Effect of norathyriol,isolated from Tripterospermum lanceolatum,on A 23 187-induced pleurisy and analgesia in mice

A 23 187-induced pleurisy in the mouse was demonstrated in this study. The protein leakage, leukocyte accumulation, LTB₄ and PGE₂ production in the pleural cavity of mice were increased by A 23 187 in a dose-dependent manner. At 7.5 nmole A 23 187 intrapleural injection, the protein level peaked at 0.5–2 h, PMN leukocytes accumulation peaked at 3–4 h, and LTB₄ and PGE₂ production peaked at 0.5–1 h. In this in vivo model we investigated the anti-inflammatory effect of norathyriol, isolated from Tripterospermum lanceolatum. A 23 187-induced protein leakage was reduced by norathyriol (ID₅₀ was about 30.6 mg/kg i.p.), indomethacin and BW 755 C. A 23 187-induced PMN leukocytes accumulation was suppressed by norathyriol (ID₅₀ was about 16.8 mg/kg, i.p.) and BW 755 C, while enhanced by indomethacin. Like BW 755 C, norathyriol reduced both LTB₄ and PGE₂ production (ID₅₀ was about 18.6and 29.1 mg/kg i.p., respectively), while indomethacin reduced PGE₂ but not LTB₄ generation. We also demonstrated the analgesic effect of norathyriol on the acetic acid-induced writhing response. Acetic acid-induced writhing response was depressed by norathyriol (ID₅₀ was about 27.9 mg/kg i.p.), indomethacin and ibuprofen. These results suggest that norathyriol, like BW 755 C, might be a dual, yet weak, cyclooxygenase and lipoxygenase pathway blocker. The inhibitory effect of norathyriol on the A 23 187-induced pleurisy and acetic acid-induced writhing response in mice is proposed to be dependent on the reduction of eicosanoids mediators formation in the inflammatory site. Correspondence to: J.-P. Wang at the above address     

Mechanisms of the influence of magnolol on eicosanoid metabolism in neutrophils

We have demonstrated that magnolol suppressed thromboxane B2 (TXB2) and leukotriene B4 (LTB4) formation in A23187-stimulated rat neutrophils. Maximum inhibition was obtained with about 10 microM magnolol. Magnolol was more effective in the inhibition of cyclooxygenase (COX) activity than in the inhibition of 5-lipoxygenase (5-LO) activity as assessed by means of enzyme activity determination in vitro and COX and 5-LO metabolic capacity analyses in vivo. Magnolol alone stimulated cytosolic phospholipase A2 (cPLA2) phosphorylation and the translocation of 5-LO and cPLA2 to the membrane, and evoked arachidonic acid (AA) release. Recruitment of both 5-LO and cPLA2 to the membranes was suppressed by EGTA. Arachidonyl trifluoromethyl ketone (AACOCF3), a PLA2 inhibitor, bromoenol lactone (BEL), a Ca2+-independent PLA2 (iPLA2) inhibitor, and EGTA suppressed the magnolol-induced AA release. However, none of the follows affected magnolol-induced AA-release: 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580), a p38 mitogen-activated protein kinase (MAPK) inhibitor, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene (U0126), a MAPK kinase (MEK) inhibitor, or 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF109203X), a protein kinase C (PKC) inhibitor. In addition, magnolol at 30 microM did not stimulate the p38 MAPK and extracellular signal-regulated kinase 2 (ERK2) enzyme activities. These results indicated that magnolol inhibits the formation of prostaglandins and leukotrienes in A23187-stimulated rat neutrophils, probably through a direct blockade of COX and 5-LO activities. The stimulatory effects of magnolol at high concentration on the membrane association of 5-LO and cPLA2 are attributable to the elevation of [Ca2+]i, and on the AA release is likely via activation of cPLA2 and iPLA2.     

Identification of 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 as functional targets of the anti-inflammatory and anti-carcinogenic garcinol

Garcinol (camboginol) from the fruit rind of Guttiferae species shows anti-carcinogenic and anti-inflammatory properties, but the underlying molecular mechanisms are unclear. Here we show that garcinol potently interferes with 5-lipoxygenase (EC 7.13.11.34) and microsomal prostaglandin (PG)E₂ synthase (mPGES)-1 (EC 5.3.99.3), enzymes that play pivotal roles in inflammation and tumorigenesis. In cell-free assays, garcinol inhibited the activity of purified 5-lipoxygenase and blocked the mPGES-1-mediated conversion of PGH₂ to PGE₂ with IC₅₀ values of 0.1 and 0.3 μM, respectively. Garcinol suppressed 5-lipoxygenase product formation also in intact human neutrophils and reduced PGE₂ formation in interleukin-1β-stimulated A549 human lung carcinoma cells as well as in human whole blood stimulated by lipopolysaccharide. Moreover, garcinol interfered with isolated cyclooxygenase (COX)-1 (EC 1.14.99.1, IC₅₀ = 12 μM) and with the formation of COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and thromboxane B₂ in human platelets. In contrast, neither Ca²⁺-ionophore (A23187)-induced arachidonic acid release in neutrophils nor COX-2 activity in A549 cells or whole blood, measured as formation of 6-keto PGF_1α, or isolated human recombinant COX-2 were significantly affected by garcinol (≤30 μM). Together, the high potency of garcinol to selectively suppress PGE₂ synthesis and 5-lipoxygenase product formation provides a molecular basis for the anti-inflammatory and anti-carcinogenic effects of garcinol and rationalizes its therapeutic use.     

The development of methodology for clinical measurement of 5-lipoxygenase pathway intermediates from human peripheral blood mononuclear cells

Recent studies have shown a correlation between 5-lipoxygenase (5-LO) pathway up-regulation and cardiovascular risk. Despite the existence of several assays for products of the 5-LO pathway, a reliable method for clinical determination of 5-LO activity remains to be established. In the present communication, we report conditions that allow measurement of 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) in peripheral blood mononuclear cells (PBMCs) isolated from the blood of atherosclerosis patients before and after stimulation by the calcium ionophore, A23187. LTB₄, a potent mediator of inflammation-linked cardiovascular disease, was measured using an existing competitive enzyme immunoassay (EIA) kit after making specific methodological improvements that allowed PBMCs to be used in this format for the first time. LTB₄ was also measured by LC/MS/MS along with 5-HETE, a direct by-product of the action of 5-LO on arachidonic acid and a molecule for which no commercial EIA kit exists. The LC/MS/MS assay was validated over a range of 0.025–25 ng/mL for LTB₄ and 0.1–25 ng/mL for 5-HETE. The EIA method has a validated range covering 0.025–4 ng/mL. When both assays were applied to analyze LTB₄ from stimulated PBMCs isolated from 25 subjects with various degrees of atherosclerosis, a high correlation was obtained (r = 0.9426, Pearson's correlation coefficient). A high correlation was also observed between the levels of LTB₄ and 5-HETE measured by LC/MS/MS after ionophore stimulation (r = 0.9159). Details are presented for optimized sample collection, processing, storage, and analysis in accordance with the logistical demands of clinical analysis.     

Inhibition of LTB4 biosynthesis in situ by CGS 23885, a potent 5-lipoxygenase inhibitor, correlates with its pleural fluid concentrations in an experimentally induced rat pleurisy model

An intrapleural injection of carrageenan in rats induced LTB₄ and LTC₄/D₄/E₄ biosynthesis, exudate formation, and cellular influx in the pleural cavity. An injection of calcium ionophore (A23187, 100nmol) 16–18h after carrageenan injection augmented leukotriene biosynthesis and exudate formation, but not cellular influx. The carrageenan-induced pleurisy model modifid by A23187 administration was used to study the oral effect of CGS 23885 (N-hydroxy-N-[(6-phenoxy-2H-1-benzopyran-3-yl)-methyl]urea), a potent 5-lipoxygenase (5-LO) inhibitor, on inflammatory parameters. CGS 23885 dose-dependently (1 to 30mg/kg) inhibited the enhanced LTB₄ and LTC₄/D₄/E₄ (1 to 10mg/kg) biosynthesis, but had no effect on enhanced exudate formation. An inhibitory effect of CGS 23885 of small magnitude on cellular influx due to carrageenan stimulation was seen at 30mg/kg. The concentrations of CGS 23885 in the pleural fluid were dose-related, and a positive correlation (r ²=0.989) between pleural fluid concentration of LTB₄ and CGS 23885 was observed. The results confirm that CGS 23885 is a specific, orally active 5-LO inhibitor which can achieve concentrations in the pleural cavity sufficient to inhibit production of LTB₄ and LTC₄/D₄/E₄ in an ongoing inflammatory response. Received: 9 February 1995 / Accepted: 20 December 1996     

857-860Anti-inflammatory Effect of Magnolol,Isolated from Magnolia officinalis,on A23187-induced Pleurisy in Mice

In the present study, A23187-induced pleurisy in mice was used to investigate the anti-inflammatory effect of magnolol, a phenolic compound isolated from Chinese medicine Hou p'u (cortex of Magnolia officinalis). A23187-induced protein leakage was reduced by magnolol (10mgkg^?1, i.p.), indomethacin (10mgkg^?1, i.p.) and BW755C (30mgkg^?1, i.p.). A23187-induced polymorphonuclear (PMN) leucocyte infiltration in the pleural cavity was suppressed by magnolol and BW755C, while enhanced by indomethacin. Like BW755C, magnolol reduced both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) levels in the pleural fluid of A23187-induced pleurisy, while indomethacin reduced PGE₂ but increased LTB₄ formation. In the rat isolated peripheral neutrophil suspension, magnolol (3.7 μM) and BW755C (10 μM) also suppressed the A23187-induced thromboxane B₂ (TXB₂) and LTB₄ formation. These results suggest that magnolol, like BW755C, might be a dual cyclo-oxygenase and lipoxygenase inhibitor. The inhibitory effect of magnolol on the A23187-induced pleurisy is proposed to be, at least partly, dependent on the reduction of the formation of eicosanoids mediators in the inflammatory site.     

U-73122, an aminosteroid phospholipase C inhibitor, may also block Ca2+ influx through phospholipase C-independent mechanism in neutrophil activation

1-[6-[[17a-3-Methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U-73122) has been proven to be a useful tool in investigation of phospholipase C (PLC)-coupled signal transduction during cell activation. In the present studies, the inhibition by U-73122 of cytosolic free Ca²⁺ concentration ([Ca 2+]i) of neutrophils was investigated. U-73122 suppressed the [Ca²⁺]i elevation of neutrophils suspended in Ca²⁺-containing medium challenged by N-formyl-Met-Leu-Phe (fMLP), cyclopiazonic acid (CPA) and ionomycin. The concentrations of U-73122 required for inhibition of CPA- and ionomycin-induced changes with IC₅₀ values 4.06 ± 0.27 µM and 4.04 ± 0.44 µM, respectively, is almost 10-times that required for inhibition of the fMLP-induced response (IC₅₀ value 0.62 ± 0.04 µM) U-73122 also reduced the intracellular Ca²⁺ mobilization of neutrophils suspended in Ca ²⁺-free medium stimulated by fMLP and CPA, but not by ionomycin, with IC₅₀ values 0.52 ± 0.02 µM and 6.82 ± 0.74 µM, respectively. 1-[6-[[17f3-Methoxyestra-1,3,5(10)-trien-l7-yl]amino]hexyl]2,5-pyrrolidinedione (U-73343), a close analog of U-73122 that does not inhibit PLC activity, suppressed the [Ca²⁺]_i elevation of neutrophils challenged by fMLP in Ca²⁺-containing medium, but not in Ca²⁺-free medium, with IC₅₀ value 22.30 ± 1.61 µM. In Mn²⁺-quench studies, U-73122 suppressed the Mn²⁺ influx in CPA-activated neutrophils (IC₅₀ value was 7.16 ± 0.28 µM) as well as in resting neutrophils (IC₅₀ value was 6.72 ± 0.30 M). U-73343 also suppressed the Mn²⁺ influx in resting neutrophils in a concentration-dependent manner. These results suggest that the inhibitory effect of U-73122 on [Ca²⁺]i of activated neutrophils is attributed partly to the suppression of Ca²⁺ release from the intracellular Ca²⁺ stores through PLC inhibition, and partly to the blockade, especially at higher concentrations, of Ca²⁺ entry from the extracellular space through PLC-independent processes.     

Serum deprivation and re-addition: effects on cyclooxygenase inhibitor sensitivity in cultured glia

A number of drugs were assessed for their ability to inhibit stimulus-evoked prostanoid synthesis in cultured glia. These drugs included non-selective cyclooxygenase (COX) inhibitors and those considered to be selective for the inducible isoform of this enzyme (COX-2). Experiments were carried out on normal cultures and those which had been maintained in serum-free growth medium for four days then re-exposed to serum for a further seven days. All of the drugs tested elicited concentration-dependent inhibitions of arachidonic acid (AA)-stimulated thromboxane B₂ (TXB₂) accumulation in normal cultures with the following rank order of potency: indomethacin > piroxicam > nimesulide = NS398 > ibuprofen ≫ aspirin > paracetamol. In cultures which had been deprived of serum for four days, basal and AA-stimulated TXB₂ production was considerably reduced, as was the amount of COX immunoreactivity determined by Western blotting. Basal and AA-stimulated TXB₂ production together with COX immunoreactivity were restored to control levels by the re-addition of serum to serum-deprived cultures for 7 days. In these cultures, the rank order of potency was: indomethacin > piroxicam ≫ ibuprofen > nimesulide = NS398 ≫ aspirin > paracetamol; however, there were marked charges in the apparent IC₅₀ values for particular drugs. Indomethacin, piroxicam and aspirin were very similar to control, but the potencies of ibuprofen (3-fold), NS398 (30-fold) and nimesulide (40-fold) were found to be decreased when compared to control. Paracetamol, on the other hand, was found to be almost 3-fold more potent under these conditions. Glia appear to express a COX with a novel sensitivity to particular inhibitors following serum deprivation and re-addition.     

On the inhibition of 5-lipoxygenase product formation by tryptanthrin: mechanistic studies and efficacy in vivo

BACKGROUND AND PURPOSE

Leukotrienes (LTs) are pro-inflammatory mediators produced by 5-lipoxygenase (5-LO). Currently available 5-LO inhibitors either lack efficacy or are toxic and novel approaches are required to establish a successful anti-LT therapy. Here we provide a detailed evaluation of the effectiveness of the plant-derived alkaloid tryptanthrin as an inhibitor of LT biosynthesis.

EXPERIMENTAL APPROACH

We analysed LT formation and performed mechanistic studies in human neutrophils stimulated with pathophysiologically relevant stimuli (LPS and formyl peptide), as well as in cell-free assays (neutrophil homogenates or recombinant human 5-LO) and in human whole blood. The in vivo effectiveness of tryptanthrin was evaluated in the rat model of carrageenan-induced pleurisy.

KEY RESULTS

Tryptanthrin potently reduced LT-formation in human neutrophils (IC₅₀ = 0.6 µM). However, tryptanthrin is not a redox-active compound and did not directly interfere with 5-LO activity in cell-free assays. Similarly, tryptanthrin did not inhibit the release of arachidonic acid, the activation of MAPKs, or the increase in [Ca²⁺]_i, but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC₅₀ = 10 µM) and reduced LTB₄ levels in the rat pleurisy model after a single oral dose of 10 mg·kg⁻¹.

CONCLUSIONS AND IMPLICATIONS

Our data reveal that tryptanthrin is a potent natural inhibitor of cellular LT biosynthesis with proven efficacy in whole blood and is effective in vivo after oral administration. Its unique pharmacological profile supports further analysis to exploit its pharmacological potential.     

Augmentation of human neutrophil and alveolar macrophage LTB4 production by N-acetylcysteine: role of hydrogen peroxide

1. The actions of N-acetylcysteine (NAC) on hydrogen peroxide (H₂O₂) and leukotriene B₄ (LTB₄) production by human resting and stimulated peripheral blood neutrophils and alveolar macrophages were investigated.
2. At a concentration of 100 μM, NAC significantly (P<0.01) suppressed the accumulation of H₂O₂ in the incubation medium of resting and opsonized zymosan (OZ; 0.5 mg ml⁻¹)- or N-formylmethionyl-leucyl-phenylalanine (fMLP; 1 μM)-stimulated neutrophils and of resting and OZ-stimulated macrophages. At concentrations of 10 μM and above, NAC augmented significantly the level of LTB₄ in the supernatants of OZ- and fMLP-stimulated neutrophils (P<0.01 and P<0.05, respectively) and OZ-stimulated macrophages (P<0.05 at 10 μM, P<0.01 at 100 μM NAC).
3. NAC (100 μM) caused a significant (P<0.01) reduction in the quantity of measurable H₂O₂ when incubated with exogenous H₂O₂ concentrations equivalent to those released from OZ-stimulated neutrophils and macrophages. At no concentration did NAC affect quantitites of measurable LTB₄ when incubated with exogenous LTB₄.
4. Superoxide dismutase (SOD), which catalyzes the conversion of superoxide anion to H₂O₂ had no significant effect on LTB₄ production by human neutrophils. In contrast, catalase, which catalyzes the conversion of H₂O₂ to H₂O and O₂, caused a pronounced, statistically significant (P<0.01) increase in the levels of LTB₄ measured in the supernatants of OZ- and fMLP-stimulated neutrophils.
5. H₂O₂ (12.5 μM and 25 μM, concentrations equivalent to those measured in the supernatants of activated neutrophils and alveolar macrophages, respectively) caused a small (13%) decrease in the quantity of measurable LTB₄ (P=0.051 and P<0.05 at 12.5 μM and 25 μM, respectively) that was inhibited by NAC (100 μM) but not by catalase (400 u ml⁻¹).
6. In conclusion, the anti-oxidant drug, NAC, increases LTB₄ production by human neutrophils and alveolar macrophages, probably through the elimination of cell-derived H₂O₂. LTB₄ undergoes a H₂O₂-dependent oxidation that is inhibited by NAC but this is unlikely to account fully for the increased levels of LTB₄, suggesting that NAC may increase LTB₄ production by blocking the H₂O₂-dependent inhibition of a synthetic enzyme, such as 5-lipoxygenase.

     

山莨菪碱对大鼠胸水中性白细胞花生四烯酸代谢的影响

本文研究了654-2对大鼠胸水中性白细胞代谢[1-¹⁴C]AA及内源性AA的影响。大鼠白细胞AA经5-LPO代谢途径形成的主要产物为LTB₄及5-HETE,经CO途径的主要产物为HHT及TXB₂。654-2对白细胞代谢[1-¹⁴C]AA无抑制作用,但显著减少白细胞从内源性AA形成的LTB₄,5-HETE,HHT及TXB₂。这种抑制作用与654-2呈剂量依赖关系。本实验结果表明,654-2抑制PG及LT的形成可能是影响了AA从胞膜的释放,而并非直接抑制CO及5-LPO。     

Leukotriene B4 receptor antagonists as therapeutics for inflammatory disease: preclinical and clinical developments

Leukotriene B₄ (LTB₄) is a lipid inflammatory mediator derived from membrane phospholipids by the sequential actions of cytosolic phospholipase A2 (PLA2), 5-lipoxygenase (5-LO) and leukotriene A₄ (LTA₄) hydrolase. Several inflammatory diseases, including asthma, chronic obstructive pulmonary disease, arthritis and inflammatory bowel disease, have been associated with elevated levels of LTB₄. As a result, pharmacological strategies to modulate the synthesis of LTB₄ (inhibition of PLA2, 5-LO or LTA₄ hydrolase) or the effects of LTB₄ itself (antagonism of LTB₄ receptors) are being developed by several companies. Two G-protein-coupled receptors mediate the effects of LTB₄, namely BLT1 and BLT2. The pharmacology, expression and function of these two receptors were last reviewed by Tager and Luster in 2004. Since then, there has been an increased understanding of the function of these receptors, in particular for the lesser understood of the two receptors, BLT2. Furthermore, since last reviewed in 1996, there have been several clinical developments in the use of BLT receptor antagonists for inflammatory diseases. This review summarizes the latest preclinical and clinical developments in BLT antagonism for inflammatory diseases and discusses potential future developments.     

Hypouricaemic action of mangiferin results from metabolite norathyriol via inhibiting xanthine oxidase activity

Context Mangiferin has been reported to possess a potential hypouricaemic effect. However, the pharmacokinetic studies in rats showed that its oral bioavailability was only 1.2%, suggesting that mangiferin metabolites might exert the action.

Objective The hypouricaemic effect and the xanthine oxidase inhibition of mangiferin and norathyriol, a mangiferin metabolite, were investigated. Inhibition of norathyriol analogues (compounds 3–9) toward xanthine oxidase was also evaluated.

Materials and methods For a dose-dependent study, mangiferin (1.5–6.0?mg/kg) and norathyriol (0.92–3.7?mg/kg) were administered intragastrically to mice twice daily for five times. For a time-course study, mice received mangiferin and norathyriol both at a single dose of 7.1?μmol/kg. In vitro, inhibition of test compounds (2.4–2.4?mM) against xanthine oxidase activity was evaluated by the spectrophotometrical method. The inhibition type was identified from Lineweaver–Burk plots.

Results Norathyriol (0.92, 1.85 and 3.7?mg/kg) dose dependently decreased the serum urate levels by 27.0, 33.6 and 37.4%, respectively. The action was more potent than that of mangiferin at the low dose, but was equivalent at the higher doses. Additionally, the hypouricaemic action of them exhibited a time dependence. In vitro, norathyriol markedly inhibited the xanthine oxidase activities, with the IC₅₀ value of 44.6?μM, but mangiferin did not. The kinetic studies showed that norathyriol was an uncompetitive inhibitor by Lineweaver–Burk plots. The structure–activity relationships exhibited that three hydroxyl groups in norathyriol at the C-1, C-3 and C-6 positions were essential for maintaining xanthine oxidase inhibition.

Discussion and conclusion Norathyriol was responsible for the hypouricaemic effect of mangiferin via inhibiting xanthine oxidase activity.     

Differential modulation of microglia superoxide anion and thromboxane B2 generation by the marine manzamines

Background

Thromboxane B₂ (TXB₂) and superoxide anion (O₂ ^-) are neuroinflammatory mediators that appear to be involved in the pathogenesis of several neurodegenerative diseases. Because activated-microglia are the main source of TXB₂ and O₂ ^- in these disorders, modulation of their synthesis has been hypothesized as a potential therapeutic approach for neuroinflammatory disorders. Marine natural products have become a source of novel agents that modulate eicosanoids and O₂ ^- generation from activated murine and human leukocytes. With the exception of manzamine C, all other manzamines tested are characterized by a complex pentacyclic diamine linked to C-1 of the β-carboline moiety. These marine-derived alkaloids have been reported to possess a diverse range of bioactivities including anticancer, immunostimulatory, insecticidal, antibacterial, antimalarial and antituberculosis activities. The purpose of this investigation was to conduct a structure-activity relationship study with manzamines (MZ) A, B, C, D, E and F on agonist-stimulated release of TXB₂ and O₂ ^- from E. coli LPS-activated rat neonatal microglia in vitro.

Results

The manzamines differentially attenuated PMA (phorbol 12-myristate 13-acetate)-stimulated TXB₂ generation in the following order of decreasing potency: MZA (IC₅₀ <0.016 μM) >MZD (IC₅₀ = 0.23 μM) >MZB (IC₅₀ = 1.6 μM) >MZC (IC₅₀ = 2.98 μM) >MZE and F (IC₅₀ >10 μM). In contrast, there was less effect on OPZ (opsonized zymosan)-stimulated TXB₂ generation: MZB (IC₅₀ = 1.44 μM) >MZA (IC₅₀ = 3.16 μM) >MZC (IC₅₀ = 3.34 μM) >MZD, MZE and MZF (IC₅₀ >10 μM). Similarly, PMA-stimulated O₂ ^- generation was affected differentially as follows: MZD (apparent IC₅₀<0.1 μM) >MZA (IC₅₀ = 0.1 μM) >MZB (IC₅₀ = 3.16 μM) >MZC (IC₅₀ = 3.43 μM) >MZE and MZF (IC₅₀ >10 μM). In contrast, OPZ-stimulated O₂ ^- generation was minimally affected: MZB (IC₅₀ = 4.17 μM) >MZC (IC₅₀ = 9.3 μM) >MZA, MZD, MZE and MZF (IC₅₀ > 10 μM). From the structure-activity relationship perspective, contributing factors to the observed differential bioactivity on TXB₂ and O₂ ^- generation are the solubility or ionic forms of MZA and D as well as changes such as saturation or oxidation of the β carboline or 8-membered amine ring. In contrast, the fused 13-membered macrocyclic and isoquinoline ring system, and any substitutions in these rings would not appear to be factors contributing to bioactivity.

Conclusion

To our knowledge, this is the first experimental study that demonstrates that MZA, at in vitro concentrations that are non toxic to E. coli LPS-activated rat neonatal microglia, potently modulates PMA-stimulated TXB₂ and O₂ ^- generation. MZA may thus be a lead candidate for the development of novel therapeutic agents for the modulation of TXB₂ and O₂ ^- release in neuroinflammatory diseases. Marine natural products provide a novel and rich source of chemical diversity that can contribute to the design and development of new and potentially useful anti-inflammatory agents to treat neurodegenerative diseases.     

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B₄ (LTB₄) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB₄ production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK among these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB₄. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB₄, subsequent MMP-9 production and plaque rupture.     

Arachidonic acid — Induced platelet aggregation and prostanoid formation in whole blood in relation to plasma concentration of indomethacin

Summary A single oral dose of indomethacin 1 mg/kg was given to 6 male and 6 female volunteers. The formation of thromboxane B₂ (TXB₂) and 6-ketoprostaglandin F₁ (6-keto-PGF₁) in clotting whole blood was measured by radioimmunoassay, and platelet aggregation induced by archidonic acid (AA) was measured with a plasma aggregometer. The results were related to the concomitant plasma concentration of indomethacin. The maximum plasma concentration ranged between 3.24 and 8.11 µg/ml and the elimination half-life between 4 and 11 h. Formation of the prostanoids was reversibly inhibited, with maximum suppression when the drug concentration in plasma exceeded 0.5–1.0 µg/ml; the IC₅₀ was approximately 0.1 µg/ml. Platelet aggregation was also reversibly inhibited. The correlation between the formation of prostanoids and the different phases of the aggregatory response to exogenous AA is discussed.     

Effects of AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide], a novel inhibitor of human microsomal prostaglandin E synthase-1, on prostanoid biosynthesis in human monocytes in vitro

Inhibitors of microsomal prostaglandin (PG) E synthase-1 (mPGES-1) are being developed for the relief of pain. Redirection of the PGH₂ substrate to other PG synthases, found both in vitro and in vivo, in mPGES-1 knockout mice, may influence their efficacy and safety. We characterized the contribution of mPGES-1 to PGH₂ metabolism in lipopolysaccharide (LPS)-stimulated isolated human monocytes and whole blood by studying the synthesis of prostanoids [PGE₂, thromboxane (TX)B₂, PGF_2α and 6-keto-PGF_1α] and expression of cyclooxygenase (COX)-isozymes and down-stream synthases in the presence of pharmacological inhibition by the novel mPGES-1 inhibitor AF3442 [N-(9-ethyl-9H-carbazol-3-yl)-2-(trifluoromethyl)benzamide]. AF3442 caused a concentration-dependent inhibition of PGE₂ in human recombinant mPGES-1 with an IC₅₀ of 0.06 μM. In LPS-stimulated monocytes, AF3442 caused a concentration-dependent reduction of PGE₂ biosynthesis with an IC₅₀ of 0.41 μM. At 1 μM, AF3442 caused maximal selective inhibitory effect of PGE₂ biosynthesis by 61 ± 3.3% (mean ± SEM, P < 0.01 versus DMSO vehicle) without significantly affecting other prostanoids (i.e. TXB₂, PGF_2α and 6-keto-PGF_1α). In LPS-stimulated whole blood, AF3442 inhibited in a concentration-dependent fashion inducible PGE₂ biosynthesis with an IC₅₀ of 29 μM. A statistically significant inhibition of mPGES-1 activity was detected at 10 and 100 μM (38 ± 14%, P < 0.05, and 69 ± 5%, P < 0.01, respectively). Up to 100 μM, the other prostanoids were not significantly affected. In conclusion, AF3442 is a selective mPGES-1 inhibitor which reduced monocyte PGE₂ generation also in the presence of plasma proteins. Pharmacological inhibition of mPGES-1 did not translate into redirection of PGH₂ metabolism towards other terminal PG synthases in monocytes. The functional relevance of this observation deserves to be investigated in vivo.     

In vivo and in vitro pharmacological activity of Aristolochia tagala (syn: Aristolochia acuminata) root extracts

Context: Aristolochia tagala Cham. (syn: Aristolochia acuminata Lam.) (Aristolochiaceae), known as Nallayishwari in Telugu, has been of interest to researchers because of its traditional uses for treating rheumatic pains and fever.

Objective: The anti-inflammatory activity of the petroleum ether, ethyl acetate, and ethanol extracts of A. tagala roots were investigated for the first time.

Materials and Methods: In vivo and in vitro anti-inflammatory effects were investigated employing the carrageenan-induced hind paw edema in rats and the macrophage cell line RAW264.7 stimulated with proinflammatory stimuli (lipopolysaccharide interferon γ or the calcium ionophore A23187) to determine PGE₂ or LTB₄ release, respectively.

Results: All the extracts exhibited anti-inflammatory effects which were found to be significant (p?<?0.001) at 200 and 400?mg/kg, p.o, in rats tested and the ethyl acetate extract inhibited the induction of PGE₂ with IC₅₀?=?39.1?mg mL^?1 and LTB₄ with IC₅₀?=?29.5?mg mL^?1.

Discussion and conclusion: These findings demonstrate that the A. tagala roots have excellent anti-inflammatory activity and validate the traditional indications of this plant in its origin country.     

Lithium ions inhibit function of lowbut not high-affinity muscarinic receptors of murine neuroblastoma cells (clone N1E-115)

Murine neuroblastoma cells (clone N1E-115) possess both high- and low-affinity muscarinic receptors. The low-affinity muscarinic receptor, when stimulated, initiates the formation of cyclic GMP by activating the enzyme guanylate cyclase; whereas stimulation of the high-affinity receptor inhibits prostaglanding E₁-mediated cyclic AMP formation by inhibiting the enzyme adenylate cyclase. We have reported that lithium ion (Li⁺) inhibits cyclic GMP formation mediated by the muscarinic receptor agonist, carbachol. in a concentration-dependent manner and that neither ammonium nor sodium ions have such an effect. We extended this study to show that Li⁺ was an apparently noncompetitive inhibitor of the low-affinity muscarinic receptor with an IC₅₀ (±SEM)=13.6±0.8 mM. In addition, Li⁺ with a similar IC₅₀ inhibited the cyclic GMP response in intact cells to sodium azide, which is thought to stimulate guanylate cyclase directly. Moreover, though Li⁺ was found to have a slight inhibitory effect on prostaglandin E₁-stimulated cyclic AMP formation (15% inhibition at 10 mM), it had no effect on the function of the high-affinity muscarinic receptor in intact murine neuroblastoma cells.     

Myrtucommulone,a natural acylphloroglucinol,inhibits microsomal prostaglandin E2 synthase-1

Background and purpose:

The selective inhibition of prostaglandin (PG)E₂ formation via interference with microsomal PGE₂ synthase (mPGES)-1 could have advantages in the treatment of PGE₂-associated diseases, such as inflammation, fever and pain, compared with a general suppression of all PG biosynthesis, provided by inhibition of cyclooxygenase (COX)-1 and 2. Here, we addressed whether the naturally occurring acylphloroglucinol myrtucommulone (MC) from Myrtus communis L. (myrtle) affected mPGES-1.

Experimental approach:

The effect of MC on PGE₂ formation was investigated in a cell-free assay by using microsomal preparations of interleukin-1β-stimulated A549 cells as the source of mPGES-1, in intact A549 cells, and in lipopolysaccharide-stimulated human whole blood. Inhibition of COX-1 and COX-2 activity in cellular and cell-free assays was assessed by measuring 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid and 6-oxo PGF_1α formation.

Key results:

MC concentration-dependently inhibited cell-free mPGES-1-mediated conversion of PGH₂ to PGE₂ (IC₅₀ = 1 µmol·L⁻¹). PGE₂ formation was also diminished in intact A549 cells as well as in human whole blood at low micromolar concentrations. Neither COX-2 activity in A549 cells nor isolated human recombinant COX-2 was significantly affected by MC up to 30 µmol·L⁻¹, and only moderate inhibition of cellular or cell-free COX-1 was evident (IC₅₀ > 15 µmol·L⁻¹).

Conclusions and implications:

MC is the first natural product to inhibit mPGES-1 that efficiently suppresses PGE₂ formation without significant inhibition of the COX enzymes. This provides an interesting pharmacological profile suitable for interventions in inflammatory disorders, without the typical side effects of coxibs and non-steroidal anti-inflammatory drugs.