Multiomics integrated analysis and experimental validation identify TLR4 and ALOX5 as oxidative stress-related biomarkers in intracranial aneurysms.

BACKGROUND: Intracranial aneurysm (IA) is a severe cerebrovascular disease, and effective gene therapy and drug interventions for its treatment are still lacking. Oxidative stress (OS) is closely associated with the IA, but the key regulatory genes involved are still unclear. Through multiomics analysis and experimental validation, we identified two diagnostic markers for IA associated with OS. METHODS: In this study, we first analyzed the IA dataset GSE75436 and conducted a joint analysis of oxidative stress-related genes (ORGs). Differential analysis, functional enrichment analysis, immune infiltration, WGCNA, PPI, LASSO, and other methods were used to identify IA diagnostic markers related to OS. Next, the functions of TLR4 and ALOX5 expression in IA and their potential targeted therapeutic drugs were analyzed. We also performed single-cell sequencing of patient IA and control (superficial temporal artery, STA) tissues. 23,342 cells were captured from 2 IA and 3 STA samples obtained from our center. Cell clustering and annotation were conducted using R software to observe the distribution of TLR4 and ALOX5 expression in IAs. Finally, the expression of TLR4 and ALOX5 were validated in IA patients and in an elastase-induced mouse IA model using experiments such as WB and immunofluorescence. RESULTS: Through bioinformatics analysis, we identified 16 key ORGs associated with IA pathogenesis. Further screening revealed that ALOX5 and TLR4 were highly expressed to activate a series of inflammatory responses and reduce the production of myocytes. Methotrexate (MTX) may be a potential targeted drug. Single-cell analysis revealed a notable increase in immune cells in the IA group, with ALOX5 and TLR4 primarily localized to monocytes/macrophages. Validation through patient samples and mouse models confirmed high expression of ALOX5 and TLR4 in IAs. CONCLUSIONS: Bioinformatics analysis indicated that ALOX5 and TLR4 are the most significant ORGs associated with the pathogenesis of IA. Single-cell sequencing and experiments revealed that the high expression of ALOX5 and TLR4 are closely related to IA. These two genes are promising new targets for IA therapy.

Structure-Activity Relationship Studies of New Sinapic Acid Phenethyl Ester Analogues Targeting the Biosynthesis of 5-Lipoxygenase Products: The Role of Phenolic Moiety, Ester Function, and Bioisosterism.

Sinapic acid is found in many edible plants and fruits, such as rapeseed, where it is the predominant phenolic compound. New sinapic acid phenethyl ester (SAPE) analogues were synthesized and screened as inhibitors of the biosynthesis of 5-lipoxygenase (5-LO) in stimulated HEK293 cells and polymorphonuclear leukocytes (PMNL). Inhibition of leukotriene biosynthesis catalyzed by 5-LO is a validated therapeutic strategy against certain inflammatory diseases and allergies. Unfortunately, the only inhibitor approved to date has limited clinical use because of its poor pharmacokinetic profile and liver toxicity. With the new analogues synthesized in this study, the role of the phenolic moiety, ester function, and bioisosterism was investigated. Several of the 34 compounds inhibited the biosynthesis of 5-LO products, and 20 compounds were 2-11 times more potent than zileuton in PMNL, which are important producers of 5-LO products. Compounds 5i (IC50: 0.20 µM), 5l (IC50: 0.20 µM), and 5o (IC50: 0.21 µM) bearing 4-trifluoromethyl, methyl, or methoxy substituent at meta-position of the phenethyl moiety were 1.5 and 11.5 times more potent than SAPE (IC50: 0.30 µM) and zileuton (IC50: 2.31 µM), respectively. Additionally, compound 9 (IC50: 0.27 µM), which was obtained after acetylation of the 4-hydroxyl of SAPE, was equivalent to SAPE and 8 times more active than zileuton. Furthermore, compound 20b (IC50: 0.27 µM) obtained after the bioisosteric replacement of the ester function of SAPE by the 1,2,4-oxadiazole heterocycle was equivalent to SAPE and 8 times more active than zileuton. Thus, this study provides a basis for the rational design of new molecules that could be developed further as anti 5-LO therapeutics.

Lipoxygenase Inhibition by Plant Extracts.

Lipoxygenases are widespread enzymes that catalyze oxidation of polyunsaturated fatty acids (linoleic, linolenic, and arachidonic acid) to produce hydroperoxides. Lipoxygenase reactions can be desirable, but also lipoxygenases can react in undesirable ways. Most of the products of lipoxygenase reactions are aromatic compounds that can affect food properties, especially during long-term storage. Lipoxygenase action on unsaturated fatty acids could result in off-flavor/off-odor development, causing food spoilage. In addition, lipoxygenases are present in the human body and play an important role in stimulation of inflammatory reactions. Inflammation is linked to many diseases, such as cancer, stroke, and cardiovascular and neurodegenerative diseases. This review summarized recent research on plant families and species that can inhibit lipoxygenase activity.

Microglia depletion diminishes key elements of the leukotriene pathway in the brain of Alzheimer's Disease mice.

Leukotrienes (LTs) contribute to the neuropathology of chronic neurodegenerative disorders including Alzheimer's Disease (AD), where they mediate neuroinflammation and neuronal cell-death. In consequence, blocking the action of Leukotrienes (LTs) ameliorates pathologies and improves cognitive function in animal models of neurodegeneration. Surprisingly, the source of Leukotrienes (LTs) in the brain is largely unknown. Here, we identified the Leukotriene (LT) synthesis rate-limiting enzyme 5-Lipoxygenase (5-Lox) primarily in neurons and to a lesser extent in a subpopulation of microglia in human Alzheimer´s Disease (AD) hippocampus brain sections and in brains of APP Swedish PS1 dE9 (APP-PS1) mice, a transgenic model for Alzheimer´s Disease (AD) pathology. The 5-Lipoxygenase (5-Lox) activating protein (FLAP), which anchors 5-Lipoxygenase (5-Lox) to the membrane and mediates the contact to the substrate arachidonic acid, was confined exclusively to microglia with the entire microglia population expressing 5-Lipoxygenase activating protein (FLAP). To define the contribution of microglia in the Leukotriene (LT) biosynthesis pathway, we ablated microglia using the colony stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 in wildtype (WT) and APP-PS1 mice. Microglia ablation not only diminished the expression of FLAP and of the Leukotriene (LT) receptor Cysteinylleukotriene receptor 1 (CysLTR1), as expected based on their microglia cell type-specific expression, but also drastically reduced 5-Lipoxygenase (5-Lox) mRNA expression in the brain and its protein expression in neurons, in particular in wildtype (WT) mice. In conclusion i) microglia are key in Leukotriene (LT) biosynthesis, and ii) they regulate neuronal 5-Lipoxygenase (5-Lox) expression implying a yet unknown signaling mechanism between neurons and microglia.

Discovery of a novel 2,5-dihydroxycinnamic acid-based 5-lipoxygenase inhibitor that induces apoptosis and may impair autophagic flux in RCC4 renal cancer cells.

The inhibition of 5-lipoxygenase (5-LO), the key enzyme for the biosynthesis of leukotrienes (LTs), has generated increasing enthusiasm as anti-inflammatory and antitumor strategies in recent years. Based on our previous studies, we synthesized a series of dihydroxycinnamic acid-based analogs that might be 5-LO inhibitors. LTs biosynthesis inhibition in HEK293 cells and polymorphonuclear leukocytes (PMNL) was measured and antitumor activities were investigated in Renal Cell Carcinoma (RCC). Results showed that the 2,5-dihydroxycinnamic acid phenethyl ester (10b) was the best 5-LO inhibitor and was 7-fold more potent than Zileuton (1), the only clinically approved 5-LO inhibitor. 2,5-Dihydroxy substitution was more favorable to 5-LO inhibition since compound 10b is twice as active as CAPE (2) which is a 3,4-dihydroxylcinnamic acid ester. Meanwhile, 10b reduced the cell viability of renal cancer cells  and was more selective toward RCC4 and 786.0 cells which are deficient for the Von Hippel-Lindau (VHL) tumor suppressor gene. As to the underlying cell-death mechanisms, 10b induced apoptosis in VHL-deficient RCC4 cells. Also, increases in LC3B and p62 expression suggest a blockage of the autophagic flux in RCC in response to 10b.

Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

Sphingosine-1-phosphate (S1P) is involved in the regulation of important cellular processes, including immune-cell trafficking and proliferation. Altered S1P signaling is strongly associated with inflammation, cancer progression, and atherosclerosis; however, the mechanisms underlying its pathophysiologic effects are only partially understood. This study evaluated the effects of S1P in vitro and in vivo on the biosynthesis of leukotrienes (LTs), which form a class of lipid mediators involved in the pathogenesis of inflammatory diseases. Here, we report for the first time that S1P potently suppresses LT biosynthesis in Ca2+-ionophore-stimulated intact human neutrophils. S1P treatment resulted in intracellular Ca2+ mobilization, perinuclear translocation, and finally irreversible suicide inactivation of the LT biosynthesis key enzyme 5-lipoxygenase (5-LO). Agonist studies and S1P receptor mRNA expression analysis provided evidence for a S1P receptor 4-mediated effect, which was confirmed by a functional knockout of S1P4 in HL60 cells. Systemic administration of S1P in wild-type mice decreased both macrophage and neutrophil migration in the lungs in response to LPS and significantly attenuated 5-LO product formation, whereas these effects were abrogated in 5-LO or S1P4 knockout mice. In summary, targeting the 5-LO pathway is an important mechanism to explain S1P-mediated pathophysiologic effects. Furthermore, agonism at S1P4 represents a novel effective strategy in pharmacotherapy of inflammation.-Fettel, J., Kühn, B., Guillen, N. A., Sürün, D., Peters, M., Bauer, R., Angioni, C., Geisslinger, G., Schnütgen, F., Meyer zu Heringdorf, D., Werz, O., Meybohm, P., Zacharowski, K., Steinhilber, D., Roos, J., Maier, T. J. Sphingosine-1-phosphate (S1P) induces potent anti-inflammatory effects in vitro and in vivo by S1P receptor 4-mediated suppression of 5-lipoxygenase activity.

TGFß/SMAD signalling modulates MLL and MLL-AF4 mediated 5-lipoxygenase promoter activation.

5-Lipoxygenase (5-LO) catalyzes the initial two steps of the conversion of arachidonic acid to leukotrienes which represent a group of pro-inflammatory lipid mediators involved in immune defense reactions as well as inflammation, allergy and cancer. Transforming growth factor-ß (TGFß) and calcitriol strongly upregulate 5-LO expression during myeloid cell differentiation and MLL-AF4 has been shown to strongly activate the 5-LO promoter. Here, we investigated the role of TGFß/SMAD signalling in 5-LO promoter activation. We identified two functional SMAD binding elements in the proximal part of the 5-LO promoter which significantly induce 5-LO promoter activity via TGFß and SMAD3/4. Since aberrant 5-LO gene expression has been linked with mixed lineage leukemia (MLL) which is characterized by the presence of MLL fusion proteins (e.g. MLL-AF4), we also investigated the influence of TGFß/SMADs on MLL- and MLL-AF4-mediated 5-LO promoter activation. Our data show that induction of 5-LO promoter activity by SMAD3/4 is MLL-dependent and that knockdown of the MLL complex component MEN1 attenuates the SMAD effect. Our data suggest that induction of 5-LO gene expression by TGFß is at least in part due to stimulation of transcript initiation.

A Novel Inhibitor of 5-Lipoxygenase (5-LOX) Prevents Oxidative Stress-Induced Cell Death of Retinal Pigment Epithelium (RPE) Cells.

PURPOSE: 5-Lipoxygenase (5-LOX) oxygenates arachidonic acid to form 5-hydroperoxyeicosatetraenoic acid, which is further converted into biologically detrimental leukotrienes, such as leukotriene B4 (LTB4). The RPE and retina express the PNPLA2 gene for pigment epithelium-derived factor receptor (PEDF-R), a lipase involved in cell survival. The purpose here was to investigate the role of PEDF-R on the 5-LOX pathway in oxidative stress of RPE. METHODS: Lipoxygenase activity assays were performed with soybean and potato lipoxygenase. Binding was evaluated by peptide-affinity chromatography and pull-down assays with PEDF-R-derived synthetic peptides or recombinant protein. Oxidative stress was induced in human ARPE-19 and primary pig RPE cells with indicated concentrations of H2O2/TNF-α. Reverse transcription-PCR of ALOX5 and PNPLA2 genes was performed. Cell viability and death rates were determined using respective biomarkers. Leukotriene B4 levels were measured by ELISA. RESULTS: Among five peptides spanning between positions Leu159 and Met325 of human PEDF-R polypeptide, only two overlapping peptides, E5b and P1, bound and inhibited lipoxygenase activity. Human recombinant 5-LOX bound specifically to peptide P1 and to His6/Xpress-tagged PEDF-R via ionic interactions. The two inhibitor peptides E5b and P1 promoted cell viability and decreased cell death of RPE cells undergoing oxidative stress. Oxidative stress decreased the levels of PNPLA2 transcripts with no effect on ALOX5 expression. Exogenous additions of P1 peptide or overexpression of the PNPLA2 gene decreased both LTB4 levels and death of RPE cells undergoing oxidative stress. CONCLUSIONS: A novel peptide region of PEDF-R inhibits 5-LOX, which intersects with RPE cell death pathways induced by oxidative stress.

Predictive Bioinformatic Assignment of Methyl-Bearing Stereocenters, Total Synthesis, and an Additional Molecular Target of Ajudazol B.

Full details on the evaluation and application of an easily feasible and generally useful method for configurational assignments of isolated methyl-bearing stereocenters are reported. The analytical tool relies on a bioinformatic gene cluster analysis and utilizes a predictive enoylreductase alignment, and its feasibility was demonstrated by the full stereochemical determination of the ajudazols, highly potent inhibitors of the mitochondrial respiratory chain. Furthermore, a full account of our strategies and tactics that culminated in the total synthesis of ajudazol B, the most potent and least abundant of these structurally unique class of myxobacterial natural products, is presented. Key features include an application of an asymmetric ortholithiation strategy for synthesis of the characteristic anti-configured hydroxyisochromanone core bearing three contiguous stereocenters, a modular oxazole formation, a flexible cross-metathesis approach for terminal allyl amide synthesis, and a late-stage Z,Z-selective Suzuki coupling. This total synthesis unambiguously proves the correct stereochemistry, which was further corroborated by comparison with reisolated natural material. Finally, 5-lipoxygenase was discovered as an additional molecular target of ajudazol B. Activities against this clinically validated key enzyme of the biosynthesis of proinflammatory leukotrienes were in the range of the approved drug zileuton, which further underlines the biological importance of this unique natural product.

Dual inhibition of COX-2/5-LOX blocks colon cancer proliferation, migration and invasion in vitro.

Inflammation is emerging as a new hallmark of cancer. Arachidonic acid (AA) metabolism, the family of cyclooxygenases (COXs) and lipoxygenase (LOX) play important roles in AA-related inflammatory cascades. In 94 colorectal cancer samples collected from the Han population, the immunohistochemical results indicated that 68% of the patients with colorectal cancer had a co-expression of both COX-2 and 5-LOX, while both displayed low expression in the matched normal tissues. In cell lines, three colorectal cancer cell lines exhibited high expression of COX-2 and 5-LOX. During stable silencing of the expression of COX-2 or 5-LOX in LoVo cancer cells, we found that downregulation of either COX-2 or 5-LOX significantly diminished the growth, migration and invasion of the colon cancer cells and specifically, downregulation of COX-2 could elicit upregulation of 5-LOX protein and vice versa. The above results suggested that the simultaneous blocking of COX-2 and 5-LOX activity may bring more potential benefits in managing the progression of colon cancer. Therefore, we sought to explore the effectiveness of a dual COX-2/5-LOX inhibitor darbufelone on the proliferation, migration, invasion and apoptosis of colon cancer cells, as well as the underlying mechanism of action. The results indicated that darbufelone significantly decreased the proliferative and invasive abilities of the colon cancer cells, in a dose-dependent manner. During the study of the related mechanisms, we found an upregulation of p27 and downregulation of cyclin D1 as well as CDK4 after darbufelone treatment, which indicated that darbufelone could arrest the cell cycle of LoVo cells at the G0/G1 phase. Furthermore, the activation of caspase-3 and -9, upregulation of Bax and downregulation of Bcl-2 demonstrated the occurrence of apoptosis by darbufelone. Finally, darbufelone also prevented the migration and invasion of LoVo cells, which may be ascribed to the upregulation of E-cadherin and ZO-1. In summary, our data suggest that the inhibition of both COX-2/5-LOX may be an effective therapeutic approach for colon cancer management, particularly for those patients with high expression of COX-2/5-LOX.

AF4 and AF4-MLL mediate transcriptional elongation of 5-lipoxygenase mRNA by 1, 25-dihydroxyvitamin D3.

The human 5-lipoxygenase (5-LO), encoded by the ALOX5 gene, is the key enzyme in the formation of pro-inflammatory leukotrienes. ALOX5 gene transcription is strongly stimulated by calcitriol (1α, 25-dihydroxyvitamin D3) and TGFß (transforming growth factor-ß). Here, we investigated the influence of MLL (activator of transcript initiation), AF4 (activator of transcriptional elongation) as well as of the leukemogenic fusion proteins MLL-AF4 (ectopic activator of transcript initiation) and AF4-MLL (ectopic activator of transcriptional elongation) on calcitriol/TGFß-dependent 5-LO transcript elongation. We present evidence that the AF4 complex directly interacts with the vitamin D receptor (VDR) and promotes calcitriol-dependent ALOX5 transcript elongation. Activation of transcript elongation was strongly enhanced by the AF4-MLL fusion protein but was sensitive to Flavopiridol. By contrast, MLL-AF4 displayed no effect on transcriptional elongation. Furthermore, HDAC class I inhibitors inhibited the ectopic effects caused by AF4-MLL on transcriptional elongation, suggesting that HDAC class I inhibitors are potential therapeutics for the treatment of t(4;11)(q21;q23) leukemia.

Cytoplasmic 5-Lipoxygenase Staining Is a Highly Sensitive Marker of Human Tumors of the Choroid Plexus.

OBJECTIVES: To determine the immunoreactivity status of 5-lipoxygenase (5-LO) in normal tissues, in tumors of the human choroid plexus, and in other brain tumors. METHODS: In total, 135 cases of various types of brain tumors were selected. Tissue samples were immunostained with a rabbit polyclonal anti-5-LO antibody. RESULTS: Nuclear reactivity was observed in most brain tumors, with most of the positive tumor cells exhibiting low-level reactivity. Cytoplasmic strong immunoreactivity for 5-LO (2+ or 3+) was only observed in 8.8% of astrocytic tumors, 0% of oligodendrogliomatous tumors, 5.6% of ependymal tumors, 0% of embryonal tumors, 3.1% of meningeal tumors, and 0% of metastatic lung adenocarcinomas. In contrast, cytoplasmic immunoreactivity for 5-LO was detected in all 27 cases of choroid plexus tumors. Twenty-five cases showed strong and diffuse cytoplasmic immunoreactivity. CONCLUSIONS: Our findings indicate that cytoplasmic 5-LO immunoreactivity is highly characteristic of human choroid plexus tumors but not other central nervous system tumor types. Cytoplasmic staining for 5-LO may prove to be a useful immunoreactive marker in the diagnosis of choroid plexus tumors.

Leukotriene B4-leukotriene B4 receptor axis promotes oxazolone-induced contact dermatitis by directing skin homing of neutrophils and CD8⁺ T cells.

Leukotriene B4 (LTB4 ) is a lipid mediator that is rapidly generated in inflammatory sites, and its functional receptor, BLT1, is mostly expressed on immune cells. Contact dermatitis is a common inflammatory skin disease characterized by skin oedema and abundant inflammatory infiltrates, primarily including neutrophils and CD8(+) T cells. The role of the LTB4 -BLT1 axis in contact dermatitis remains largely unknown. In this study, we found up-regulated gene expression of 5-lipoxygenase and leukotriene A4 hydrolase, two critical enzymes for LTB4 synthesis, BLT1 and elevated LTB4 levels in skin lesions of oxazolone (OXA)-induced contact dermatitis. BLT1 deficiency or blockade of LTB4 and BLT1 by the antagonists, bestatin and U-75302, respectively, in the elicitation phase caused significant decreases in ear swelling and skin-infiltrating neutrophils and CD8(+) T cells, which was accompanied by significantly reduced skin expression of CXCL1, CXCL2, interferon-γ and interleukin-1ß. Furthermore, neutrophil depletion during the elicitation phase of OXA-induced contact dermatitis also caused significant decreases in ear swelling and CD8(+) T-cell infiltration accompanied by significantly decreased LTB4 synthesis and gene expression of CXCL2, interferon-γ and interleukin-1ß. Importantly, subcutaneous injection of exogenous LTB4 restored the skin infiltration of CD8(+) T cells in neutrophil-depleted mice following OXA challenge. Collectively, our results demonstrate that the LTB4 -BLT1 axis contributes to OXA-induced contact dermatitis by mediating skin recruitment of neutrophils, which are a major source of LTB4 that sequentially direct CD8(+) T-cell homing to OXA-challenged skin. Hence, LTB4 and BLT1 could be potential therapeutic targets for the treatment of contact dermatitis.

Progesterone rapidly down-regulates the biosynthesis of 5-lipoxygenase products in human primary monocytes.

5-Lipoxygenase (5-LO), the key enzyme in the biosynthesis of pro-inflammatory leukotrienes (LTs) from arachidonic acid, is regulated by androgens in human neutrophils and monocytes accounting for sex differences in LT formation. Here we show that progesterone suppresses the synthesis of 5-LO metabolites in human primary monocytes. 5-LO product formation in monocytes stimulated with Ca(2+)-ionophore A23187 or with lipopolysaccharide/formyl peptide was suppressed by progesterone at concentrations of 10-100 nM in cells from females and at 1 µM in cells from males. Progesterone down-regulated 5-LO product formation in a rapid and reversible manner, but did not significantly inhibit 5-LO activity in cell-free assays using monocyte homogenates. Also, arachidonic acid release and its metabolism to other eicosanoids in monocytes were not significantly reduced by progesterone. The inhibitory effect of progesterone on LTs was still observed when mitogen-activated protein kinases were pharmacologically blocked, stimulatory 1-oleoyl-2-acetyl-sn-glycerol was exogenously supplied, or extracellular Ca(2+) was removed by chelation. Instead, suppression of PKA by means of two different pharmacological approaches (i.e. H89 and a cell-permeable PKA inhibitor peptide) prevented inhibition of 5-LO product generation by progesterone, to a similar extent as observed for the PKA activators prostaglandin E2 and 8-Br-cAMP, suggesting the involvement of PKA. In summary, progesterone affects the capacity of human primary monocytes to generate 5-LO products and, in addition to androgens, may account for sex-specific effects on pro-inflammatory LTs.

5-lipoxygenase is a direct target of miR-19a-3p and miR-125b-5p.

5-Lipoxygenase (5-LO) is the key enzyme in leukotriene biosynthesis. Leukotrienes are mediators of the innate immune system and inflammatory processes, and they might also be involved in cancer development. MicroRNAs (miRNAs) are important translational regulators and have been shown to be involved in development, differentiation, and cancer. Unraveling the miRNA network is important for understanding the cellular regulation processes. We identified two new miRNAs, miR-19a-3p and miR-125b-5p, regulating 5-LO and confirmed direct interaction by reporter gene assays. Furthermore, we investigated the regulation of 5-LO by these two miRNAs in several cell types. Inhibition of both miRNAs by antagomirs during differentiation of the myeloid cell line Mono Mac 6 led to a significant increase in 5-LO protein expression. Stimulation of human T lymphocytes with PHA resulted in a strong downregulation of 5-LO mRNA expression and in the induction of miR-19a-3p. The inhibition of miR-19a-3p with an antagomir led to a significant increase in 5-LO mRNA expression in T lymphocytes. Taken together, our data reveal that miR-19a-3p and miR-125b-5p target 5-LO in a cell type- and stimulus-specific manner.

5-Lipoxygenase activating protein (FLAP) dependent leukotriene biosynthesis inhibition (MK591) attenuates Lipid A endotoxin-induced inflammation.

The Lipid A moiety of endotoxin potently activates TLR-4 dependent host innate immune responses. We demonstrate that Lipid-A mediated leukotriene biosynthesis regulates pathogen-associated molecular patterns (PAMP)-dependent macrophage activation. Stimulation of murine macrophages (RAW264.7) with E. coli 0111:B4 endotoxin (LPS) or Kdo2-lipid A (Lipid A) induced inflammation and Lipid A was sufficient to induce TLR-4 mediated macrophage inflammation and rapid ERK activation. The contribution of leukotriene biosynthesis was evaluated with a 5-lipoxygenase activating protein (FLAP) inhibitor, MK591. MK591 pre-treatment not only enhanced but also sustained ERK activation for up to 4 hours after LPS and Lipid A stimulation while inhibiting cell proliferation and enhancing cellular apoptosis. Leukotriene biosynthesis inhibition attenuated inflammation induced by either whole LPS or the Lipid A fraction. These responses were regulated by inhibition of the key biosynthesis enzymes for the proinflammatory eicosanoids, 5-lipoxygenase (5-LO), and cyclooxygenase-2 (COX-2) quantified by immunoblotting. Inhibition of leukotriene biosynthesis differentially regulated TLR-2 and TLR-4 cell surface expression assessed by flow cytometry, suggesting a close mechanistic association between TLR expression and 5-LO associated eicosanoid activity in activated macrophages. Furthermore, MK591 pre-treatment enhanced ERK activation and inhibited cell proliferation after LPS or Lipid A stimulation. These effects were regulated in part by increased apoptosis and modulation of cell surface TLR expression. Together, these data clarify the mechanistic association between 5-lipoxygenase activating protein-mediated leukotriene biosynthesis and 5-LO dependent eicosanoid metabolites in mediating the TLR-dependent inflammatory response after endotoxin exposure typical of bacterial sepsis.

Neuroprotective effect of melatonin against kainic acid-induced oxidative injury in hippocampal slice culture of rats.

Endogenous melatonin is a known free radical scavenger that removes reactive oxygen species (ROS), thus, alleviating oxidative stress. The purpose of this study was to demonstrate its effect against kainic acid (KA)-induced oxidative stress in organotypic hippocampal slice cultures (OHSCs). To observe neuroprotective effects of melatonin, different concentrations (0.01, 0.1 and 1 mM) of melatonin were administrated after KA treatment for 18 h in OHSCs of rat pups. Dose-response studies showed that neuronal cell death was significantly reduced after 0.1 and 1 mΜ melatonin treatments based on propidium iodide (PI) uptake and cresyl violet staining. The dichlorofluorescein (DCF) fluorescence which indicates ROS formation decreased more in the melatonin-treated group than in the KA group. The expression of 5-lipoxigenase (5-LO) and caspase-3 were reduced in the melatonin-treated groups compared to the KA group. These results suggest that melatonin may be an effective agent against KA-induced oxidative stress in the OHSC model.

Interleukin-1ß inhibits synthesis of 5-lipooxygenase in lipopolysaccharide-stimulated equine whole blood.

Interleukin-1ß (IL-1ß) is a pro-inflammatory cytokine. It induces the synthesis of prostaglandin E2 (PGE2) catalyzed by cyclooxygenase (COX) and microsomal prostaglandin E synthase (m-PGES). Besides its pro-inflammatory properties, PGE2 also exhibits anti-inflammatory properties by inhibiting synthesis of 5-lipooxygenase (5-LO) products which are in themselves, pro-inflammatory mediators. Thus, inhibition of 5-LO products is beneficial in regulating immune-responses and pro-inflammatory processes. To investigate the hypothesis that IL-1ß is responsible for the increase in the synthesis of PGE2 and in the reduction of 5-LO products, equine whole blood (EWB) was treated with lipopolysaccharide (LPS). In vitro treatment of EWB with LPS resulted in increased expression of IL-1ß while expression of 5-LO was suppressed. Quantification of eicosanoids using liquid-chromatography-mass spectrometry/multiple reaction monitoring (LC-MS/MRM) showed increased concentrations of prostaglandins and decreased 5-LO products in LPS-treated EWB. Pretreatment of EWB with IL-1ß followed by calcium ionophore A23187 (CI) reduced synthesis of 5-LO products. However, pretreatment of EWB with COX-2 inhibitor (NS-398) or m-PGES-1 inhibitor (CAY 10526) and IL-1ß followed with CI resulted in a significant (p<0.0001) increase in 5-LO products. Pretreatment of EWB with phospholipase C inhibitor (U73122) followed with LPS reduced PGE2 production but increased 5-LO products. The result of this study indicated that increased PGE2 production led to reduction in 5-LO products in LPS-treated EWB via IL-1ß. However, other pathways, cytokines and mediators may be involved in inhibiting 5-LO products but the present study did not include those other potential pathways. Inhibition of 5-LO products by PGE2 in EWB may regulate the initiation and pathogenesis of inflammatory responses in the horse.

Augmentation of 5-lipoxygenase activity and expression during dengue serotype-2 infection.

BACKGROUND: Leukotriene B4, a 5-lipoxygenase product of arachidonic acid with potent chemotactic effects on neutrophils, has not been assessed in dengue patients. In this study, plasma leukotriene B4 and serum high-sensitivity C-reactive protein levels were determined in adult patients during the febrile, convalescent and defervescent stages of dengue serotype-2 (DENV-2) infection, and compared with those of age-matched healthy and non-dengue febrile subjects. In vitro studies were performed to examine the effects of live and heat-inactivated DENV-2 on the activities and expression of 5-lipoxygenase in human neutrophils. RESULTS: Plasma leukotriene B4 was elevated during the febrile stages of dengue infection compared to levels during convalescence and in study controls. Plasma leukotriene B4 also correlated with serum high-sensitivity C-reactive protein in dengue patients (febrile, r = 0.91, p < 0.001; defervescence, r = 0.87, p < 0.001; convalescence, r = 0.87, p < 0.001). Exposure of human neutrophils to DENV-2 resulted in a significant rise in leukotriene B4; the extent of increase, however, did not differ between exposure to live and heat-inactivated DENV-2. Pre-incubation of either live or heat-inactivated DENV-2 resulted in reduced leukotriene B4 release by neutrophils, indicating that contact with dengue antigens (and not replication) triggers the neutrophil response. Production of leukotriene B4 was associated with an increase in 5-lipoxygenase expression in human neutrophils; addition of MK886 (a 5-lipoxygenase activating protein inhibitor) attenuated further increase in leukotriene B4 production. CONCLUSION: These findings provide important clinical and mechanistic data on the involvement of 5-lipoxygenase and its metabolites in dengue infection. Further studies are needed to elucidate the therapeutic implications of these findings.