Leukotriene Formation Research Articles

Aspergillus fumigatus-derived gliotoxin impacts innate immune cell activation through modulating lipid mediator production in macrophages.

Gliotoxin (GT), a secondary metabolite and virulence factor of the fungal pathogen Aspergillus fumigatus, suppresses innate immunity and supports the suppression of host immune responses. Recently, we revealed that GT blocks the formation of the chemotactic lipid mediator leukotriene (LT)B4 in activated human neutrophils and monocytes, and in rodents in vivo, by directly inhibiting LTA4 hydrolase. Here, we elucidated the impact of GT on LTB4 biosynthesis and the entire lipid mediator networks in human M1- and M2-like monocyte-derived macrophages (MDMs) and in human tissue-resident alveolar macrophages. In activated M1-MDMs with high capacities to generate LTs, the formation of LTB4 was effectively suppressed by GT, connected to attenuated macrophage phagocytic activity as well as human neutrophil movement and migration. In resting macrophages, especially in M1-MDMs, GT elicited strong formation of prostaglandins, while bacterial exotoxins from Staphylococcus aureus evoked a broad spectrum of lipid mediator biosynthesis in both MDM phenotypes. We conclude that GT impairs functions of activated innate immune cells through selective suppression of LTB4 biosynthesis, while GT may also prime the immune system by provoking prostaglandin formation in macrophages.

Phytochemical Constituents and Evaluation of Lipoxygenase Activity of Tetracera Macrophylla Twigs Wall.Ex Hook.f.& Thoms

Lipoxygenase is an enzyme that catalyzes the formation of leukotriene, which is involved in various diseases such as asthma, osteoporosis and atherosclerosis. Inhibition of this enzyme can overcome inflammatory diseases. Tetracera macrophylla Wall.ex Hook. f.& Thoms plant has shown various biological activities, especially as an anti-inflammatory. This study aims to analyze phytochemical constituents and evaluate the lipoxygenase activity of Tetracera macrophylla twig extract. The sample was gradually macerated using n-hexane, ethyl acetate, and methanol. Phytochemical analysis of the extract was carried out using the LC-MS/MS method. Lipoxygenase assay was carried out in vitro using RAW 264.7 macrophage cells induced with lipopolysaccharide using spectrofluorometry. As a comparison, zileuton, which has been established as a 5-LOX inhibitor and quercetin, which has the potential as a 5-LOX inhibitor. The results showed that methanol extract of Tetracera macrophylla twigs had lower specific LOX activity than standard zileuton and quercetin. This plant contains phenol and flavonoid compounds, which can be developed as lipoxygenase inhibitors to prevent inflammation.

Highly potent and selective 5-lipoxygenase inhibition by new, simple heteroaryl-substituted catechols for treatment of inflammation

5-Lipoxygenase (LO) catalyzes the first steps in the formation of pro-inflammatory leukotrienes (LT) that are pivotal lipid mediators contributing to allergic reactions and inflammatory disorders. Based on its key role in LT biosynthesis, 5-LO is an attractive drug target, demanding for effective and selective inhibitors with efficacy in vivo, which however, are still rare. Encouraged by the recent identification of the catechol 4-(3,4-dihydroxyphenyl)dibenzofuran 1 as 5-LO inhibitor, simple structural modifications were made to yield even more effective and selective catechol derivatives. Within this new series, the two most potent compounds 3,4-dihydroxy-3′-phenoxybiphenyl (6b) and 2-(3,4-dihydroxyphenyl)benzo[b]thiophene (6d) potently inhibited human 5-LO in cell-free (IC506b and 6d = 20 nM) and cell-based assays (IC506b = 70 nM, 6d = 60 nM). Inhibition of 5-LO was reversible, unaffected by exogenously added substrate arachidonic acid, and not primarily mediated via radical scavenging and antioxidant activities. Functional 5-LO mutants expressed in HEK293 cells were still prone to inhibition by 6b and 6d, and docking simulations revealed distinct binding of the catechol moiety to 5-LO at an allosteric site. Analysis of 5-LO nuclear membrane translocation and intracellular Ca2+ mobilization revealed that these 5-LO-activating events are hardly affected by the catechols. Importantly, the high inhibitory potency of 6b and 6d was confirmed in human blood and in a murine zymosan-induced peritonitis model in vivo. Our results enclose these novel catechol derivatives as highly potent, novel type inhibitors of 5-LO with high selectivity and with marked effectiveness under pathophysiological conditions.

Inhibitors of Human 5-Lipoxygenase Potently Interfere With Prostaglandin Transport.

5-Lipoxygenase (5-LO) is the key enzyme in the formation of pro-inflammatory leukotrienes (LT) which play an important role in a number of inflammatory diseases. Accordingly, 5-LO inhibitors are frequently used to study the role of 5-LO and LT in models of inflammation and cancer. Interestingly, the therapeutic efficacy of these inhibitors is highly variable. Here we show that the frequently used 5-LO inhibitors AA-861, BWA4C, C06, CJ-13,610 and the FDA approved compound zileuton as well as the pan-LO inhibitor nordihydroguaiaretic acid interfere with prostaglandin E2 (PGE2) release into the supernatants of cytokine-stimulated (TNFα/IL-1β) HeLa cervix carcinoma, A549 lung cancer as well as HCA-7 colon carcinoma cells with similar potencies compared to their LT inhibitory activities (IC50 values ranging from 0.1–9.1 µM). In addition, AA-861, BWA4C, CJ-13,610 and zileuton concentration-dependently inhibited bacterial lipopolysaccharide triggered prostaglandin (PG) release into human whole blood. Western Blot analysis revealed that inhibition of expression of enzymes involved in PG synthesis was not part of the underlying mechanism. Also, liberation of arachidonic acid which is the substrate for PG synthesis as well as PGH2 and PGE2 formation were not impaired by the compounds. However, accumulation of intracellular PGE2 was found in the inhibitor treated HeLa cells suggesting inhibition of PG export as major mechanism. Further, experiments showed that the PG exporter ATP-binding cassette transporter multidrug resistance protein 4 (MRP-4) is targeted by the inhibitors and may be involved in the 5-LO inhibitor-mediated PGE2 inhibition. In conclusion, the pharmacological effects of a number of 5-LO inhibitors are compound-specific and involve the potent inhibition of PGE2 export. Results from experimental models on the role of 5-LO in inflammation and pain using 5-LO inhibitors may be misleading and their use as pharmacological tools in experimental models has to be revisited. In addition, 5-LO inhibitors may serve as new scaffolds for the development of potent prostaglandin export inhibitors.

Ethoxy acetalated dextran-based nanocarriers accomplish efficient inhibition of leukotriene formation by a novel FLAP antagonist in human leukocytes and blood

Leukotrienes are pro-inflammatory lipid mediators generated by 5-lipoxygenase aided by the 5-lipoxygenase-activating protein (FLAP). BRP-201, a novel benzimidazole-based FLAP antagonist, inhibits leukotriene biosynthesis in isolated leukocytes. However, like other FLAP antagonists, BRP-201 fails to effectively suppress leukotriene formation in blood, which limits its therapeutic value. Here, we describe the encapsulation of BRP-201 into poly(lactide-co-glycolide) (PLGA) and ethoxy acetalated dextran (Ace-DEX) nanoparticles (NPs), aiming to overcome these detrimental pharmacokinetic limitations and to enhance the bioactivity of BRP-201. NPs loaded with BRP-201 were produced via nanoprecipitation and the physicochemical properties of the NPs were analyzed in-depth using dynamic light scattering (size, dispersity, degradation), electrophoretic light scattering (effective charge), NP tracking analysis (size, dispersity), scanning electron microscopy (size and morphology), UV–VIS spectroscopy (drug loading), an analytical ultracentrifuge (drug release, degradation kinetics), and Raman spectroscopy (chemical attributes). Biological assays were performed to study cytotoxicity, cellular uptake, and efficiency of BRP-201-loaded NPs versus free BRP-201 to suppress leukotriene formation in primary human leukocytes and whole blood. Both PLGA- and Ace-DEX-based NPs were significantly more efficient to inhibit leukotriene formation in neutrophils versus free drug. Whole blood experiments revealed that encapsulation of BRP-201 into Ace-DEX NPs strongly increases its potency, especially upon pro-longed (≥ 5 h) incubations and upon lipopolysaccharide-challenge of blood. Finally, intravenous injection of BRP-201-loaded NPs significantly suppressed leukotriene levels in blood of mice in vivo. These results reveal the feasibility of our pharmacological approach using a novel FLAP antagonist encapsulated into Ace-DEX-based NPs with improved efficiency in blood to suppress leukotriene biosynthesis.

EFFECT OF ANDROGENS ON IMMUNE CELLS

I recent years, much attention of researchers has been paid to the role of testosterone in the regulation of immune cells and its influence on the course of autoimmune diseases and inflammatory diseases. Better understanding of testosterone-regulated cellular and molecular pathways and the role of androgens in stimulating tissue-specific differences in inflammation susceptibility can lead to the development of innovative treatment strategies for a variety of diseases depending on gender. The aim of this study is to analyze the latest research on the effect of androgens on immunocompetent cells. Sex differences in the functions of the immune system are genetically determined and mediated by the action of male and female sex hormones. Myeloid immune cells express sex hormone receptors and are sensitive to sex hormones. Androgens, including dihydrotestosterone and testosterone, inhibit the activity of immune cells. Effects of testosterone restricts the activity of NK cells, the surface expression of TLR4 on macrophages, the synthesis of proinflammatory products, including TNF-α and nitric oxide synthase, and the formation of leukotriene in neutrophils. Androgens also affect inducing stimuli that regulate the activity of monocyte-macrophages. Testosterone controls complement activation. Androgens have a suppressive effect on the expression of proinflammatory cytokines, reduce the production of prostaglandin E2 in monocytes. The presence of androgen receptors is important factor in regulating neutrophil differentiation. Androgen receptors regulate, first of all, the transition between the proliferation of precursors (myeloblasts, promyelocytes and myelocytes) and the maturation of neutrophils (metamyelocytes, band and segmented neutrophils). The number and activity of cells associated with innate immunity differ between the sexes. Males have a higher natural killer (NK) percentage than females. Phagocytic activity of neutrophils and macrophages in women is higher than in men. Antigen-presenting cells in women are more efficiently presented by peptides than in men. Sex differences are also observed in innate lymphoid cells, which are lymphocytes that regulate a number of tissue immune responses by producing effector cytokines.

Analysis of Boswellic Acid Contents and Related Pharmacological Activities of Frankincense-Based Remedies That Modulate Inflammation.

Extracts of frankincense, the gum resin of Boswellia species, have been extensively used in traditional folk medicine since ancient times and are still of great interest as promising anti-inflammatory remedies in Western countries. Despite their common therapeutic use and the intensive pharmacological research including studies on active ingredients, modes of action, bioavailability, pharmacokinetics, and clinical efficacy, frankincense preparations are available as nutraceuticals but have not yet approved as a drug on the market. A major issue of commercially available frankincense nutraceuticals is the striking differences in their composition and quality, especially related to the content of boswellic acids (BAs) as active ingredients, mainly due to the use of material from divergent Boswellia species but also because of different work-up and extraction procedures. Here, we assessed three frequently used frankincense-based preparations for their BA content and the interference with prominent pro-inflammatory actions and targets that have been proposed, that is, 5-lipoxygenase and leukotriene formation in human neutrophils, microsomal prostaglandin E2 synthase-1, and inflammatory cytokine secretion in human blood monocytes. Our data reveal striking differences in the pharmacological efficiencies of these preparations in inflammation-related bioassays which obviously correlate with the amounts of BAs they contain. In summary, high-quality frankincense extracts display powerful anti-inflammatory effectiveness against multiple targets which can be traced back to BAs as bioactive ingredients.

Anti-inflammatory celastrol promotes a switch from leukotriene biosynthesis to formation of specialized pro-resolving lipid mediators

The pentacyclic triterpenoid quinone methide celastrol (CS) from Tripterygium wilfordii Hook. F. effectively ameliorates inflammation with potential as therapeutics for inflammatory diseases. However, the molecular mechanisms underlying the anti-inflammatory and inflammation-resolving features of CS are incompletely understood. Here we demonstrate that CS potently inhibits the activity of human 5-lipoxygenase (5-LOX), the key enzyme in pro-inflammatory leukotriene (LT) formation, in cell-free assays with IC50 = 0.19–0.49 µM. Employing metabololipidomics using ultra-performance liquid chromatography coupled to tandem mass spectrometry in activated human polymorphonuclear leukocytes or M1 macrophages we found that CS (1 µM) potently suppresses 5-LOX-derived products without impairing the formation of lipid mediators (LM) formed by 12-/15-LOXs as well as fatty acid substrate release. Intriguingly, CS induced the generation of 12-/15-LOX-derived LM including the specialized pro-resolving mediator (SPM) resolvin D5 in human M2 macrophages. Finally, intraperitoneal pre-treatment of mice with 10 mg/kg CS strongly impaired zymosan-induced LT formation and simultaneously elevated the levels of SPM and related 12-/15-LOX-derived LM in peritoneal exudates, spleen and plasma in vivo. Conclusively, CS promotes a switch from LT biosynthesis to formation of SPM which may underlie the anti-inflammatory and inflammation-resolving effects of CS, representing an interesting pharmacological strategy for intervention with inflammatory disorders.

LPS induces ALOX5 promoter activation and 5-lipoxygenase expression in human monocytic cells

5-lipoxygenase (5-LO), coded by the ALOX5 gene, is expressed in leukocytes and catalyzes the formation of leukotrienes, pro-inflammatory lipid mediators. Leukotrienes are central to immune responses, but are also involved in inflammatory disorders and 5-LO expression is associated with leukemia stem cell survival. It is therefore important to understand mechanisms that control 5-LO expression. This study investigated the control of 5-LO expression and leukotriene biosynthesis following the maturation of human monocytic cells. MonoMac-1 (MM1) and THP-1 cells were incubated for up to 72 h with or without LPS and TGF-β. LPS, but not TGF-β, increased CD14 expression in both MM1 and THP-1 cells. Incubation with LPS (100 ng/ml) and TGF-β (1 ng/ml) synergistically increased the capacity of MM1 cells to produce 5-LO products from undetectable levels to 40±5 pmol/106 cells. 5-LO product biosynthesis in THP-1 cells increased 25-fold. A synergistic effect of LPS and TGF-β was measured with increases in 5-LO mRNA of 54- and 13-fold in MM1 and THP-1 cells, respectively. 5-LO protein expression increased significantly in both MM1 and THP-1 cells. ALOX5 promoter activity was significantly elevated >2-fold in both cell lines following LPS treatment, but TGF-β was without effect. The main 5-LO products were cysteinyl-leukotrienes, however LPS and TGF-β did not impact on the capacity of the cells to metabolize leukotriene A4. Overall, this study demonstrates that receptor-mediated stimulation of MM1 and THP-1 cells by LPS is associated with increased 5-LO expression. This represents a new mechanism by which leukotriene biosynthesis can be modulated by pathological agents.

Improved Bioactivity of the Natural Product 5-Lipoxygenase Inhibitor Hyperforin by Encapsulation into Polymeric Nanoparticles.

Hyperforin, a highly hydrophobic prenylated acylphloroglucinol from the medical plant St. John's Wort, possesses anti-inflammatory properties and suppresses the formation of proinflammatory leukotrienes by inhibiting the key enzyme 5-lipoxygenase (5-LO). Despite its strong effectiveness and the unique molecular mode of interference with 5-LO, the high lipophilicity of hyperforin hampers its efficacy in vivo and, thus, impairs its therapeutic value, especially because of poor water solubility and strong plasma (albumin) protein binding. To overcome these hurdles that actually apply to many other hydrophobic 5-LO inhibitors, we have encapsulated hyperforin into nanoparticles (NPs) consisting of acetalated dextran (AcDex) to avoid plasma protein binding and thus improve its cellular supply under physiologically relevant conditions. Encapsulated hyperforin potently suppressed 5-LO activity in human neutrophils, but it failed to interfere with 5-LO activity in a cell-free assay, as expected. In the presence of human serum albumin (HSA), hyperforin was unable to inhibit cellular 5-LO activity, seemingly because of strong albumin binding. However, when encapsulated into NPs, hyperforin caused strong inhibition of 5-LO activity in the presence of HSA. Together, encapsulation of the highly hydrophobic hyperforin as a representative of lipophilic 5-LO inhibitors into AcDex-based NPs allows for efficient inhibition of 5-LO activity in neutrophils in the presence of albumin because of effective uptake and circumvention of plasma protein binding.

Review on the Bioactivities of Quercetin

Quercetin, the most active bioflavonoid which is produced as a secondary metabolite by plants, is a polyphenol with a wide spectrum of bioactivities. This bioflavonoid is the ―nature‘s biological response modifier‖ as it interferes with the various allergens and other reactive compounds. Apple, oranges, tomatoes, onions, black tea and green tea are good sources of quercetin and it is also available commercially. After absorption in the small intestine and colon, quercetin conjugates with glucuronic acid and binds to albumin and passes to liver and benefits the body by its various bioactivities. Quercetin‘s antioxidant activity enhances the radical scavenging activity and metal chelation of the ions but the prooxidant activity depends on its high concentration. Further, quercetin interferes with the formation of leukotrienes from arachidonic acid showing its anti-inflammatory effect. A combined effect of quercetin and bromelain effectively suppresses the allergic reactions and the excessive inflammation resulting from bruising and tissue damage. The mutualistic effect of vitamin C and quercetin protects each other from getting oxidized. A direct relationship was also found to exist between quercetin's antiviral activity and enhancement of cyclic adenosine monophosphate (cAMP), which is a second messenger involved in many biological processes. Quercetin helps in down regulation of mutant gene p53 and inhibits the growth of cancerous cells by putting a check at G1 phase. This also controls the surpassing of the normal regulatory growth by the tumor cells and inhibits the production of heat shock proteins and thus showing its anticancer properties. Owing to the potential pharmaceutical properties of quercetin, the bioactivities, principle uses and mechanisms involved in the treatment of various diseases were reviewed in this paper. In addition, safety issues involved in the partake of quercetin by humans have also been discussed.

ATEROSCLEROSIS AND INFLAMMATION: GENERAL PATHOGENETIC MECHANISMS

According to modern notions, atherosclerosis is a complex multifactorial process in which the mechanisms of development involved endothelial dysfunction, inflammation, dyslipidemia, disorders of the blood coagulation system, and the like. Polyunsaturated fatty acids are a source of biologically active substances that regulate metabolic processes in the body. It is known that during the development of inflammation, biologically active agents are synthesized and accumulated, which both provide a protective function, and play the role of triggers or lead to the development of additional alteration and the formation of pathological systems.
 Purpose of the study. The purpose of the work is to study the functional state of the lipid transport system and to ascertain the role of polyunsaturated fatty acids in the mechanisms of atherosclerosis.
 The analysis of the functional state of the lipid-transport system of 221 patients with diffuse cardiosclerosis with different nosological forms revealed a disturbance of direct and reverse transport of cholesterol, but with varying degrees of orientation. Significant increase in the content of saturated fatty acids by 7.51% and reduction of unsaturated.
 Reducing the level of polyunsaturated fatty acids is a consequence of increased formation of prostaglandins and leukotrienes in inflammatory processes of the vascular wall, and indicates a disbalance in the regulation of lipid homeostasis. The deficiency in polyunsaturated fatty acid cells for many years models the high potential of endothelial dysfunction, which can be a pathogenetic mechanism not only for atherosclerotic lesions, but also for inflammation of the vessels.
 Conclusion. The recognition of atherogenesis as an active process rather than a cholesterol storage disease has highlighted some key inflammatory mechanisms. In our opinion, the local violation of the transport of lipids in the tissue is at the heart of the atherosclerotic lesion of the vascular wall, which develops irrespective of inflammation in the vessel wall, but substantially potentiates them due to the action of inflammatory mediators.

ATEROSCLEROSIS AND INFLAMMATION: GENERAL PATHOGENETIC MECHANISMS

According to modern notions, atherosclerosis is a complex multifactorial process in which the mechanisms of development involved endothelial dysfunction, inflammation, dyslipidemia, disorders of the blood coagulation system, and the like. Polyunsaturated fatty acids are a source of biologically active substances that regulate metabolic processes in the body. It is known that during the development of inflammation, biologically active agents are synthesized and accumulated, which both provide a protective function, and play the role of triggers or lead to the development of additional alteration and the formation of pathological systems.
 Purpose of the study. The purpose of the work is to study the functional state of the lipid transport system and to ascertain the role of polyunsaturated fatty acids in the mechanisms of atherosclerosis.
 The analysis of the functional state of the lipid-transport system of 221 patients with diffuse cardiosclerosis with different nosological forms revealed a disturbance of direct and reverse transport of cholesterol, but with varying degrees of orientation. Significant increase in the content of saturated fatty acids by 7.51% and reduction of unsaturated.
 Reducing the level of polyunsaturated fatty acids is a consequence of increased formation of prostaglandins and leukotrienes in inflammatory processes of the vascular wall, and indicates a disbalance in the regulation of lipid homeostasis. The deficiency in polyunsaturated fatty acid cells for many years models the high potential of endothelial dysfunction, which can be a pathogenetic mechanism not only for atherosclerotic lesions, but also for inflammation of the vessels.
 Conclusion. The recognition of atherogenesis as an active process rather than a cholesterol storage disease has highlighted some key inflammatory mechanisms. In our opinion, the local violation of the transport of lipids in the tissue is at the heart of the atherosclerotic lesion of the vascular wall, which develops irrespective of inflammation in the vessel wall, but substantially potentiates them due to the action of inflammatory mediators.

Green leaf volatile-burst in Arabidopsis is governed by galactolipid oxygenation by a lipoxygenase that is under control of calcium ion

Plants form green leaf volatiles (GLVs) almost instantly after tissue disruption caused by damages, such as herbivore damage. This rapid formation of GLVs, namely GLV-burst, is an essential factor for the plants’ GLV-dependent direct and indirect defenses. However, mechanism of GLV-burst remains unknown. We observed that the formation of monogalactosyldiacylglycerol hydroperoxides (MGDG-OOHs) by Arabidopsis lipoxygenase 2 (AtLOX2) governs GLV-burst in Arabidopsis. Addition of a Ca2+ selective chelating reagent, BAPTA, during tissue disruption effectively suppressed the formation of MGDG-OOHs as well as GLV-burst. This suppression was relieved by the addition of Ca2+. Therefore, we propose that Ca2+-dependent activation of AtLOX2 facilitates GLV-burst formation as observed in leukotriene formation, which is regulated by Ca2+-dependent activation of LOXs in animal cells.

Cyclooxygenase pathway mediates the inhibition of Na-glutamine co-transporter B0AT1 in rabbit villus cells during chronic intestinal inflammation.

In the mammalian intestine, glutamine assimilation by the absorptive villus cells is mediated by Na-glutamine co-transport, specifically by B0AT1. In a rabbit model of chronic intestinal inflammation, B0AT1 is inhibited secondary to a decrease in the number of co-transporters in the brush border membrane (BBM). This inhibition can be reversed by treatment with a broad-spectrum immune modulator such as glucocorticoid suggesting that immune inflammatory mediators may regulate B0AT1 during chronic intestinal inflammation. Arachidonic acid (AA) metabolites (AAM) are increased during chronic intestinal inflammation. However, whether AAM may regulate B0AT1 during chronic intestinal inflammation is unknown. Treatment of rabbits with ATK, to prevent the release of AAM reversed the inhibition of B0AT1. AAM are products of cyclooxygenase (COX) and/or lipoxygenase (LOX) pathways. Inhibition of COX with piroxicam, therefore reduction of prostaglandin formation in the chronically inflamed intestine, reversed the inhibition of B0AT1 to its normal levels. In contrast, inhibition of LOX with MK886, thus reduction of leukotriene formation during chronic enteritis, did not affect the inhibition of B0AT1. Kinetic studies showed that the mechanism of restoration of B0AT1 by ATK or piroxicam was secondary to the restoration of BBM co-transporter numbers. Western Blot analysis also demonstrated restoration of BBM B0AT1 co-transporter numbers. In conclusion, this study demonstrates that Na-glutamine co-transport mediated by B0AT1 in villus cells is regulated by prostaglandins rather than leukotrienes in the chronically inflamed intestine.

Antileukotriene therapy by reducing tau phosphorylation improves synaptic integrity and cognition of P301S transgenic mice.

SummaryThe 5‐lipoxygenase (5LO) is a source of inflammatory leukotrienes and is upregulated in Alzheimer's disease and related tauopathies. However, whether it directly modulates tau phosphorylation and the development of its typical neuropathology in the absence of Aβ or is a secondary event during the course of the disease pathogenesis remains to be fully elucidated. The goal of this study was to evaluate the effect that pharmacologic blockade of this inflammatory pathway has on the phenotype of a transgenic mouse model of tauopathy, the P301S mice. Starting at 3 months of age, P301S mice were randomized to receive zileuton, a specific 5LO blocker, for 7 months; then, its effect on their behavioral deficits and neuropathology was assessed. Inhibition of leukotrienes formation was associated with a reduction in tau phosphorylation and an amelioration of memory and learning as well as synaptic integrity, which were secondary to a downregulation of the cdk5 kinase pathway. Our results demonstrate that the 5LO enzyme is a key player in modulating tau phosphorylation and pathology and that blockade of its enzymatic activity represents a desirable disease‐modifying therapeutic approach for tauopathy.

A tiered approach to investigate the mechanism of anti-inflammatory activity of an herbal medicinal product containing a fixed combination of thyme herb and primula root extracts

BackgroundThe herbal medicinal product Bronchipret® TP film-coated tablets contains a fixed combination of thyme and primula dry extracts (BRO) and has long and successfully been used for the treatment of acute bronchitis. However, the underlying pharmacological mechanisms of action have not been determined so far. We report a tiered approach applying in vivo and in vitro studies to investigate the pharmacodynamic activity and underlying mechanisms of action, and to identify possible active ingredients contributing to the product’s pharmacodynamic activity.ResultsIn an LPS-induced rat model of bronchoalveolitis oral administration of BRO effectively ameliorated the influx of leukocytes into the lung. This was accompanied by reduced levels of leukotriene (LT) B4, cysteinyl-LTs (cysLT), and prostaglandin (PG) E2. We also found that BRO potently reduced the production of LTB4 in vitro in rat whole blood stimulated with the Ca2+-ionophore A23187 whereas the effect on PGE2 was much less pronounced. The transferability of these findings to human cells was assessed by measuring the effects of BRO on A23187-stimulated human monocytes and neutrophils. BRO and thyme extract were potent inhibitors of LTB4 and cysLT production. We further investigated the effects of BRO, thyme extract as well as single compounds of thyme extract on enzymatic activity of 5-lipoxygenase (5-LO). 5-LO activity was potently reduced by BRO and thyme extract. Of the single ingredients, thymoquinone and rosmarinic acid showed most potent inhibitory activity against 5-LO, with lower potency for thymol and carvacrol.ConclusionBRO attenuates inflammation-induced leukotriene formation in vivo and affects leukotriene biosynthesis in vitro via 5-LO inhibition. This inhibitory activity appears to be primarily related to the thyme extract component. However, none of the single ingredients tested seems to fully account for the activity of thyme extract alone. Rather, the interaction of different compounds seems to be required for its overall inhibitory effect on leukotriene production.

Comparative evaluation of the efficacy of topical amlexanox 5% oral paste and triamcinolone acetonide 0.1% oral paste in the treatment of Recurrent Aphthous Stomatitis (RAS)

Background: A double-blind randomized control trial was conducted to compare the clinical efficacy of topical triamcinolone acetonide 0.1% and amlexanox 5% in recurrent apthous stomatitis (minor). Materials and Methods: Forty adult patients were assessed for erythema, ulcer size, pain scores, and ulcer healing on three evaluation visits within 24 h of ulcer formation, third day, and fifth day. All participants were monitored for compliance with treatment protocols of four times daily application of calibrated quantity of medication. Results and Discussion: Ninety percent of controls when compared with 65% of the participants in the amlexanox group showed complete improvement of peri-ulcer erythema. Pain reduction was marked from the first to the fifth day (F = 15.249, P = 0.000). The control participants showed 90% reduction in pain in comparison to 70% in the amlexanox group. The mean ulcer size was shown to reduce from the first to fifth day in both groups (F = 18.611, P = 0.000). Completely healed ulcers were seen in 70% of the participants in the control group compared with 75% of the participants in the amlexanox group. Conclusion: The data suggest that the clinical efficacy of topical amlexanox 5% is comparable to topical 0.1% triamcinolone acetonide and may be considered a substitute except in pain control, which was marginally less for the treatment for recurrent apthous stomatitis (minor).

Unique regulation of Na-glutamine cotransporter SN2/SNAT5 in rabbit intestinal crypt cells during chronic enteritis.

The only Na‐nutrient cotransporter described in mammalian small intestinal crypt cells is SN2/SNAT5, which facilitates glutamine uptake. In a rabbit model of chronic intestinal inflammation, SN2 stimulation is secondary to an increase in affinity of the cotransporter for glutamine. However, the immune regulation of SN2 in the crypt cells during chronic intestinal inflammation is unknown. We sought to determine the mechanism of regulation of Na‐nutrient cotransporter SN2 by arachidonic acid metabolites in crypt cells. The small intestines of New Zealand white male rabbits were inflamed via inoculation with Eimeria magna oocytes. After 2‐week incubation, control and inflamed rabbits were subjected to intramuscular injections of arachidonyl trifluoromethyl ketone (ATK), piroxicam and MK886 for 48 hrs. After injections, the rabbits were euthanized and crypt cells from small intestines were harvested and used. Results: Treatment of rabbits with ATK prevented the release of AA and reversed stimulation of SN2. Inhibition of cyclooxygenase (COX) with piroxicam did not affect stimulation of SN2. However, inhibition of lipoxygenase (LOX) with MK886, thus reducing leukotriene formation during chronic enteritis, reversed the stimulation of SN2. Kinetic studies showed that the mechanism of restoration of SN2 by ATK or MK886 was secondary to the restoration of the affinity of the cotransporter for glutamine. For all treatment conditions, Western blot analysis revealed no change in SN2 protein levels. COX inhibition proved ineffective at reversing the stimulation of SN2. Thus, this study provides evidence that SN2 stimulation in crypt cells is mediated by the leukotriene pathway during chronic intestinal inflammation.

Beneficial effect of n-3 polyunsaturated fatty acids on inflammation and analgesic use in psoriatic arthritis: a randomized, double blind, placebo-controlled trial

Objective: This study aimed to investigate the effects of marine n-3 polyunsaturated fatty acids (PUFAs) on disease activity, use of analgesics, and inflammatory biomarkers in patients with psoriatic arthritis (PsA).Method: Patients with established PsA (n = 145) were investigated in a randomized, double-blind, placebo-controlled study. The participants received a supplement of 3 g n-3 PUFA/day or 3 g olive oil/day (control) for 24 weeks. Outcome measures for disease activity, use of analgesics, and leukotriene formation from activated granulocytes were assessed at baseline and at study end.Results: In total, 145 patients were included and 133 completed the study. After 24 weeks, the n-3 PUFA group showed a decrease in Disease Activity Score (DAS28-CRP), 68 tender joint count, enthesitis score, and psoriasis area and severity index, although not significantly different from the controls. There was a significant reduction in non-steroidal anti-inflammatory drug (NSAID) and paracetamol use compared with controls (p = 0.04). In addition, the participants in the n-3 PUFA group had significantly lower formation of leukotriene B4 (p = 0.004) from stimulated granulocytes and significantly higher formation of leukotriene B5 (p < 0.001) compared with controls.Conclusion: The n-3 PUFA-supplemented group showed improvement in outcome measures for disease activity, although the difference between the groups was not statistically significant. However, use of NSAIDs and paracetamol was significantly reduced in the n-3 PUFA group compared to the control group. Finally, there was a significant decrease in leukotriene B4 formation in the n-3 PUFA group compared with controls.