Lipopolysaccharide-induced Cyclooxygenase-2 Expression Research Articles

Heat-Killed Lactic Acid Bacteria Inhibit Nitric Oxide Production via Inducible Nitric Oxide Synthase and Cyclooxygenase-2 in RAW 264.7 Cells

Heat-killed lactic acid bacteria perform immunomodulatory functions and are advantageous as probiotics, considering their long product shelf-life, easy storage, and convenient transportation. In this study, we aimed to develop appropriate heat treatments for industrial preparation of probiotics with antioxidant activity. Among 75 heat-killed strains, Lactococcus lactis MG5125 revealed the highest nitric oxide inhibition (86.2%), followed by Lactobacillus acidophilus MG4559 (86.0%), Lactobacillus plantarum MG5270 (85.7%), Lactobacillus fermentum MG4510 (85.3%), L. plantarum MG5239 (83.9%), L. plantarum MG5289 (83.2%), and L. plantarum MG5203 (81.8%). Moreover, the heat-killed selected strains markedly inhibited lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression. The use of heat-killed bacteria with intact bio-functionality can elongate the shelf-life and simplify the food processing steps of probiotic foods, given their high stability. The antioxidant and immune-modulatory activities of the heat-killed strains selected in this study indicate a strong potential for their utilization probiotic products manufacturing.

Effect of thiol antioxidants on lipopolysaccharide-induced cyclooxygenase-2 expression in pulmonary epithelial cells.

Cyclooxygenase-2 (COX-2) plays an important role in pulmonary inflammatory response, and its expression is regulated by several transcription factors including nuclear factor kappa-B (NF-κB), activator protein-1 (AP-1), and signal transducer and activator of transcription-3 (STAT-3), which are activated by oxidative stress. Glutathione (GSH) and N-acetyl cysteine (NAC) are thiol antioxidants that scavenge reactive oxygen species (ROS). The present study investigated whether lipopolysaccharide (LPS) induces COX-2 expression through ROS generation and the activation of oxidant-sensitive transcription factors such as NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells. The cells were pretreated with GSH or NAC for 1 hour prior to LPS stimulation. Intracellular ROS levels, DNA-binding activities of NF-κB, AP-1, and STAT-3, and mRNA and protein levels of COX-2 were determined. Our results showed that LPS increased ROS levels that peaked at 2 hours. LPS activated NF-κB, AP-1, and STAT-3 and induced the expression of COX-2 in A549 cells in a time-dependent manner. Pretreatment of thiol antioxidants GSH and NAC reduced ROS levels and attenuated the increase in ROS, the activation of NF-κB, AP-1, and STAT-3, and the expression of COX-2 in LPS-treated A549 cells. In conclusion, GSH and NAC suppress COX-2 expression by reducing ROS levels and inhibiting the activation of NF-κB, AP-1, and STAT-3 in pulmonary epithelial A549 cells exposed to LPS. Pretreatment with thiol antioxidants GSH and NAC may be beneficial for the treatment of pulmonary inflammation associated with oxidative stress.

Candesartan ameliorates brain inflammation associated with Alzheimer's disease.

Alzheimer's disease (AD) pathology is associated with brain inflammation involving microglia and astrocytes. The renin-angiotensin system contributes to brain inflammation associated with AD pathology. This study aimed to investigate the role of candesartan, an angiotensin II type 1 receptor blocker, in modulation of glial functions associated with AD. Focusing on the role of candesartan in glial inflammation, we evaluated inflammatory mediators' levels, secreted by lipopolysaccharide-induced microglia following candesartan treatment. Also, short-term intranasal candesartan effects on amyloid burden and microglial activation were investigated in 5 familial AD mice. Candesartan showed anti-inflammatory effects and shifted microglial activation toward a more neuroprotective phenotype. Candesartan decreased the lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression levels, which was accompanied by an induction of arginase-1 expression levels and enhanced Aβ1-42 uptake by microglia. Moreover, intranasally administered candesartan to AD mice model significantly reduced the amyloid burden and microglia activation in the hippocampus. These results thus shed light on the neuroprotective role of candesartan in the early stage of AD, which might relate to modulation of microglial activation states.

Store-operated Ca2+ Entry Facilitates the Lipopolysaccharide-induced Cyclooxygenase-2 Expression in Gastric Cancer Cells

Helicobacter pylori has been identified as one of the major causes of chronic gastritis, gastric and duodenal ulcers, and gastric cancer. Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria, and H. pylori LPS might play an exclusively important role in activating inflammatory pathways in monocytes and macrophages. To study the role of LPS in the underlying mechanism of inflammatory responses, we established an in vitro model using the human AGS gastric cancer cell line. We found that LPS mediates inflammation through setting off a cascade of events: activation of the store-operated calcium (SOC) channel, initiation of downstream NF-κB signaling, and phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2). Phosphorylated ERK1/2 promotes the nuclear translocation of NF-κB, and eventually elevates the expression level of COX-2, a major inflammatory gene.

Bioactive Constituents from the Termite Nest-Derived Medicinal Fungus Xylaria nigripes

Six new eremophilane-type sesquiterpenes, namely, nigriterpenes A-F (1-6), and one new phenolic compound, named 2-hydroxymethyl-3-pentylphenol (7), along with fomannoxin alcohol, 3-butyl-7-hydroxyphthalide, scytalone, and fomannoxin were isolated from the ethyl acetate extracts of the fermented broths of termite nest-derived Xylaria nigripes, which has long been used as a traditional Chinese medicine for treating insomnia and depression. Their structures were elucidated on the basis of spectroscopic data analysis and compared with the literature. All the pure isolates were evaluated against lipopolysaccharide-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) expression, and NO production in murine brain microglial BV-2 cells. Of the compounds tested, nigriterpene C (3) and fomannoxin alcohol exerted significant inhibitory effects on two induced enzymes and NO production without any significant cellular toxicity. The most potent compound, 3, exhibited concentration-dependent inhibition on NO production and iNOS and COX-2 expression with IC50 values of 21.7 ± 4.9, 8.1 ± 2.3, and 16.6 ± 5.5 μM, respectively. These results indicated that the potential anti-inflammatory effects of nigriterpene C (3) and fomannoxin alcohol on murine brain microglial BV-2 cells may provide a rationale for the traditional medical uses of X.nigripes for treating insomnia and depression.

Bioactive constituents from the termite nest-derived medicinal fungus Xylaria nigripes

Six new eremophilane-type sesquiterpenes, namely, nigriterpenes A-F (1-6), and one new phenolic compound, named 2-hydroxymethyl-3-pentylphenol (7), along with fomannoxin alcohol, 3-butyl-7-hydroxyphthalide, scytalone, and fomannoxin were isolated from the ethyl acetate extracts of the fermented broths of termite nest-derived Xylaria nigripes, which has long been used as a traditional Chinese medicine for treating insomnia and depression. Their structures were elucidated on the basis of spectroscopic data analysis and compared with the literature. All the pure isolates were evaluated against lipopolysaccharide-induced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) expression, and NO production in murine brain microglial BV-2 cells. Of the compounds tested, nigriterpene C (3) and fomannoxin alcohol exerted significant inhibitory effects on two induced enzymes and NO production without any significant cellular toxicity. The most potent compound, 3, exhibited concentration-dependent inhibition on NO production and iNOS and COX-2 expression with IC50 values of 21.7 ± 4.9, 8.1 ± 2.3, and 16.6 ± 5.5 μM, respectively. These results indicated that the potential anti-inflammatory effects of nigriterpene C (3) and fomannoxin alcohol on murine brain microglial BV-2 cells may provide a rationale for the traditional medical uses of X. nigripes for treating insomnia and depression.

Marine soft corals as source of lead compounds for anti-inflammatories

Marine soft corals are known to produce a wide array of secondary metabolites, particularly diterpenoids and steroids, and often characterized by uncommon structural features and potent bioactivities. The remarkable abundance and diversity of bioactive small molecule which have been isolated from soft corals have made these organisms an important source of new drug candidates for human diseases, particularly for their anti-inflammatory activity. In this paper, the authors reported anti-inflammatory marine natural products isolated from diverse species of soft corals determined in vitro by their inhibition of lipopolysaccharide-induced expression of inducible NO synthase and cyclooxygenase-2 in murine macrophage cells (RAW 264.7).

Protein Phosphatase 2A in Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Murine Lymphatic Endothelial Cells.

The lymphatic endothelium plays an important role in the maintenance of tissue fluid homeostasis. It also participates in the pathogenesis of several inflammatory diseases. However, little is known about the underlying mechanisms by which lymphatic endothelial cell responds to inflammatory stimuli. In this study, we explored the mechanisms by which lipopolysaccharide (LPS) induces cyclooxygenase (COX)-2 expression in murine lymphatic endothelial cells (SV-LECs). LPS caused increases in cox-2 mRNA and protein levels, as well as in COX-2 promoter luciferase activity in SV-LECs. These actions were associated with protein phosphatase 2A (PP2A), apoptosis signal-regulating kinase 1 (ASK1), JNK1/2 and p38MAPK activation, and NF-κB subunit p65 and C/EBPβ phosphorylation. PP2A-ASK1 signaling blockade reduced LPS-induced JNK1/2, p38MAPK, p65 and C/EBPβ phosphorylation. Transfection with PP2A siRNA reduced LPS’s effects on p65 and C/EBPβ binding to the COX-2 promoter region. Transfected with the NF-κB or C/EBPβ site deletion of COX-2 reporter construct also abrogated LPS’s enhancing effect on COX-2 promoter luciferase activity in SV-LECs. Taken together, the induction of COX-2 in SV-LECs exposed to LPS may involve PP2A-ASK1-JNK and/or p38MAPK-NF-κB and/or C/EBPβ cascade.

Eupartoium makinoi suppresses lipopolysaccharide-induced inducible nitric oxide synthase and cyclooxygenase-2 expression

Inflammation is involved in numerous diseases including cancer. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) play important roles in the development of certain inflammatory diseases. Eupatorium makinoi, which belongs to a family of Asteraceae plants, is used medicinally in East Asia. We investigated the effects of an ethanol extract of E. makinoi (EEM) on nuclear factor-kappa B (NF-κB) activation and the expression of iNOS and COX-2 with lipopolysaccharide (TLR4 agonist) in murine macrophages. EEM suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. These results suggest that EEM may regulate TLR4 signalling pathways and this may be a useful strategy for anti-inflammatory therapies.

Valproic acid suppresses lipopolysaccharide-induced cyclooxygenase-2 expression via MKP-1 in murine brain microvascular endothelial cells

Inflammation and vascular perturbations are increasingly implicated in the pathogenesis of neurodegenerative diseases. Prevailing evidence suggests that valproic acid (VPA), an antiepileptic and mood stabilizer, exhibits not only neuro-protective effects, but also anti-inflammatory effects in neurodegenerative diseases. However, the underlying mechanism contributing to VPA's suppression of inflammatory responses remains unclear. In this study, we explored the inhibitory action of VPA on cyclooxygenase (COX)-2 expression in bEnd.3 mouse brain microvascular endothelial cells exposed to lipopolysaccharide (LPS), a pro-inflammatory stimulus. The LPS-induced increases in COX-2 protein level and COX-2 promoter-luciferase activity were significantly suppressed by VPA. VPA inhibited p38MAPK and JNK phosphorylation in LPS-stimulated bEnd.3 cells. Treatment of cells with a p38MAPK inhibitor (p38MAPK inhibitor III) or a JNK signaling inhibitor (JNK inhibitor II) significantly inhibited LPS-induced COX-2 expression. VPA inhibited LPS-induced NF-κB subunit p65 phosphorylation and κB-luciferase activity. LPS-increased p65 and C/EBPβ binding to the COX-2 promoter region was attenuated in the presence of VPA. In addition, VPA suppression of p38MAPK, JNK and p65 phosphorylation, and subsequent COX-2 expression was restored in cells transfected with mitogen-activated protein kinase phosphatase-1 (MKP-1) dominant negative (DN) mutant. VPA also caused increases in MKP-1 acetylation and MKP-1 phosphatase activity in bEnd.3 cells. In conclusion, VPA may cause MKP-1 activation to dephosphorylate p38MAPK and JNK, leading to decrease in p65 and C/EBPβ binding to the COX-2 promoter region and COX-2 down-regulation in LPS-stimulated bEnd.3 cells. The present study therefore supports the therapeutic value of VPA in alleviating brain inflammatory processes.

Inhibition of lipopolysaccharide-induced expression of cyclooxygenase-2 by Zingiber cassumunar Roxb. constituents in human dental pulp cells

Dental pulp inflammation expresses many pro-inflammatory cytokines; interleukins, cyclooxygenase (COX)-2 and prostaglandin E2 (PGE2). Inflammatory reduction and promoting regeneration of dental pulp cells are advantage for dental pulp healing. Zingiber cassumunar Roxb. (Phlai) has anti-inflammatory activity which may be applied to decrease inflammation in dental pulp tissues. The present study had aimed to evaluate inhibitory effect of Phlai constituents on COX-1, COX-2 and PGE2 expression inhuman dental pulp cells stimulated with lipopolysaccharide (LPS). After cells were inhibited with dexamethasone (cox-inhibitor) or Phlai constituents for 2 h, LPS were added and concentration adjusted to 100 µg/ml. COX-1 and COX-2 gene expressions were obtained by reverse-transcription polymerase chain reaction (RT-PCR) and realtime RT-PCR after incubating for 24 h. COX-1 and COX-2 protein expressions were identified by Western blot and PGE2 level was detected by enzyme linked immunosorbent assay (ELISA) after incubating for 72 h. These Phlai constituents as cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-3',4'-dimethoxystyryl]cyclohex-1-ene (compound B), cis-3-(2',4',5'-Trimethoxyphenyl)-4-[(E)-2',4',5'-trimethoxystyryl]cyclohex-1-ene (compound C) and (E)-1-(3',4'-Dimethoxyphenyl)but-1,3-diene (DMPBD) could reduce PGE2 level and COX-2 expression.cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-2',4',5'-trimethoxystyryl]cyclohex-1-ene (compound C¢) reduced PGE2 levels but had less effect on COX-2. All constituents showed no significant change on COX-1. Our results suggest that some constituents of Phlai can reduce inflammation in human dental pulp cells by reducing COX-2 and PGE2 production. Key words: Zingiber cassumunar, Phlai, cyclooxygenase-1, cyclooxygenase-2, lipopolysaccharide, anti-inflammation, dental pulp cells, prostaglandin E2.

Dichloromethane fraction of Laminaria japonica ethanolic extract inhibits lipopolysaccharide-induced nitric oxide synthase and cyclooxygenase-2 expression in RAW 264.7 cells via NF-κB pathway.

Strong anti-inflammatory activity has been found in Laminaria japonica dichloromethane fraction (LDF); however, the molecular mechanisms underlying its anti-inflammatory activity are not reported. Our results indicated that LDF inhibited LPS-induced nitric oxide and prostaglandin E(2) production in a dose-dependent manner and suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) in RAW 264.7 cells. Also, levels of pro-inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1β and IL-6 were remarkably reduced by LDF in LPS-treated RAW 264.7 cells. LDF greatly inhibited promoter activity of nuclear factor-κB (NF-κB) and translocation of NF-κB subunits by prevention of the degradation of inhibitor κB-α in LPS-treated RAW 264.7 cells (p < 0.05). Moreover, LDF inhibited activation of mitogen-activated protein kinases and AKT in LPS-treated RAW 264.7 cells. These results indicate that the LDF downregulates iNOS and COX-2 expressions through the suppression of NF-κB pathway associated with inhibition of multiple signaling proteins.

Lipopolysaccharide-induced Cyclooxygenase-2 Expression in Mouse Transformed Clara Cells

Background/Aims: Exacerbation of innate immune responses can contribute to development of acute lung injury. Multiple cell populations, including the bronchiolar epithelium, coordinate these inflammatory responses. Clara cells, non-ciliated epithelial cells, are located in the distal airways in humans and conducting airways in mice. These cells actively participate in innate immune responses but their precise contributions remain poorly defined. Methods: To test the hypothesis that E. coli lipopolysaccaride (LPS) treatment stimulates production of pro-inflammatory mediators in mouse transformed Clara cells (MTCC), MTCC were treated with E. coli lipopolysaccaride (LPS). Results: LPS increased COX-2 expression and stimulated production of prostaglandins, including prostaglandin E₂ (PGE₂). Enhanced mitogen activated protein kinase (MAPK) activation, nuclear factor-ĸB (NFĸB) activation, and chemokine production were observed in MTCC in response to LPS treatment. Conclusions: While the role for Clara cells in the regulation of host defense and the progression of acute lung injury needs further characterization, our data suggests the importance of this unique cell population in the pathogenesis of LPS-induced acute lung injury.

Extract from &lt;i&gt;Nandina domestica&lt;/i&gt; Inhibits Lipopolysaccharide-Induced Cyclooxygenase-2 Expression in Human Pulmonary Epithelial A549 Cells

Extract from fruits of Nandina domestica THUNBERG (NDE) has been used to improve cough and breathing difficulty in Japan for many years. To explore whether NDE may alleviate respiratory inflammation, we investigated its effect on expression of cyclooxygenase-2 (COX-2) and production of prostaglandin E₂ (PGE₂) in human pulmonary epithelial A549 cells in culture. Treatment with lipopolysaccharide (LPS; 6 µg/mL) resulted in an increase of COX-2 expression and PGE₂ production in A549 cells. Both the LPS-induced COX-2 expression and PGE₂ production were significantly inhibited by NDE (1-10 µg/mL) in a concentration-dependent manner. NDE did not affect COX-1 expression nor COX activity. These results suggest that NDE downregulates LPS-induced COX-2 expression and inhibits PGE₂ production in pulmonary epithelial cells. Furthermore, higenamine and nantenine, two major constituents responsible for tracheal relaxing effect of NDE, did not mimic the inhibitory effect of NDE on LPS-induced COX-2 expression in A549 cells. To identify active constituent(s) of NDE responsible for the anti-inflammatory effect, NDE was introduced in a polyaromatic absorbent resin column and stepwise eluted to yield water fraction, 20% methanol fraction, 40% methanol fraction, 99.8% methanol fraction, and 99.5% acetone fraction. However, none of these five fractions alone inhibited LPS-induced COX-2 expression. On the other hand, exclusion of water fraction from NDE abolished the inhibitory effect of NDE on LPS-induced COX-2 expression. These results suggest that constituent(s) present in water fraction is required but not sufficient for the anti-inflammatory activity of NDE, which may result from interactions among multiple constituents.

Inhibition of Lipopolysaccharide-Induced Cyclooxygenase-2 and Inducible Nitric Oxide Synthase Expression by 4-[(2′-O-acetyl-α-L-Rhamnosyloxy)Benzyl]Isothiocyanate from Moringa oleifera

Moringa oleifera Lamarck is commonly consumed for nutritional or medicinal properties. We recently reported the isolation and structure elucidation of novel bioactive phenolic glycosides, including 4-[(2′-O-acetyl-α-L-rhamnosyloxy)benzyl]isothiocyanate (RBITC), which was found to suppress inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in lipopolysaccharide-stimulated RAW 264.7 mouse macrophage cells. Inhibitors of proteins such as cyclooxygenase-2 (COX-2) and iNOS are potential antiinflammatory and cancer chemopreventive agents. The inhibitory activity of RBITC on NO production (IC50 = 0.96 ± 0.23 μM) was greater than that mediated by other well-known isothiocyanates such as sulforaphane (IC50 = 2.86 ± 0.39 μM) and benzyl isothiocyanate (IC50 = 2.08 ± 0.28 μM). RBITC inhibited expression of COX-2 and iNOS at both the protein and mRNA levels. Major upstream signaling pathways involved mitogen-activated protein kinases and nuclear factor-κB (NF-κB). RBITC inhibited phosphorylation of extracellular signal-regulated kinase and stress-activated protein kinase, as well as ubiquitin-dependent degradation of inhibitor κBα (IκBα). In accordance with IκBα degradation, nuclear accumulation of NF-κB and subsequent binding to NF-κB cis-acting element was attenuated by treatment with RBITC. These data suggest RBITC should be included in the dietary armamentarium of isothiocyanates potentially capable of mediating antiinflammatory or cancer chemopreventive activity.

Withaferin A down-regulates lipopolysaccharide-induced cyclooxygenase-2 expression and PGE2 production through the inhibition of STAT1/3 activation in microglial cells

Microglia are the major immune effector cells in the brain, and microglia activated by injury and infection can produce inflammatory mediators. A number of studies have reported that withaferin A has anti-inflammatory functions. However, the effects of withaferin A on the microglial inflammatory response have not been investigated. Our results show that withaferin A inhibited lipopolysaccharide (LPS)-induced cyclooxygenase (COX)-2 mRNA and protein expression and prostaglandin E2 (PGE 2) production in BV2 murine microglial cells. Withaferin A had no effect on LPS-induced Akt and ERK phosphorylation, but phosphorylation of p38 and JNK was slightly decreased by withaferin A. Withaferin A significantly inhibited LPS-induced STAT1 and STAT3 phosphorylation in a dose-dependent manner. Furthermore, withaferin A inhibited nuclear translocation of STAT1 and interferon-gamma activated sequence (GAS)-promoter activity. Taken together, these results suggest that withaferin A inhibits LPS-induced PGE 2 production and COX-2 expression, at least in part, by blocking STAT1 and STAT3 activation.

Heme oxygenase-1 mediates nicotine- and lipopolysaccharide-induced expression of cyclooxygenase-2 and inducible nitric oxide synthase in human periodontal ligament cells

Although heme oxygenase-1 (HO-1) plays a key role in inflammation, its anti-inflammatory effects and mechanism of action in periodontitis are still unknown. This study aimed to identify the effects of HO-1 on the proinflammatory mediators activated by nicotine and lipopolysaccharide (LPS) stimulation in human periodontal ligament (PDL) cells. The production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) was evaluated using Griess reagent and an enzyme immunoassay, respectively. The expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and HO-1 proteins was evaluated by Western blot analysis. Lipopolysaccharide and nicotine synergistically induced the production of NO and PGE(2) and increased the protein expression of iNOS, COX-2 and HO-1. Treatment with an HO-1 inhibitor and HO-1 small interfering RNAs blocked the LPS- and nicotine-stimulated NO and PGE(2) release as well as the expression of iNOS and COX-2. Our data suggest that the nicotine- and LPS-induced inflammatory effects on PDL cells may act through a novel mechanism involving the action of HO-1. Thus, HO-1 may provide a potential therapeutic target for the treatment of periodontal disease associated with smoking and dental plaque.

Induction of heme oxygenase-1 attenuates lipopolysaccharide-induced cyclooxygenase-2 expression in mouse brain endothelial cells

BackgroundProstaglandin E2 (PGE2), an arachidonic acid metabolite converted by cyclooxygenase-2 (COX-2), plays important roles in the regulation of endothelial functions in response to bacterial infection. The enzymatic activity of COX-2 can be down-regulated by heme oxygenase-1 (HO-1) induction. However, the mechanisms underlying HO-1 modulating COX-2 protein expression are not known.ObjectiveThe aim of the present study was to investigate whether the up-regulation of HO-1 regulates COX-2 expression induced by lipopolysaccharide (LPS), an endotoxin produced by Gram negative bacteria, in mouse brain endothelial cells (bEnd.3)MethodsCultured bEnd.3 cells were used to investigate LPS-induced COX-2 expression and PGE2 production. Cobalt protoporphyrin IX (CoPP, an HO-1 inducer), infection with a recombinant adenovirus carried with HO-1 gene (Adv-HO-1), or zinc protoporphyrin (ZnPP, an HO-1 inhibitor) was used to stimulate HO-1 induction or inhibit HO-1 activity. The expressions of COX-2 and HO-1 were evaluated by western blotting. PGE2 levels were detected by an enzyme-linked immunoassay. Hemoglobin (a chelator of carbon monoxide, CO, one of metabolites of HO-1) and CO-RM2 (a CO releasing molecule) were used to investigate the mechanisms of HO-1 regulating COX-2 expression.ResultsWe found that LPS-induced COX-2 expression and PGE2 production were mediated through NF-κB (p65) via activation of Toll-like receptor 4 (TLR4). LPS-induced COX-2 expression was inhibited by HO-1 induction by pretreatment with CoPP or infection with Adv-HO-1. This inhibitory effect of HO-1 was reversed by pretreatment with either ZnPP or hemoglobin. Pretreatment with CO-RM2 also inhibited TLR4/MyD88 complex formation, NF-κB (p65) activation, COX-2 expression, and PGE2 production induced by LPS.ConclusionsWe show here a novel inhibition of HO-1 on LPS-induced COX-2/PGE2 production in bEnd.3. Our results reinforce the emerging role of cerebral endothelium-derived HO-1 as a protector against cerebral vascular inflammation triggered by bacterial infection.

Comparative metabolomics approach coupled with cell- and gene-based assays for species classification and anti-inflammatory bioactivity validation of Echinacea plants

Echinacea preparations were the top-selling herbal supplements or medicines in the past decade; however, there is still frequent misidentification or substitution of the Echinacea plant species in the commercial Echinacea products with not well chemically defined compositions in a specific preparation. In this report, a comparative metabolomics study, integrating supercritical fluid extraction, gas chromatography/mass spectrometry and data mining, demonstrates that the three most used medicinal Echinacea species, Echinacea purpurea, E. pallida, and E. angustifolia, can be easily classified by the distribution and relative content of metabolites. A mitogen-induced murine skin inflammation study suggested that alkamides were the active anti-inflammatory components present in Echinacea plants. Mixed alkamides and the major component, dodeca-2 E,4 E,8 Z,10 Z( E)-tetraenoic acid isobutylamides ( 8/ 9), were then isolated from E. purpurea root extracts for further bioactivity elucidation . In macrophages, the alkamides significantly inhibited cyclooxygenase 2 (COX-2) activity and the lipopolysaccharide-induced expression of COX-2, inducible nitric oxide synthase and specific cytokines or chemokines [i.e., TNF-α, interleukin (IL)-1α, IL-6, MCP-1, MIP-1β] but elevated heme oxygenase-1 protein expression. Cichoric acid, however, exhibited little or no effect. The results of high-performance liquid chromatography/electron spray ionization/mass spectrometry metabolite profiling of alkamides and phenolic compounds in E. purpurea roots showed that specific phytocompound (i.e., alkamides, cichoric acid and rutin) contents were subject to change under certain post-harvest or abiotic treatment. This study provides new insight in using the emerging metabolomics approach coupled with bioactivity assays for medicinal/nutritional plant species classification, quality control and the identification of novel botanical agents for inflammatory disorders.

Chlorogenic acid inhibits lipopolysaccharide-induced cyclooxygenase-2 expression in RAW264.7 cells through suppressing NF-κB and JNK/AP-1 activation

Chlorogenic acid (CGA) is a naturally occurring phenolic acid in human diet. Data obtained from in vivo and in vitro experiments demonstrate that CGA mostly presents anti-oxidant and anti-carcinogenic activities. Here we show that CGA also inhibits lipopolysaccharide (LPS)-induced inflammatory response[AU1] in RAW 264.7 cells. Our results indicated that CGA significantly decreased LPS-induced up-regulation of cyclooxygenase (COX-2) at protein and mRNA levels in RAW 264.7 cells and as a result it inhibited prostaglandin E 2 (PGE 2) release from LPS-treated RAW 264.7 cells. In the further experiments, LPS-induced activation of nuclear factor-kappaB (NF-κB) and c-Jun N-terminal kinase (JNK)-c-Jun-activator protein (AP-1) pathway were suppressed significantly by CGA. In addition, CGA did not affect phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38. In conclusion, CGA suppresses LPS-induced COX-2 expression via attenuating the activation of NF-κB and JNK/AP-1 signaling pathways suggesting that CGA, the polyphenol compound in our food, could exert anti-inflammatory effects through inhibiting PGE 2 production.