Discovery of novel MIF inhibitors that attenuate microglial inflammatory activation by structures-based virtual screening and in vitro bioassays.

Macrophage migration inhibitory factor (MIF) is a pluripotent pro-inflammatory cytokine and is related to acute and chronic inflammatory responses, immune disorders, tumors, and other diseases. In this study, an integrated virtual screening strategy and bioassays were used to search for potent MIF inhibitors. Twelve compounds with better bioactivity than the prototypical MIF-inhibitor ISO-1 (IC50 = 14.41 µM) were identified by an in vitro enzymatic activity assay. Structural analysis revealed that these inhibitors have novel structural scaffolds. Compound 11 was then chosen for further characterization in vitro, and it exhibited marked anti-inflammatory efficacy in LPS-activated BV-2 microglial cells by suppressing the activation of nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs). Our findings suggest that MIF may be involved in the regulation of microglial inflammatory activation and that small-molecule MIF inhibitors may serve as promising therapeutic agents for neuroinflammatory diseases.

Early glycolytic reprogramming controls microglial inflammatory activation.

BACKGROUND: Microglial activation-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases. Inflammatory activation of microglial cells is often accompanied by a metabolic switch from oxidative phosphorylation to aerobic glycolysis. However, the roles and molecular mechanisms of glycolysis in microglial activation and neuroinflammation are not yet fully understood. METHODS: The anti-inflammatory effects and its underlying mechanisms of glycolytic inhibition in vitro were examined in lipopolysaccharide (LPS) activated BV-2 microglial cells or primary microglial cells by enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, immunoprecipitation, flow cytometry, and nuclear factor kappa B (NF-κB) luciferase reporter assays. The anti-inflammatory and neuroprotective effects of glycolytic inhibitor, 2-deoxoy-D-glucose (2-DG) in vivo were measured in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-or LPS-induced Parkinson's disease (PD) models by immunofluorescence staining, behavior tests, and Western blot analysis. RESULTS: We found that LPS rapidly increased glycolysis in microglial cells, and glycolysis inhibitors (2-DG and 3-bromopyruvic acid (3-BPA)), siRNA glucose transporter type 1 (Glut-1), and siRNA hexokinase (HK) 2 abolished LPS-induced microglial cell activation. Mechanistic studies demonstrated that glycolysis inhibitors significantly inhibited LPS-induced phosphorylation of mechanistic target of rapamycin (mTOR), an inhibitor of nuclear factor-kappa B kinase subunit beta (IKKß), and NF-kappa-B inhibitor alpha (IκB-α), degradation of IκBα, nuclear translocation of p65 subunit of NF-κB, and NF-κB transcriptional activity. In addition, 2-DG significantly inhibited LPS-induced acetylation of p65/RelA on lysine 310, which is mediated by NAD-dependent protein deacetylase sirtuin-1 (SIRT1) and is critical for NF-κB activation. A coculture study revealed that 2-DG reduced the cytotoxicity of activated microglia toward MES23.5 dopaminergic neuron cells with no direct protective effect. In an LPS-induced PD model, 2-DG significantly ameliorated neuroinflammation and subsequent tyrosine hydroxylase (TH)-positive cell loss. Furthermore, 2-DG also reduced dopaminergic cell death and microglial activation in the MPTP-induced PD model. CONCLUSIONS: Collectively, our results suggest that glycolysis is actively involved in microglial activation. Inhibition of glycolysis can ameliorate microglial activation-related neuroinflammatory diseases.

Knockdown of milk-fat globule EGF factor-8 suppresses glioma progression in GL261 glioma cells by repressing microglial M2 polarization.

Tumor-associated microglial cells promote glioma growth, invasion, and chemoresistance by releasing inflammatory factors. Milk fat globule EGF factor 8 protein (MFG-E8), a secreted glycoprotein, is closely related to tissue homeostasis and anti-inflammation. In the present study, we investigated the role of MFG-E8 in microglial polarization and glioma progression in vitro and in vivo. We found that glioma cells secrete comparable amounts of MFG-E8 in culture media to astrocytes. Recombinant MFG-E8 triggered microglia to express the M2 polarization markers, such as arginase-1 (ARG-1), macrophage galactose-type C-type lectin-2 (MGL-2), and macrophage mannose receptor (CD206). Forced expression of MFG-E8 in BV-2 microglia cells not only promoted IL-4-induced M2 polarization but also inhibited lipopolysaccharide (LPS)-induced M1 microglial polarization. Mechanistic studies demonstrated that recombinant MFG-E8 markedly induced signal transducer and activator of transcription 3 (STAT3) phosphorylation, and the STAT3 inhibitor stattic significantly blocked MFG-E8-induced ARG-1 expression. Administration of antibody against MFG-E8 and knockdown of its receptor, integrin ß3, significantly attenuated MFG-E8-induced ARG-1 expression. Similarly, knockdown of MFG-E8 also markedly reduced IL-4-induced M2 marker expression and increased LPS-induced M1 marker expression in microglia cells. Moreover, the knockdown of MFG-E8 in GL261 glioma cells inhibited cell proliferation and enhanced chemosensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), which was likely associated with the downregulation of FAK/AKT activation and STAT3/cyclin D1 signaling. The murine GL261 glioma experimental model demonstrated that knockdown of MFG-E8 significantly reduced tumor size and extended survival times. Additionally, attenuated CD11b+ cell infiltration and reduced CD206+ expression in CD11b+ cells were also observed in an MFG-E8 knockdown GL261 murine glioma model. These results suggested that inhibition of MFG-E8 might hamper the immunosuppressive microenvironment in gliomas and therefore ameliorate tumor progression.

PHLDA1 promotes microglia-mediated neuroinflammation via regulating K63-linked ubiquitination of TRAF6.

Microglia-mediated neuroinflammation plays an important role in the progression of neurodegenerative diseases including Parkinson's disease (PD). Pleckstrin homology-like domain family A member 1 (PHLDA1) plays an important role in immunological regulation, particularly in the Toll-like receptor-mediated immune response. Here, we explored the potential roles of PHLDA1 in microglia-mediated inflammation and neuronal protection. We found that PHLDA1 expression was rapidly increased in response to inflammatory stimuli in microglia cells in vivo or in vitro. Knockdown of PHLDA1 using adeno-associated virus serotype (AAV) ameliorated MPTP-induced motor deficits and inhibited neuroinflammation in mice. In support of this observation in vivo, we found that LPS-induced proinflammatory gene expression, including TNF-α, IL-1ß, iNOS, and COX-2, was decreased in PHLDA1-deficient microglial cells. Mechanistic studies demonstrated that increased expression of PHLDA1, upon LPS stimulation in microglia, led to direct interaction with TRAF6 and enhanced its K63-linked ubiquitination-mediated NF-κB signaling activation. PHLDA1 deficiency interfered with TRAF6 K63-linked ubiquitination and inhibited microglial inflammatory responses. These findings reveal the first evidence that PHLDA1 is an important modulator of microglial function that is associated with microglia-mediated dopaminergic neurotoxicity. The data therefore provided the first evidence that PHLDA1 may be a potent modulator for neuroinflammation, and PHLDA1 may be a novel drug target for treatment of neuroinflammation-related diseases such as PD.

Development and characterization of an inducible Dicer conditional knockout mouse model of Parkinson's disease: validation of the antiparkinsonian effects of a sigma-1 receptor agonist and dihydromyricetin.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by motor impairment and progressive loss of dopamine (DA) neurons. At present, the acute application of neurotoxic drugs such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and 6-hydroxydopamine (6-OHDA) are commonly used to simulate the pathology of PD; however, it is difficult to induce the progressive pathogenesis of PD with these models. In this study, we employed DAT promoter-mediated Cre transgenic mice to establish tamoxifen-inducible Dicer conditional knockout (cKO) mice in an effort to mimic the progressive loss of DA neurons and the development of PD-like behavioral phenotypes. The results showed that Dicer cKO mice exhibited progressive loss of DA neurons in the substantia nigra (SN) following tamoxifen administration. Significant DA loss was observed 6 weeks after tamoxifen administration; accordingly, progressive motor function impairment was also observed. We also found that a significant neuroinflammatory response, as evidenced by microglial proliferation, another hallmark of PD pathogenesis, accompanied the loss of DA neurons. The acute application of levo-DOPA (L-DOPA) relieved the PD-like motor impairments in Dicer cKO mice to exert its antiparkinsonian action, indicating that the model can be used to evaluate the antiparkinsonian efficacy of PD drugs. To further elucidate the potential application of this novel PD animal model for PD drug development, we employed the powerful neuroprotective agent dihydromyricetin (DHM) (10 mg/kg) and the selective sigma-1 receptor agonist PRE-084 (1 mg/kg), both of which were previously shown to produce antiparkinsonian effects. The results indicated that the chronic administration of either DHM or PRE-084 attenuated the Dicer cKO-induced loss of DA neurons and motor impairments, although the two drugs acted through different mechanisms. These data indicate that the Dicer cKO mouse model may be a useful model for investigating the pathological development of PD and intervention-mediated changes. In conclusion, this transgenic mouse model appears to simulate the progressive pathogenesis of PD and may be a potentially useful model for PD drug discovery.

The oncometabolite 2-hydroxyglutarate inhibits microglial activation via the AMPK/mTOR/NF-κB pathway.

Microglia, the brain-resident macrophage, is known as the innate immune cell type in the central nervous system. Microglia is also the major cellular component of tumor mass of gliomas that plays a key role in glioma development. Mutations of isocitrate dehydrogenases 1 and 2 (IDH1/2) frequently occur in gliomas, which leads to accumulation of oncometabolic product 2-hydroxyglutarate (2HG). Moreover, IDH1/2 mutations were found to correlate with better prognosis in glioma patients. In the present study, we investigated the effects of the 2HG on microglial inflammatory activation. We showed that the conditioned media (CM) from GL261 glioma cells stimulated the activation of BV-2 microglia cells, evidenced by markedly increased expression of interleukin-6 (IL-6), IL-1ß, tumor necrosis factor-α (TNF-α), CCL2 (C-C motif chemokine ligand 2) and CXCL10 (C-X-C motif chemokine 10). CM-induced expression of proinflammatory genes was significantly suppressed by pretreatment with a synthetic cell-permeable 2HG (1 mM) or a nuclear factor-κB (NF-κB) inhibitor BAY11-7082 (10 µM). In lipopolysaccharide (LPS)- or TNF-α-stimulated BV-2 microglia cells and primary microglia, pretreatment with 2HG (0.25-1 mM) dose-dependently suppressed the expression of proinflammatory genes. We further demonstrated that 2HG significantly suppressed LPS-induced phosphorylation of IκB kinase α/ß (IKKα/ß), IκBα and p65, IκB degradation, and nuclear translocation of p65 subunit of NF-κB, as well as NF-κB transcriptional activity. Similarly, ectopic expression of mutant isocitrate dehydrogenase 1 (IDH1) (R132H) significantly decreased TNF-α-induced activation of NF-κB signaling pathway. Finally, we revealed that activation of adenosine 5'-monophosphate-activated protein kinase (AMPK) and subsequent inhibition of mammalian target of rapamycin (mTOR) signaling contributed to the inhibitory effect of 2HG on NF-κB signaling pathway in BV-2 cells. Taken together, these results, for the first time, show that oncometabolite 2HG inhibits microglial activation through affecting AMPK/mTOR/NF-κB signaling pathway and provide evidence that oncometabolite 2HG may regulate glioma development via modulating microglial activation in tumor microenvironment.

Macrophage migration inhibitory factor (MIF) inhibitor, Z-590 suppresses cartilage destruction in adjuvant-induced arthritis via inhibition of macrophage inflammatory activation.

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pleiotropic pro-inflammatory mediator that is involved in the progression of rheumatoid arthritis (RA). Previously, we demonstrated a small molecule compound 3-[(biphenyl-4-ylcarbonyl) carbamothioyl] amino benzoic acid (Z-590) could inhibit MIF activity with docking-based virtual screening and experimental evaluation. METHODS: The LPS activated RAW264.7 macrophage cells were used to determine the anti-inflammatory effects of Z-590 in vitro. A rat adjuvant-induced arthritis (AIA) model was used to determine the anti-arthritic effects of Z-590 in vivo. RESULTS: MIF inhibitor Z-590 significantly inhibited the production of NO, TNF-α and IL-6 in LPS-activated RAW 264.7 macrophage cells and markedly inhibited LPS-induced expression of TNF-α, IL-6, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Z-590 also significantly reduced paw edema, serum level of TNF-α, IL-6 and spleen index in the adjuvant-induced arthritis (AIA) rat model. Furthermore, Z-590 markedly ameliorated joint inflammation and articular cartilage damage in AIA rat model. CONCLUSION: MIF inhibitor Z-590 possesses potent anti-arthritic activity through suppression of macrophage activation, and could be a potential therapeutic treatment for RA.

Clk1-regulated aerobic glycolysis is involved in glioma chemoresistance.

Chemoresistance remains a major challenge for the treatment of glioma. In this study, we investigated the role of Clock 1 (Clk1), which encodes an enzyme that is necessary for ubiquinone biosynthesis in glioma chemoresistance in vitro. The results showed that Clk1 was highly expressed in GL261 mouse glioma cells which were most sensitive to 1,3Bis (2-chloroethyl) 1 nitrosourea (BCNU) while was low expressed in BCNU resistant cells such as glioma cancer stem cells, T98G, U87MG and U251 glioma cells. Knockdown of Clk1 in GL261 glioma cells significantly reduced BCNU- or cisplatin-induced cell apoptosis, whereas the proliferative activity and the expression of multidrug resistance-related genes including MDR1, O6-methylguanine-DNA methyltransferase, and GSTP1 were not changed. When Clk1 was re-expressed in Clk1 knockdown GL261 glioma cells, the BCNU sensitivity was restored. The mechanistic study revealed that knockdown of Clk1 in GL261 glioma cells increased aerobic glycolysis including high glucose consumption, lactate production, and up-regulation of glycolysis-associated genes. Inhibition of glycolysis can reverse the chemoresistance elicited by Clk1 knockdown in GL261 cells. Moreover, knockdown of Clk1 induced HIF-1α expression in GL261 glioma cells which was found to be mediated by AMP-activated protein kinase (AMPK)/mechanistic target of rapamycin (mTOR) signaling pathway. Both metformin and rapamycin reversed the chemoresistance of Clk1 knockdown GL261 glioma cells. Over-expression of Clk1 significantly increased the sensitivity of T98G or U251 human glioblastoma cells to BCNU which was accompanied by decreased lactate secretion, decreased expression of HIF-1α, AMPK activation, and inhibition of mTOR pathway. Inhibition of glycolysis or activation of AMPK did not alter Clk1 expression in variant glioma cell lines suggesting that aerobic glycolysis is not an upstream event of Clk1 expression in glioma cells. Taken together, our results revealed, for the first time, that mitochondrial Clk1 regulated chemoresistance in glioma cells through AMPK/mTOR/HIF-1α mediated glycolysis pathway.

Activation of Nur77 in microglia attenuates proinflammatory mediators production and protects dopaminergic neurons from inflammation-induced cell death.

Microglia-mediated neuroinflammation plays a critical role in the pathological development of Parkinson's disease (PD). Orphan nuclear receptor Nur77 (Nur77) is abundant in neurons, while its role in microglia-mediated neuroinflammation remains unclear. The present data demonstrated that the expression of Nur77 in microglia was reduced accompanied by microglia activation in response to lipopolysaccharide (LPS) in vitro and in experimental 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-PD mouse model. Nur77 over-expression or application of Nur77 agonist cytosporone B suppressed the expression of proinflammatory genes, such as inducible nitric oxide NOS, cyclooxygenase-2, IL-1ß, and tumor necrosis factor-α in the activated microglia, while silenced Nur77 exaggerated the inflammatory responses in microglia. Moreover, activation of Nur77 suppressed the LPS-induced NF-κB activation which was partly dependent on p38 MAPK activity, since inhibition of p38 MAPK by SB203580 abolished the LPS-activated NF-κB in microglia. On the other hand, inhibition of p38 MAPK attenuated LPS-induced Nur77 reduction. Furthermore, in a microglia-conditioned cultured media system, Nur77 ameliorated the cytotoxicity to MN9D dopaminergic cells. Lastly, cytosporone B attenuated microglia activation and loss of dopaminergic neuron in the substantia nigra pars compacta (SNpc) of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-PD mouse model. Taken together, these findings revealed the first evidence that Nur77 was an important modulator in microglia function that associated with microglia-mediated dopaminergic neurotoxicity, and thus modulation of Nur77 may represent a potential novel target for treatment for neurodegenerative disease.

Clk1 deficiency promotes neuroinflammation and subsequent dopaminergic cell death through regulation of microglial metabolic reprogramming.

Clock (Clk)1/COQ7 is a mitochondrial hydroxylase that is necessary for the biosynthesis of ubiquinone (coenzyme Q or UQ). Here, we investigate the role of Clk1 in neuroinflammation and consequentially dopaminergic (DA) neuron survival. Reduced expression of Clk1 in microglia enhanced the LPS-induced proinflammatory response and promoted aerobic glycolysis. Inhibition of glycolysis abolished Clk1 deficiency-induced hypersensitivity to the inflammatory stimulation. Mechanistic studies demonstrated that mTOR/HIF-1α and ROS/HIF-1α signaling pathways were involved in Clk1 deficiency-induced aerobic glycolysis. The increase in neuronal cell death was observed following treatment with conditioned media from Clk1 deficient microglia. Increased DA neuron loss and microgliosis were observed in Clk1+/- mice after treatment with MPTP, a rodent model of Parkinson's disease (PD). This increase in DA neuron loss was due to an exacerbated microglial inflammatory response, rather than direct susceptibility of Clk1+/- DA cells to MPP+, the active species of MPTP. Exaggerated expressions of proinflammatory genes and loss of DA neurons were also observed in Clk1+/- mice after stereotaxic injection of LPS. Our results suggest that Clk1 regulates microglial metabolic reprogramming that is, in turn, involved in the neuroinflammatory processes and PD.

Inhibition of macrophage migration inhibitory factor (MIF) tautomerase activity suppresses microglia-mediated inflammatory responses.

Macrophage migration inhibitory factor (MIF), a pleiotropic pro-inflammatory cytokine, is a key regulator in both innate and acquired immunity systems. MIF has become a promising drug target for inflammatory diseases. Apart from its cytokine activities, MIF is known to act as a d-dopachrome tautomerase. Our previous work has identified that 3-[(biphenyl-4-ylcarbonyl)carbamothioyl]amino benzoic acid (Z-590) exhibited a potent inhibitory activity against MIF. In this study, we investigate the effect of Z-590 on lipopolysaccharide (LPS)-activated microglial cell activation. Our results demonstrate that Z-590 significantly decreases the production of nitric oxide (NO), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-1ß, cyclooxygenase (COX-2), inducible nitric oxide synthase (iNOS) as well as reactive oxygen species (ROS) involved in inhibiting MAKPs signalling pathway in LPS-stimulated microglia cells. Furthermore, Z-590 reduced cytotoxicity of activated microglia toward HT-22 hippocampal cells in a microglia-conditioned medium system. Taken together, these results indicate that MIF inhibitor Z-590 elicits a potent inhibitor for microglia-mediated neuroinflammation.

Allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation.

Recent studies have shown that sigma-1 receptor orthodox agonists can inhibit neuroinflammation. SKF83959 (3-methyl-6-chloro-7,8-hydroxy-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1H-3-benzazepine), an atypical dopamine receptor-1 agonist, has been recently identified as a potent allosteric modulator of sigma-1 receptor. Here, we investigated the anti-inflammatory effects of SKF83959 in lipopolysaccharide (LPS)-stimulated BV2 microglia. Our results indicated that SKF83959 significantly suppressed the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS), and inhibited the generation of reactive oxygen species. All of these responses were blocked by selective sigma-1 receptor antagonists (BD1047 or BD1063) and by ketoconazole (an inhibitor of enzyme cytochrome c17 to inhibit the synthesis of endogenous dehydroepiandrosterone, DHEA). Additionally, we found that SKF83959 promoted the binding activity of DHEA with sigma-1 receptors, and enhanced the inhibitory effects of DHEA on LPS-induced microglia activation in a synergic manner. Furthermore, in a microglia-conditioned media system, SKF83959 inhibited the cytotoxicity of conditioned medium generated by LPS-activated microglia toward HT-22 neuroblastoma cells. Taken together, our study provides the first evidence that allosteric modulation of sigma-1 receptors by SKF83959 inhibits microglia-mediated inflammation. SKF83959 is a potent allosteric modulator of sigma-1 receptor. Our results indicated that SKF83959 enhanced the activity of endogenous dehydroepiandrosterone (DHEA) in a synergic manner, and inhibited the activation of BV2 microglia and the expression/release of the pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), inducible nitric oxide synthase (iNOS).

Anti-inflammatory effects of glaucocalyxin B in microglia cells.

Over-activated microglia is involved in various kinds of neurodegenerative process including Parkinson, Alzheimer and HIV dementia. Suppression of microglial over activation has emerged as a novel strategy for treatment of neuroinflammation-based neurodegeneration. In the current study, anti-inflammatory and neuroprotective effects of the ent-kauranoid diterpenoids, which were isolated from the aerial parts of Rabdosia japonica (Burm. f.) var. glaucocalyx (Maxim.) Hara, were investigated in cultured microglia cells. Glaucocalyxin B (GLB), one of five ent-kauranoid diterpenoids, significantly decreased the generation of nitric oxide (NO), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) in the lipopolysaccharide (LPS)-activated microglia cells. In addition, GLB inhibited activation of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and generation of reactive oxygen species (ROS) in LPS-activated microglia cells. Furthermore, GLB strongly induced the expression of heme oxygenase (HO)-1 in BV-2 microglia cells. Finally, GLB exhibited neuroprotective effect by preventing over-activated microglia induced neurotoxicity in a microglia/neuron co-culture model. Taken together, the present study demonstrated that the GLB possesses anti-nueroinflammatory activity, and might serve as a potential therapeutic agent for treating neuroinflammatory diseases.

Synthesis of 5α-cholestan-6-one derivatives and their inhibitory activities of NO production in activated microglia: discovery of a novel neuroinflammation inhibitor.

Glial activation-mediated neuroinflammation plays a pivotal role in the process of several neuroinflammatory diseases including stroke, Alzheimer's diseases, Parkinson's diseases, multiple sclerosis and ischemia. Inhibition of microglial activation may ameliorate neuronal degeneration under the inflammatory conditions. In the present study, a number of 5α-cholestan-6-one derivatives were prepared and the anti-inflammatory effects of these compounds were evaluated in LPS-stimulated BV-2 microglia cells. Those derivatives were synthesized from readily available hyodeoxycholic acid (1). Among the tested compounds, several analogs (16-18, 25, 35, 38) exhibited potent inhibitory activities on nitric oxide production with no or weak cell toxicity. Compound 16 also significantly suppressed the expression of TNF-α, interleukin (IL)-1ß, cyclooxygenase (COX-2) as well as inducible nitric oxide synthase (iNOS) in LPS-stimulated BV-2 microglia cells. In addition, compound 16 markedly reduced infarction volume in a focal ischemic mice model.

1-O-tigloyl-1-O-deacetyl-nimbolinin B inhibits LPS-stimulated inflammatory responses by suppressing NF-κB and JNK activation in microglia cells.

Overactivation of microglia may contribute to the pathogenesis of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and HIV dementia. Thus, regulating microglial activation has been an important therapeutic strategy for treating neurodegenerative diseases. In this research, we compared three limonoids compounds extracted from Melia toosendan by a cell-based assay to investigate their anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated microglia cells. Our study indicated that 1-O-tigloyl-1-O-deacetyl-nimbolinin B (TNB) markedly suppressed the production of nitric oxide (NO) and tumor necrosis factor (TNF)-α in LPS-stimulated microglia cells. TNB also inhibited the gene expression of inducible nitric oxide synthase (iNOS), TNF-α, cyclooxygenase (COX-2), and interleukin (IL)-1ß. In addition, TNB inhibited generation of intracellular reactive oxygen species (ROS). We found that TNB significantly attenuated the nuclear translocation of NF-κB, inhibiting the activation of c-jun N-terminal kinase (JNK) in LPS-stimulated BV-2 cells. Furthermore, TNB reduced cytotoxicity of activated microglia toward HT-22 hippocampal cells in a co-culture system. Taken together, our experimental results reveal, for the first time, that TNB is a potent inhibitor of microglia-mediated inflammation, and it might be a potential candidate for the treatment of neurodegenerative diseases.

Antiinflammatory effects of orientin-2"-O-galactopyranoside on lipopolysaccharide-stimulated microglia.

Microglia activation-mediated neuroinflammation plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, multiple sclerosis, and human immunodeficiency virus (HIV)-associated dementia. Inhibition of microglia activation may alleviate neurodegeneration under neuroinflammatory conditions. In the present study, we compared three flavone C-glycosides extracted from Trollius chinensis BUNGE using a cell-based assay to evaluate their antiinflammatory effects on microglial cells. The results showed that orientin-2"-O-galactopyranoside (OGA) significantly inhibited the production of nitric oxide and tumor necrosis factor (TNF)-α in lipopolysaccharide (LPS)-stimulated microglial cells. OGA also markedly inhibited the LPS-induced expression of TNF-α, interleukin-1ß, inducible nitric oxide (NO) synthase, and cyclooxygenase-2, which was accompanied by suppression of the activation of nuclear factor (NF)-κB and the extracellular signal-regulated kinase (ERK) signal pathway. In addition, OGA decreased LPS-induced reactive oxygen species generation, which appears to be related to the activation of the NF-E2-related factor2 (NRF2)/ heme oxygenase-1 (HO-1) pathway in BV-2 microglial cells. Furthermore, OGA reduced the cytotoxicity of activated microglia toward HT-22 neuroblastoma cells in a co-culture system. Taken together, the present study demonstrated that the induction of HO-1-mediated inhibition of the NF-κB and ERK pathways contributes significantly to the antineuroinflammatory and neuroprotective effects elicited by OGA.

Comparative analysis of the role of small G proteins in cell migration and cell death: cytoprotective and promigratory effects of RalA.

Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.

Pro-apoptotic role of integrin ß3 in glioma cells.

Malignant gliomas are the most destructive type of brain cancer. In order to gain a better understanding of the molecular mechanisms of glioma cell death and survival, we previously established an alkylating agent 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant variant of C6 rat glioma cells. Proteomic analysis indicated a significant down-regulation of integrin beta 3 (ITGB3) in the BCNU-resistant C6R cells. Re-expression of ITGB3 in C6R cells restored the BCNU sensitivity. In U87MG, U373MG, and T98G human glioma cells, there was a positive correlation between ITGB3 expression and the sensitivity to BCNU and etoposide, suggesting an important role of ITGB3 in glioma cell death. Over-expression of ITGB3 cDNA significantly increased the sensitivity of the human glioma cells to the anticancer drug-induced apoptosis. Nitric oxide showed an additive effect on the anticancer drug-induced glioma cell death by increasing ITGB3 expression. Subsequent dissection of signaling pathways indicated that extracellular signal-regulated kinase and unligated integrin-mediated cell death pathway may be involved in the pro-apoptotic role of ITGB3 in glioma cells. These results implicate ITGB3 in glioma cell death/survival and drug resistance.

Inhibition of neuroinflammation by MIF inhibitor 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}1methyl)benzoic acid (Z-312).

Microglial overactivation-mediated neuroinflammation contributes greatly to the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. Macrophage migration inhibitory factor (MIF) is a pleiotropic proinflammatory cytokine that is involved in the pathophysiology of various inflammatory diseases by inducing various proinflammatory cytokines. Compound 3-({[4-(4-methoxyphenyl)-6-methyl-2-pyrimidinyl]thio}methyl)benzoic acid (Z-312) is a novel small -molecule inhibitor of MIF tautomeric activity. In this study, we investigated the anti-inflammatory effects of Z-312 on liposaccharide (LPS)-induced neuroinflammation in vitro and in vivo. The results showed that Z-312 significantly decreased the production of nitric oxide (NO), interleukin (IL)-1ß, tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated microglial cells. Mechanistically, nuclear translocation of the p65 subunit of nuclear factor (NF)-κB, degradation and phosphorylation of IκBα, NF-κB transcriptional activity and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and JNK were markedly attenuated by pretreatment with Z-312 in BV-2 microglial cells. In addition, Z-312 suppressed the neurotoxic effects of cell culture medium of LPS-activated BV-2 microglia on cocultured mouse HT22 neuroblastoma cells. An in vivo study demonstrated that Z-312 markedly ameliorated microglial activation and subsequent DA neuron loss in an LPS-induced Parkinson's disease (PD) mouse model. These results suggest that MIF inhibitor Z-312 may be a promising neuroprotective agent for the treatment of neuroinflammation-mediated neurological diseases.

Down-regulation of lipocalin 2 contributes to chemoresistance in glioblastoma cells.

Malignant gliomas are the most common primary brain tumor and have a poor clinical prognosis. 1, 3-Bis (2-chloroethyl)-1-nitrosourea (BCNU) is an alkylating agent that is commonly used in glioma therapy. However, BCNU chemotherapy often fails due to drug resistance. To gain better understanding of molecular mechanisms underlying the drug resistance of glioma, a BCNU-resistant variant (C6R) of C6 rat glioma cells was selected and characterized. The established C6R cells were resistant to BCNU-induced cell death and cell cycle arrest as confirmed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide reduction assay and flow cytometric analysis of DNA content. C6R cells showed an increased expression of common drug resistance-related genes such as O6-methylguanine-DNA methyltransferase and multiple drug resistance 1. In contrast, C6R cells showed a decreased expression of glial fibrillary acidic protein, therefore, displaying shorter cellular processes compared with parental C6 cells. More importantly, in conjunction with the morphological changes, the expression of lipocalin-2 (lcn2), a 25-kDa secreted proapoptotic protein, was markedly reduced in the BCNU-resistant C6R cells. However, there was no significant change in the expression of lcn2 receptors. Addition of recombinant LCN2 protein or introduction of lcn2 cDNA significantly increased the sensitivity of C6 cells and human glioma cells to BCNU or other anticancer drugs, while knockdown of lcn2 expression by antisense cDNA transfection decreased the sensitivity. When lcn2 was re-expressed in C6R cells, the BCNU sensitivity was restored. Lcn2 enhanced BCNU-induced Akt dephosphorylation providing a molecular basis of apoptosis sensitization. These results suggest that LCN2 protein may be involved in glioma drug resistance and may provide a new approach to sensitizing glioblastoma to chemotherapy.