Chrysanthemum boreale Makino Inhibits Oxidative Stress-Induced Neuronal Damage in Human Neuroblastoma SH-SY5Y Cells by Suppressing MAPK-Regulated Apoptosis.

Oxidative stress has been demonstrated to play a pivotal role in the pathological processes of many neurodegenerative diseases. In the present study, we demonstrated that Chrysanthemum boreale Makino extract (CBME) suppresses oxidative stress-induced neurotoxicity in human neuroblastoma SH-SY5Y cells and elucidated the underlying molecular mechanism. Our observations revealed that CBME effectively protected neuronal cells against H2O2-induced cell death by preventing caspase-3 activation, Bax upregulation, Bcl-2 downregulation, activation of three mitogen-activated protein kinases (MAPKs), cAMP response element-binding protein (CREB) and NF-κB phosphorylation, and iNOS induction. These results provide evidence that CBME has remarkable neuroprotective properties in SH-SY5Y cells against oxidative damage, suggesting that the complementary or even alternative role of CBME in preventing and treating neurodegenerative diseases is worth further studies.

Therapeutic Applications of Type 2 Diabetes Mellitus Drug Metformin in Patients with Osteoarthritis.

Type 2 diabetes mellitus (T2DM) and osteoarthritis (OA) are common chronic diseases that frequently co-exist. The link between OA and T2DM is attributed to common risk factors, including age and obesity. Several reports suggest that hyperglycemia and accumulated advanced glycosylation end-products might regulate cartilage homeostasis and contribute to the development and progression of OA. Metformin is used widely as the first-line treatment for T2DM. The drug acts by regulating glucose levels and improving insulin sensitivity. The anti-diabetic effects of metformin are mediated mainly via activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK), which is an energy sensing enzyme activated directly by an increase in the AMP/ATP ratio under conditions of metabolic stress. Dysregulation of AMPK is strongly associated with development of T2DM and metabolic syndrome. In this review, we discuss common risk factors, the association between OA and T2DM, and the role of AMPK. We also address the adaptive use of metformin, a known AMPK activator, as a new drug for treatment of patients with OA and T2DM.

Investigation of the Factors Responsible for the Poor Oral Bioavailability of Acacetin in Rats: Physicochemical and Biopharmaceutical Aspects.

Acacetin, an important ingredient of acacia honey and a component of several medicinal plants, exhibits therapeutic effects such as antioxidative, anticancer, anti-inflammatory, and anti-plasmodial activities. However, to date, studies reporting a systematic investigation of the in vivo fate of orally administered acacetin are limited. Moreover, the in vitro physicochemical and biopharmaceutical properties of acacetin in the gastrointestinal (GI) tract and their pharmacokinetic impacts remain unclear. Therefore, in this study, we aimed to systematically investigate the oral absorption and disposition of acacetin using relevant rat models. Acacetin exhibited poor solubility (≤119 ng/mL) and relatively low stability (27.5-62.0% remaining after 24 h) in pH 7 phosphate buffer and simulated GI fluids. A major portion (97.1%) of the initially injected acacetin dose remained unabsorbed in the jejunal segments, and the oral bioavailability of acacetin was very low at 2.34%. The systemic metabolism of acacetin occurred ubiquitously in various tissues (particularly in the liver, where it occurred most extensively), resulting in very high total plasma clearance of 199 ± 36 mL/min/kg. Collectively, the poor oral bioavailability of acacetin could be attributed mainly to its poor solubility and low GI luminal stability.

Incidence of Occult Spinal Dysraphism Among Infants With Cutaneous Stigmata and Proportion Managed With Neurosurgery: A Systematic Review and Meta-analysis.

Importance: Occult spinal dysraphism (OSD) is the most common congenital spinal anomaly. Cutaneous anomalies such as skin dimples or deviated gluteal folds are well known as stigmata of OSD and are indicators for further evaluation; however, the association between cutaneous anomalies and OSD has not been systemically evaluated. Objective: To evaluate the incidence of OSD and the proportion of OSD cases managed with a neurosurgical intervention among neonates or infants with various cutaneous stigmata. Data Sources: PubMed and Embase databases were searched for studies published up to July 25, 2018, that evaluated the proportion of OSD cases in neonates or infants with cutaneous stigmata. Search terms included ultrasound, dysraphism, dimple, and infant or neonate. The search was limited to English-language publications. Study Selection: Two reviewers selected the studies evaluating the incidence of OSD among neonates or infants with cutaneous stigmata. Data Extraction and Synthesis: The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) guidelines for data extraction were followed. Pooled proportions of OSD cases and OSD cases that were managed with a neurosurgical intervention were obtained using the generalized linear mixed model and maximum likelihood method. Main Outcome and Measures: The pooled incidence of OSD and OSD cases managed with neurological surgery among patients with cutaneous stigmata was the primary outcome. This outcome was also evaluated in each subgroup, and heterogeneity was explored using subgroup analysis. Results: A total of 15 studies, involving 6558 neonate or infant patients with various cutaneous stigmata, were included. The pooled proportion of OSD cases among the patients with cutaneous stigmata was 2.8% (95% CI, 2.1%-3.8%; I2 = 51.6%), and the proportion managed with neurological surgery was 0.6% (95% CI, 0.3%-1.3%; I2 = 66.4%). Cases with combined stigmata showed a significantly higher association with OSD than those with a single stigma (10.5% [95% CI, 6.9%-15.8%] vs 2.3% [%, 95% CI, 1.5%-3.5%]; P < .001). The pooled proportion of OSD cases among patients with an atypical dimple was significantly higher than among those with simple dimple (8.8% [95% CI, 4.5%-16.6%] vs 0.6% [95% CI of 1.4%-2.1%]; P = .001). Conclusions and Relevance: The proportion of OSD in healthy, asymptomatic patients with midline cutaneous stigmata was low, and the proportion of patients who underwent a neurosurgical intervention was even lower. However, a careful evaluation as well as potential spinal magnetic resonance imaging is recommended for neonates or infants with combined stigmata or an atypical dimple for possible high-risk lesions.

Correction to: Cinnamon extract induces tumor cell death through inhibition of NFκB and AP1.

Following publication of the original article [1], the authors have re-evaluated the authorship for this article. The updated author group is.

Discovery of Potent, Selective, and Orally Bioavailable Estrogen-Related Receptor-γ Inverse Agonists To Restore the Sodium Iodide Symporter Function in Anaplastic Thyroid Cancer.

An inverse agonist of estrogen-related receptor-γ (ERRγ), an orphan nuclear receptor encoded by E srrg, enhances sodium iodide symporter-mediated radioiodine uptake in anaplastic thyroid cancer (ATC) cells, thereby facilitating responsiveness to radioiodine therapy in vitro. We synthesized potent, selective, and orally bioavailable ERRγ-inverse agonists and evaluated their activity by analyzing in vitro pharmacology and absorption, distribution, metabolism, excretion, and toxicity profiles. X-ray crystallographic analysis of the ligand and ERRγ complex showed that 35 completely binds to the target protein (PDB 6A6K ). Our results showed improved radioiodine avidity in ATC cells through compound 35-mediated upregulation of iodide-handling genes, leading to enhanced responsiveness to radioiodine therapy in vitro. Importantly, in vivo 124I-positron emission tomography/computed tomography imaging revealed that 35 increases radioiodine avidity in CAL62 tumors. Collectively, these results demonstrated that 35 can be developed as a promising treatment for ERRγ-related cancer in the future.

Glucosamine improves survival in a mouse model of sepsis and attenuates sepsis-induced lung injury and inflammation.

The aim of the current study was to investigate the effects of glucosamine (GlcN) on septic lethality and sepsis-induced inflammation using animal models of mice and zebrafish. GlcN pretreatment improved survival in the cecal ligation and puncture (CLP)-induced sepsis mouse model and attenuated lipopolysaccharide (LPS)-induced septic lung injury and systemic inflammation. GlcN suppressed LPS-induced M1-specific but not M2-specific gene expression. Furthermore, increased expressions of inflammatory genes in visceral tissue of LPS-injected zebrafish were suppressed by GlcN. GlcN suppressed LPS-induced activation of mitogen-activated protein kinase (MAPK) and NF-κB in lung tissue. LPS triggered a reduction in O-GlcNAc levels in nucleocytoplasmic proteins of lung, liver, and spleen after 1 day, which returned to normal levels at day 3. GlcN inhibited LPS-induced O-GlcNAc down-regulation in mouse lung and visceral tissue of zebrafish. Furthermore, the O-GlcNAcase (OGA) level was increased by LPS, which were suppressed by GlcN in mouse and zebrafish. OGA inhibitors suppressed LPS-induced expression of inflammatory genes in RAW264.7 cells and the visceral tissue of zebrafish. Stable knockdown of Oga via short hairpin RNA led to increased inducible nitric oxide synthase (iNOS) expression in response to LPS with or without GlcN in RAW264.7 cells. Overall, our results demonstrate a protective effect of GlcN on sepsis potentially through modulation of O-GlcNAcylation of nucleocytoplasmic proteins.

Antartin, a Cytotoxic Zizaane-Type Sesquiterpenoid from a Streptomyces sp. Isolated from an Antarctic Marine Sediment.

Antartin (1), a new zizaane-type sesquiterpene, was isolated from Streptomyces sp. SCO736. The chemical structure of 1 was assigned from the interpretation of 1D and 2D NMR in addition to mass spectrometric data. The relative stereochemistry of 1 was determined by analysis of NOE data, while the absolute stereochemistry was decided based on a comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Antartin (1) showed cytotoxicity against A549, H1299, and U87 cancer cell lines by causing cell cycle arrest at the G1 phase.

Identification of Antiangiogenic Potential and Cellular Mechanisms of Napyradiomycin A1 Isolated from the Marine-Derived Streptomyces sp. YP127.

Angiogenesis is the process of new blood vessel formation. Excessive angiogenesis is a critical factor in the progression of cancer, macular degeneration, and other chronic inflammatory diseases. When investigating the effects of crude extracts of cultured marine microorganisms, an extract of the cultured Streptomyces sp. YP127 strain was found to inhibit human umbilical vein endothelial cell (HUVEC) tube formation. Bioassay-guided fractionation and spectroscopic data analyses led to the identification of napyradiomycin A1 (1) as an antiangiogenic component of the extract. Compound 1 inhibited HUVEC tube formation in a concentration-dependent manner. It inhibited endothelial cell proliferation but did not affect human dermal fibroblast proliferation. Compound 1 also suppressed migration and invasion of vascular endothelial cells. In addition, compound 1 suppressed vascular endothelial cadherin expression and increased the permeability of the endothelial cell membrane. These results suggested that compound 1 modulates cell permeability and inhibits the angiogenesis of endothelial cells.

Lipopolysaccharide (LPS)-stimulated iNOS Induction Is Increased by Glucosamine under Normal Glucose Conditions but Is Inhibited by Glucosamine under High Glucose Conditions in Macrophage Cells.

We investigated the regulatory effect of glucosamine (GlcN) for the production of nitric oxide (NO) and expression of inducible NO synthase (iNOS) under various glucose conditions in macrophage cells. At normal glucose concentrations, GlcN dose dependently increased LPS-stimulated production of NO/iNOS. However, GlcN suppressed NO/iNOS production under high glucose culture conditions. Moreover, GlcN suppressed LPS-induced up-regulation of COX-2, IL-6, and TNF-α mRNAs under 25 mm glucose conditions yet did not inhibit up-regulation under 5 mm glucose conditions. Glucose itself dose dependently increased LPS-induced iNOS expression. LPS-induced MAPK and IκB-α phosphorylation did not significantly differ at normal and high glucose conditions. The activity of LPS-induced nuclear factor-κB (NF-κB) and DNA binding of c-Rel to the iNOS promoter were inhibited under high glucose conditions in comparison with no significant changes under normal glucose conditions. In addition, we found that the LPS-induced increase in O-GlcNAcylation as well as DNA binding of c-Rel to the iNOS promoter were further increased by GlcN under normal glucose conditions. However, both O-GlcNAcylation and DNA binding of c-Rel decreased under high glucose conditions. The NF-κB inhibitor, pyrrolidine dithiocarbamate, inhibited LPS-induced iNOS expression under high glucose conditions but it did not influence iNOS induction under normal glucose conditions. In addition, pyrrolidine dithiocarbamate inhibited NF-κB DNA binding and c-Rel O-GlcNAcylation only under high glucose conditions. By blocking transcription with actinomycin D, we found that stability of LPS-induced iNOS mRNA was increased by GlcN under normal glucose conditions. These results suggest that GlcN regulates inflammation by sensing energy states of normal and fuel excess.

Glioma-secreted soluble factors stimulate microglial activation: The role of interleukin-1ß and tumor necrosis factor-α.

We aimed to elucidate the effect of soluble factors secreted by glioma on microglial activation. Conditioned medium (CM) from glioma cells, CRT-MG and C6, significantly induced nitric oxide (NO) production and stimulated the mRNA expression of inducible NO synthase (iNOS), interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-α) and cyclooxygenase 2 (COX-2) in BV2 cells. Glioma CM stimulated p38 mitogen-activated protein kinase (MAPK) phosphorylation, and a p38 MAPK inhibitor, SB203580, suppressed CM-induced NO production in BV2 cells. In addition, CM stimulated nuclear factor-kappaB (NF-κB) DNA binding and transcriptional activity, which was repressed by SB203580. Gliomas displayed higher mRNA expression and release of TNF-α and IL-1ß than primary astrocyte cells. Neutralization of TNF-α and IL-1ß in C6-CM using a neutralizing antibody inhibited NO/iNOS expression in BV-2 cells. These results indicate potential contribution of diffusible tumor-derived factors to regulate microglial activation and subsequent tumor microenvironment.

A dopamine-alpha-lipoic acid hybridization compound and its acetylated form inhibit LPS-mediated inflammation.

In the present study, we synthesized and evaluated the anti-inflammatory effects of dopamine and alpha-lipoic acid (ALA) hybrid compounds, ALA-dopamine (HBU-199) and its acetylated derivative, ALA-acetyl dopamine (HBU-200), in BV2 microglia and RAW264.7 macrophage cells. HBU-199 and HBU-200 both significantly and dose-dependently inhibited LPS-induced nitric oxide (NO) productions, NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-6 and interleukin-1ß mRNA expressions and iNOS and COX-2 protein expressions. Furthermore, HBU-199 and HBU-200 protected RAW264.7 cells from activation-induced cell death. However, at same concentrations, dopamine or ALA did not inhibit LPS-mediated production of inflammatory molecules and activation-induced cell death. HBU-199 and HBU-200 inhibited LPS-induced inhibition of inhibitory kappa-B-alpha (IκB-α) phosphorylation and nuclear factor-kappa B (NF-κB) activation. Furthermore, LPS-mediated DNA binding of p65 and p50 to the NF-κB binding site of the iNOS promoter was inhibited by HBU-199 and HBU-200, whereas dopamine and ALA did not inhibit LPS-induced NF-κB activation and IκB-α phosphorylation. Moreover, HBU-199 and HBU-200 suppressed LPS-stimulated phosphorylation of Akt, but not glycogen synthase kinase 3 beta. Overall, our data suggest that the ALA-dopamine hybrid compounds down-regulate inflammatory responses via inhibition of NF-κB and NF-κB-dependent gene expression, suggesting that it is a promising therapeutic agent for both systemic inflammatory diseases and inflammatory diseases of central nervous system.

Glucosamine enhances body weight gain and reduces insulin response in mice fed chow diet but mitigates obesity, insulin resistance and impaired glucose tolerance in mice high-fat diet.

OBJECTIVE: This study investigated the potential of glucosamine (GlcN) to affect body weight gain and insulin sensitivity in mice normal and at risk for developing diabetes. METHODS: Male C57BL/6J mice were fed either chow diet (CD) or a high fat diet (HFD) and the half of mice from CD and HFD provided with a solution of 10% (w/v) GlcN. Total cholesterol and nonesterified free fatty acid levels were determined. Glucose tolerance test and insulin tolerance test were performed. HepG2 human hepatoma cells or differentiated 3T3-L1 adipocytes were stimulated with insulin under normal (5 mM) or high glucose (25 mM) conditions. Effect of GlcN on 2-deoxyglucose (2-DG) uptake was determined. JNK and Akt phosphorylation and nucleocytoplasmic protein O-GlcNAcylation were assayed by Western blotting. RESULTS: GlcN administration stimulated body weight gain (6.58±0.82 g vs. 11.1±0.42 g), increased white adipose tissue fat mass (percentage of bodyweight, 3.7±0.32 g vs. 5.61±0.34 g), and impaired the insulin response in livers of mice fed CD. However, GlcN treatment in mice fed HFD led to reduction of body weight gain (18.02±0.66 g vs. 16.22±0.96 g) and liver weight (2.27±0.1 vs. 1.85±0.12 g). Furthermore, obesity-induced insulin resistance and impaired Akt insulin signaling in the liver were alleviated by GlcN administration. GlcN inhibited the insulin response under low (5 mM) glucose conditions, whereas it restored the insulin response for Akt phosphorylation under high (25 mM) glucose conditions in HepG2 and 3T3-L1 cells. Uptake of 2-DG increased upon GlcN treatment under 5 mM glucose compared to control, whereas insulin-stimulated 2-DG uptake decreased under 5 mM and increased under 25 mM glucose in differentiated 3T3-L1 cells. CONCLUSION: Our results show that GlcN increased body weight gain and reduced the insulin response for glucose maintenance when fed to normal CD mice, whereas it alleviated body weight gain and insulin resistance in HFD mice. Therefore, the current data support the integrative function of the HBP reflecting the nutrient status of lipids or glucose and further implicate the importance of the pathway in insulin signaling for the regulation of metabolism.

A novel caffeic acid-1-piperonylpiperazine hybridization compound HBU-47 inhibits LPS-mediated inflammation in RAW264.7 macrophage cells.

In the present study, we synthesized a new hybrid compound by coupling caffeic acid and 1-piperonylpiperazine. The synthetic compound, acetyl-caffeic acid-1-piperonylpiperazine (HBU-47), showed potent anti-inflammatory effects inhibiting lipopolysaccharide (LPS)-induced production of nitric oxide (NO) in RAW264.7 macrophage cells. HBU-47 inhibited LPS-caused induction of inducible NO synthase (iNOS), cyclooxygenase-2, interleukin-6 and interleukin-1ß in RAW264.7 cells in time- and dose-dependent manner. Compared to HBU-47, neither caffeic acid nor 1-piperonylpiperazine displayed significant inhibition of LPS responses. HBU-47 did not affect LPS-caused activation of mitogen-activated kinases (MAPKs) or IκB-α degradation. Instead, LPS-mediated NF-κB activation and DNA bindings of p65, p50 and c-Rel to the NF-κB binding site of iNOS promoter were inhibited by HBU-47. Overall, our data suggest that the novel caffeic acid hybrid compound downregulates inflammatory responses through inhibition of NF-κB and NF-κB-dependent gene expressions, thus, further suggesting its efficacy as a promising therapeutic agent.

Tunicamycin inhibits Toll-like receptor-activated inflammation in RAW264.7 cells by suppression of NF-κB and c-Jun activity via a mechanism that is independent of ER-stress and N-glycosylation.

In this study, we investigated the effect of tunicamycin on the production of pro-inflammatory molecules in RAW264.7 macrophage cells in response to lipopolysaccharide (LPS) and Toll-like receptor (TLR) agonists. Tunicamycin caused a reduction in LPS-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1ß) and tumor necrosis factor alpha (TNF-α). In contrast, other ER stress-inducing chemicals, such as A23187 and thapsigargin (TG), increased LPS-induced COX-2 expression and had no effect on LPS-induced iNOS, TNF-α or IL-1ß expression. Furthermore, the inhibitory effect of tunicamycin on LPS-induced inflammation was not influenced by salubrinal, an ER stress inhibitor, suggesting that the anti-inflammatory effect of tunicamycin is independent of ER stress. Tunicamycin also inhibited the expression of inflammatory molecule mRNAs induced by stimulation of TLR2 (with lipoteichoic acid) or TLR3 (with polyinosinic:polycytidylic acid), which do not require myeloid differentiation protein-2 (MD2) for their activation. Moreover, inhibition of LPS-induced iNOS expression was not inhibited by castanospermine, another N-glycosylation inhibitor, suggesting that the inhibitory effect of tunicamycin on LPS-induced iNOS induction is likely independent of MD2 N-glycosylation. Tunicamycin inhibited nuclear factor-kappaB (NF-κB) activity by suppressing LPS-induced nuclear translocation of p50 and subsequent DNA binding of p50 and p65 to the NF-κB site of the iNOS promoter. Tunicamycin also inhibited the transcriptional activity of a cAMP-response element (CRE) reporter, possibly by inhibiting c-Jun activation. Therefore, we conclude that tunicamycin represses TLR-induced inflammation through suppression of NF-κB and CRE activity via a mechanism that is independent of ER-stress and N-glycosylation.

A novel glucosamine derivative exerts anti-inflammatory actions via inhibition of nuclear factor-kappaB.

Glucosamine suppresses lipopolysaccharide (LPS)-induced upregulation of pro-inflammatory mediators both in vivo and in culture systems of mouse microglia or macrophage. In the present study, we show that the novel glucosamine derivative, 2-deoxy-2-[(o-methylbenzylidene)]-ß-glucopyranoside (NK-4), significantly reduced LPS-induced production of nitric oxide (NO) in BV2 microglia, RAW264.7 macrophage, and primary cultured peritoneal macrophages cells. NK-4 inhibited LPS-induced upregulation of inducible NO synthase (iNOS), cyclooxygenase-2, interleukin-6, tumor necrosis factor-α, and interleukin-1ß in RAW264.7 cells in a time- and concentration-dependent manner. Furthermore, administering NK-4 significantly inhibited the induction of inflammatory cytokine mRNAs in the brains of LPS-injected mice. Although NK-4 inhibited LPS-induced nuclear factor-kappaB (NF-κB) activation, IκB-α degradation was not changed. Instead, NK-4 inhibited LPS-induced DNA-binding activity of NF-κB by suppressing p50 and c-Rel binding to NF-κB binding site of the iNOS promoter.

O-GlcNAc transferase inhibits LPS-mediated expression of inducible nitric oxide synthase through an increased interaction with mSin3A in RAW264.7 cells.

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT), which catalyzes the addition of a single ß-N-GlcNAc unit to target proteins, has been shown to act as a transcriptional regulator. In the current study, we discovered that OGT exerted inhibitory effects on the LPS-driven activation of NF-κB and inducible nitric oxide synthase (iNOS). In response to LPS, OGT exhibited an increased interaction with the transcriptional corepressor mammalian Sin3A (mSin3A). Furthermore, mSin3A, histone deacetylase (HDAC)1, and HDAC2 displayed increased binding to the iNOS promoter in response to LPS. Treatment with GlcN, in contrast, inhibits LPS-induced inflammation and decreased LPS-mediated recruitment of OGT, mSin3A, and HDACs. LPS treatment also resulted in the hypo-O-GlcNAcylation of mSin3A, which was reversed by GlcN. When the effect of the HDAC inhibitor trichostatin A (TSA) on LPS- and/or GlcN-mediated iNOS protein/mRNA induction was investigated, the results revealed that TSA dose dependently enhanced iNOS expression in response to LPS and/or GlcN. In addition, histone acetyltransferases, p300, and cAMP response element-binding protein-binding protein (CBP) enhanced LPS- and/or GlcN-induced iNOS protein expression. These results collectively suggest that OGT inhibits LPS-driven NF-κB activation and subsequent iNOS transcription by modulating histone acetylation either directly or indirectly.

O-GlcNAcylation and p50/p105 binding of c-Rel are dynamically regulated by LPS and glucosamine in BV2 microglia cells.

BACKGROUND AND PURPOSE: Previously, we demonstrated that glucosamine (GlcN) exerts a suppressive effect on LPS-induced inducible NOS (iNOS) through the inhibition of NF-κB activation in BV2 mouse microglial cells. The purpose of the present study was to examine the mechanisms by which GlcN inhibits NF-κB activation. EXPERIMENTAL APPROACH: BV2 cells were stimulated with LPS with or without GlcN. NF-κB/c-Rel activities were studied by EMSA, nuclear translocation, reporter assay or chromatin immunoprecipitation. Wheat germ agglutinin precipitation or galactosyltransferase assay were used to measure O-linked N-acetylglucosamine (O-GlcNAc) modification (O-GlcNAcylation) of c-Rel. Protein-protein interactions were examined by co-immunoprecipitation. KEY RESULTS: LPS stimulated the activation of c-Rel, increased the O-GlcNAcylation of c-Rel and enhanced the binding of c-Rel to the NF-κB site in the iNOS promoter; GlcN attenuated these effects of LPS. O-GlcNAcylation of both nuclear and cytosolic forms of c-Rel was increased by LPS and reduced by GlcN. LPS increased the interaction of c-Rel with O-GlcNAc transferase (OGT) and p50/p105, and GlcN suppressed these interactions. Knockdown of OGT reduced the c-Rel O-GlcNAcylation and c-Rel-p50 interaction in response to LPS, but did not affect either the binding of c-Rel to the iNOS promoter or the transcriptional activity of c-Rel. CONCLUSIONS AND IMPLICATIONS: In BV2 microglial cells, the anti-inflammatory effect of GlcN is mediated by prevention of the prolonged activation of transcription factors, c-Rel and NF-κB. Further clarification of the mechanism by which GlcN exerts this effect will facilitate the development of pharmacological strategies for preventing excessive NO formation when targeting inflammatory diseases of the periphery or CNS.

Glucosamine inhibits lipopolysaccharide-stimulated inducible nitric oxide synthase induction by inhibiting expression of NF-kappaB/Rel proteins at the mRNA and protein levels.

Expression of inducible nitric oxide synthase (iNOS) protein by lipopolysaccharide (LPS) in BV2 microglia cells increased in a biphasic manner. Glucosamine (GlcN) selectively suppressed the late- but not early-stage iNOS response to LPS. Prolonged induction of iNOS expression by LPS was inhibited by cycloheximide, suggesting that de novo protein synthesis was required. Late-phase activation of nuclear factor-kappaB (NF-κB) activity required for sustained iNOS induction. Nuclear translocation and DNA binding of NF-κB, and Rel proteins expressions were inhibited by GlcN at later time points but not upon immediate early-stage activation by LPS. We show that GlcN selectively inhibits sustained iNOS induction by inhibiting Rel protein expression at both the mRNA and protein levels; such expression is required for prolonged iNOS induction by LPS. Our results provide mechanistic evidence that GlcN regulates inflammation, represented by iNOS. The implication of these results is that GlcN may be a potent transcriptional regulator of iNOS and other genes involved in the general inflammation process.