l-663536 and Inflammation

Inflammation comprises the reaction of the body to injury, in which a series of changes of the terminal vascular bed, blood, and connective tissue tends to eliminate the injurious agent and to repair the damaged tissue. It is a complex process, which involves the release of diverse regulatory mediators. The current anti-inflammatory agents are challenged by multiple side effects and thus, new effective therapies are highly needed. The aim of this review is to summarize the described microsomal prostaglandin E synthase-1 (mPGES-1) inhibitors or transcriptional suppressors from medicinal plants, which could be an ideal approach in the management of inflammatory disorders, but need further clinical trials in order to be ultimately validated.

Topics: Animals; Anti-Inflammatory Agents; Biological Products; Drug Discovery; Enzyme Inhibitors; Humans; Inflammation; Plants, Medicinal; Prostaglandin-E Synthases

Until recently, studies on the role of the metabolites of arachidonic acid (AA), eicosanoids in fever have primarily focused on prostaglandins, prostaglandin E2 (PGE2) in particular, derived from the pathway related to cyclooxygenases (COX). COX exists in two known isoforms; a constitutive COX-1, and COX-2, which is inducible upon the action of pyrogens. Data accumulated in our laboratories suggest a thermoregulatory role for two other pathways of arachidonate metabolism; 5-lipoxygenase (5-LOX) and cytochrome P-450 (epoxygenase). We have demonstrated that leukotrienes (LTs; 5-LOX-derived eicosanoids) and various isomers of epoxyeicosatrienoic acids (EETs; epoxygenase-derived eicosanoids) contribute to the process of endogenous antipyresis or cryogenesis, which limits the height of fever. In support of this are several lines of evidence based on both in vivo and in vitro experiments. 1) Intracerebroventricular (icv) injections of LTC4 at nanomolar concentrations cause a dose-dependent decrease of body temperature (Tb) in mice. 2) Lipopolysaccharide (LPS)-induced anapyrexia in mice is preceded and accompanied by elevation in hypothalamic cysteinyl-LT (CysLT) production. 3) The inhibitor of LT synthesis MK-886 suppresses both of these processes. 4) EETs as well as inducers of the epoxygenase attenuate, whereas inhibitors of epoxygenase enhance the LPS-induced fever in rats. 5) One of the isomers of EET, 11,12-EET, in in vitro studies inhibited both the generation of PGE2 and IL-6 in monocytes stimulated with LPS. These results, together with a well-established pyrogenic role of PGE2, indicate that AA cascade may be regarded as an endogenous system to regulate the temperature response upon disease. COX, 5-LOX, and epoxygenase products may act at the level of hypothalamus as proximal mediators of, respectively, fever (PGE2) or cryogenesis (CysLTs and EETs), or indirectly by influencing the other endogenous cryogens and pyrogens.

Topics: 8,11,14-Eicosatrienoic Acid; Animals; Arachidonate 5-Lipoxygenase; Body Temperature; Cyclooxygenase 1; Cyclooxygenase 2; Cytokines; Eicosanoids; Feedback, Physiological; Fever; Humans; Hypothalamus; Indoles; Inflammation; Leukotrienes; Lipopolysaccharides; Macrophages; Mice; Protein Isoforms; Rats

In conclusion, an effective modulator of the AA cascade for the treatment of asthma and other inflammatory diseases may require 5-LO inhibitory activity as well as LTD4 antagonism in order to limit the effects of LTB4, LTD4, and 5-HPETE. The unknown role of LTC4 with respect to bronchoconstriction and mucus production could mask the efficacy of a pure LTD4 antagonist in man, whereas the chemotactic property of LTB4 for eosinophils can contribute to lung inflammation. Indeed, it is observed that the blood of patients with bronchial asthma has increased numbers of hypodense eosinophils. In addition, the formation of lipid-derived peroxide radicals, such as 5-HPETE, are believed to be responsible for various types of cellular injuries associated with the inflammatory disease process. Because inhibition of the CO pathway is thought to explain the therapeutic effects of nonsteroidal antiinflammatory agents in rheumatic diseases, a 5-LO inhibitor with CO inhibitory activity may also be desirable profile for an antiasthma agent. The validation of the LT hypothesis of disease had to wait for the demonstration of a clinical effect by either a LTD4 receptor antagonist or a LT synthesis inhibitor (5-LO inhibitor). Only very recently has this evidence become available and it is now apparent that compounds that antagonize LTD4 receptors or inhibit LT synthesis have shown clinical efficacy in a wide range of diseases. Due to the breakthrough nature of this approach, certain of these compounds are being considered for expedited development. The absence of side effects seen in the clinical trials of selective 5-LO inhibitors is gratifying and argues that LTs are not important in homeostasis. Only time will tell whether 5-LO inhibitors will take their place in the therapeutic armamentarium; however, the recent demonstration of clinical efficacy by a number of these compounds is a significant step in this direction.

Topics: Animals; Arachidonate 5-Lipoxygenase; Asthma; Eosinophils; Humans; Inflammation; Leukotriene Antagonists; Lipoxygenase Inhibitors; Lung Diseases; Neutrophils; Quinolines

Topics: Animals; Antigens; Dicarboxylic Acids; Eicosanoids; Indazoles; Indoles; Inflammation; Leukotrienes; SRS-A

Our previous studies have indicated that osthole may ameliorate the hepatic lipid metabolism and inflammatory response in nonalcoholic steatohepatitic rats, but the underlying mechanisms remain unclear. This study aimed to determine whether the effects of osthole were mediated by the activation of hepatic peroxisome proliferator-activated receptor α/γ (PPARα/γ). A rat model with steatohepatitis was induced by orally feeding high-fat and high-sucrose emulsion for 6 weeks. These experimental rats were then treated with osthole (20 mg/kg), PPARα antagonist MK886 (1 mg/kg) plus osthole (20 mg/kg), PPARγ antagonist GW9662 (1 mg/kg) plus osthole (20 mg/kg) and MK886 (1 mg/kg) plus GW9662 (1 mg/kg) plus osthole (20 mg/kg) for 4 weeks. The results showed that after osthole treatment, the hepatic triglycerides, free fatty acids, tumor necrosis factor-α, monocyte chemotactic protein-1, interleukin-6 (IL-6), IL-8 and liver index decreased by 52.3, 31.0, 32.4, 28.9, 36.3, 29.3 and 29.9%, respectively, and the score of steatohepatitis also decreased by 70.0%, indicating that osthole improved the hepatic steatosis and inflammation. However, these effects of osthole were reduced or abrogated after simultaneous addition of the specific PPARα antagonist MK886 or/and the PPARγ antagonist GW9662, especially in the co-PPARα/γ antagonists-treated group. Importantly, the osthole-induced hepatic expressions of PPARα/γ proteins were decreased, and the osthole-regulated hepatic expressions of lipogenic and inflammatory gene proteins were also reversed by PPARα/γ antagonist treatment. These findings demonstrated that the ameliorative effect of osthole on nonalcoholic steatohepatitis was mediated by PPARα/γ activation, and osthole might be a natural dual PPARα/γ activator.

Topics: Anilides; Animals; Coumarins; Fatty Acids, Nonesterified; Fatty Liver; Indoles; Inflammation; Lipid Metabolism; Liver; Male; PPAR alpha; PPAR gamma; Rats; Rats, Sprague-Dawley; Triglycerides

Microsomal prostaglandin E2 synthase (mPGES)-1 is responsible for the massive prostaglandin E2 (PGE2) formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to nonsteroidal anti-inflammatory drugs. The first selective mPGES-1 inhibitors recently entered clinical trials. Major challenges for drug development have been the high plasma protein binding of lead structures, interspecies discrepancies, nuisance inhibition, sophisticated enzyme assays, and limited structural information about the mPGES-1 inhibitor binding site. Since most of these drawbacks could be solved during the past few years, we are standing at the threshold of a new era of mPGES-1-targeting anti-inflammatory drugs. This perspective introduces mPGES-1 as a key player within the network of eicosanoid biosynthesis and summarizes our current understanding of its structure and mechanism. Moreover, we present high-throughput and in silico screening techniques and discuss the structure-activity relationship and pharmacological potential of major mPGES-1 inhibitor classes in light of recent insights from pharmacophore models and cocrystallization studies.

Topics: Animals; Anti-Inflammatory Agents; Dinoprostone; Drug Design; Drug Evaluation, Preclinical; Enzyme Inhibitors; High-Throughput Screening Assays; Humans; Inflammation; Models, Molecular; Prostaglandin-E Synthases; Structure-Activity Relationship

Peroxisome proliferator-activated receptor (PPAR) α/γ may control lipid metabolism and inflammatory response by regulating the downstream target genes, and play a crucial role in the process of non-alcoholic steatohepatitis (NASH) formation, but the difference and interaction between PPARα and PPARγ are poorly understood. The rat model with NASH was established by orally feeding high-fat and high-sucrose emulsion for 6weeks. The results shown that after the model rats were simultaneously treated with PPARα/γ agonists, the total cholesterol (TC), triglyceride (TG) and inflammatory cytokine levels in serum and hepatic tissue, the hepatic steatosis and inflammatory cellular infiltration were decreased, and were consistent with the results of hepatic lipogenic gene and nuclear factor (NF)-κB protein expressions. Conversely, these indexes were increased by PPARα/γ antagonist treatment. Compared with the model group, the serum free fatty acid (FFA) level was increased in the PPARα agonist-treated group, decreased in the PPARγ agonist-treated group, and unchanged in the PPARα/γ agonists-treated group. The hepatic FFA level was low in the PPARα/γ agonists-treated groups, but no significant variation in the PPARα/γ antagonists-treated groups. The increments of hepatic reduced glutathione (GSH) and superoxide dismutase (SOD) contents in the PPARα/γ agonists-treated groups were accompanied by decreased hepatic malondialdehyde (MDA) content. These findings demonstrated that PPARα/γ activation might decrease the hepatic lipid accumulation, oxidative stress and inflammatory cytokine production, and PPARγ could counterbalance the adverse effect of PPARα on circulating FFA. It was concluded that the integrative application of PPARα and PPARγ agonists might exert a synergic inhibitory effect on NASH formation through the modulation of PPARα/γ-mediated lipogenic and inflammatory gene expressions.

Topics: Anilides; Animals; Chemokine CCL2; Cytokines; Disease Models, Animal; Fatty Acids, Nonesterified; Fenofibrate; Gene Expression Regulation; Glutathione; Indoles; Inflammation; Interleukin-6; Interleukin-8; Lipid Metabolism; Liver; Male; Malondialdehyde; NF-kappa B; Non-alcoholic Fatty Liver Disease; Oxidative Stress; PPAR alpha; PPAR gamma; Rats; Rats, Sprague-Dawley; Rosiglitazone; Superoxide Dismutase; Thiazolidinediones; Tumor Necrosis Factor-alpha

We present the synthesis and biological evaluation of a collection of s-triazine derivatives as a novel scaffold of compounds with the capability to inhibit the PGE₂ production in LPS-induced RAW 264.7 macrophage cells. A total of 12 derivatives were synthesized and assayed for PGE₂ reduction at 10 μM concentration. Two compounds (7b and 7i) exhibiting >90% inhibition of PGE₂ production were found to have IC₅₀ values of 5.76 and 5.52 μM, respectively. They were counter screened for inhibition on COX-2 activity in a cell free assay. Specifically, compound 7i (R¹ = 4-Bn-Ph, R² = Cl, R³ = Ph, R⁵ = CO₂Me) was highly active in cells while maintaining little COX-2 inhibition (∼0% at 10 μM). Molecular docking study provides the possibility that compound 7i could inhibit PGE₂ production by blocking the PGH₂ binding site of mPGES-1 instead of COX-2 enzyme. Based on this result, our synthetic efforts will focus on intensive structure-activity relationship (SAR) study of s-triazine scaffold to discovery a potential PGE₂ synthesis inhibitor.

Topics: Animals; Dinoprostone; Inflammation; Intramolecular Oxidoreductases; Macrophages; Mice; Prostaglandin-E Synthases; Structure-Activity Relationship; Triazines

Previously studies have demonstrated that Cl(-)/HCO(3)(-) exchange was inhibited during chronic intestinal inflammation secondary to decrease in the affinity of the exchanger for Cl(-) rather than the number of transporters. Arachidonic acid metabolites (AAM) are elevated in the mucosa of the chronically inflamed small intestine. However, their role in the alteration of Cl(-)/HCO(3)(-) during chronic enteritis was unknown. Inhibition of AAM formation with arachidonyl trifluoro methylketone (ATMK) in chronically inflamed rabbit intestine reversed the diminished Cl(-)/HCO(3)(-) exchange activity. Kinetics studies showed that the reversal was secondary to restoration of the altered affinity of transporter. Downstream regulation of Cl(-)/HCO(3)(-) inhibition by AAM was determined to be by the cyclooxygenase pathway since only inhibition of cyclooxygenase with piroxicam treatment reversed the inhibited Cl(-)/HCO(3)(-) exchange. Further, DRA was shown to be the primary Cl(-)/HCO(3)(-) exchanger in villus cells. Kinetics and molecular studies indicated that the mechanism of inhibition of Cl(-)/HCO(3)(-) exchange by cyclooxygenase pathway metabolites was secondary to diminished affinity of the transporter for Cl(-) without a change in DRA BBM expression. Thus our data indicated that cyclooxygenase pathway metabolites mediate the inhibition of DRA during chronic intestinal inflammation.

Topics: Animals; Arachidonic Acid; Chloride-Bicarbonate Antiporters; Dose-Response Relationship, Drug; Ileum; Immunohistochemistry; Indoles; Inflammation; Intestinal Mucosa; Ketones; Kinetics; Leukotrienes; Male; Microvilli; Prostaglandins; Rabbits

Inflammatory mediators are thought to be involved in the systemic and local immune response induced by the Tityus serrulatus scorpion envenomation. New functional aspects of lipid mediators have recently been described. Here, we examine the unreported role of lipid mediators in cell recruitment to the peritoneal cavity after an injection with Ts2 or Ts6 toxins isolated from the T. serrulatus scorpion venom. In this report, we demonstrate that following a single intraperitoneal (i.p.) injection of Ts2 or Ts6 (250 μg/kg) in mice, there was an induction of leukocytosis with a predominance of neutrophils observed at 4, 24, 48 and 96 h. Moreover, total protein, leukotriene (LT)B(4), prostaglandin (PG)E(2) and pro-inflammatory cytokine levels were increased. We also observed an increase of regulatory cytokines, including interleukin (IL)-10, after the Ts2 injection. Finally, we observed that Ts2 or Ts6 injection in 5-lipoxygenase (LO) deficient mice and in wild type (WT) 129sv mice pre-treated with LTs and PGs inhibitors (MK-886 and celecoxib, respectively) a reduction the influx of leukocytes occurs in comparison to WT. The recruitment of these cells demonstrated a phenotype characteristic of neutrophils, macrophages, CD4 and CD8 lymphocytes expressing GR1+, F4/80+, CD3+/CD4+ and CD3+/CD8+, respectively. In conclusion, our data demonstrate that Ts2 and Ts6 induce inflammation by mechanisms dependent on lipid mediators and cytokine production. Ts2 may play a regulatory role whereas Ts6 exhibits pro-inflammatory activity exclusively.

Topics: Animals; Arachidonate 5-Lipoxygenase; Celecoxib; Cell Movement; Cyclooxygenase 2 Inhibitors; Cytokines; Immunohistochemistry; Indoles; Inflammation; Inflammation Mediators; Leukocytes; Leukotrienes; Lipids; Lipoxygenase Inhibitors; Male; Mice; Peritoneal Cavity; Prostaglandins; Pyrazoles; Scorpion Venoms; Sulfonamides

Microsomal prostaglandin E(2) synthase-1 (mPGES-1) is an enzyme, which is induced during the inflammatory response. Therefore, inhibitors of this enzyme are considered to be potential anti-inflammatory drugs. We have identified 3-(4-dodecanoyl-1,3,5-trimethylpyrrol-2-yl)propionic acid (12) as submicromolar inhibitor of mPGES-1. Surprisingly, structural variations made around this lead only resulted in a relatively small change of enzyme inhibitory potency. Such flat structure-activity relationships are reported to be typical for so called nuisance inhibitors, which exert their action not by directly binding to the enzyme, but by forming colloid-like aggregates at micromolar and sometimes submicromolar concentrations, which somehow sequester and inhibit enzyme targets without specificity. Since aggregate-based inhibition is highly sensitive to non-ionic detergents such as Triton X-100, we investigated some of our compounds for inhibition of human recombinant mPGES-1 also in presence of this detergent. The pyrrole derivatives 12, 67 and 81, which exhibited IC(50) values in absence of Triton X-100 in the range of 0.1 and 1μM, were virtually inactive at the highest test concentration of 10μM when 0.1% of the detergent was added. In the same way, the published mPGES-1 inhibitor 2-[(4-{[(1,1'-biphenyl)-4-ylmethyl]amino}-6-chloropyrimidin-2-yl)thio]octanoic acid (Cay10589) (6) totally lost its activity under these conditions. Therefore, these compounds have to be judged as nuisance inhibitors of the enzyme. In contrast, the known indole derivative 3-[3-(tert-butylthio)-1-(4-chlorobenzyl)-5-isopropylindol-2-yl]-2,2-dimethylpropionic acid (MK-886) (2) showed a considerable activity (75% inhibition at 10μM) also in the presence of Triton X-100.

Topics: Cell Line, Tumor; Enzyme Inhibitors; Humans; Inflammation; Inhibitory Concentration 50; Intramolecular Oxidoreductases; Magnetic Resonance Spectroscopy; Microsomes; Molecular Structure; Octoxynol; Propionates; Prostaglandin-E Synthases; Pyrroles; Spectrometry, Mass, Electrospray Ionization; Structure-Activity Relationship

Inflammation is a multi-staged process whose expansive phase is thought to be driven by acutely released arachidonic acid (AA) and its metabolites. Inhibition of cyclooxygenase (COX), lipoxygenase (LOX), or soluble epoxide hydrolase (sEH) is known to be anti-inflammatory. Inhibition of sEH stabilizes the cytochrome P450 (CYP450) products epoxyeicosatrienoic acids (EETs). Here we used a non-selective COX inhibitor aspirin, a 5-lipoxygenase activation protein (FLAP) inhibitor MK886, and a sEH inhibitor t-AUCB to selectively modulate the branches of AA metabolism in a lipopolysaccharide (LPS)-challenged murine model. We used metabolomic profiling to simultaneously monitor representative AA metabolites of each branch. In addition to the significant crosstalk among branches of the AA cascade during selective modulation of COX, LOX, or sEH, we demonstrated that co-administration of t-AUCB enhanced the anti-inflammatory effects of aspirin or MK886, which was evidenced by the observations that co-administration resulted in favorable eicosanoid profiles and better control of LPS-mediated hypotension as well as hepatic protein expression of COX-2 and 5-LOX. Targeted disruption of the sEH gene displayed a parallel profile to that produced by t-AUCB. These observations demonstrate a significant level of crosstalk among the three major branches of the AA cascade and that they are not simply parallel pathways. These data illustrate that inhibition of sEH by both pharmacological intervention and gene knockout enhances the anti-inflammatory effects of aspirin and MK886, suggesting the possibility of modulating multiple branches to achieve better therapeutic effects.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Arachidonic Acid; Aspirin; Benzoates; Blood Pressure; Dose-Response Relationship, Drug; Enzyme Activation; Epoxide Hydrolases; Gene Expression Regulation, Enzymologic; Hydroxyeicosatetraenoic Acids; Indoles; Inflammation; Lipopolysaccharides; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Prostaglandin-Endoperoxide Synthases; Urea

Dual inhibition of the prostaglandin (PG) and leukotriene (LT) biosynthetic pathway is supposed to be superior over single interference, both in terms of efficacy and side effects. Here, we present a novel class of dual microsomal PGE(2) synthase-1/5-lipoxygenase (5-LO) inhibitors based on the structure of pirinixic acid [PA, 2-(4-chloro-6-(2,3-dimethylphenylamino)pyrimidin-2-ylthio)acetic acid, compound 1]. Target-oriented structural modification of 1, particularly alpha substitution with extended n-alkyl or bulky aryl substituents and concomitant replacement of the 2,3-dimethylaniline by a biphenyl-4-yl-methane-amino residue, resulted in potent suppression of mPGES-1 and 5-LO activity, exemplified by 2-(4-(biphenyl-4-ylmethylamino)-6-chloropyrimidin-2-ylthio)octanoic acid (7b, IC(50) = 1.3 and 1 microM, respectively). Select compounds also potently reduced PGE(2) and 5-LO product formation in intact cells. Importantly, inhibition of cyclooxygenases-1/2 was significantly less pronounced. Taken together, these pirinixic acid derivatives constitute a novel class of dual mPGES-1/5-LO inhibitors with a promising pharmacological profile and a potential for therapeutic use.

Topics: Cell Line, Tumor; Cell Survival; Chemistry, Pharmaceutical; Cyclooxygenase 1; Cyclooxygenase 2; Drug Design; Enzyme Activation; Enzyme Inhibitors; Humans; Inflammation; Inhibitory Concentration 50; Intramolecular Oxidoreductases; Lipoxygenase Inhibitors; Models, Chemical; Prostaglandin-E Synthases; Pyrimidines

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class II expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4+ (but not CD4-) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4+ cells ex vivo. MK886 blocked induction of CCL17. Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalbumin-restimulated CD4+ cells initiate eosinophil recruitment which is strictly dependent on LTB4 production.

Topics: Allergens; Animals; CD4-Positive T-Lymphocytes; Cell Movement; Chemokines; Chronic Disease; Dexamethasone; Eosinophilia; Eosinophils; Indoles; Inflammation; Leukotriene B4; Male; Mice; Mice, Inbred BALB C; Ovalbumin

The peroxisome proliferator-activated receptor system is exciting much interest as a novel point of therapeutic intervention in inflammation. Here, the effect of a peroxisome proliferator-activated receptor alpha agonist, [4-chloro-6-(2,3-xylidine)-pyrimidinylthio]acetic acid (Wy14,643), was examined in arachidonic acid-induced murine ear inflammation. 3-[1-(4-Chlorobenzyl)-3-t-butyl-thio-5-isopropylindol-2-yl]-2,2-dimethylpropanoic acid (MK886, a 5-lipoxygenase inhibitor) and indomethacin (a cyclo-oxygenase inhibitor) were used as reference compounds. Wy14,643 dose dependently inhibited ear swelling and polymorphonuclear leukocyte influx, as did MK886, associated with reduced tissue leukotriene B4 but not prostaglandin E2 levels. Unlike MK886, Wy14,643 did not inhibit ex vivo leukotriene B4 production. However, Wy14,643, but not MK886, induced peroxisomal enzyme activity. Indomethacin was less effective, though tissue prostaglandin E2 but not leukotriene B4 levels were reduced. Again, unlike indomethacin, Wy14,643 did not reduce ex vivo prostaglandin E2 production. However, indomethacin did increase peroxisomal enzyme activity but to a lesser extent than Wy14,643. This study demonstrates that peroxisome proliferator-activated receptor alpha activation can inhibit arachidonic acid-induced inflammation in part by enhancing degradation of leukotriene B4.

Topics: Administration, Oral; Animals; Anti-Inflammatory Agents; Arachidonate 5-Lipoxygenase; Dinoprostone; Disease Models, Animal; Dose-Response Relationship, Drug; Ear, External; Edema; Female; Indoles; Indomethacin; Inflammation; Leukotriene B4; Lipoxygenase Inhibitors; Mice; Neutrophils; Palmitoyl Coenzyme A; Peroxisomes; PPAR alpha; Prostaglandin-Endoperoxide Synthases; Pyrimidines

The cellular and molecular mechanisms for the development of tendinopathy are not clear, but inflammatory mediators produced by tendon fibroblasts in response to repetitive mechanical loading may be an important factor.. (1) Cyclic stretching of tendon fibroblasts affects the production of leukotriene B(4) and the expression of 5-lipoxygenase; and (2) the production level of leukotriene B(4) is inversely related to that of prostaglandin E(2).. Controlled laboratory study.. Human patellar tendon fibroblasts were uniaxially stretched in the presence of indomethacin (25 micro M) or MK-886 (10 micro M). After stretching for 4 hours, followed by 4 hours rest, levels of prostaglandin E(2), leukotriene B(4), and expression of 5-lipoxygenase were measured.. Stretched tendon fibroblasts increased the levels of leukotriene B(4) but did not appreciably change the expression of 5-lipoxygenase. Indomethacin decreased the cellular production of prostaglandin E(2) but caused increased leukotriene B(4) levels. MK-886 caused decreased production of leukotriene B(4) but increased production of prostaglandin E(2).. Cyclic stretching of human tendon fibroblasts increases the production of prostaglandin E(2) and leukotriene B(4). Blocking prostaglandin E(2) production leads to increased leukotriene B(4) levels and vice versa.. The use of nonsteroidal anti-inflammatory drugs for the treatment of tendon inflammation might increase the levels of leukotriene B(4) within the tendon, potentially contributing to the development of tendinopathy.

Topics: Adult; Anti-Inflammatory Agents, Non-Steroidal; Arachidonate 5-Lipoxygenase; Bicycling; Cell Culture Techniques; Dinoprostone; Fibroblasts; Humans; Indoles; Indomethacin; Inflammation; Leukotriene B4; Lipoxygenase Inhibitors; Tendons; Tensile Strength

Prostaglandin E synthase (PGES) including isoenzymes of membrane-associated PGES (mPGES)-1, mPGES-2, and cytosolic PGES (cPGES) is the recently identified terminal enzyme of the arachidonic acid cascade. PGES converts prostaglandin (PG)H2 to PGE2 downstream of cyclooxygenase (COX). We investigated the expression of PGES isoenzyme in articular chondrocytes from patients with osteoarthritis (OA). Chondrocytes were treated with various cytokines and the expression of PGES isoenzyme mRNA was analyzed by the reverse transcription-polymerase chain reaction and Northern blotting, whereas Western blotting was performed for protein expression. The subcellular localization of mPGES-1 was determined by immunofluorescent microscopy. Conversion of arachidonic acid or PGH2 to PGE2 was measured by enzyme-linked immunosorbent assay. Finally, the expression of mPGES-1 protein in OA articular cartilage was assessed by immunohistochemistry. Expression of mPGES-1 mRNA in chondrocytes was significantly induced by interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha, whereas other cytokines, such as IL-4, IL-6, IL-8, IL-10, and interferon-gamma, had no effect. COX-2 was also induced under the same conditions, although its pattern of expression was different. Expression of cPGES, mPGES-2, and COX-1 mRNA was not affected by IL-1beta or TNF-alpha. The subcellular localization of mPGES-1 and COX-2 almost overlapped in the perinuclear region. In comparison with 6-keto-PGF1alpha and thromboxane B2, the production of PGE2 was greater after chondrocytes were stimulated by IL-1beta or TNF-alpha. Conversion of PGH2 to PGE2 (PGES activity) was significantly increased in the lysate from IL-1beta-stimulated chondrocytes and it was inhibited by MK-886, which has an inhibitory effect on mPGES-1 activity. Chondrocytes in articular cartilage from patients with OA showed positive immunostaining for mPGES-1. These results suggest that mPGES-1 might be important in the pathogenesis of OA. It might also be a potential new target for therapeutic strategies that specifically modulate PGE2 synthesis in patients with OA.

Topics: 6-Ketoprostaglandin F1 alpha; Cartilage, Articular; Cells, Cultured; Chondrocytes; Cyclooxygenase 2; Cytokines; Humans; Indoles; Inflammation; Interleukin-1; Intramolecular Oxidoreductases; Isoenzymes; Membrane Proteins; Osteoarthritis; Phenotype; Prostaglandin-E Synthases; Prostaglandin-Endoperoxide Synthases; RNA, Messenger; Thromboxane B2; Time Factors; Up-Regulation

Proinflammatory eicosanoids formed from arachidonic acid (AA) by lipoxygenase (LO) and cyclooxygenase (COX) pathways have been shown to inhibit apoptosis in certain cell types. This study determined whether inhibition of LO and COX increased apoptosis in AA-treated HL-60 cells in vitro.. HL-60 cells were incubated with 50 micromol/L AA and an enzyme inhibitor (1-10 micromol/L) for COX, LO, 12-LO, and 5-LO for 12 hours. Flow cytometry was used to assess viability, apoptosis, and necrosis. Apoptosis was further assessed using terminal dUTP nick end-labeling and DNA fragmentation.. The highest concentration of LO inhibitors, but not COX inhibitors, decreased viability and increased apoptosis and necrosis in the presence of exogenous AA.. These results suggest that disruption of the metabolism of AA by LO, in particular 5-LO, decreases cell survival and increases apoptosis. Thus, downstream metabolic processing of AA by LO but not COX plays a critical role in the regulation of HL-60 cell apoptosis.

Topics: Apoptosis; Arachidonate 5-Lipoxygenase; Benzoquinones; Cyclooxygenase Inhibitors; DNA Fragmentation; Fatty Acids, Unsaturated; Fish Oils; Flavanones; Flavonoids; Flow Cytometry; HL-60 Cells; Humans; Ibuprofen; In Situ Nick-End Labeling; Indoles; Inflammation; Lipoxygenase Inhibitors; Necrosis; Neutrophils; Prostaglandin-Endoperoxide Synthases; Respiratory Distress Syndrome

Neutrophil migration to an infectious focus is essential for control and resolution of infection. Early studies demonstrated that the failure of such migration is observed in lethal sepsis induced by cecal ligation and puncture (L-CLP), whereas intense neutrophil migration is seen in sublethal CLP (SL-CLP). In this study, we found that inhibition of synthesis of prostaglandins or leukotriene B4 (LTB4) did not modify the failure of neutrophil migration or the survival rate of L-CLP mice. In addition, pretreatment of L-CLP mice with a platelet activating factor (PAF) receptor antagonist (UK74505), despite not interfering with the failure process, significantly increased (33%) the survival rate of the animals. Inhibitors of prostaglandin synthesis (indomethacin and meloxican) and UK74505 did not modify the neutrophil migration observed in SL-CLP. On the other hand, the blockade of LTB4 synthesis (MK886, a 5-lipoxygenase-activating protein inhibitor) or of its receptors (CP-105,696) resulted in reduced neutrophil migration to the peritoneal cavity in SL-CLP mice (62% and 60%, respectively), a consequent increase in the number of bacteria in the inflammatory focus, and a reduced survival rate of the animals (43% and 38%, respectively). Both SL-CLP and L-CLP animals presented significant levels of LTB4 in the peritoneal exudate (3- and 8-fold higher than sham group, respectively) and these were reduced by the pretreatment of mice with LTB4 inhibitors. In conclusion, our results suggest that LTB4, but not prostaglandins or PAF, is an important chemoattractant involved in neutrophil recruitment to infection sites in SL-CLP, a crucial event in confining the invading pathogens to a restricted area. However, in circumstances in which the infection turns to a lethal sepsis, LTB4 is not involved in the observed failure of neutrophil migration to the infectious focus.

Topics: Animals; Cecum; Cell Movement; Dihydropyridines; Eicosanoids; Imidazoles; Indoles; Inflammation; Leukotriene B4; Leukotrienes; Lipoxygenase Inhibitors; Male; Mice; Mice, Inbred C57BL; Neutrophils; Peritoneum; Platelet Activating Factor; Prostaglandins; Time Factors; Wounds, Penetrating

Leukotrienes (LTs) are considered important for antibacterial defense in the lung. Multidrug resistance protein 1 (mrp1) is a transmembrane protein responsible for the cellular extrusion of LTC(4). To determine the role of mrp1 in host defense against pneumonia, mrp1(-/-) and wild-type mice were intranasally inoculated with Streptococcus pneumoniae. mrp1(-/-) mice displayed a diminished outgrowth of pneumococci in lungs and a strongly reduced mortality. These findings were related to an effect of mrp1 on LT metabolism, because survival was similar in mrp1(-/-) and wild-type mice treated with the 5-lipoxygenase-activating protein inhibitor MK-886. Although LTC(4) levels remained low in the bronchoalveolar lavage fluid of mrp1(-/-) mice, LTB(4) concentrations were higher than in wild-type mice. These elevated LTB(4) concentrations were important for the relative protection of mrp1(-/-) mice, because the LTB(4) antagonist LTB(4)-dimethyl amide abolished their survival advantage. In vitro experiments suggested that the intracellullar accumulation of LTC(4) in mrp1(-/-) mice results in product inhibition of LTC(4)-synthase, diminishing substrate competition between LTA(4)-hydrolase (which yields LTB(4)) and LTC(4)-synthase for the available LTA(4). We conclude that mrp1(-/-) mice are resistant against pneumococcal pneumonia by a mechanism that involves increased release of LTB(4). These results identify mrp1 as a novel target for adjunctive therapy in pneumonia.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; Female; Genetic Predisposition to Disease; Immunity, Innate; Indoles; Inflammation; Injections, Intraperitoneal; Intracellular Fluid; Leukotriene B4; Lipoxygenase Inhibitors; Lung; Mice; Mice, Inbred Strains; Mice, Knockout; Pneumonia, Pneumococcal; Streptococcus pneumoniae; Up-Regulation

We analyzed DNA-fragmentation in human polymorphonuclear neutrophil granulocytes (PMNs) from healthy donors after addition of exogenous arachidonic acid (AA) and eicosapentaenoic acid (EPA) by flow cytometry (propidium iodide staining of DNA and DNA strand break detection). The PMNs were incubated from 30 min up to 48 hours in RPMI 1640 which was supplemented with different concentrations of AA or EPA (5-40 microM). In contrast to EPA the addition of AA led to a significant increase in apoptosis up to 67.8% compared to the RPMI-control whereas the addition of EPA led to an inhibition of DNA-fragmentation. When the cells were incubated with MK 886 (1 microM, inhibitor of leukotriene biosynthesis) an increase in DNA-fragmentation (up to 63.3%) was observed. Conversely, in the presence of indomethacin (1 microM, inhibitor of prostanoid synthesis) an inhibition in DNA-fragmentation (up to 60.9%) occurred. Furthermore, preincubation of PMNs with pentoxifylline (1mM, phosphodiesterase inhibitor) reduced AA-stimulated DNA-fragmentation up to 43.4%. These data provide evidence for the involvement of AA and distinct AA metabolites in the regulation of apoptosis in human PMNs.

Topics: Apoptosis; Arachidonic Acid; Coloring Agents; Cyclooxygenase Inhibitors; DNA Fragmentation; Eicosapentaenoic Acid; Flow Cytometry; Humans; In Vitro Techniques; Indoles; Indomethacin; Inflammation; Leukotrienes; Lipoxygenase Inhibitors; Neutrophils; Pentoxifylline; Phosphodiesterase Inhibitors; Propidium

Chronically elevated shear stress and inflammation are important in hypertensive lung vessel remodeling. We postulate that 5-lipoxygenase (5-LO) is a molecular determinant of these processes. Immunohistology localized the 5-LO to macrophages of normal and chronically hypoxic rat lungs and also to vascular endothelial cells in chronically hypoxic lungs only. In situ hybridization of normal and chronically hypoxic lungs demonstrated that 5-LO mRNA is expressed in macrophages. Rats hypoxic for 4 wk-developed pulmonary hypertension increased translocation of the lung 5-LO from the cytosol to the membrane fraction and increased levels of lung tissue 5-lipoxygenase-activating protein (FLAP). A FLAP ligand, 3-[l-(4-chlorobenzyl)-3-t-butyl-thio-t-isopropylindol-2-yl]-2,2- dimethylpropanoic acid (MK-886), inhibited the acute angiotensin II and hypoxia-induced pulmonary vasoconstriction in vitro and the development of chronic hypoxic pulmonary hypertension in rats in vivo. Mice bred with the deletion of the 5-LO enzyme (5-LO knockout) developed less right heart hypertrophy than age-matched 5-LO competent mice. Our results support the hypothesis that the 5-LO is involved in lung vascular tone regulation and in the development of chronic pulmonary hypertension in hypoxic rodent models.

Topics: 5-Lipoxygenase-Activating Proteins; Altitude; Angiotensin II; Animals; Arachidonate 5-Lipoxygenase; Cardiomegaly; Carrier Proteins; Endothelium, Vascular; Gene Expression; Hypertension, Pulmonary; Hypoxia; Immunohistochemistry; In Situ Hybridization; Indoles; Inflammation; Lipoxygenase Inhibitors; Male; Membrane Proteins; Mice; Mice, Knockout; Pulmonary Artery; Rats; Rats, Sprague-Dawley; Vasoconstriction

alpha-naphthylisothiocyanate (ANIT) administration to rats results in periportal hepatic inflammation and injury. Glutathione (GSH) appears to be necessary for the liver injury to occur. The leukotrienes (LTs) are metabolites of arachidonic acid and potent mediators of inflammation that have been implicated in certain liver injury models. Inasmuch as GSH is a cofactor for the synthesis of cysteinyl-LTs and since inflammation is a prominent component of ANIT injury, we hypothesized that LTs are involved in producing the hepatic insult that results from ANIT administration. To test this hypothesis, rats were treated with one of several inhibitors of LT biosynthesis, A63162, Zileuton or MK-886. Each of these agents prevented the formation of LTB4 in Ca++ ionophore-stimulated whole blood from rats treated with the inhibitors. A63162 attenuated the hepatic parenchymal injury caused by ANIT and resulted in a modest decrease in ANIT-induced cholestasis. In contrast, neither Zileuton nor MK-886 attenuated liver injury. AT-125 (Acivicin) inhibits gamma-glutamyl transferase (GGT), the enzyme that catalyzes the formation of LTD4 from LTC4. AT-125 pretreatment did not prevent ANIT-induced hepatic parenchymal insult. It did, however, ameliorate the cholestasis caused by ANIT. In conclusion, the partial protection afforded by A63162 and AT-125 likely results from effects unrelated to the formation of LTs, since Zileuton and MK-886 inhibited LT synthesis without affording protection. The lack of protection by Zileuton and MK-886 in the face of LT synthesis inhibition suggests that LTs are not necessary for the expression of injury after ANIT administration.

Topics: 1-Naphthylisothiocyanate; Acetamides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Calcimycin; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Glutathione; Hydroxyurea; Indoles; Inflammation; Leukotriene Antagonists; Leukotrienes; Lipoxygenase Inhibitors; Liver; Liver Diseases; Male; Phenyl Ethers; Rats; Rats, Sprague-Dawley

In mouse skin in vivo the irritant and hyperplasiogenic tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) strongly increased the epidermal content of the cysteinyl leukotrienes LTC4, LTD4 and LTE4, but not of leukotriene LTB4. This effect was completely suppressed by the selective leukotriene biosynthesis inhibitor MK-886. Intragastric administration of MK-886 prevented phorbol ester-induced ear edema, but not epidermal hyperproliferation and tumor promotion. These data indicate that leukotrienes are involved in the pro-inflammatory effects of the phorbol ester, whereas its hyperproliferative and tumor-promoting activities do not depend on 5-lipoxygenase-catalyzed leukotriene formation. This action differs from several non-selective inhibitors of lipoxygenases that were found to inhibit tumor promotion in initiated mouse skin.

Topics: Animals; Ear, External; Edema; Epidermis; Female; Hyperplasia; Indoles; Inflammation; Leukotriene B4; Mice; Skin Neoplasms; SRS-A; Tetradecanoylphorbol Acetate

The liver modulates host responses to endotoxemia by production and clearance of tumor necrosis factor alpha (TNF-alpha) and eicosanoid lipoxygenation products. Reductions in liver blood flow (QL) are common during endotoxemia, but it is unknown whether the kinetics of TNF-alpha and leukotrienes (LTs) are thereby altered to amplify lung inflammation. To test this hypothesis, reductions in QL were modeled by an end-to-side portacaval shunt (PCS) in Sprague-Dawley rats. Conscious animals received 2.5 mg/kg of intravenous E. coli lipopolysaccharide (LPS) serotype 055:B5 (PCS + LPS; n = 17) or saline (n = 5). Responses were compared with those in sham-operated rats (sham + LPS; n = 13) and NSS-challenged control rats (n = 5). Cardiopulmonary changes, serum TNF-alpha, and formed elements were determined at t = 0, 1.5, 3.5, and 24 h, when organ wet/dry ratios (W/D) were measured with TNF-alpha, LTB4, and polymorphonuclear neutrophils (PMN) in bronchoalveolar lavage fluid (BALF). In PCS + LPS rats, mortality was 59% and serum TNF-alpha peaked at 1.5 h (2,784 +/- 658 U/ml, mean +/- SEM) coincident with the onset of hypotension. Despite equivalent endotoxemia and liver- and lung-associated TNF-alpha in sham + LPS rats at 1.5 h, peak serum TNF-alpha was 38% less and mortality was 15% (p < 0.05). Cardiac, hepatic, and cecal W/D were likewise increased in PCS + LPS versus sham + LPS rats, as were BALF PMNs (p < 0.05). In parallel studies, the disappearance kinetics of infused rTNF-alpha were not altered in nonendotoxemic PCS animals, implicating enhanced lung uptake of LPS and systemic export of TNF-alpha in PCS + LPS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Bacteremia; Bronchoalveolar Lavage Fluid; Diethylcarbamazine; Endotoxins; Escherichia coli Infections; Hemodynamics; Indoles; Inflammation; Leukotriene Antagonists; Leukotrienes; Liver; Liver Circulation; Lung; Male; Metabolic Clearance Rate; Neutrophils; Organ Size; Portacaval Shunt, Surgical; Rats; Rats, Sprague-Dawley; Survival Rate; Tumor Necrosis Factor-alpha

Leukotrienes (LTs) have been implicated as mediators of the inflammation and ulceration associated with ulcerative colitis and Crohn's disease. In the present study, the effects of a novel, orally active inhibitor of LT synthesis (MK-886) were examined in a rat model of chronic colitis. Colitis was induced by intracolonic administration of trinitrobenzenesulfonic acid. Colonic LTB4 synthesis was measured after incubation of tissue samples in vitro and by in vivo equilibrium dialysis. A single dose of MK-886 (10 mg/kg) significantly inhibited colonic LTB4 synthesis for greater than 24 h. Daily treatment with this dose significantly reduced colonic damage, as assessed macroscopically and histologically, when the treatment was performed 2 h before induction of colitis and daily thereafter for 1 wk, but not when treatment was performed during the second week after induction of colitis. A less marked beneficial effect of MK-886 was observed when the pretreatment dose was excluded, suggesting a role for LTs in the early events of the inflammatory process. Inhibition of LT synthesis during the first 24 h after induction of colitis did not alter the extent of infiltration of neutrophils into the colon, as measured by tissue myeloperoxidase activity. Daily treatment with sulfasalazine (100 mg/kg po) either during the first or second week after induction of colitis did not significantly affect the rates of healing. At the dose used, sulfasalazine only produced a transient inhibition of colonic LTB4 synthesis. This study therefore demonstrates that a specific, orally active inhibitor of LT synthesis can significantly accelerate healing in this animal model of colitis when the treatment is performed during the early phase of the inflammatory response.

Topics: Administration, Oral; Animals; Colitis, Ulcerative; Colon; Disease Models, Animal; Indoles; Inflammation; Leukotriene B4; Male; Rats; Rats, Inbred Strains; Sulfasalazine; Trinitrobenzenesulfonic Acid

Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.

Topics: Affinity Labels; Amino Acid Sequence; Animals; Azides; Electrophoresis, Polyacrylamide Gel; Humans; Indoles; Inflammation; Kinetics; Leukotriene Antagonists; Leukotrienes; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Neutrophils; Rats