Search

Lipoxygenase (LOX) activity provides oxidative lipid metabolites, which are involved in inflammatory disorders and tumorigenesis. Activity‐based probes to detect the activity of LOX enzymes in their cellular context provide opportunities to explore LOX biology and LOX inhibition. Here, we developed Labelox B as a potent covalent LOX inhibitor for one‐step activity‐based labeling of proteins with LOX activity. Labelox B was used to establish an ELISA‐based assay for affinity capture and antibody‐based detection of specific LOX isoenzymes. Moreover, Labelox B enabled efficient activity‐based labeling of endogenous LOXs in living cells. LOX proved to localize in the nucleus, which was rationalized by identification of a functional bromodomain‐like consensus motif in 15‐LOX‐1. This indicates that 15‐LOX‐1 is not only involved in oxidative lipid metabolism, but also in chromatin binding, which suggests a potential role in chromatin modifications., Activity‐based labeling of lipoxygenase (LOX) activity using the novel one‐step labeling probe Labelox B enables detection of isoenzyme inhibitory selectivity in an ELISA‐based assay and visualization of the localization of LOX activity in living cells. The observed nuclear localization was rationalized by identification of a bromodomain‐like region in the 15‐LOX‐1 isoenzyme.

In this work, the design, synthesis, and structure-activity relationships of a novel array of geranyloxy and farnesyloxy 3-acetylcoumarins were reported as potent soybean 15-lipoxygenase inhibitors. Among the prepared coumarins, 7-farnesyloxy-3-acetylcoumarin (12b) was found to be the most potent inhibitor by IC50 = 0.68 μM while O-geranyl substituents at positions 5 and 6 of 3-acetylcoumarin (10a and 11a) were not inhibitors. Using docking studies, the binding affinity and the preferred pose of synthetic compounds were considered. It was found that lipoxygenase inhibitory activity and prenyl length chain were directly related. The hydrophobic cavity of the enzyme was more effectively occupied by the farnesyl moiety of the potent inhibitor 12b rather than other derivatives. Also, with this pose of farnesyl chain in 7-farnesyloxy-3-acetylcoumarins, the acetyl group could be directed to the hydrophilic pocket in the active site.

In this study, a series of mono- and diallylphenol derivative were designed, synthesized, and evaluated as potential human 15-lipoxygenase-1 (15-hLOX-1) inhibitors. Radical scavenging potency of the synthetic allylphenol derivatives was assessed and the results were in accordance with lipoxygenase (LOX) inhibition potency. It was found that the electronic natures of allyl moiety and para substituents play the main role in radical scavenging activity and subsequently LOX inhibition potency of the synthetic inhibitors. Among the synthetic compounds, 2,6-diallyl-4-(hexyloxy)phenol (42) and 2,6-diallyl-4-aminophenol (47) showed the best results for LOX inhibition (IC50 = 0.88 and 0.80 μM, respectively).

Damage-associated endogenous molecules induce innate immune response, thus making sterile inflammation medically relevant. Stress-derived extracellular vesicles (stressEVs) released during oxidative stress conditions were previously found to activate Toll-like receptor 4 (TLR4), resulting in expression of a different pattern of immune response proteins in comparison to lipopolysaccharide (LPS), underlying the differences between pathogen-induced and sterile inflammation. Here we report that synergistic activities of 15-lipoxygenase (15-LO) and secreted phospholipase A(2) (sPLA(2)) are needed for the formation of TLR4 agonists, which were identified as lysophospholipids (lysoPLs) with oxidized unsaturated acyl chain. Hydroxy, hydroperoxy, and keto products of 2-arachidonoyl-lysoPI oxidation by 15-LO were identified by mass spectrometry (MS), and they activated the same gene pattern as stressEVs. Extracellular PLA(2) activity was detected in the synovial fluid from rheumatoid arthritis and gout patients. Furthermore, injection of sPLA(2) promoted K/BxN serum-induced arthritis in mice, whereby ankle swelling was partially TLR4 dependent. Results confirm the role of oxidized lysoPL of stressEVs in sterile inflammation that promotes chronic diseases. Both 15-LO and sPLA(2) enzymes are induced during inflammation, which opens the opportunity for therapy without compromising innate immunity against pathogens.

Lipoxygenases (ALOX-isoforms) are lipid peroxidizing enzymes, which have been implicated in cell differentiation and maturation but also in the biosynthesis of lipid mediators playing important roles in the pathogenesis of inflammatory, hyperproliferative and neurological diseases. In mammals these enzymes are widely distributed and the human genome involves six functional genes encoding for six distinct human ALOX paralogs. In mice, there is an orthologous enzyme for each human ALOX paralog but the catalytic properties of human and mouse ALOX orthologs show remarkable differences. ALOX inhibitors are frequently employed for deciphering the biological role of these enzymes in mouse models of human diseases but owing to the functional differences between mouse and human ALOX orthologs the uncritical use of such inhibitors is sometimes misleading. In this study we evaluated the paralog- and ortholog-specificity of 13 frequently employed ALOX-inhibitors against four recombinant human and mouse ALOX paralogs (ALOX15, ALOX15B, ALOX12, ALOX5) under different experimental conditions. Our results indicated that except for zileuton, which exhibits a remarkable paralog-specificity for mouse and human ALOX5, no other inhibitor was strictly paralog specific but some compounds exhibit an interesting ortholog-specificity. Because of the variable isoform specificities of the currently available ALOX inhibitors care must be taken when the biological effects of these compounds observed in complex in vitro and in vivo systems are interpreted.

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 MF15 stimulate the activity of 15-LOX-1, supported by experiments with HEK293 cells transfected with either 5-LOX, 15-LOX-1 and 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.

15-lipoxygenase is one of the key enzymes for the metabolism of unsaturated fatty acids that its manipulation has been proposed recently as a new molecular target for regulating cancer cell growth. Aberrant expression of 15-lipoxygenase enzyme seems to play an indicative role in the pathology of different cancer types, tumor progression, metastasis, or apoptosis. Based on the fact that breast cancer is one of the most common cancers that imposes a burden of mortality in women also, on the other hand, evidence in experimental models and human studies indicate the emerging role of the 15-lipoxygenase pathway in breast cancer pathogenesis, we present a review of recent findings related to the role of 15- lipoxygenase enzyme and metabolites in breast cancer growth, apoptosis, metastasis, and invasion as well as their local and circulating expression pattern in patients with breast cancer. Our review supports the emerging role of 15- lipoxygenase in molecular and cellular processes regulating breast tumor cell fate with both positive and negative effects.

Catalyzed peroxidation of unsaturated lipid in animals and plants intimately is linked to the activity of 15-Lipoxygenase enzymes. Lipoxygenases (LOXs) are well known to play an important role in many acute and chronic syndromes such as inflammation, asthma, cancer, and allergy. In this study, a series of mono prenyloxycarbostyrils were synthesized and evaluated as potential inhibitors of soybean 15-Lipoxygenase (SLO) and their inhibitory potencies were compared to mono prenyloxycoumarins which had been reported in the previous works. The synthetic compounds inhibit lipoxygenase enzyme by competitive mechanism like the prenyloxy coumarins. The results showed that position and length of the prenyl moiety play the important role in lipoxygenase inhibitory activity. Among all of the synthetic compounds (coumarin and carbostyril derivatives), 5-farnesyloxycoumarin and 8-farnesyloxycarbostyril demonstrated the best inhibitory activity by IC50 values of 1.1 µM and 0.53 µM, respectively.

Recent studies suggest a potential role of bioactive lipids in acute kidney injury induced by lipopolysaccharide (LPS). The current study was designed to determine the profiling activities of various polyunsaturated fatty acid (PUFA) metabolizing enzymes, including lipoxygenases (LO), cyclooxygenase, and cytochrome P450 in the plasma of LPS-injected mice using LC-MS. Heat map analysis revealed that out of 126 bioactive lipids screened, only the 12/15-LO metabolite, 12-HETE, had a significant (2.24 ± 0.4) fold increase relative to control (P = 0.0001) after Bonferroni Correction (BCF α = 0.003). We then determined the role of the 12/15-LO in LPS-induced acute kidney injury using genetic and pharmacological approaches. Treatment of LPS injected mice with the 12/15-LO inhibitor, baicalein, significantly reduced levels of renal injury and inflammation markers including urinary thiobarbituric acid reactive substance (TBARs), urinary monocyte chemoattractant protein-1 (MCP-1), renal interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Similarly, knocking-out of 12/15-LO reduced levels of renal inflammation and injury markers elicited by LPS injection. Next, we tested whether exogenous supplementation with docosahexaenoic acid (DHA) as a substrate would divert the role of 12/15-LO from being pro-inflammatory to anti-inflammatory via increased production of the anti-inflammatory metabolite. DHA treatment restored the decreased in plasma level of resolvin D2 (RvD2) and reduced renal injury in LPS-injected mice whereas DHA treatment failed to provide any synergistic effects in reducing renal injury in LPS injected 12/15-LO knock-out mice. The ability of RvD2 to protect kidney against LPS-induced renal injury was further confirmed by exogenous RvD2 which significantly reduced the elevation in renal injury in LPS injected mice. These data suggest a double-edged sword role of 12/15-LO in LPS-induced acute renal inflammation and injury, depending on the type of substrate available for its activity.

2-Arachidonoyl-glycerol (2-AG) is an endocannabinoid with anti-inflammatory properties. Blocking 2-AG hydrolysis to enhance CB2 signaling has proven effective in mouse models of inflammation. However, the expression of 2-AG lipases has never been thoroughly investigated in human leukocytes. Herein, we investigated the expression of seven 2-AG hydrolases by human blood leukocytes and alveolar macrophages (AMs) and found the following protein expression pattern: monoacylglycerol (MAG lipase; eosinophils, AMs, monocytes), carboxylesterase (CES1; monocytes, AMs), palmitoyl-protein thioesterase (PPT1; AMs), α/β-hydrolase domain (ABHD6; mainly AMs), ABHD12 (all), ABHD16A (all), and LYPLA2 (lysophospholipase 2; monocytes, lymphocytes, AMs). We next found that all leukocytes could hydrolyze 2-AG and its metabolites derived from cyclooxygenase-2 (prostaglandin E2-glycerol [PGE2-G]) and the 15-lipoxygenase (15-hydroxy-eicosatetraenoyl-glycerol [15-HETE-G]). Neutrophils and eosinophils were consistently better at hydrolyzing 2-AG and its metabolites than monocytes and lymphocytes. Moreover, the efficacy of leukocytes to hydrolyze 2-AG and its metabolites was 2-AG ≥ 15-HETE-G >> PGE2-G for each leukocyte. Using the inhibitors methylarachidonoyl-fluorophosphonate (MAFP), 4-nitrophenyl-4-(dibenzo[d][1,3]dioxol-5-yl(hydroxy)methyl)piperidine-1-carboxylate (JZL184), Palmostatin B, 4′-carbamoylbiphenyl-4-yl methyl(3-(pyridin-4-yl)benzyl)carbamate, N-methyl-N-[[3-(4-pyridinyl)phenyl]methyl]-4′-(aminocarbonyl)[1,1′-biphenyl]-4-yl ester carbamic acid (WWL70), 4′-[[[methyl[[3-(4-pyridinyl)phenyl]methyl]amino]carbonyl]oxy]-[1,1′-biphenyl]-4-carboxylic acid, ethyl ester (WWL113), tetrahydrolipstatin, and ML349, we could not pinpoint a specific hydrolase responsible for the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by these leukocytes. Furthermore, JZL184, a selective MAG lipase inhibitor, blocked the hydrolysis of 2-AG, PGE2-G, and 15-HETE-G by neutrophils and the hydrolysis of PGE2-G and 15-HETE-G by lymphocytes, two cell types with limited/no MAG lipase. Using an activity-based protein profiling (ABPP) probe to label hydrolases in leukocytes, we found that they express many MAFP-sensitive hydrolases and an unknown JZL184-sensitive hydrolase of ∼52 kDa. Altogether, our results indicate that human leukocytes are experts at hydrolyzing 2-AG and its metabolites via multiple lipases and probably via a yet-to-be characterized 52 kDa hydrolase. Blocking 2-AG hydrolysis in humans will likely abrogate the ability of human leukocytes to degrade 2-AG and its metabolites and increase their anti-inflammatory effects in vivo.

Human 15-lipoxygenase-1 (15-LOX-1) is a mammalian lipoxygenase which plays an important regulatory role in several CNS and inflammatory lung diseases. To further explore the role of this enzyme in drug discovery, novel potent inhibitors with favorable physicochemical properties are required. In order to identify such new inhibitors, we established a combinatorial screening method based on acylhydrazone chemistry. This represents a novel application of combinatorial chemistry focusing on the improvement of physicochemical properties, rather than on potency. This strategy allowed us to efficiently screen 44 reaction mixtures of different hydrazides and our previously reported indole aldehyde core structure, without the need for individual synthesis of all possible combinations of building blocks. Our approach afforded three new inhibitors with IC50 values in the nanomolar range and improved lipophilic ligand efficiency.

In type 1 diabetes, an autoimmune event increases oxidative stress in islet β cells, giving rise to cellular dysfunction and apoptosis. Lipoxygenases are enzymes that catalyze the oxygenation of polyunsaturated fatty acids that can form lipid metabolites involved in several biological functions, including oxidative stress. 12-Lipoxygenase and 12/15-lipoxygenase are related but distinct enzymes that are expressed in pancreatic islets, but their relative contributions to oxidative stress in these regions are still being elucidated. In this study, we used mice with global genetic deletion of the genes encoding 12-lipoxygenase (arachidonate 12-lipoxygenase, 12S type [Alox12]) or 12/15-lipoxygenase (Alox15) to compare the influence of each gene deletion on β cell function and survival in response to the β cell toxin streptozotocin. Alox12(−/−) mice exhibited greater impairment in glucose tolerance following streptozotocin exposure than WT mice, whereas Alox15(−/−) mice were protected against dysglycemia. These changes were accompanied by evidence of islet oxidative stress in Alox12(−/−) mice and reduced oxidative stress in Alox15(−/−) mice, consistent with alterations in the expression of the antioxidant response enzymes in islets from these mice. Additionally, islets from Alox12(−/−) mice displayed a compensatory increase in Alox15 gene expression, and treatment of these mice with the 12/15-lipoxygenase inhibitor ML-351 rescued the dysglycemic phenotype. Collectively, these results indicate that Alox12 loss activates a compensatory increase in Alox15 that sensitizes mouse β cells to oxidative stress.

In this paper, human platelet 12-lipoxygenase [h12-LOX (ALOX12)], human reticulocyte 15-lipoxygenase-1 [h15-LOX-1 (ALOX15)], and human epithelial 15-lipoxygenase-2 [h15-LOX-2 (ALOX15B)] were observed to react with docosahexaenoic acid (DHA) and produce 17S-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid (17S-HpDHA). The k(cat)/K(M) values with DHA for h12-LOX, h15-LOX-1, and h15-LOX-2 were 12, 0.35, and 0.43 s(−1) μM(−1), respectively, which demonstrate h12-LOX as the most efficient of the three. These values are comparable to their counterpart k(cat)/K(M) values with arachidonic acid (AA), 14, 0.98, and 0.24 s(−1) μM(−1), respectively. Comparison of their product profiles with DHA demonstrates that the three LOX isozymes produce 11S-HpDHA, 14S-HpDHA, and 17S-HpDHA, to varying degrees, with 17S-HpDHA being the majority product only for the 15-LOX isozymes. The effective k(cat)/K(M) values (k(cat)/K(M) × percent product formation) for 17S-HpDHA of the three isozymes indicate that the in vitro value of h12-LOX was 2.8-fold greater than that of h15-LOX-1 and 1.3-fold greater than that of h15-LOX-2. 17S-HpDHA was an effective substrate for h12-LOX and h15-LOX-1, with four products being observed under reducing conditions: protectin DX (PDX), 16S,17S-epoxy-4Z,7Z,10Z,12E,14E,19Z-docosahexaenoic acid (16S,17S-epoxyDHA), the key intermediate in neuroprotection D1 biosynthesis [NPD1, also known as protectin D1 (PD1)], 11,17S-diHDHA, and 16,17S-diHDHA. However, h15-LOX-2 did not react with 17-HpDHA. With respect to their effective k(cat)/K(M) values, h12-LOX was markedly less effective than h15-LOX-1 in reacting with 17S-HpDHA, with a 55-fold lower effective k(cat)/K(M) in producing 16S,17S-epoxyDHA and a 27-fold lower effective k(cat)/K(M) in generating PDX. This is the first direct demonstration of h15-LOX-1 catalyzing this reaction and reveals an in vitro pathway for PDX and NPD1 intermediate biosynthesis. In addition, epoxide formation from 17S-HpDHA and h15-LOX-1 was negatively affected via allosteric regulation by 17S-HpDHA (K(d) = 5.9 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K(d) = 2.5 μM), and 17S-hydroxy-13Z,15E,19Z-docosatrienoic acid (17S-HDTA) (K(d) = 1.4 μM), suggesting a possible regulatory pathway in reducing epoxide formation. Finally, 17S-HpDHA and PDX inhibited platelet aggregation, with EC(50) values of approximately 1 and 3 μM, respectively. The in vitro results presented here may help advise in vivo PDX and NPD1 intermediate (i.e., 16S,17S-epoxyDHA) biosynthetic investigations and support the benefits of DHA rich diets.

In search for new anti-inflammatory agents that inhibit the enzymes of arachidonic acid pathway as the drug targets, the present article describes the screening of 1,3,4-oxadiazole analogues against lipoxygenase (LOX) enzyme. The work is based on the synthesis of new N-alkyl/aralky/aryl derivatives (6a-o) of 2-(4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,3,4-oxadiazol-3-ylthio)acetamide which were obtained by the reaction of 1,3,4-oxadiazole (3) with various electrophiles (5a-o), in KOH. The synthesized analogues showed potent to moderate inhibitory activity against the soybean 15-LOX enzyme; especially 6g, 6b, 6a and 6l displayed the potent inhibitory potential with IC50 values 7.15 ± 0.26, 9.32 ± 0.42, 15.83 ± 0.45 & 18.37 ± 0.53 µM, respectively, while excellent to moderate inhibitory profiles with IC50 values in the range of 26.13–98.21 µM were observed from the compounds 6k, 6m, 6j, 6o, 6h, 6f, 6n and 6c. Most of the active compounds exhibited considerable cell viability against blood mononuclear cells (MNCs) at 0.25 mM by MTT assay except 6f, 6h, 6k and 6m which showed around 50% cell viability. Flow cytometry studies of the selected compounds 6a, 6j and 6n revealed that these caused 79.5–88.51% early apoptotic changes in MNCs compared with 4.26% for control quercetin at their respective IC50 values. The relative expression of 5-LOX gene was monitored in MNCs after treatment with these three molecules and all down-regulated the enzyme activity. In silico ADME and molecular docking studies further supported these studies of oxadiazole derivatives and considered it as potential ‘lead’ compounds in drug discovery and development.

Resolvin D1, a specialized pro-resolving lipid mediator produced from docosahexaenoic acid by 15- and 5-lipoxygenase, exerts anti-inflammatory effects driving to the resolution of inflammation. The present study aimed to elucidate its role in small intestinal damage induced by nonsteroidal anti-inflammatory drug (NSAID). Indomethacin was administered orally to C57BL/6J male mice, which were sacrificed 24 h later to collect small intestine specimens. Before administration of indomethacin, mice were subjected to intraperitoneal treatment with resolvin D1 or oral administration of baicalein, a 15-lipoxygenase inhibitor. Small intestinal damage induced by indomethacin was attenuated by pretreatment with resolvin D1. Furthermore, resolvin D1 reduced the gene expression levels of interleukin-1β, tumor necrosis factor-α, and CXCL1/keratinocyte chemoattractant. Conversely, the inhibition of 15-lipoxygenase activity by baicalein increased the expression of genes coding for these inflammatory cytokines and chemokine, leading to exacerbated small intestinal damage, and reduced the concentration of resolvin D1 in the small intestinal tissue. Exogenous treatment with resolvin D1 negated the deleterious effect of baicalein. 15-lipoxygenase was mainly expressed in the epithelium and inflammatory cells of the small intestine, and its gene and protein expression was not affected by the administration of indomethacin. Inhibition of the resolvin D1 receptor, lipoxin A4 receptor /formyl peptide receptor 2, by its specific inhibitors Boc-1 and WRW4 aggravated indomethacin-induced small intestinal damage. Collectively, these results indicate that resolvin D1 produced by 15-lipoxygenase contributes to mucoprotection against NSAID-induced small intestinal damage through its anti-inflammatory effect.

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.

N-Arachidonoyl-ethanolamine (AEA) is an endocannabinoid (eCB) and endogenous lipid mimicking many of the effects of Δ9-tetrahydrocannabinol, notably on brain functions, appetite, pain and inflammation. The eCBs and eCB-like compounds contain fatty acids, the main classes being the monoacylglycerols and the N-acyl-ethanolamines (NAEs). Thus, each long chain fatty acid likely exists under the form of a monoacylglycerol and NAE, as it is the case for arachidonic acid (AA) and linoleic acid (LA). Following their biosynthesis, AA and AEA can be further metabolized into additional eicosanoids, notably by the 15-lipoxygenase pathway. Thus, we postulated that NAEs possessing a 1Z,4Z-pentadiene motif, near their omega end, would be transformed into their 15-lipoxygenase metabolites. As a proof of concept, we investigated N-linoleoyl-ethanolamine (LAE). We successfully synthesized LEA and LEA-d4 as well as their 15-lipoxygenase-derived derivatives, namely 13-hydroxy-9Z,11E-octadecadienoyl-N-ethanolamine (13-HODE-EA) and 13-HODE-EA-d4, using Novozyme 435 immobilized on acrylic resin and soybean lipoxygenase respectively. We also show that both human 15-lipoxygenase-1 and -2 can biosynthesize 13-HODE-EA. Co-incubation of LEA and LA with either human 15-lipoxygenase led to the biosynthesis of 13-HODE-EA and 13-HODE in a ratio equal to or greater than 3:1, indicating that LEA is preferred to LA by these enzymes. Finally, we show that 13-HODE-EA is found in human saliva and skin and is a weak although selective TRPV1 agonist. The full biological importance of 13-HODE-EA remains to be explored.

The oxylipins, 5S,12S-dihydroxy-6E,8Z,10E,14Z-eicosatetraenoic acid (5S,12S-diHETE) and 5S,15S-dihydroxy-6E,8Z,11Z,13E-eicosatetraenoic acid (5S,15S-diHETE), have been identified in cell exudates and have chemotactic activity toward eosinophils and neutrophils. Their biosynthesis has been proposed to occur by sequential oxidations of arachidonic acid (AA) by lipoxygenase enzymes, specifically through oxidation of AA by h5-LOX followed by h12-LOX, h15-LOX-1, or h15-LOX-2. In this work, h15-LOX-1 demonstrates altered positional specificity when reacting with 5S-HETE, producing 90% 5S,12S-diHETE, instead of 5S,15S-diHETE, with kinetics 5-fold greater than that of h12-LOX. This is consistent with previous work in which h15-LOX-1 reacts with 7S-HDHA, producing the noncanonical, DHA-derived, specialized pro-resolving mediator, 7S,14S-diHDHA. It is also determined that oxygenation of 5S-HETE by h15-LOX-2 produces 5S,15S-diHETE and its biosynthetic k(cat)/K(M) flux is 2-fold greater than that of h15-LOX-1, suggesting that h15-LOX-2 may have a greater role in lipoxin biosynthesis than previously thought. In addition, it is shown that oxygenation of 12S-HETE and 15S-HETE by h5-LOX is kinetically slow, suggesting that the first step in the in vitro biosynthesis of both 5S,12S-diHETE and 5S,15S-diHETE is the production of 5S-HETE.

Made available in DSpace on 2020-12-12T02:01:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-28 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Ethnopharmacological relevance: Terminalia catappa L. (Combretaceae), known as “amendoeira da praia” in Brazil, has been recognized as a medicinal plant in folk medicine for the treatment of gastrointestinal disorders and other inflammatory conditions. The present study aimed to investigate the preventive and healing effects of the infusion of leaves of T. catappa (ILTC) against gastric lesions caused by ischemia and reperfusion (I/R) injury and characterize its mechanism of action in the gastric mucosa of rats. Materials and methods: Different doses (30, 100, and 300 mg/kg) of ILTC were orally administered as acute and subacute treatments against I/R-induced gastric lesion in rats. After treatment, the stomach of rats was collected to measure the lesion area, redox parameters malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and reduced glutathione (GSH) and inflammatory parameters myeloperoxidase activity (MPO), interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). The activities of matrix metalloproteinases 2 and 9 (MMPs 2 and 9) were assessed by zymography method to clarify the mechanisms of the healing acceleration promoted by ILTC. Results: Pretreatment with ILTC (100 mg/kg) was effective in preventing the aggravation of lesions in the acute model by reducing MPO activity by 38% relative to control group, despite the lack of clarity of this action at the macroscopical level at the lesion area (p < 0.05). After three days of treatment with ILTC (30 and 100 mg/kg), this infusion significantly reduced the lesion area by 95% and 89%, respectively, compared the control (p < 0.05). The gastric healing effect of all doses of ILTC was followed by a reduction in MPO activity (decrease by 70–78%). Compared to the negative control, an improvement in gastric healing owing to treatment with ILTC was observed and this was followed by an increase in MMP-2 (20–47%) (p < 0.05). Conclusion: Three days of treatment with ILTC could accelerate the healing process in I/R-induced lesions in rats. By decreasing MPO levels, ILTC enabled the action of MMP-2, which led to tissue recovery in the gastric mucosa. Department of Physiology Biosciences Institute UNESP-São Paulo State University, São Paulo Snyder Institute for Chronic Diseases Cumming School of Medicine University of Calgary Biosciences Institute UNESP-São Paulo State University, São Vicente, São Paulo Institute of Chemistry UNESP-São Paulo State University, São Paulo Laboratory of Extracellular Matrix UNESP- São Paulo State University, São Paulo Department of Physiology Biosciences Institute UNESP-São Paulo State University, São Paulo Biosciences Institute UNESP-São Paulo State University, São Vicente, São Paulo Institute of Chemistry UNESP-São Paulo State University, São Paulo Laboratory of Extracellular Matrix UNESP- São Paulo State University, São Paulo FAPESP: 2015/14797-3 CNPq: 301397/2017-1 CNPq: 306209/2016-0 CAPES: 88882.183585/2018-01 CAPES: 88882.183586/2018-01

The two oxylipins 7S,14S-dihydroxydocosahexaenoic acid (diHDHA) and 7S,17S-diHDHA [resolvin D5 (RvD5)] have been found in macrophages and infectious inflammatory exudates and are believed to function as specialized pro-resolving mediators (SPMs). Their biosynthesis is thought to proceed through sequential oxidations of DHA by lipoxygenase (LOX) enzymes, specifically, by human 5-LOX (h5-LOX) first to 7(S)-hydroxy-4Z,8E,10Z,13Z,16Z,19Z-DHA (7S-HDHA), followed by human platelet 12-LOX (h12-LOX) to form 7(S),14(S)-dihydroxy-4Z,8E,10Z,12E,16Z,19Z-DHA (7S,14S-diHDHA) or human reticulocyte 15-LOX-1 (h15-LOX-1) to form RvD5. In this work, we determined that oxidation of 7(S)-hydroperoxy-4Z,8E,10Z,13Z,16Z,19Z-DHA to 7S,14S-diHDHA is performed with similar kinetics by either h12-LOX or h15-LOX-1. The oxidation at C14 of DHA by h12-LOX was expected, but the noncanonical reaction of h15-LOX-1 to make over 80% 7S,14S-diHDHA was larger than expected. Results of computer modeling suggested that the alcohol on C7 of 7S-HDHA hydrogen bonds with the backbone carbonyl of Ile399, forcing the hydrogen abstraction from C12 to oxygenate on C14 but not C17. This result raised questions regarding the synthesis of RvD5. Strikingly, we found that h15-LOX-2 oxygenates 7S-HDHA almost exclusively at C17, forming RvD5 with faster kinetics than does h15-LOX-1. The presence of h15-LOX-2 in neutrophils and macrophages suggests that it may have a greater role in biosynthesizing SPMs than previously thought. We also determined that the reactions of h5-LOX with 14(S)-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-DHA and 17(S)-hydroperoxy-4Z,7Z,10Z,13Z,15E,19Z-DHA are kinetically slow compared with DHA, suggesting that these reactions may be minor biosynthetic routes in vivo. Additionally, we show that 7S,14S-diHDHA and RvD5 have anti-aggregation properties with platelets at low micromolar potencies, which could directly regulate clot resolution.

Human reticulocyte 15-lipoxygenase-1 (h15-LOX-1 or ALOX15) and platelet 12-lipoxygenase (h12-LOX or ALOX12) catalysis of docosahexaenoic acid (DHA) and the maresin precursor, 14S-hydroperoxy-4Z,7Z,10Z,12E,16Z,19Z-docosahexaenoic acid (14S-HpDHA), were investigated to determine their product profiles and relative rates in the biosynthesis of the key maresin intermediate, 13S,14S-epoxy-4Z,7Z,9E,11E,16Z,19Z-docosahexaenoic acid (13S,14S-epoxy-DHA). Both enzymes converted DHA to 14S-HpDHA, with h12-LOX having a 39-fold greater k(cat)/K(M) value (14.0 ± 0.8 s(−1) μM(−1)) than that of h15-LOX-1 (0.36 ± 0.08 s(−1) μM(−1)) and a 1.8-fold greater 14S-HpDHA product selectivity, 81 and 46%, respectively. However, h12-LOX was markedly less effective at producing 13S,14S-epoxy-DHA from 14S-HpDHA than h15-LOX-1, with a 4.6-fold smaller k(cat)/K(M) value, 0.0024 ± 0.0002 and 0.11 ± 0.006 s(−1) μM(−1), respectively. This is the first evidence of h15-LOX-1 to catalyze this reaction and reveals a novel in vitro pathway for maresin biosynthesis. In addition, epoxidation of 14S-HpDHA is negatively regulated through allosteric oxylipin binding to h15-LOX-1 and h12-LOX. For h15-LOX-1, 14S-HpDHA (K(d) = 6.0 μM), 12S-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic acid (12S-HETE) (K(d) = 3.5 μM), and 14S-hydroxy-7Z,10Z,12E,16Z,19Z-docosapentaenoic acid (14S-HDPA(ω-3)) (K(d) = 4.0 μM) were shown to decrease 13S,14S-epoxy-DHA production. h12-LOX was also shown to be allosterically regulated by 14S-HpDHA (K(d) = 3.5 μM) and 14S-HDPA(ω-3) (K(d) = 4.0 μM); however, 12S-HETE showed no effect, indicating for the first time an allosteric response by h12-LOX. Finally, 14S-HpDHA inhibited platelet aggregation at a submicrololar concentration, which may have implications in the benefits of diets rich in DHA. These in vitro biosynthetic pathways may help guide in vivo maresin biosynthetic investigations and possibly direct therapeutic interventions.

Autoimmune diseases are defined by an immune response against a specific autoantigen, driven by antigen-specific T cells or antibodies. While the mechanisms resolving brief episodes of acute inflammation elicited by microbial components or tissue injury are well understood, the mechanisms resolving tissue inflammation in autoimmune diseases are still largely elusive. We have, therefore, addressed the mechanisms of resolution in IgG-mediated autoimmune diseases using a mouse model of the pemphigoid disease ""bullous pemphigoid-like epidermolysis bullosa acquisita"" (BP-like EBA) as prototypical example. We found that 12/15-LO is induced in skin lesions of BP-like EBA and is predominantly expressed in eosinophils. Dependent on the expression of 12/15-LO, large amounts of proresolving lipid mediators, are biosynthesized in the skin by the point disease peaks. Their production is timely correlated to the gradual reversal of tissue inflammation. Genetic deficiency in Alox15, the gene encoding 12/15-LO, disrupts this process significantly protracting and aggravating disease. This protraction is associated reduced recruitment of regulatory T cells (Tregs) into lesional skin. Intriguingly, Alox15−/− mice also exhibit reduced recruitment of eosinophils into the skin, and the chemotaxis of cultured Alox15−/− eosinophils towards CCL11/eotaxin-1 is compromised. Finally, we demonstrate that 15-lipoxygenase-1, the human homologue of 12/15-LO is induced in granulocytes in lesional skin of patients suffering from a pemphigoid disease. Collectively, our result uncover key mechanisms resolving IgG-mediated skin inflammation. These mechanisms are orchestrated by 12/15-LO expressed in eosinophils promoting the recruitment of eosinophils and Tregs, which in turn inhibit neutrophils.

Ferroptotic death is the penalty for losing control over three processes-iron metabolism, lipid peroxidation and thiol regulation-that are common in the pro-inflammatory environment where professional phagocytes fulfill their functions and yet survive. We hypothesized that redox reprogramming of 15-lipoxygenase (15-LOX) during the generation of pro-ferroptotic signal 15-hydroperoxy-eicosa-tetra-enoyl-phosphatidylethanolamine (15-HpETE-PE) modulates ferroptotic endurance. Here, we have discovered that inducible nitric oxide synthase (iNOS)/NO•-enrichment of activated M1 (but not alternatively activated M2) macrophages/microglia modulates susceptibility to ferroptosis. Genetic or pharmacologic depletion/inactivation of iNOS confers sensitivity on M1 cells, whereas NO• donors empower resistance of M2 cells to ferroptosis. In vivo, M1 phagocytes, in comparison to M2 phagocytes, exert higher resistance to pharmacologically induced ferroptosis. This resistance is diminished in iNOS-deficient cells in the pro-inflammatory conditions of brain trauma or the tumour microenvironment. The nitroxygenation of eicosatetraenoyl (ETE)-PE intermediates and oxidatively truncated species by NO• donors and/or suppression of NO• production by iNOS inhibitors represent a novel redox mechanism of regulation of ferroptosis in pro-inflammatory conditions.

Type 1 diabetes (T1D) is a chronic autoimmune disease caused by proinflammatory T cell infiltration associated cytokine secretion and reactive oxygen species (ROS) production. In this study, our objective was to determine whether co-delivery of sunitinib, a member of tyrosine kinase inhibitor (TKI) family, and siRNA against Alox15 (siAlox15) could reverse the new-onset of T1D in non-obese diabetic (NOD)/ShiLtJ female mice which spontaneously develop diabetes. A cationic copolymer methoxy poly(ethylene glycol)-block-poly(2-methyl-2-carboxyl-propylene carbonate-graft-dodecanol-graft-tetraethylenepentamine) (mPEG-b-PCC-g-DC-g-TEPA) was used to encapsulate sunitinib with 8.32-9.39% loading efficacy and form complex with siAlox15 at the N/P ratio of 16:1. These micellar formulations exhibited sustained release of sunitinib for around 50% at 24 h and protected siAlox15 from serum degradation for at least 10 h. Further, both sunitinib and siAlox15 inhibited human peripheral blood mononuclear cell (PBMC) proliferation and activation by downregulating the phosphorylation of Akt, platelet-derived growth factor receptor (PDGFR) at protein levels and Alox15 in mRNA levels, respectively. Besides, siAlox15 treatment reversed the cytokine induced ROS upregulation in rat INS-1E β cells. Finally, systemic administration of these micelles carrying sunitinib and siAlox15 efficiently reversed T1D in NOD mice as indicated by decreased blood glucose concentration to below 250 mg/dL. The mice showed fast response to high glucose which was determined by intraperitoneal glucose tolerance test and promoted serum insulin production. Tissue staining indicated that sunitinib and siAlox15 combination could inhibit T cell infiltration to mouse islets, thereby promoting islet survival and function. In summary, our results demonstrated that combination therapy of sunitinib and siAlox15 could be a novel, reliable and safe approach for patients who were recently diagnosed with T1D.

The reaction of 5 S,15 S-dihydroperoxyeicosatetraenoic acid (5,15-diHpETE) with human 5-lipoxygenase (LOX), human platelet 12-LOX, and human reticulocyte 15-LOX-1 was investigated to determine the reactivity and relative rates of producing lipoxins (LXs). 5-LOX does not react with 5,15-diHpETE, although it can produce LXA4 when 15-HpETE is the substrate. In contrast, both 12-LOX and 15-LOX-1 react with 5,15-diHpETE, forming specifically LXB4. For 12-LOX and 5,15-diHpETE, the kinetic parameters are kcat = 0.17 s-1 and kcat/ KM = 0.011 μM-1 s-1 [106- and 1600-fold lower than those for 12-LOX oxygenation of arachidonic acid (AA), respectively]. On the other hand, for 15-LOX-1 the equivalent parameters are kcat = 4.6 s-1 and kcat/ KM = 0.21 μM-1 s-1 (3-fold higher and similar to those for 12-HpETE formation by 15-LOX-1 from AA, respectively). This contrasts with the complete lack of reaction of 15-LOX-2 with 5,15-diHpETE [Green, A. R., et al. (2016) Biochemistry 55, 2832-2840]. Our data indicate that 12-LOX is markedly inferior to 15-LOX-1 in catalyzing the production of LXB4 from 5,15-diHpETE. Platelet aggregation was inhibited by the addition of 5,15-diHpETE, with an IC50 of 1.3 μM; however, LXB4 did not significantly inhibit collagen-mediated platelet activation up to 10 μM. In summary, LXB4 is the primary product of 12-LOX and 15-LOX-1 catalysis, if 5,15-diHpETE is the substrate, with 15-LOX-1 being 20-fold more efficient than 12-LOX. LXA4 is the primary product with 5-LOX but only if 15-HpETE is the substrate. Approximately equal proportions of LXA4 and LXB4 are produced by 12-LOX but only if LTA4 is the substrate, as described previously [Sheppard, K. A., et al. (1992) Biochim. Biophys. Acta 1133, 223-234].

The reaction specificity of lipoxygenases (ALOX) impacts the biological activity of these enzymes and also constitutes the decisive parameter for enzyme classification. ALOX15 orthologs, commonly known as 12/15-lipoxygenases, have recently been suggested to exhibit mainly arachidonic acid 12-lipoxygenating specificity in mammals ranked in evolution lower than gibbons and 15-lipoxygenating specificity in the higher ranking primates (Adel et al., PNAS 113, E4266–75). This hypothesis was worked out on the basis of the functional characteristics of 15 mammalian ALOX15 orthologs with a focus on three sequence determinants affecting substrate binding in the active site (Triad Concept). This review is aimed at summarizing the currently available functional data on the reaction specificity of mammalian ALOX15 orthologs and at analyzing the primary structures of 97 mammalian ALOX15 isoforms that have been deposited in public sequence databases. Our conclusions suggest that the reaction specificity of ALOX15 orthologs can be predicted by analyzing their amino acid sequence and that 98% of all mammalian ALOX15 orthologs follow the Evolutionary Hypothesis.

Bioactive lipid mediators derived from n-3 and n-6 fatty acids are known to modulate leukocytes. Metabolic transformation of essential fatty acids to endogenous bioactive molecules plays a major role in human health. Here we tested the potential of substrates; linoleic acid (LA) and docosahexaenoic acid (DHA) and their bioactive products; resolvin D1 (RvD1) and 12- S-hydroxyeicosatetraenoic acids (HETE) to modulate macrophage plasticity and cardiac fibroblast phenotype in presence or absence of lipid metabolizing enzyme 12/15-lipoxygenase (LOX). Peritoneal macrophages and cardiac fibroblasts were isolated from wild-type (C57BL/6J) and 12/15LOX -/- mice and treated with DHA, LA, 12(S)-HETE, and RvD1 for 4, 8, 12, and 24 hr. LA, DHA, 12(S)-HETE, and RvD1 elicited mRNA expression of proinflammatory markers; tumor necrosis factor-α ( Tnf-α), interleukin 6 ( IL-6), chemokine (C-C motif) ligand 2 (Ccl2), and IL-1β in wild type (WT) and in 12/15LOX -/- macrophages at early time point (4 hr). Bioactive immunoresolvent RvD1 lowered the levels of Tnf-α, IL-6, and IL-1β at 24 hr time point. Both DHA and RvD1 stimulated the proresolving markers such as arginase 1 ( Arg-1), chitinase-like protein 3 ( Ym-1), and mannose receptor C-type 1 in WT macrophage. RvD1 induced proresolving phenotype Arg-1 expression in both WT 12/15LOX -/- macrophages even in presence of 12(S)-HETE. RvD1 peaked 5LOX expression in both WT and 12/15LOX -/- at 24 hr time point compared with DHA. RvD1 diminished cyclooxygenase-2 but upregulated 5LOX expression in fibroblast compared with DHA. In summary, the feed-forward enzymatic interaction with fatty acids substrates and direct mediators (RvD1 and 12(S)-HETE) are responsive in determining macrophages phenotype and cardiac fibroblast plasticity. Particularly, macrophages and fibroblast phenotypes are responsive to milieu and RvD1 governs the milieu-dependent chemokine signaling in presence or absence of 12/15LOX enzyme to resolve inflammation.

15-Lipoxygenase-2 (15-LOX-2) and 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) have been considered as latent mediators of diverse biological processes including cancer. However, their functions in lung adenocarcinoma (LAC) are unclear. In this study, we aimed to determine whether 15-LOX-2/15(S)-HETE is involved in the proliferation and migration of A549 cells and to identify the signaling pathways that participate in this process. We used immunohistochemistry to identify the expression levels of 15-LOX-2 in lung cancer tissue samples. The effects of 15(S)-HETE on the proliferation and migration of A549 cells under hypoxic conditions were assessed by cell viability assays, immunofluorescence, western blotting, scratch wound assays and transwell assays. We found that the expression of 15-LOX-2 was significantly up-regulated in LAC tissue samples compared with adjacent normal tissue samples. The content of 15(S)-HETE in A549 cells was increased under hypoxic conditions. Moreover, 15(S)-HETE could stimulate the expression of PCNA, cyclin A and cyclin D. In addition, siRNA of 15-LOX-2 inhibited the proliferation and migration of A549 cells in vitro. Our data also provide novel evidence demonstrating that the STAT3 pathway participates in the 15(S)-HETE-induced proliferation and migration of A549 cells. This study may provide a greater understanding of LAC metastasis and shed new light on the mechanisms by which the 15(S)-HETE/STAT3 pathway is related to this disease.

We studied the effect of SIRT1 deacetylase and PPARγ receptor activators on proinflammatory (M1), anti-inflammatory (M2) polarization of RAW264.7 macrophages and their modulating effects on insulin sensitivity of adipocytes. In M1 macrophages, the expression of TNFα and CXCL9, secretion of CXCL11, ROS generation, and content of dendritic-like cells were elevated. In M2 macrophages, expression of IGF-1 and ALOX15 factors was enhanced. SIRT1 activator (DCHC) and PPARγ receptor ligand (rosiglitazone) reduced expression of inflammatory markers TNFα and CXCL9 and increased expression of IGF-1 and ALOX15. SIRT1 inhibitor Ex527 increased the proportion of dendritic cells in macrophage populations. The paracrine effect of M1-macrophage-conditioned media attenuated insulin-dependent phosphorylation of threonine (Thr308) in Akt kinase and enhanced phosphorylation of serine (Ser473). This effect was attenuated by DCHC and rosiglitazone.

12/15 lipoxygenase (LOX) directs inflammation and lipid remodeling. However, the role of 12/15LOX in post-myocardial infarction (MI) left ventricular remodeling is unclear. To determine the role of 12/15LOX, 8–12 week-old C57BL/6 J wild-type (WT; n = 93) and 12/15LOX−/− (n = 97) mice were subjected to permanent coronary artery ligation and monitored at day (d)1 and d5 post-operatively. Post-MI d28 survival was measured in male and female mice. No-MI surgery mice were maintained as d0 naive controls. 12/15LOX−/− mice exhibited higher survival rates with lower cardiac rupture and improved LV function as compared with WT post-MI. Compared to WT, neutrophils and macrophages in 12/15LOX−/− mice were polarized towards N2 and M2 phenotypes, respectively, with increased of expression mrc-1, ym-1, and arg-1 post-MI. 12/15LOX−/− mice exhibited lower levels of pro-inflammatory 12-(S)-hydroperoxyeicosatetraenoic acid (12(S)-HETE) and higher CYP2J-derived epoxyeicosatrienoic acids (EETs) levels. CYP2J-derived 5,6-, 8,9-, 11,12-, and 14,15-EETs activated macrophage-specific hemeoxygenase (HO)-1 marked with increases in F4/80+/Ly6Clow and F4/80+/CD206high cells at d5 post-MI in 12/15LOX−/− mice. In contrast, inhibition of HO-1 led to total mortality in 12/15LOX−/− mice by post-MI d5. 12/15LOX−/− mice exhibited reduced collagen density and lower α-smooth muscle actin (SMA) expression at d5 post-MI, indicating delayed or limited fibroblast-to-myofibroblast differentiation. In conclusion, genetic deletion of 12/15LOX reduces 12(S)-HETE and activates CYP2J-derived EETs to promote effective resolution of inflammation post-MI leading to reduced cardiac rupture, improved LV function, and better survival.

Vascular structure and integrity are at the forefront of blood pressure regulation. However, there are many factors that affect the responses of the vessels. One of these is the inflammatory processes associated with high cholesterol and its modification. 15-lipoxygenase (15-LOX) is the critical enzyme in cholesterol oxidation, but this enzyme is also responsible for the synthesis of specialized proresoving lipid mediators (SPMs) called Lipoxin (Lxs) and Resolvin (Rvs). In this study, we determined serum LXA4, RvD1 and RvE1 levels in newly diagnosed hypertension (HT) and normotension (NT) cases. We evaluated how the presence of hypercholesterolemia (HC) in the follow-up changes the levels of these SPMs. We found that the three SPMs we measured decreased significantly in the presence of HC. In addition, we found a negative and significant correlation with systolic blood pressure and total cholesterol levels for the three SPMs. In conclusion, HT and HC are independent risk factors for cardiovascular death. However, the presence of HC may be an important factor for the development of HT. Increasing cholesterol levels may cause 15-LOX to shift towards LDL oxidation, thus leading to inflammation. This situation may negatively affect the vascular functions in the regulation of blood pressure. Serum LXA4, RvD1 and RvE1 measurements may provide clues that represent a shift of 15-LOX enzyme activity towards cholesterol.

The presistent increase of 12/15 lipoxygenase enzyme activity is correlated with uncontrolled inflammation, leading to organ dysfunction. ML351, a potent 12/15 lipoxygenase (12/15LOX) inhibitor, was reported to reduce infarct size and inflammation in a murine ischemic stroke model. In the presented work, we have applied three complementary experimental approaches, in-vitro, ex-vivo, and in-vivo, to determine whether pharmacological inhibition of 12/15LOX could dampen the inflammatory response in adult mice after Kdo2-Lipid A (KLA) as an endotoxin stimulator or post myocardial infarction (MI). Male C57BL/6 (8–12 weeks) mice were subjected to permanent coronary ligation thereby inducing acute heart failure (MI-d1 and MI-d5) for in-vivo studies. 12/15LOX antagonist ML351 (50 mg/kg) was subcutaneously injected 2 h post-MI, while MI-controls received saline. For ex-vivo experiments, ML351 (25 mg/kg) was injected as bolus after 5 min of inflammatory stimulus (KLA 1 μg/g) injection. Peritoneal macrophages (PMɸ) were harvested after 4 h post KLA. For in-vitro studies, PMɸ were treated with KLA (100 ng/mL), ML351 (10 µM), or KLA + ML351 for 4 h, and inflammatory response was evaluated. In-vivo, 5LOX expression was reduced after ML351 administration, inducing a compensatory increase of 12LOX that sensitized PMɸ toward a proinflammatory state. This was marked by higher inflammatory cytokines and dysregulation of the splenocardiac axis post-MI. ML351 treatment increased CD11b+ and Ly6Chigh populations in spleen and Ly6G+ population in heart, with a decrease in F4/80+ macrophage population at MI-d1. In-vitro results indicated that ML351 suppressed initiation of inflammation while ex-vivo results suggested ML351 overactivated inflammation consequently delaying the resolution process. Collectively, in-vitro, ex-vivo, and in-vivo results indicated that pharmacological blockade of lipoxygenases using ML351 impaired initiation of inflammation thereby dysregulated acute immune response in cardiac repair.

Ebselen is a potent competitive inhibitor of the active form of rabbit 15-lipoxygenase, an enzyme involved in many inflammatory diseases. Light-induced Z-to-E isomerization of the ebselen-like 2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester (BODTCM) molecule was used to convert the weak (Z)-BOTDCM inhibitor into the (E)-isomer with much higher inhibitory capacity. In this study, the binding modes of ebselen, (E)-BOTDCM and (Z)-BOTDCM, have been analyzed to provide molecular insights on the inhibitory potency of ebselen and on the geometric-isomer specificity of (E)- and (Z)-BOTDCM inhibitors. The inhibitor-enzyme structures obtained from docking and molecular dynamics simulations as well as from QM/MM calculations show that the inhibitor molecules are not coordinated to the nonheme iron in the active site. Thermal motion allows ebselen and (E)-BOTDCM to visit a wide range of the configurational space competing with the polyunsaturated fatty acid for binding at the active site. Both molecules present similar MM/PBSA binding free energies. The energy penalty for the bigger geometric deformation undergone by (E)-BODTCM would explain its lower inhibitor potency. The (Z)-isomer is the weakest inhibitor because thermal motion moves it to a region very far from the first coordination sphere of Fe, where it could not compete with the fatty acid substrate.

The omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA) is enriched in neural membranes of the CNS, and recent studies have shown a role of DHA metabolism by 15-lipoxygenase-1 (Alox15) in prefrontal cortex resolvin D1 formation, hippocampo-prefrontal cortical long-term-potentiation, spatial working memory, and anti-nociception/anxiety. In this study, we elucidated epigenetic regulation of Alox15 via histone modifications in neuron-like cells. Treatment of undifferentiated SH-SY5Y human neuroblastoma cells with the histone deacetylase (HDAC) inhibitors trichostatin A (TSA) and sodium butyrate significantly increased Alox15 mRNA expression. Moreover, Alox15 expression was markedly upregulated by Class I HDAC inhibitors, MS-275 and depsipeptide. Co-treatment of undifferentiated SH-SY5Y cells with the p300 histone acetyltransferase (HAT) inhibitor C646 and TSA or sodium butyrate showed that p300 HAT inhibition modulated TSA or sodium butyrate-induced Alox15 upregulation. Differentiation of SH-SY5Y cells with retinoic acid resulted in increased neurite outgrowth and Alox15 mRNA expression, while co-treatment with the p300 HAT inhibitor C646 and retinoic acid modulated the increases, indicating a role of p300 HAT in differentiation-associated Alox15 upregulation. Increasing Alox15 expression was found in primary murine cortical neurons during development from 3 to 10 days-in-vitro, reaching high levels of expression by 10 days-in-vitro-when Alox15 was not further upregulated by HDAC inhibition. Together, results indicate regulation of Alox15 mRNA expression in neuroblastoma cells by histone modifications, and increasing Alox15 expression in differentiating neurons. It is possible that one of the environmental influences on the immature brain that can affect cognition and memory, may take the form of epigenetic effects on Alox15 and metabolites of DHA.

Free and ester forms of unsaturated fatty acids and cholesterol are oxidized in vivo by multiple oxidants to give diverse products. Some lipid oxidation is mediated by enzymes to selectively give specific products, while others proceed randomly to produce mixtures of many kinds of regioisomers and stereoisomers. The efficacy of antioxidants against lipid oxidation depends on the nature of the oxidants and therefore the identification of oxidant is important for understanding the roles and effects of lipid oxidation and antioxidants in vivo. In the present study, the isomer distribution of hydro(pero)xyoctadecadienoates (H(p)ODEs) and hydro(pero)xyeicosatetraenoates (H(p)ETEs), the most abundant lipid oxidation products found in human plasma, produced in the oxidation of plasma by peroxyl radicals, peroxynitrite, hypochlorite, 15-lipoxygenase, and singlet oxygen were examined. It was shown that 9- and 13-(E,E)-HODEs, 13(S)-(Z,E)-HODE, and 10- and 12-(Z,E)-HODEs were specific lipid oxidation products by free radical, 15-lipoxygenase, and singlet oxygen, respectively. The isomer distribution of HODEs produced by peroxynitrite was similar to that by peroxyl radical, suggesting that the peroxynitrite mediated lipid oxidation proceeds by free radical mechanisms. The production of HODEs and HETEs by hypochlorite was very small. HODEs may be a better biomarker than HETEs since linoleates are oxidized by simpler mechanisms than arachidonates and all the HODEs isomers can be quantified more easily. These products may be used as specific biomarkers for the identification of responsible oxidants and for the assessment of oxidant-specific lipid oxidation levels and effects of antioxidants in vivo.

Alcoholic liver disease is a major human health problem leading to significant morbidity and mortality in the United States and worldwide. Dietary fat plays an important role in alcoholic liver disease pathogenesis. Herein, we tested the hypothesis that a combination of ethanol and a diet rich in linoleic acid (LA) leads to the increased production of oxidized LA metabolites (OXLAMs), specifically 9- and 13-hydroxyoctadecadienoic acids (HODEs), which contribute to a hepatic proinflammatory response exacerbating liver injury. Mice were fed unsaturated (with a high LA content) or saturated fat diets (USF and SF, respectively) with or without ethanol for 10 days, followed by a single binge of ethanol. Compared to SF+ethanol, mice fed USF+ethanol had elevated plasma alanine transaminase levels, enhanced hepatic steatosis, oxidative stress, and inflammation. Plasma and liver levels of 9- and 13-HODEs were increased in response to USF+ethanol feeding. We demonstrated that primarily 9-HODE, but not 13-HODE, induced the expression of several proinflammatory cytokines in vitro in RAW264.7 macrophages. Finally, deficiency of arachidonate 15-lipoxygenase, a major enzyme involved in LA oxidation and OXLAM production, attenuated liver injury and inflammation caused by USF+ethanol feeding but had no effect on hepatic steatosis. This study demonstrates that OXLAM-mediated induction of a proinflammatory response in macrophages is one of the potential mechanisms underlying the progression from alcohol-induced steatosis to alcoholic steatohepatitis.

In cancer cells the small compounds erastin and RSL3 promote a novel type of cell death called ferroptosis, which requires iron‐dependent accumulation of lipid reactive oxygen species. Here we assessed the contribution of lipid peroxidation activity of lipoxygenases (LOX) to ferroptosis in oncogenic Ras‐expressing cancer cells. Several 12/15‐LOX inhibitors prevented cell death induced by erastin and RSL3. Furthermore, siRNA‐mediated silencing of ALOX15 significantly decreased both erastin‐induced and RSL3‐induced ferroptotic cell death, whereas exogenous overexpression of ALOX15 enhanced the effect of these compounds. Immunofluorescence analyses revealed that the ALOX15 protein consistently localizes to cell membrane during the course of ferroptosis. Importantly, treatments of cells with ALOX15‐activating compounds accelerated cell death at low, but not high doses of erastin and RSL3. These observations suggest that tumor ferroptosis is promoted by LOX‐catalyzed lipid hydroperoxide generation in cellular membranes.

Eicosanoids are inflammatory signaling lipids that are biosynthesized in response to cellular injury or threat. They were originally thought to be pro-inflammatory molecules, but members of at least one subclass, the lipoxins, are able to resolve inflammation. One step in lipoxin synthesis is the oxygenation of arachidonic acid by 15-lipoxygenase (15-LOX). 15-LOX contains two domains: a Ca2+ binding PLAT domain and a catalytic domain. 15-LOX is a soluble cytosolic protein until binding of Ca2+ to the PLAT domain promotes translocation to the membrane surface. The role of 15-LOX structural dynamics in this translocation has remained unclear. We investigated the dynamics of 15-LOX isoform B (15-LOX-2) upon binding of Ca2+ and ligands, as well as upon membrane association using hydrogen–deuterium exchange mass spectrometry (HDX-MS). We used HDX-MS to probe the solvent accessibility and backbone flexibility of 15-LOX-2, revealing significant differences in deuterium incorporation between the PLAT and catalytic domains, with the PLAT domain demonstrating higher flexibility. Comparison of HDX for 15-LOX-2 in the presence and absence of Ca2+ indicates there are few differences in structural dynamics. Furthermore, our HDX results involving nanodisc-associated 15-LOX-2 suggest that significant structural and dynamic changes in 15-LOX-2 are not required for membrane association. Our results also show that a substrate lipid binding to the active site in the catalytic domain does induce changes in incorporation of deuterium into the PLAT domain. Overall, our results challenge the previous hypothesis that Ca2+ binding induces major structural changes in the PLAT domain and support the hypothesis that is interdomain communication in 15-LOX-2.

The metabolic transformation of fatty acids to form oxylipids using 12/15-lipoxygenase (LOX) can promote either resolving or nonresolving inflammation. However, the mechanism of how 12/15-LOX interacts with polyunsaturated fatty acids (PUFA) in postmyocardial infarction (post-MI) healing is unclear. Here, we reported the role of 12/15-LOX in post-MI cardiac remodeling in a PUFA [10% (wt/wt), 22 kcal]-enriched environment. Wild-type (WT; C57BL/6J) and 12/15-LOX-null (12/15-LOX−/−) male mice of 8–12 wk of age were fed a PUFA-enriched diet for 1 mo and subjected to permanent coronary artery ligation. Post-MI mice were monitored for day 1 or until day 5 along with standard diet-fed MI controls. No-MI surgery mice served as naïve controls. PUFA-fed WT and 12/15-LOX−/−mice improved ejection fraction and reduced lung edema greater than WT mice at day 5 post-MI ( P < 0.05). Post-MI, neutrophil density was decreased in PUFA-fed WT and 12/15-LOX−/−mice at day 1 ( P < 0.05). Deletion of 12/15-LOX in mice led to increased cytochrome P-450-derived bioactive lipid mediator epoxyeicosatrienoic acids (EETs), i.e., 11,12-EpETrE and 14,15-EpETrE, which were further enhanced by acute PUFA intake post-MI. Macrophage density was decreased in WT + PUFA and 12/15-LOX−/−mice compared with their respective standard diet-fed WT controls at day 5 post-MI. 12/15-LOX−/−+ PUFA mice displayed an increased expression of chemokine (C-C motif) ligand 2 and reparative macrophages markers ( Ym-1, Mrc-1, and Arg-1, all P < 0.05) in the infarcted area. Furthermore, 12/15-LOX−/−mice, with or without PUFA, showed reduced collagen deposition at day 5 post-MI compared with WT mice. In conclusion, deletion of 12/15-LOX and short-term exposure of PUFA promoted leukocyte clearance, thereby limiting cardiac remodeling and promoting an effective resolution of inflammation.NEW & NOTEWORTHY This study determined that 1) deletion of 12/15-lipoxygenase (LOX) promotes the generation of epoxyeicosatrienoic acids, the cytochrome P-450-derived metabolites in postmyocardial infarction (post-MI) healing; 2) acute exposure of fatty acids to 12/15-LOX−/−mice drives leukocyte (neutrophils and macrophages) clearance post-MI; and 3) metabolic transformation of fats is the significant contributor in leukocyte clearance to drive either resolving or nonresolving inflammation post-MI.

Aims Our previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & methods We first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC–MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. Results Analysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. Conclusion Our data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.

The function of δ opioid receptors (DOR) expressed by peripheral pain-sensing neurons (nociceptors) is regulated by both cyclooxygenase- and lipoxygenase (LOX)-dependent arachidonic acid (AA) metabolites. Whereas cyclooxygenase metabolites enhance responsiveness, LOX metabolites elicit a refractory, nonsignaling state of the DOR receptor system for antinociceptive signaling. In this study, using high-performance liquid chromatography-tandem mass spectrometry analyses, we have found that the 12-/15-LOX metabolites, 12-hydroxyeicosatetraenoic acid (HETE) and 15-HETE, were elevated after treatment of adult rat primary sensory neuron cultures with AA. Exogenously applied 12-HETE and 15-HETE, but not 5-HETE, completely prevented DOR and κ opioid receptor (KOR) agonist–mediated inhibition of prostaglandin E2 (PGE2)-stimulated cAMP accumulation, but not inhibition, by the 5-HT1 receptor agonist 5-carboxamidotryptamine in cultured peripheral sensory neurons and in Chinese hamster ovary (CHO) cells heterologously expressing DOR or KOR. Similarly, intraplantar injection of 12- or 15-HETE, either alone or in combination, prevented DOR agonist-mediated inhibition of PGE2-evoked thermal allodynia. Further, both AA- and carrageenan-mediated induction of the nonresponsive state of the DOR system was blocked by an intraplantar coinjection of the 12-/15-LOX inhibitors baicalein and luteolin. In contrast to the regulation of cAMP signaling, pretreatment with 12- and 15-HETE had no effect on either DOR or KOR agonist- mediated activation of extracellular signal-regulated kinase in peripheral sensory neurons or CHO cells. These results suggest that the analgesic efficacy of peripherally restricted opioids for treatment of inflammatory pain may be enhanced by adjunct inhibition of LOX activity.

Two series of diaza derivatives were prepared by solvent-free condensation of benzoic acid and 4-substituted phenylhydrazines in order to obtain phenylhydrazides (HYD series) and, by oxidation of these compounds, the corresponding benzoyldiazenes (DIA series). Both sets were evaluated as inhibitors of soybean 15-lipoxygenase activity and antioxidant capability in the FRAP and CUPRAC assays. The most potent inhibitors of both series exhibited IC50 values in the low micromolar range. Kinetic studies showed that at least the more active compounds were competitive inhibitors. Docking results indicated that the most potent inhibitor interacts strongly with Ile-839 and iron in the active site.

Cerebral ischemia exhibits a multiplicity of pathophysiological mechanisms. Taurine (Tau), an endogenous substance, possesses a number of cytoprotective properties. The aim of the present study was to examine the neuroprotective effect of Tau, through affecting 12/15 lipoxygenase (12/15-LOX) signal pathway in an acute permanent middle cerebral artery occlusion (MCAO) model of rats.Sprague-Dawley rats were randomly divided into 3 groups (n = 10), namely the sham-operated group, MCAO group and Tau group. Tau was intraperitoneally administrated immediately after cerebral ischemia. At 24 h after MCAO, neurological function score, brain water content and infarct volume were assessed. The expression of 12/15-lipoxygenase (12/15-LOX), p38 mitogen-activated protein kinase (p38 MAPK), and cytosolic phospholipase A2 (cPLA2) was measured by Western blot. Enzyme-linked immunosorbent assay was used to evaluate the inflammatory factors TNF-α, IL-1β and IL-6 in serum.Compared with MCAO group, taurine significantly improved neurological function and significantly reduced brain water content (p 0.05) and infarct volume (p 0.05). Consistent with these indices, the overexpressions of 12/15-LOX, p38 MAPK, cPLA2, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were significantly decreased in Tau group (p 0.05).Taurine protected the brain from damage caused by MCAO; this effect may be through down-regulation of 12/15-LOX, p38 MAPK, and cPLA2.

Background The 12/15-lipoxygenase (12/15-LO) enzyme is upregulated in the brains of patients with Alzheimer’s disease (AD), and its expression levels influence the onset of the AD-like phenotype in mouse models. However, whether targeting this pathway after the neuropathology and behavioral impairments have been established remains to be investigated. Methods Triple transgenic (3xTg) mice received either PD146176—a selective and specific pharmacological inhibitor of 12/15-LO—or placebo starting at 12 months of age for 12 weeks. They were then assessed for the effect of the treatment on neuropathologies and behavioral impairments. Results At the end of the study, mice in the control group showed a worsening of memory and learning abilities, whereas mice receiving PD146176 were undistinguishable from wild-type mice. The same group also had significantly lower amyloid beta levels and deposition, less tau neuropathology, increased synaptic integrity, and autophagy activation. Ex vivo and in vitro genetic and pharmacological studies found that the mechanism involved in these effects was the activation of neuronal autophagy. Conclusions Our findings provide new insights into the disease-modifying action of 12/15-LO pharmacological inhibition and establish it as a viable therapeutic approach for patients with AD.

Cancer is a multifactorial disorder involving multiplicity of interrelated signaling pathways and molecular targets. To that end, a multi-target design strategy was adopted to develop some 1,2,3-triazoles hybridized with some pharmacophoric anticancer fragments, as first-in-class simultaneous inhibitors of COX-2, 15-LOX and tumor associated carbonic anhydrase enzymes. Results revealed that compounds 5a, 5d, 8b and 8c were potent inhibitors of COX-2 and 15-LOX enzymes. COX-2 inhibitory activity was further demonstrated by the inhibition of the accumulation of 6-keto-PGF1α, a metabolite of COX-2 products in two cancer cell lines. The sulfonamide bearing derivatives 5d and 8c were effective nanomolar and submicromolar inhibitors of tumor associated hCA XII isoform, respectively. Strong to moderate inhibitory activities were observed in the in vitro antiproliferative assay on lung (A549), liver (HepG2) and breast (MCF7) cancer cell lines (IC50 2.37–28.5 μM) with high safety margins on WI-38 cells. A cytotoxic advantage of CA inhibition was observed as an increased activity against tumor cell lines expressing CA IX/XII. Further mechanistic clues for the anticancer activities of compound 5a and its sulfonamide analog 5d were derived from induction of cell cycle arrest at G2/M phase. They also triggered apoptosis via increasing expression levels of caspase-9 and Bax together with suppressing that of Bcl-2. The in vitro anti-tumor activity was reflected as reduced tumor size upon treatment with 8c in an in vivo cancer xenograft model. Docking experiments on the target enzymes supported their in vitro data and served as further molecular evidence. In silico calculations and ligand efficiency indices were promising. In light of these data, such series could offer new structural insights into the understanding and development of multi-target COX-2/15-LOX/hCA inhibitors for anticancer outcomes.

Herin we report the design, synthesis, full characterization and biological investigation of new 15-LOX/COX dual inhibitors based on 1,3-thiazolidin-4-one (15-lipoxygenase pharmacophore) and 1,3,4-thiadiazole (COX pharmacophore) scaffolds. This series of molecular modifications is an extension of a previously reported series to further explore the structural activity relationship. Compounds 3a, 4e, 4n, 4q, 7 and 8 capable of inhibiting 15-LOX at (2.74, 4.2, 3.41, 10.21, 3.71 and 3.36 µM, respectively) and COX-2 at (0.32, 0.28, 0.28, 0.1, 0.28 and 0.27 µM, respectively). The results revealed that binding to 15-LOX and COX is sensitive to the bulkiness of the substituents at the 5 positions. 15-LOX bind better with small substituents, while COXs bind better with bulky substituents. Compounds 3a, 4r and 4q showed comparable in vivo anti-inflammatory activity to the reference drug (celecoxib). The ulcer liability test showed no sign of ulceration which ensures the safe gastric profile. Docking study was performed to explore the possible mode of interaction of the new compounds with the active site of human 15-LOX and COX-2. This study discloses some structural features for binding to 15-LOX and COX, thus pave the way to design anti-inflammatory agents with balanced dual inhibition of these enzymes.

His596 of human ALOX12 has been suggested to interact with the COO--group of arachidonic acid during ALOX catalysis. In mammalian ALOX15 orthologs Gln596 occupies this position and this amino acid exchange might contribute to the functional differences between the two ALOX-isoforms. To explore the role of Gln596 for ALOX15 functionality we mutated this amino acid to different residues in rabbit and human ALOX15 and investigated the impact of these mutations on structural, catalytic and allosteric enzyme properties. To shed light on the molecular basis of the observed functional alterations we performed in silico substrate docking studies and molecular dynamics simulations and also explored the impact of Gln596 exchange on the protein structure. The combined theoretical and experimental data suggest that Gln596 may not directly interact with the COO--group of arachidonic acid. In contrast, mutations at Gln596 destabilize the secondary and tertiary structure of ALOX15 orthologs, which may be related to a disturbance of the electrostatic interaction network with other amino acids in the immediate surrounding. Moreover, our MD-simulations suggest that the geometry of the dimer interface depends on the structure of substrate bound inside the substrate-binding pocket and that Gln596Ala exchange impairs the allosteric properties of the enzyme. Taken together, these data indicate the structural and functional importance of Gln596 for ALOX15 catalysis.

Aspirin-exacerbated respiratory disease (AERD) is characterized by asthma, chronic rhinosinusitis with nasal polyps (CRSwNP), and an intolerance of medications that inhibit cyclooxygenase-1. Patients with AERD have more severe upper and lower respiratory tract disease than do aspirin-tolerant patients with CRSwNP. A dysregulation in arachidonic acid metabolism is thought to contribute to the enhanced sinonasal inflammation in AERD.Our aim was to utilize an unbiased approach investigating arachidonic acid metabolic pathways in AERD.Single-cell RNA sequencing (10× Genomics, Pleasanton, Calif) was utilized to compare the transcriptional profile of nasal polyp (NP) cells from patients with AERD and patients with CRSwNP and map differences in the expression of select genes among identified cell types. Findings were confirmed by traditional real-time PCR. Lipid mediators in sinonasal tissue were measured by mass spectrometry. Localization of various proteins within NPs was assessed by immunofluorescence.The gene encoding for 15-lipooxygenase (15-LO), ALOX15, was significantly elevated in NPs of patients with AERD compared to NPs of patients with CRSwNP (P .05) or controls (P .001). ALOX15 was predominantly expressed by epithelial cells. Expression levels significantly correlated with radiographic sinus disease severity (r = 0.56; P .001) and were associated with asthma. The level of 15-oxo-eicosatetraenoic acid (15-Oxo-ETE), a downstream product of 15-LO, was significantly elevated in NPs from patients with CRSwNP (27.93 pg/mg of tissue) and NPs from patients with AERD (61.03 pg/mg of tissue) compared to inferior turbinate tissue from controls (7.17 pg/mg of tissue [P .001]). Hydroxyprostaglandin dehydrogenase, an enzyme required for 15-Oxo-ETE synthesis, was predominantly expressed in mast cells and localized near 15-LOEpithelial and mast cell interactions, leading to the synthesis of 15-Oxo-ETE, may contribute to the dysregulation of arachidonic acid metabolism via the 15-LO pathway and to the enhanced sinonasal disease severity observed in AERD.

The polarization of macrophages is critical to inflammation and tissue repair, with unbalanced macrophage polarization associated with critical dysfunctions of the immune system. Cytochrome P450 1A1 (CYP1A1) is a hydroxylase mainly controlled by the inflammation-limiting aryl hydrocarbon receptor (AhR), which plays a critical role in mycoplasma infection, oxidative stress injury, and cancer. Arginase-1 (Arg-1) is a surrogate for polarized alternative macrophages and is important to the production of nitric oxide (NO) by the modulation of arginine. In the present study, we found CYP1A1 to be upregulated in IL-4-stimulated mouse peritoneal macrophages (PMs) and human peripheral blood monocytes. Using CYP1A1-overexpressing RAW264.7 cells (CYP1A1/RAW) we found that CYP1A1 augmented Arg-1 expression by strengthening the activation of the JAK1/STAT6 signaling pathway in macrophages treated with IL-4. 15(S)-HETE, a metabolite of CYP1A1 hydroxylase, was elevated in IL-4-induced CYP1A1/RAW cells. Further, in macrophages, the loss-of-CYP1A1-hydroxylase activity was associated with reduced IL-4-induced Arg-1 expression due to impaired 15(S)-HETE generation. Of importance, CYP1A1 overexpressing macrophages reduced the inflammation associated with LPS-induced peritonitis. Taken together, these findings identified a novel signaling axis, CYP1A1-15(S)-HETE-JAK1-STAT6, that may be a promising target for the proper maintenance of macrophage polarization and may also be a means by which to treat immune-related disease due to macrophage dysfunction.

The present study includes design and synthesis of new molecular hybrids of 2-methylthiobenzimidazole linked to various anti-inflammatory pharmacophores through 2-aminothiazole linker, to investigate the effect of such molecular variation on cyclooxygenase (COX) and 15-lipoxygenase (15-LOX) enzymes inhibition as well as in vivo anti-inflammatory activity. The chemical structures of new hybrids were confirmed using different spectroscopic tools and elemental analyses. Benzimidazole-thiazole hybrids linked to acetyl moiety 13, phenyl thiosemicarbazone 14, 1,3-thiazolines 15a-c and 4-thiazolidinone 16 exhibited significant COX-2 inhibition (IC50 = 0.045–0.075 µM) with significant COX-2 selectivity indices (SI = 142–294). All hybrids revealed potent 15-LOX inhibitory activity (IC50 = 1.67–6.56 µM). Benzimidazole-thiazole hybrid 15b was the most potent dual COX-2 (IC50 = 0.045 µM, SI = 294) inhibitor approximate to celecoxib (COX-2; IC50 = 0.045 µM, SI = 327), with double inhibitory activity versus 15-LOX enzyme (IC50 = 1.67 µM) relative to quercetin (IC50 = 3.34 µM). Three hybrids (14, 15b & 16) were selected for in vivo screening using carrageenan-induced paw edema method. Benzimidazole-thiazole hybrid linked to 4-thiazolidinone 16 showed the maximum edema inhibition at both 3 h and 4 h intervals as well (~119% and 102% relative to indomethacin, respectively). The gastric ulcerogenic effect of benzimidazole-thiazole hybrid 16 was estimated compared with indomethacin showing superior gastrointestinal safety profile. In bases of molecular modeling; all new active hybrids were subjected to docking simulation into active sites of COX-2 and 15-LOX enzymes to study the binding mode of these novel potent dual COX-2/15-LOX inhibitors.