein/*pharmacology ; Animals ; Autocrine Communication/*drug effects/physiology ; Cells, Cultured ; Microglia/*drug effects ; Peptide Fragments/*pharmacology ; Pyridoxal Phosphate/analogs & derivatives/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/*metabolism ; Receptors, Purinergic P2/physiology

OBJECTIVETo observe the effect of normobaric hyperoxia exposure on the functions of N9 microglia and explore the underlying mechanism of hyperoxia-induced immature brain injury.

METHODSN9 microglial cells were exposed to 900 ml/L O(2) for 2, 6, 12, 24 or 48 h, and the cell apoptotic rate was assessed using flow cytometry. The intracellular oxidative stress was measured using a fluorescent DCFH-DA probe, and the expression of Toll-like receptor 4 (TLR4) mRNA was detected using RT-PCR. Interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) concentrations in the supernatant of the cell cultures were tested with ELISA following the exposures. TLR4 protein expression was observed using immunofluorescence staining.

RESULTSSignificant cell apoptosis was detected after oxygen exposures for 12-24 h. Accumulation of reactive oxygen species (ROS) were detected after a 2-h exposure. After prolonged hyperoxia exposure, TLR4 expression and IL-1β and TNF-α levels significantly increased in the cells.

CONCLUSIONHyperoxia exposure activates TLR4 signaling pathway in N9 microglial cells in vitro, leading to massive production of ROS, IL-1β, and TNF-α and thus triggering cell apoptosis.

Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; Cells, Cultured ; Interleukin-1beta ; metabolism ; Mice ; Mice, Inbred ICR ; Microglia ; cytology ; drug effects ; physiology ; Oxygen ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Reactive Oxygen Species ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism

Excessive production of nitric oxide (NO) and proinflammatory cytokines from activated microglia play an important role in human neurodegenerative disorders. Here, we investigated whether celastrol, which has been used as a potent anti-inflammatory and anti-oxidative agent in Chinese medicine, attenuates excessive production of NO and proinflammatory cytokines such as TNF-alpha and IL-1beta in LPS-stimulated BV-2 cells, a mouse microglial cell line. We report here that the LPS-elicited excessive production of NO, TNF-alpha, and IL-1beta in BV-2 cells was largely inhibited in the presence of celastrol, and the attenuation of inducible iNOS and these cytokines resulted from the reduced expression of mRNAs of iNOS and these cytokines, respectively. The molecular mechanisms that underlie celastrol-mediated attenuation were the inhibition of LPS-induced phosphorylation of MAPK/ERK1/2 and the DNA binding activity of NF-kappaB in BV-2 cells. The results indicate that celastrol effectively attenuated NO and proinflammatory cytokine production via the inhibition of ERK1/2 phosphorylation and NF-kappaB activation in LPS-activated microglia. Thus, celastrol may be an effective therapeutic candidate for use in the treatment of neurodegenerative human brain disorders.
Animals ; Cell Line ; Cytokines/*biosynthesis/drug effects ; Gene Expression Regulation, Enzymologic/drug effects/immunology ; Inflammation/immunology ; Inflammation Mediators/immunology ; Mice ; Microglia/*drug effects/immunology ; Mitogen-Activated Protein Kinases/*physiology ; NF-kappa B/metabolism/*physiology ; Nitric Oxide/*metabolism ; Nitric Oxide Synthase Type II/biosynthesis/drug effects ; RNA, Messenger/analysis ; Signal Transduction/*drug effects/physiology ; Transcription, Genetic/drug effects/immunology ; Triterpenes/*pharmacology

Occupational exposure to 1-bromopropane (1-BP) induces learning and memory deficits. However, no therapeutic strategies are currently available. Accumulating evidence has suggested that N-methyl-D-aspartate receptors (NMDARs) and neuroinflammation are involved in the cognitive impairments in neurodegenerative diseases. In this study we aimed to investigate whether the noncompetitive NMDAR antagonist MK801 protects against 1-BP-induced cognitive dysfunction. Male Wistar rats were administered with MK801 (0.1 mg/kg) prior to 1-BP intoxication (800 mg/kg). Their cognitive performance was evaluated by the Morris water maze test. The brains of rats were dissected for biochemical, neuropathological, and immunological analyses. We found that the spatial learning and memory were significantly impaired in the 1-BP group, and this was associated with neurodegeneration in both the hippocampus (especially CA1 and CA3) and cortex. Besides, the protein levels of phosphorylated NMDARs were increased after 1-BP exposure. MK801 ameliorated the 1-BP-induced cognitive impairments and degeneration of neurons in the hippocampus and cortex. Mechanistically, MK801 abrogated the 1-BP-induced disruption of excitatory and inhibitory amino-acid balance and NMDAR abnormalities. Subsequently, MK801 inhibited the microglial activation and release of pro-inflammatory cytokines in 1-BP-treated rats. Our findings, for the first time, revealed that MK801 protected against 1-BP-induced cognitive dysfunction by ameliorating NMDAR function and blocking microglial activation, which might provide a potential target for the treatment of 1-BP poisoning.
Animals ; Brain ; drug effects ; metabolism ; pathology ; Cognitive Dysfunction ; drug therapy ; metabolism ; pathology ; Disease Models, Animal ; Dizocilpine Maleate ; pharmacology ; Excitatory Amino Acid Antagonists ; pharmacology ; Hydrocarbons, Brominated ; Inflammasomes ; drug effects ; metabolism ; Male ; Maze Learning ; drug effects ; physiology ; Microglia ; drug effects ; metabolism ; pathology ; NLR Family, Pyrin Domain-Containing 3 Protein ; metabolism ; Neurons ; drug effects ; metabolism ; pathology ; Nootropic Agents ; pharmacology ; Random Allocation ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; metabolism ; Spatial Memory ; drug effects ; physiology ; Specific Pathogen-Free Organisms

To elucidate the roles of 8-hydroxydeoxyguanosine (oh8dG), the nucleoside of 8-hydroxyguanine (oh8Gua), we examined the effects of oh8dG upon LPS-induced intercellular adhesion molecule-1 (ICAM-1) expression and the underlying mechanisms in brain microglial cells. We found that oh8dG reduces LPS-induced reactive oxygen species (ROS) production, STAT3 activation, and ICAM-1 expression. oh8dG also suppresses pro-inflammatory cytokines, such as TNF-alpha, IL-6 and IFN-gamma. Overexpression of dominant negative STAT3 completely diminshed STAT3-mediated ICAM-1 transcriptional activity. Chromatin immunoprecipitation studies revealed that oh8dG inhibited recruitment of STAT3 to the ICAM-1 promoter, followed by a decrease in ICAM-1 expression. Using mice lacking a functional Toll-like receptor 4 (TLR4), we demonstrated that, while TLR4+/+ microglia were activated by LPS, TLR4-/-microglia exhibited inactivated STAT3 in response to LPS. Evidently, LPS modulates STAT3-dependent ICAM-1 induction through TLR4-mdiated cellular responses. Oh8dG apparently plays a role in anti-inflammatory actions via suppression of ICAM-1 gene expression by blockade of the TLR4-STAT3 signal cascade in inflammation-enhanced brain microglia. Therefore, oh8dG in the cytosol probably functions as an anti-inflammatory molecule and should be considered as a candidate for development of anti-inflammatory agents.
Toll-Like Receptor 4/genetics ; STAT3 Transcription Factor/physiology ; Reactive Oxygen Species/metabolism ; Microglia/*drug effects/metabolism ; Mice, Knockout ; Mice, Inbred C57BL ; Mice ; Male ; Lipopolysaccharides/*pharmacology ; Intercellular Adhesion Molecule-1/*metabolism ; Inflammation Mediators/metabolism ; Encephalitis/drug therapy ; Deoxyguanosine/*analogs & derivatives/pharmacology/therapeutic use ; Cytokines/biosynthesis ; Cell Survival/drug effects ; Brain/cytology/drug effects ; Anti-Inflammatory Agents, Non-Steroidal/*pharmacology ; Animals

OBJECTIVETo evaluate the role of thrombin-activated microglia in the neurodegeneration of nigral dopaminergic neurons in the rat substantia nigra (SN) in vivo.

METHODSAfter stereotaxic thrombin injection into unilateral SN of rats, immunostaining, reverse transcription polymerase chain reaction (RT-PCR) and biochemical methods were used to observe tyrosine hydroxylase (TH) immunoreactive positive cells, microglia activation, nitric oxide (NO) amount and inducible nitric-oxide synthase (iNOS) expression.

RESULTS(1) Selective damage to dopaminergic neurons was produced after thrombin injection, which was evidenced by loss of TH immunostaining in time-dependent manner; (2) Strong microglial activation was observed in the SN; (3) RT-PCR demonstrated the early and transient expression of neurotoxic factors iNOS mRNA in the SN. Immunofluorescence results found that thrombin induced expression of iNOS in microglia. The NO production in the thrombin-injected rats was significantly higher than that of controls (P < 0.05).

CONCLUSIONThrombin intranigral injection can injure the dopaminergic neurons in the SN. Thrombin-induced microglia activation precedes dopaminergic neuron degeneration, which suggest that activation of microglia and release of NO may play important roles in dopaminergic neuronal death in the SN.

Animals ; Disease Progression ; Dopamine ; biosynthesis ; Encephalitis ; chemically induced ; metabolism ; physiopathology ; Female ; Gliosis ; chemically induced ; metabolism ; physiopathology ; Immunohistochemistry ; Inflammation Mediators ; toxicity ; Injections ; Microglia ; drug effects ; metabolism ; Nerve Degeneration ; chemically induced ; metabolism ; physiopathology ; Neurons ; drug effects ; metabolism ; pathology ; Nitric Oxide ; biosynthesis ; Nitric Oxide Synthase Type II ; drug effects ; metabolism ; Oxidative Stress ; drug effects ; physiology ; Parkinsonian Disorders ; chemically induced ; metabolism ; physiopathology ; RNA, Messenger ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Substantia Nigra ; drug effects ; metabolism ; physiopathology ; Thrombin ; toxicity ; Time Factors ; Tyrosine 3-Monooxygenase ; drug effects ; genetics ; metabolism ; Up-Regulation ; drug effects ; physiology

A degree of brain inflammation is required for repair of damaged tissue, but excessive inflammation causes neuronal cell death. Here, we observe that IL-10 is expressed in LPS-injected rat cerebral cortex, contributing to neuronal survival. Cells immunopositive for IL-10 were detected as early as 8 h post-injection and persisted for up to 3 d, in parallel with the expression of IL-1beta, TNF-alpha, and iNOS. Double immunofluorescence staining showed that IL-10 expression was localized mainly in activated microglia. Next, we examined the neuroprotective effects of IL-10 using IL-10 neutralizing antibody (IL-10NA). Blockade of IL-10 action caused a significant loss of neurons both 3 d and 7 d after LPS injection. Further, the induction of mRNA species encoding IL-1beta, TNF-alpha, and iNOS, reactive oxygen species (ROS) production, and NADPH oxidase activation, increased after co-injection of LPS and IL-10NA, compared to the levels seen after injection of LPS alone. Taken together, these results clearly suggest that LPS-induced endogenous expression of IL-10 in microglia contributes to neuronal survival by inhibiting brain inflammation.
Animals ; Cerebral Cortex/drug effects/*pathology ; Fluorescent Antibody Technique ; Interleukin-10/immunology/*physiology ; Lipopolysaccharides/*pharmacology ; Microglia/cytology/*metabolism ; Nerve Degeneration/pathology/*prevention & control ; Neurons/cytology/drug effects/*metabolism ; Nitric Oxide Synthase/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism

Microglia are the principal immune effectors in brain and participate in a series ofneurodegenerative diseases. The microglial shapes are highly plastic. The morphology is closely related with their activation status and biological functions. Cerebral ischemia could induce microglial activation, and microglial activation is subjected to precise regulation. Microglia could play either protective or neurotoxic roles in cerebral ischemia. Therefore, regulating the expression of receptors or protein molecules on microglia, inhibiting the excessive activation of microglia and production of pro-inflammatory factors, promoting the release of neuroprotective substances might be beneficial to the treatment of cerebral ischemia. The study about relationship between microglia and cerebral ischemia will shed a light on the treatment of cerebral ischemia. This paper is a review of microglial activation and regulation during cerebral ischemia as well as related therapeutic methods.
Animals ; Brain Ischemia ; metabolism ; pathology ; Class Ib Phosphatidylinositol 3-Kinase ; metabolism ; Humans ; Inflammation ; metabolism ; Microglia ; cytology ; drug effects ; metabolism ; physiology ; Neuroprotective Agents ; pharmacology ; Nitric Oxide Synthase ; metabolism ; Receptors, Purinergic P2X7 ; metabolism ; Regeneration ; TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Toll-Like Receptors ; metabolism

Chronic neuroinflammation is an integral pathological feature of major neurodegenerative diseases. The recruitment of microglia to affected brain regions and the activation of these cells are the major events leading to disease-associated neuroinflammation. In a previous study, we showed that neuron-released alpha-synuclein can activate microglia through activating the Toll-like receptor 2 (TLR2) pathway, resulting in proinflammatory responses. However, it is not clear whether other signaling pathways are involved in the migration and activation of microglia in response to neuron-released alpha-synuclein. In the current study, we demonstrated that TLR2 activation is not sufficient for all of the changes manifested by microglia in response to neuron-released alpha-synuclein. Specifically, the migration of and morphological changes in microglia, triggered by neuron-released alpha-synuclein, did not require the activation of TLR2, whereas increased proliferation and production of cytokines were strictly under the control of TLR2. Construction of a hypothetical signaling network using computational tools and experimental validation with various peptide inhibitors showed that beta1-integrin was necessary for both the morphological changes and the migration. However, neither proliferation nor cytokine production by microglia was dependent on the activation of beta1-integrin. These results suggest that beta1-integrin signaling is specifically responsible for the recruitment of microglia to the disease-affected brain regions, where neurons most likely release relatively high levels of alpha-synuclein.
Animals ; Antigens, CD29/genetics/*metabolism ; Cell Line, Tumor ; *Cell Movement ; Cells, Cultured ; Culture Media, Conditioned/*pharmacology ; Gene Regulatory Networks ; Humans ; Mice ; Mice, Inbred C57BL ; Microglia/drug effects/metabolism/*physiology ; Neurons/*metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Toll-Like Receptor 2/metabolism ; alpha-Synuclein/*pharmacology