Qijiao Shengbai Capsule alleviated leukopenia by interfering leukotriene pathway: Integrated network study of multi-omics.

BACKGROUND: Leukopenia could be induced by chemotherapy, which leads to bone marrow suppression and even affects the therapeutic progression of cancer. Qijiao Shengbai Capsule (QSC) has been used for the treatment of leukopenia in clinic, but its bioactive components and mechanisms have not yet been elucidated clearly. PURPOSE: This study aimed to elucidate the molecular mechanisms of QSC in treating leukopenia. STUDY DESIGN: Serum pharmacochemistry, multi-omics, network pharmacology, and validation experiment were combined to study the effect of QSC in murine leukopenia model. METHODS: First, UPLC-QTOF-MS was used to clarify the absorbed components of QSC. Then, cyclophosphamide (CTX) was used to induce mice model with leukopenia, and the therapeutic efficacy of QSC was assessed by an integrative approach of multi-omics and network pharmacology strategy. Finally, molecular mechanisms and potential therapeutic targets were identified by validated experiments. RESULTS: 121 compounds absorbed in vivo were identified. QSC significantly increase the count of white blood cells (WBCs) in peripheral blood of leukopenia mice with 15 days treatment. Multi-omics and network pharmacology revealed that leukotriene pathway and MAPK signaling pathway played crucial roles during the treatment of leukopenia with QSC. Six targets (ALOX5, LTB4R, CYSLTR1, FOS, JUN, IL-1ß) and 13 prototype compounds were supposed to be the key targets and potential active components, respectively. The validation experiment further confirmed that QSC could effectively inhibit the inflammatory response induced by leukopenia. The inhibitors of ALOX5 activity can significantly increase the number of WBCs in leukopenia mice. Molecular docking of ALOX5 suggested that calycosin, daidzein, and medicarpin were the potentially active compounds of QSC. CONCLUSION: Leukotriene pathway was found for the first time to be a key role in the development of leukopenia, and ALOX5 was conformed as the potential target. QSC may inhibit the inflammatory response and interfere the leukotriene pathway, it is able to improve hematopoiesis and achieve therapeutic effects in the mice with leukopenia.

Natural chalcones elicit formation of specialized pro-resolving mediators and related 15-lipoxygenase products in human macrophages.

Specialized pro-resolving mediators (SPMs) comprise lipid mediators (LMs) produced from polyunsaturated fatty acids (PUFAs) via stereoselective oxygenation particularly involving 12/15-lipoxygenases (LOXs). In contrast to pro-inflammatory LMs such as leukotrienes formed by 5-LOX and prostaglandins formed by cyclooxygenases, the SPMs have anti-inflammatory and inflammation-resolving properties. Although glucocorticoids and non-steroidal anti-inflammatory drugs (NSAIDs) that block prostaglandin production are still prime therapeutics for inflammation-related diseases despite severe side effects, novel concepts focus on SPMs as immunoresolvents for anti-inflammatory pharmacotherapy. Here, we studied the natural chalcone MF-14 and the corresponding dihydrochalcone MF-15 from Melodorum fruticosum, for modulating the biosynthesis of LM including leukotrienes, prostaglandins, SPM and their 12/15-LOX-derived precursors in human monocyte-derived macrophage (MDM) M1- and M2-like phenotypes. In MDM challenged with Staphylococcus aureus-derived exotoxins both compounds (10 µM) significantly suppressed 5-LOX product formation but increased the biosynthesis of 12/15-LOX products, especially in M2-MDM. Intriguingly, in resting M2-MDM, MF-14 and MF-15 strikingly evoked generation of 12/15-LOX products and of SPMs from liberated PUFAs, along with translocation of 15-LOX-1 to membranous compartments. Enhanced 12/15-LOX product formation by the chalcones was evident also when exogenous PUFAs were supplied, excluding increased substrate supply as sole underlying mechanism. Rather, MF-14 and MF-15 stimulate the activity of 15-LOX-1, supported by experiments with HEK293 cells transfected with either 5-LOX, 15-LOX-1 or 15-LOX-2. Together, the natural chalcone MF-14 and the dihydrochalcone MF-15 favorably modulate LM biosynthesis in human macrophages by suppressing pro-inflammatory leukotrienes but stimulating formation of SPMs by differential interference with 5-LOX and 15-LOX-1.

The Role of Leukotrienes Inhibitors in the Management of Chronic Inflammatory Diseases.

BACKGROUND: Leukotrienes are powerful mediators of inflammation and interact with specific receptors in target cell membrane to initiate an inflammatory response. Thus, Leukotrienes (LTs) are considered to be potent mediators of inflammatory diseases including allergic rhinitis, inflammatory bowel disease and asthma. Leukotriene B4 and the series of cysteinyl leukotrienes (C4, D4, and E4) are metabolites of arachidonic acid metabolism that cause inflammation. The cysteinyl LTs are known to increase vascular permeability, bronco-constriction and mucus secretion. OBJECTIVES: To review the published data for leukotriene inhibitors of plant origin and the recent patents for leukotriene inhibitors, as well as their role in the management of inflammatory diseases. METHODS: Published data for leukotrienes antagonists of plant origin were searched from 1938 to 2019, without language restrictions using relevant keywords in both free text and Medical Subject Headings (MeSH terms) format. Literature and patent searches in the field of leukotriene inhibitors were carried out by using numerous scientific databases including Science Direct, PubMed, MEDLINE, Google Patents, US Patents, US Patent Applications, Abstract of Japan, German Patents, European Patents, WIPO and NAPRALERT. Finally, data from these information resources were analyzed and reported in the present study. RESULTS: Currently, numerous anti-histaminic medicines are available including chloropheneremine, brompheniramine, cetirizine, and clementine. Furthermore, specific leukotriene antagonists from allopathic medicines are also available including zileuton, montelukast, pranlukast and zafirlukast and are considered effective and safe medicines as compared to the first generation medicines. The present study reports leukotrienes antagonistic agents of natural products and certain recent patents that could be an alternative medicine in the management of inflammation in respiratory diseases. CONCLUSION: The present study highlights recent updates on the pharmacology and patents on leukotriene antagonists in the management of inflammation respiratory diseases.

Omega-6 fatty acids and inflammation.

Inflammation is a normal process that is part of host defence and tissue healing. However, excessive or unresolved inflammation can lead to uncontrolled tissue damage, pathology and disease. In humans on a Western diet, the omega-6 polyunsaturated fatty acid arachidonic acid (ARA) makes a significant contribution to the fatty acids present in the membrane phospholipids of cells involved in inflammation. ARA is a precursor to a number of potent pro-inflammatory mediators including well described prostaglandins and leukotrienes, which has led to the development of anti-inflammatory pharmaceuticals that target the ARA pathway to successfully control inflammation. Hence, it is commonly believed that increasing dietary intake of the omega-6 fatty acids ARA or its precursor linoleic acid (LA) will increase inflammation. However, studies in healthy human adults have found that increased intake of ARA or LA does not increase the concentrations of many inflammatory markers. Epidemiological studies have even suggested that ARA and LA may be linked to reduced inflammation. Contrastingly, there is also evidence that a high omega-6 fatty acid diet inhibits the anti-inflammatory and inflammation-resolving effect of the omega-3 fatty acids. Thus, the interaction of omega-3 and omega-6 fatty acids and their lipid mediators in the context of inflammation is complex and still not properly understood.

In vivo modulation of LPS induced leukotrienes generation and oxidative stress by sesame lignans.

The role of inflammation and oxidative stress is critical during onset of metabolic disorders and this has been sufficiently established in literature. In the present study, we evaluated the effects of sesamol and sesamin, two important bioactive molecules present in sesame oil, on the generation of inflammatory and oxidative stress factors in LPS injected rats. Sesamol and sesamin lowered LPS induced expression of cPLA2 (61 and 56%), 5-LOX (44 and 51%), BLT-1(32 and 35%) and LTC4 synthase (49 and 50%), respectively, in liver homogenate. The diminished serum LTB4 (53 and 64%) and LTC4 (67 and 44%) levels in sesamol and sesamin administered groups, respectively, were found to be concurrent with the observed decrease in the expression of cPLA2 and 5-LOX. The serum levels of TNF-α (29 and 19%), MCP-1 (44 and 57%) and IL-1ß (43 and 42%) were found to be reduced in sesamol and sesamin group, respectively, as given in parentheses, compared to LPS group. Sesamol and sesamin offered protection against LPS induced lipid peroxidation in both serum and liver. Sesamol, but not sesamin, significantly restored the loss of catalase and glutathione reductase activity due to LPS (P<.05). However, both sesamol and sesamin reverted SOD activities by 92 and 98%, respectively. Thus, oral supplementation of sesamol and sesamin beneficially modulated the inflammatory and oxidative stress markers, as observed in the present study, in LPS injected rats. Our report further advocates the potential use of sesamol and sesamin as an adjunct therapy wherein, inflammatory and oxidative stress is of major concern.

The effects of trans-fatty acids on TAG regulation in mice depend on dietary unsaturated fatty acids.

The aim of this study was to investigate the effects of trans-fatty acids (TFA) on liver and serum TAG regulation in mice fed diets containing different proportions of n-3, n-6 and n-9 unsaturated fatty acids (UFA) from olive (O), maize (C) or rapeseed (R) oils partially substituted or not with TFA (Ot, Ct and Rt, respectively). Male CF1 mice were fed (30 d) one of these diets. The effects of the partial substitution (1 %, w/w) of different UFA with TFA on the activity and expression of hepatic enzymes involved in lipogenesis and fatty acids oxidation were evaluated, as well as their transcription factor expressions. Some of the mechanisms involved in the serum TAG regulation, hepatic VLDL rich in TAG (VLDL-TAG) secretion rate and lipoprotein lipase (LPL) activity were assessed. In liver, TFA induced an increase in TAG content in the Ot and Rt groups, and this effect was associated with an imbalance between lipogenesis and ß-oxidation. In the Ot group, exacerbated lipogenesis may be one of the mechanisms responsible for the liver steatosis induced by TFA, whereas in Rt it has been related to a decreased ß-oxidation, compared with their respective controls. The enhanced hepatic VLDL-TAG secretion in the Ot and Rt groups was compensated with a differential removal of TAG by LPL enzyme in extrahepatic tissues, leading to unchanged serum TAG levels. In brief, the effects of low levels of TFA on liver and serum TAG regulation in mice depend on the dietary proportions of n-3, n-6 and n-9 UFA.

Leukotriene production profiles and actions in the bovine endometrium during the oestrous cycle.

We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.

Regulation of atherosclerotic plaque inflammation.

The immune reactions that regulate atherosclerotic plaque inflammation involve chemokines, lipid mediators and costimulatory molecules. Chemokines are a family of chemotactic cytokines that mediate immune cell recruitment and control cell homeostasis and activation of different immune cell types and subsets. Chemokine production and activation of chemokine receptors form a positive feedback mechanism to recruit monocytes, neutrophils and lymphocytes into the atherosclerotic plaque. In addition, chemokine signalling affects immune cell mobilization from the bone marrow. Targeting several of the chemokines and/or chemokine receptors reduces experimental atherosclerosis, whereas specific chemokine pathways appear to be involved in plaque regression. Leukotrienes are lipid mediators that are formed locally in atherosclerotic lesions from arachidonic acid. Leukotrienes mediate immune cell recruitment and activation within the plaque as well as smooth muscle cell proliferation and endothelial dysfunction. Antileukotrienes decrease experimental atherosclerosis, and recent observational data suggest beneficial clinical effects of leukotriene receptor antagonism in cardiovascular disease prevention. By contrast, other lipid mediators, such as lipoxins and metabolites of omega-3 fatty acids, have been associated with the resolution of inflammation. Costimulatory molecules play a central role in fine-tuning immunological reactions and mediate crosstalk between innate and adaptive immunity in atherosclerosis. Targeting these interactions is a promising approach for the treatment of atherosclerosis, but immunological side effects are still a concern. In summary, targeting chemokines, leukotriene receptors and costimulatory molecules could represent potential therapeutic strategies to control atherosclerotic plaque inflammation.

Anti-platelet activity of water dispersible curcuminoids in rat platelets.

Curcuminoids are active principle of turmeric with plethora of health beneficial properties. In this study, we have evaluated for the first time the effect of water dispersible curcuminoids on rat platelet aggregation. Curcuminoids (10-30 µg/mL) significantly inhibited platelet aggregation induced by agonists viz., collagen, ADP and arachidonic acid. Curcuminoids were found to be two-fold more potent than curcumin in inhibiting platelet aggregation. Intracellular curcuminoid concentration was relatively higher than curcumin in rat platelets. Curcuminoids significantly attenuated thromboxane A2 , serotonin levels in rat platelets which play an important role in platelet aggregation. Curcuminoid treatment increased nitric oxide (NO) levels in platelets treated with agonists. Curcuminoids inhibited free radicals such as superoxide anion released from activated platelets, which ultimately inhibits platelet aggregation. Further, curcuminoids inhibited 12-lipoxygenase activity and formation of 12-hydroperoxyeicosatetraenoic acid (12-HPETE) in activated rat platelets which regulates platelet aggregation. The results suggest that curcuminoids have remarkable anti-platelet activity by modulating multiple mechanisms involved in platelet aggregation. Thus curcuminoids may have a therapeutic potential to prevent platelet activation related disorders.

The arachidonic acid metabolome serves as a conserved regulator of cholesterol metabolism.

Cholesterol metabolism is closely interrelated with cardiovascular disease in humans. Dietary supplementation with omega-6 polyunsaturated fatty acids including arachidonic acid (AA) was shown to favorably affect plasma LDL-C and HDL-C. However, the underlying mechanisms are poorly understood. By combining data from a GWAS screening in >100,000 individuals of European ancestry, mediator lipidomics, and functional validation studies in mice, we identify the AA metabolome as an important regulator of cholesterol homeostasis. Pharmacological modulation of AA metabolism by aspirin induced hepatic generation of leukotrienes (LTs) and lipoxins (LXs), thereby increasing hepatic expression of the bile salt export pump Abcb11. Induction of Abcb11 translated in enhanced reverse cholesterol transport, one key function of HDL. Further characterization of the bioactive AA-derivatives identified LX mimetics to lower plasma LDL-C. Our results define the AA metabolomeasconserved regulator of cholesterol metabolism, and identify AA derivatives as promising therapeutics to treat cardiovascular disease in humans.

Selenium downregulates oxidative stress-induced activation of leukotriene pathway in experimental rats with diabetic cardiac hypertrophy.

Cardiac hypertrophy has been considered as an important risk factor of morbidity and mortality. It is characterized as thickening of ventricle wall of the heart and consequent reduction in the contracting ability of the heart to pump the blood. Hyperglycemia-induced reactive oxygen species act as major mediators of diabetic complications. Inflammation plays an essential role in the development of diabetic cardiac hypertrophy. Selenium has been shown to induce insulin-like and anti-inflammatory effects in human and experimental animals. But, its mechanism of action has not been elucidated. Hence, in order to probe into its mechanism at molecular level, we designed an experiment to study the effect of selenium as sodium selenite in streptozotocin-induced diabetic rats. The rats were divided into four groups and maintained as follows: (1) controls, (2) sodium selenite-treated controls, (3) diabetic, and (4) sodium selenite-treated diabetic rats. Duration of the experiment was 30 days. Selenium supplementation enhanced the streptozotocin-induced reduction in the activities of antioxidant enzymes, decreased the serum glucose level, glycated hemoglobin content, concentration of high-sensitivity C-reactive protein, levels of lipid peroxidation products, as well as inflammatory parameters. Decrease in the phospholipase activity by selenium supplementation also contributed to the downregulation of leukotriene pathway. It also downregulated the expressions of nuclear transcription factor κB (NFκB), lipoxygenase, cyclooxygenase, 5-lipoxygenase-activating protein, and receptor for leukotriene B4. Hence, selenium decreased the production of reactive oxygen species and inhibited the activation of NFκB-mediated transcription of pro-inflammatory mediators which resulted in the downregulation of leukotriene pathway in diabetic cardiac hypertrophy.

Adjuvant effects of aluminium hydroxide-adsorbed allergens and allergoids - differences in vivo and in vitro.

Allergen-specific immunotherapy (SIT) is a clinically effective therapy for immunoglobulin (Ig)E-mediated allergic diseases. To reduce the risk of IgE-mediated side effects, chemically modified allergoids have been introduced. Furthermore, adsorbance of allergens to aluminium hydroxide (alum) is widely used to enhance the immune response. The mechanisms behind the adjuvant effect of alum are still not completely understood. In the present study we analysed the effects of alum-adsorbed allergens and allergoids on their immunogenicity in vitro and in vivo and their ability to activate basophils of allergic donors. Human monocyte derived dendritic cells (DC) were incubated with native Phleum pratense or Betula verrucosa allergen extract or formaldehyde- or glutaraldehyde-modified allergoids, adsorbed or unadsorbed to alum. After maturation, DC were co-cultivated with autologous CD4(+) T cells. Allergenicity was tested by leukotriene and histamine release of human basophils. Finally, in-vivo immunogenicity was analysed by IgG production of immunized mice. T cell proliferation as well as interleukin (IL)-4, IL-13, IL-10 and interferon (IFN)-γ production were strongly decreased using glutaraldehyde-modified allergoids, but did not differ between alum-adsorbed allergens or allergoids and the corresponding unadsorbed preparations. Glutaraldehyde modification also led to a decreased leukotriene and histamine release compared to native allergens, being further decreased by adsorption to alum. In vivo, immunogenicity was reduced for allergoids which could be partly restored by adsorption to alum. Our results suggest that adsorption of native allergens or modified allergoids to alum had no consistent adjuvant effect but led to a reduced allergenicity in vitro, while we observed an adjuvant effect regarding IgG production in vivo.

Impact of botanical oils on polyunsaturated fatty acid metabolism and leukotriene generation in mild asthmatics.

BACKGROUND: Dietary supplementation with botanical oils that contain n-6 and n-3 eighteen carbon chain (18C)-PUFA such as γ linolenic acid (GLA, 18:3n-6), stearidonic acid (SDA, 18:4n-3) and α linolenic acid (ALA, 18:3n-3) have been shown to impact PUFA metabolism, alter inflammatory processes including arachidonic acid (AA) metabolism and improve inflammatory disorders. METHODS: The diet of mild asthmatics patients was supplemented for three weeks with varying doses of two botanical seed oils (borage oil [Borago officinalis, BO] and echium seed oil [Echium plantagineum; EO]) that contain SDA, ALA and GLA. A three week wash out period followed. The impact of these dietary manipulations was evaluated for several biochemical endpoints, including in vivo PUFA metabolism and ex vivo leukotriene generation from stimulated leukocytes. RESULTS: Supplementation with several EO/BO combinations increased circulating 20-22 carbon (20-22C) PUFAs, including eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), and dihommo-gammalinolenic acid (DGLA), which have been shown to inhibit AA metabolism and inflammation without impacting circulating AA levels. BO/EO combinations also inhibited ex vivo leukotriene generation with some combinations attenuating cysteinyl leukotriene generation in stimulated basophils by >50% and in stimulated neutrophils by >35%. CONCLUSIONS: This study shows that dietary supplementation with BO/EO alters 20-22C PUFA levels and attenuates leukotriene production in a manner consistent with a reduction in inflammation.

Effects of the rhizomes of Atractylodes japonica and atractylenolide I on allergic response and experimental atopic dermatitis.

Although some anti-allergic activities of the rhizome of Atractylodes japonica have been previously reported, the active principle(s) for anti-allergic action is not fully elucidated and the effect of this plant material on atopic dermatitis (AD) is not known. In this study, the 70% ethanol extract of the rhizome of A. japonica was found to significantly inhibit 5-lipoxygenase (5-LOX)-catalyzed leukotrienes (LT) production from rat basophilic leukemia (RBL)-1 cells. From the extract of A. japonica, three major sesquiterpene derivatives including atractylenolide I, atractylenolide III and eudesma-4,7-dien-8-one were successfully isolated. Among these compounds, only atractylenolide I was shown to strongly inhibit 5-LOX from RBL-1 cells (IC(50) = 18.6 µM). To evaluate the effects of experimental AD, the ethanol extract of A. japonica (200 mg/day) was administered orally to hapten-treated NC/Nga mice which is an animal model of AD. It was firstly found that the extract significantly inhibited AD-like symptoms in mice, as judged by severity score and scratching behavior. Taken together, it is concluded that A. japonica possesses the inhibitory activity on 5-LOX and an animal model of AD, and atractylenolide I may contribute, at least in part, to these anti-allergic actions of A. japonica.

Reduced in vitro T-cell responses induced by glutaraldehyde-modified allergen extracts are caused mainly by retarded internalization of dendritic cells.

Although allergen-specific immunotherapy is a clinically effective therapy for IgE-mediated allergic diseases, the risk of IgE-mediated adverse effects still exists. For this reason, chemically modified allergoids have been introduced, which may destroy IgE-binding sites while T-cell activation should be retained. The aim of the study was to analyse the differences between intact allergens and differently modified/aggregated allergoids concerning their internalization as well as T-cell and basophil activation. For this purpose human monocyte-derived immature dendritic cells (DC) were incubated with Phleum pratense or Betula verrucosa pollen extract or with the corresponding allergoids, modified with formaldehyde or glutaraldehyde. After an additional maturation process, the antigen-loaded mature DC were co-cultured with autologous CD4(+) T cells. Allergenicity was tested by leukotriene release from basophils. In addition, the uptake of intact allergens and allergoids by immature DC was analysed. The proliferation of, as well as the interleukin-4 (IL-4), IL-10, IL-13 and interferon-γ production by, CD4(+) T cells which had been stimulated with glutaraldehyde allergoid-treated DC was reduced compared with CD4(+) T cells stimulated with intact allergen-treated or formaldehyde allergoid-treated DC. In line with this, glutaraldehyde-modified allergoids were more aggregated and were internalized more slowly. Furthermore, only the allergoids modified with glutaraldehyde induced a decreased leukotriene release by activated basophils. These findings suggest that IgE-reactive epitopes were destroyed more efficiently by modification with glutaraldehyde than with formaldehyde under the conditions chosen for these investigations. Glutaraldehyde-modified allergoids also displayed lower T-cell stimulatory capacity, which is mainly the result of greater modification/aggregation and diminished uptake by DC.

Anti-inflammatory effects of benfotiamine are mediated through the regulation of the arachidonic acid pathway in macrophages.

Benfotiamine, a lipid-soluble analogue of vitamin B1, is a potent antioxidant that is used as a food supplement for the treatment of diabetic complications. Our recent study (U.C. Yadav et al., Free Radic. Biol. Med. 48:1423-1434, 2010) indicates a novel role for benfotiamine in the prevention of bacterial endotoxin, lipopolysaccharide (LPS)-induced cytotoxicity and inflammatory response in murine macrophages. Nevertheless, it remains unclear how benfotiamine mediates anti-inflammatory effects. In this study, we investigated the anti-inflammatory role of benfotiamine in regulating arachidonic acid (AA) pathway-generated inflammatory lipid mediators in RAW264.7 macrophages. Benfotiamine prevented the LPS-induced activation of cPLA2 and release of AA metabolites such as leukotrienes, prostaglandin E2, thromboxane 2 (TXB2), and prostacyclin (PGI2) in macrophages. Further, LPS-induced expression of AA-metabolizing enzymes such as COX-2, LOX-5, TXB synthase, and PGI2 synthase was significantly blocked by benfotiamine. Furthermore, benfotiamine prevented the LPS-induced phosphorylation of ERK1/2 and expression of transcription factors NF-κB and Egr-1. Benfotiamine also prevented the LPS-induced oxidative stress and protein-HNE adduct formation. Most importantly, compared to specific COX-2 and LOX-5 inhibitors, benfotiamine significantly prevented LPS-induced macrophage death and monocyte adhesion to endothelial cells. Thus, our studies indicate that the dual regulation of the COX and LOX pathways in AA metabolism could be a novel mechanism by which benfotiamine exhibits its potential anti-inflammatory response.

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 (IC(50) = 0.6µM). 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 [Ca(2+) ](i) , but it modified the subcellular localization of 5-LO. Moreover, tryptanthrin potently suppressed LT formation in human whole blood (IC(50) = 10µM) and reduced LTB(4) levels in the rat pleurisy model after a single oral dose of 10mg·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.

Inhibition of 5-lipoxygenase and skin inflammation by the aerial parts of Artemisia capillaris and its constituents.

The aerial parts of Artemisia capillaris Thunberg (Compositae) have been used in Chinese medicine as a liver protective agent, diuretic, and for amelioration of skin inflammatory conditions. This study was conducted to establish the scientific rationale for treating skin inflammation and to find active principles from A. capillaris. To accomplish these goals, the 70% ethanol extract of the aerial parts of A. capillaris (AR) was prepared and its 5-lipoxygenase (5-LOX) inhibitory action was studied since 5-LOX products are known to be involved in several allergic and skin inflammatory disorders. AR showed potent inhibitory activity against 5-LOX-catalyzed leukotriene production by ionophore-induced rat basophilic leukemia-1 cells, with an IC(50) of < 1.0 µg/mL. Nine major compounds, scopoletin, scopolin, scoparone, esculetin, quercetin, capillarisin, isorhamnetin, 3-O-robinobioside, isorhamnetin 3-O-galactoside and chlorogenic acid, were isolated from A. capillaris, and their effects were examined to identify the active principle(s). Several coumarin and flavonoid derivatives were found to be 5-LOX inhibitors. In particular, esculetin and quercetin were potent inhibitors, with IC(50) values of 6.6 and 0.7 µM, respectively. Against arachidonic acid-induced ear edema in mice, AR, and esculetin strongly inhibited edematic response. AR and esculetin also inhibited delayed-type hypersensitivity response in mice. In conclusion, AR and some of their major constituents are 5-LOX inhibitors, and these in vitro and in vivo activities may contribute to the therapeutic potential of AR in skin inflammatory disorders in traditional medicine.

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation.

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.

Potential role of the lipoxygenase derived lipid mediators in atherosclerosis: leukotrienes, lipoxins and resolvins.

Atherogenesis is an inflammatory process with leukocytes infiltrating the arterial intima. The lipoxygenase pathways play a role in leukocyte recruitment through the generation of two classes of arachidonic acid lipid mediators, the leukotrienes and the lipoxins, and one class of omega-3 fatty acid metabolites, the resolvins. There is evidence from animal studies and human genetic studies that the leukotrienes and the enzymes necessary for their generation play a role in atherosclerosis, and possibly even in the development of the vulnerable plaque. Less is known about the effect of the anti-inflammatory lipid mediators in atherosclerosis, the lipoxins and the resolvins. Studies modulating the activity of an enzyme necessary for the production of these lipid mediators, 12/15-lipoxygenase, showed discrepant results in several animal models. Also, human genetic studies have not clearly dissected the effect of the enzyme on atherosclerosis. However, stable forms of the lipoxins and the resolvins protect animals from inflammatory diseases. Whether blocking the leukotrienes or applying anti-inflammatory lipoxins and resolvins will be effective in attenuating human atherosclerosis needs to be demonstrated in future studies. In this review, the biosynthesis of these lipid mediators, their biological effects and the evidence for their possible role in atherosclerosis are discussed with an emphasis on human disease.