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Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH.

Accumulation of senescent cells is associated with the progression of pulmonary fibrosis, but mechanisms accounting for this linkage are not well understood. To explore this issue, we investigated whether a class of biologically active profibrotic lipids, the leukotrienes (LT), is part of the senescence-associated secretory phenotype. The analysis of conditioned medium (CM), lipid extracts, and gene expression of LT biosynthesis enzymes revealed that senescent cells secreted LT, regardless of the origin of the cells or the modality of senescence induction. The synthesis of LT was biphasic and followed by antifibrotic prostaglandin (PG) secretion. The LT-rich CM of senescent lung fibroblasts (IMR-90) induced profibrotic signaling in naive fibroblasts, which were abrogated by inhibitors of ALOX5, the principal enzyme in LT biosynthesis. The bleomycin-induced expression of genes encoding LT and PG synthases, level of cysteinyl LT in the bronchoalveolar lavage, and overall fibrosis were reduced upon senescent cell removal either in a genetic mouse model or after senolytic treatment. Quantification of ALOX5+ cells in lung explants obtained from idiopathic pulmonary fibrosis (IPF) patients indicated that half of these cells were also senescent (p16Ink4a+). Unlike human fibroblasts from unused donor lungs made senescent by irradiation, senescent IPF fibroblasts secreted LTs but failed to synthesize PGs. This study demonstrates for the first time to our knowledge that senescent cells secrete functional LTs, significantly contributing to the LT pool known to cause or exacerbate IPF.

Ischemia–reperfusion (I/R) injury is a crucial factor causing liver injury in the clinic. Recent research has confirmed that human adipose‐derived stem cells (ADSCs) can differentiate into functional hepatocytes. However, the mechanism of the effects of ADSCs in the treatment of liver injury remains unclear. The characteristics of ADSCs were first identified, and exosome‐derived ADSCs were isolated and characterized. The function and mechanism of action of miR‐183 and arachidonate 5‐lipoxygenase (ALOX5) were investigated by functional experiments in HL‐7702 cells with I/R injury and in I/R rats. Our data disclosed that exosome release from ADSCs induced proliferation and inhibited apoptosis in HL‐7702 cells with I/R injury. The effect of miR‐183 was similar to that of exosomes derived from ADSCs. In addition, ALOX5, as a target gene of miR‐183, was involved in the related functions of miR‐183. Moreover, in vivo experiments confirmed that miR‐183 and exosomes from ADSCs could improve liver injury in rats and inhibit the MAPK and NF‐κB pathways. All of these findings demonstrate that exosomes derived from ADSCs have a significant protective effect on hepatic I/R injury by regulating the miR‐183/ALOX5 axis, which might provide a therapeutic strategy for liver injury. [ABSTRACT FROM AUTHOR]

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 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].

ObjectivesN-acetylcysteine (NAC) is a clinically approved thiol-containing redox modulatory compound currently in trials for many neurological and psychiatric disorders. Although generically labeled as an "antioxidant," poor understanding of its site(s) of action is a barrier to its use in neurological practice. Here, we examined the efficacy and mechanism of action of NAC in rodent models of hemorrhagic stroke.MethodsHemin was used to model ferroptosis and hemorrhagic stroke in cultured neurons. Striatal infusion of collagenase was used to model intracerebral hemorrhage (ICH) in mice and rats. Chemical biology, targeted lipidomics, arachidonate 5-lipoxygenase (ALOX5) knockout mice, and viral-gene transfer were used to gain insight into the pharmacological targets and mechanism of action of NAC.ResultsNAC prevented hemin-induced ferroptosis by neutralizing toxic lipids generated by arachidonate-dependent ALOX5 activity. NAC efficacy required increases in glutathione and is correlated with suppression of reactive lipids by glutathione-dependent enzymes such as glutathione S-transferase. Accordingly, its protective effects were mimicked by chemical or molecular lipid peroxidation inhibitors. NAC delivered postinjury reduced neuronal death and improved functional recovery at least 7 days following ICH in mice and can synergize with clinically approved prostaglandin E2 (PGE2 ).InterpretationNAC is a promising, protective therapy for ICH, which acted to inhibit toxic arachidonic acid products of nuclear ALOX5 that synergized with exogenously delivered protective PGE2 in vitro and in vivo. The findings provide novel insight into a target for NAC, beyond the generic characterization as an antioxidant, resulting in neuroprotection and offer a feasible combinatorial strategy to optimize efficacy and safety in dosing of NAC for treatment of neurological disorders involving ferroptosis such as ICH. Ann Neurol 2018;84:854-872.

As pro-inflammatory lipid mediators, leukotrienes have pathophysiological activities in several inflammatory diseases, including psoriasis. In the biosynthesis of leukotrienes from arachidonic acid, 5-lipoxygenase catalyzes the first two steps. In the present study, we showed that nutmeg (Myristica fragrans) strongly inhibited the catalytic activity of 5-lipoxygenase. To characterize the bioactive component(s) of nutmeg, we performed 5-lipoxygenase inhibitory activity-guided fractionation of aqueous ethanol extract of nutmeg, resulting in the isolation of malabaricone C having antioxidant activity. Malabaricone C exhibited potent competitive inhibition of 5-lipoxygenase with an IC 50 value of 0.2 μM. In mice with imiquimod-induced psoriasis-like skin lesions, topical application of 2 mM malabaricone C significantly ameliorated hyperplasia and inflammatory cell infiltration, and suppressed the expression of the psoriasis-associated genes S100a9 , Krt1 , Il17a , and Il22. Lipid metabolome analysis of these psoriasis-like skin lesions showed that malabaricone C markedly decreased the level of leukotriene B 4 but did not significantly increase the other pro-inflammatory lipid mediators. These findings suggest that malabaricone C decreases LTB 4 by the 5-lipoxygenase inhibition and ameliorates the symptoms of psoriasis-like skin inflammation. [Display omitted] • 5-LOX inhibitory activity-guided fractionation of nutmeg led to the isolation of MLB-C. • MLB-C competitively inhibited 5-LOX activity with an IC 50 of 0.2 μM. • MLB-C markedly reduced LTB 4 production in mouse psoriasis-like skin. • MLB-C markedly suppressed psoriasis-associated genes in the lesions. [ABSTRACT FROM AUTHOR]

Summary: Background: Arachidonate 5-lipoxygenase (Alox5) belongs to a class of nonheme iron-containing dioxygenases involved in the catalysis of leukotriene biosynthesis. However, the effects of Alox5 itself on pathological cardiac remodeling and heart failure remain elusive. Methods: The role of Alox5 in pathological cardiac remodeling was investigated by Alox5 genetic depletion, AAV9-mediated overexpression in cardiomyocytes, and a bone marrow (BM) transplantation approach. Neonatal rat cardiomyocytes were used to explore the effects of Alox5 in vitro. Molecular and signaling pathways were revealed by CUT &Tag, IP-MS, RNA sequencing and bioinformatic analyses. Findings: Untargeted metabolomics showed that serum 5-HETE (a primary product of Alox5) levels were little changed in patients with cardiac hypertrophy, while Alox5 expression was significantly upregulated in murine hypertensive cardiac samples and human cardiac samples of hypertrophy, which prompted us to test whether high Alox5 levels under hypertensive stimuli were directly associated with pathologic myocardium in an enzymatic activity-independent manner. Herein, we revealed that Alox5 deficiency significantly ameliorated transverse aortic constriction (TAC)-induced hypertrophy. Cardiomyocyte-specific Alox5 depletion attenuated hypertensive ventricular remodeling. Conversely, cardiac-specifical Alox5 overexpression showed a pro-hypertrophic cardiac phenotype. Ablation of Alox5 in bone marrow-derived cells did not affect pathological cardiac remodeling and heart failure. Mechanically, Runx2 was identified as a target of Alox5. In this regard, Alox5 PEST domain could directly bind to Runx2 PTS domain, promoting nuclear localization of Runx2 in an enzymatic activity-independent manner, simultaneously contributed to liquid–liquid phase separation (LLPS) of Runx2 at specific domain in the nucleus and increased transcription of EGFR in cardiomyocytes. Runx2 depletion alleviated hypertrophy in Ang II-pretreated Alox5-overexpressing cardiomyocytes. Interpretation: Overall, our study demonstrated that targeting Alox5 exerted a protective effect against cardiac remodeling and heart failure under hypertensive stimuli by disturbing LLPS of Runx2 and substantial reduction of EGFR transcription activation in cardiomyocytes. Our findings suggest that negative modulation of Alox5-Runx2 may provide a therapeutic approach against pathological cardiac remodeling and heart failure. Funding: National Natural Science Foundation of China.

Green marine macroalgae, particularly Ulva lactuca, is an essential constituent of the cuisines in many Asian countries. The present work aims to separate a bioactive pyrone attached benzochromene analogue, named as ulvapyrone from the organic extract of U. lactuca, followed by its structural characterisation as 2-{(6a′-hydroxyethyl-4′-methyltetrahydro-2H-pyran-2′-one)-6′-yl}-4-methyl-7-ethylacetate-8-hydroxy-7, 8-dihydrobenzo [de]chromene. Ulvapyrone exhibited prospective inhibition property against arachidonate 5-lipoxygenase (IC50 ∼1 mg mL−1) comparable to that demonstrated by ibuprofen (IC50 0.9 mg mL−1), which connoted its anti-inflammatory activity. The studied benzochromene exhibited promising antioxidant potential (IC50 0.5–0.6 mg mL−1), which further reinforced its attenuation property against 5-lipoxygenase. Bioactivities of ulvapyrone were linearly correlated with electronic parameter (topological polar surface area ∼102) along with less binding energy (–8.22 kcal mol−1) with the allosteric site of 5-lipoxygenase. In silico predictions of physicochemical parameters along with absorption, distribution, metabolism and excretion could recognise the acceptable oral bioavailability of ulvapyrone. [ABSTRACT FROM AUTHOR]

Objective: To determine the pharmacodynamic material basis and mechanism of Danbei Yifei formula on pulmonary fibrosis. Methods: Starting with the clear absorbed components of Danbei Yifei formula or the potential effective components in line with the five rules of Ribinsky, the network pharmacology method and technology of traditional Chinese medicine were used to predict and analyze the action targets of Danbei Yifei formula in vivo, such as Salvia miltiorrhiza, PINBEI, Taoren, etc. On the basis of enrichment analysis, the core pathway of Danbei Yifei formula in the treatment of pulmonary fibrosis was identified, and the binding energy of drug ligand and protein target was determined through molecular docking technology simulation and verification, and its affinity and stability were evaluated. To clarify the material basis and mechanism of Danbei Yifei formula in the treatment of pulmonary fibrosis. Result: The results of network pharmacology prediction of traditional Chinese medicine showed that Danbei Yifei formula contained 72 potential pharmacodynamic components and 26 corresponding targets, including CHRM1、 MAPK14、 CCL2、 ADRB1、 PTGS1、 PPARG、 ALOX5、 Pde3a、 CHRM2、 Adrb2、 TNF、 JUN、 Adora2a、 LTA4H、 CYP1A2、 OPRD1、 CHRM3、 DRD2、 OPRM1、 ARG1、 EDNRA、 Il6st、 TACR1、 MMP1、 MMP8、 Ptgs2, which were related to pulmonary fibrosis and pulmonary fibrosis Lung related diseases are highly correlated. There were 26 Go items (P < 0.05) in go functional enrichment analysis, including 22 biological process (BP), 9 cellular component (CC) and 3 molecular function (MF) categories. The results of network pharmacology showed that many components, such as protocatechuic acid and aminosuccinic acid, had direct effects on known targets of pulmonary fibrosis. Conclusion: Danbei Yifei formula contains many effective components which have inhibitory effect on pulmonary fibrosis, and it may play its role through the mechanism of multi-component and multi-target synergistic effect. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a complex, multifactorial disease in which different neuropathological mechanisms are likely involved, including those associated with pathological tau and Aβ species as well as neuroinflammation. In this context, the development of single multitargeted therapeutics directed against two or more disease mechanisms could be advantageous. Starting from a series of 1,5-diarylimidazoles with microtubule (MT)-stabilizing activity and structural similarities with known NSAIDs, we conducted structure-activity relationship studies that led to the identification of multitargeted prototypes with activities as MT-stabilizing agents and/or inhibitors of the cyclooxygenase (COX) and 5-lipoxygenase (5-LOX) pathways. Several examples are brain-penetrant and exhibit balanced multitargeted in vitro activity in the low μM range. As brain-penetrant MT-stabilizing agents have proven effective against tau-mediated neurodegeneration in animal models, and because COX- and 5-LOX-derived eicosanoids are thought to contribute to Aβ plaque deposition, these 1,5-diarylimidazoles provide tools to explore novel multitargeted strategies for AD and other neurodegenerative diseases.

Gasdermin B (GSDMB) on chromosome 17q21 demonstrates a strong genetic linkage to asthma, but its function in asthma is unknown. Here we identified that GSDMB is highly expressed in lung bronchial epithelium in human asthma. Overexpression of GSDMB in primary human bronchial epithelium increased expression of genes important to both airway remodeling [TGF-β1, 5-lipoxygenase (5-LO)] and airway-hyperresponsiveness (AHR) (5-LO). Interestingly, hGSDMBZp3-Cre mice expressing increased levels of the human GSDMB transgene showed a significant spontaneous increase in AHR and a significant spontaneous increase in airway remodeling, with increased smooth muscle mass and increased fibrosis in the absence of airway inflammation. In addition, hGSDMBZp3-Cre mice showed increases in the same remodeling and AHR mediators (TGF-β1, 5-LO) observed in vitro in GSDMB-overexpressing epithelial cells. GSDMB induces TGF-β1 expression via induction of 5-LO, because knockdown of 5-LO in epithelial cells overexpressing GSDMB inhibited TGF-β1 expression. These studies demonstrate that GSDMB, a gene highly linked to asthma but whose function in asthma is previously unknown, regulates AHR and airway remodeling without airway inflammation through a previously unrecognized pathway in which GSDMB induces 5-LO to induce TGF-β1 in bronchial epithelium.

Biochemical analysis of secondary metabolites of marine old-woman octopus Cistopus indicus (family Octopodidae) led to the identification of two sixteen-membered spiro-linked macrocyclic bislactones, named as cistobislactone A and cistobislactone B with unprecedented feature of henicos framework, based on extensive spectroscopic analyses. Cistobislactone B exhibited potential inhibition property against arachidonate 5-lipoxygenase (IC50 2.18 mM) than that demonstrated by cistobislactone A (IC50 2.54 mM) and standard non-steroidal anti-inflammatory agent ibuprofen (IC50 4.50 mM) thus signifying the higher anti-inflammatory activity of the cistobislactone B analogue. The studied macrocyclic bislactones exhibited promising antioxidant potential, in which cistobislactone B exhibited potential radical quenching (IC50 2.33 mM) and hydrogen peroxide scavenging (IC50 1.81 mM) activities that were proximal to the commercial anti-oxidant α-tocopherol (IC50 ~ 1.60 mM). This further reinforced its attenuation property against arachidonate 5-lipoxygenase. Considerably greater electronic properties coupled with balanced hydrophobicity of cistobislactone B could ascribe the superior ligand-receptor interfaces leading to its anti-inflammatory activity. Molecular docking analysis of cistobislactone B with 5-lipoxygenase recorded lesser docking score (−12.24 kcal mol−1) and binding energy (−11.24 kcal mol−1), which further supported its anti-inflammatory activity. Cistobislactone B, with six fold lesser value of inhibition constant (Ki 5.76 nM) towards 5-lipoxygenase than that displayed by cistobislactone A, could describe the superior protein-ligand interactions of the former. The undescribed cistobislactone B might be a potential natural anti-inflammatory lead to moderate the odds of inflammatory pathologies. [ABSTRACT FROM AUTHOR]

Propolis was collected from honeybee hives in three geographically distinct Algerian climates and extracts were characterized for composition and bioactivity. Bees were identified as native subspecies using an in-silico DraI mtDNA COI-COII test. Over 20 compounds were identified in extracts by LC-MS. Extracts from the Medea region were more enriched in phenolic content (302±28 mg GAE/g of dry extract) than those from Annaba and Ghardaia regions. Annaba extracts had the highest flavonoid content (1870±385 mg QCE/g of dry extract). Medea extracts presented the highest free-radical scavenging activity (IC 50 =13.5 μg/mL) using the DPPH radical assay while Ghardaia extracts from the desert region were weak (IC 50 >100 μg/mL). Antioxidant activities measured using AAPH oxidation of linoleic acid were similar in all extracts with IC 50 values ranging from 2.9 to 4.9 μg/mL. All extracts were cytotoxic (MTT assay) and proapoptotic (Annexin-V) against human leukemia cell lines in the low μg/mL range, although the Annaba extract was less active against the Reh cell line. Extracts inhibited cellular 5-lipoxygenase product biosynthesis with IC 50 values ranging from 0.6 to 3.2 μg/mL. Overall, examined propolis extracts exhibited significant biological activity that warrant further characterization in cellular and in vivo models., (© 2024 The Authors. Chemistry & Biodiversity published by Wiley‐VHCA AG.)

Continuing our search to find more potent and selective 5-LOX inhibitors, we present now the enzymatic evaluation of seventeen isoflavones (IR) and nine isoflavans (HIR), and their in vitro and in cellulo potency against human leukocyte 5-LOX. Of the 26 compounds tested, 10 isoflavones and 9 isoflavans possessed micromolar potency, but only three were selective against 5-LOX (IR-2, HIR-303, and HIR-309), with IC50 values at least 10 times lower than those of 12-LOX, 15-LOX-1, and 15-LOX-2. Of these three, IR-2 (6,7-dihydroxy-4-methoxy-isoflavone, known as texasin) was the most selective 5-LOX inhibitor, with over 80-fold potency difference compared with other isozymes; Steered Molecular Dynamics (SMD) studies supported these findings. The presence of the catechol group on ring A (6,7-dihydroxy versus 7,8-dihydroxy) correlated with their biological activity, but the reduction of ring C, converting the isoflavones to isoflavans, and the substituent positions on ring B did not affect their potency against 5-LOX. Two of the most potent/selective inhibitors (HIR-303 and HIR-309) were reductive inhibitors and were potent against 5-LOX in human whole blood, indicating that isoflavans can be potent and selective inhibitors against human leukocyte 5-LOX in vitro and in cellulo.

5-Lipoxygenase (5-LOX) reacts with arachidonic acid (AA) to first generate 5(S)-hydroperoxy-6(E),8(Z),11(Z),14(Z)-eicosatetraenoic acid [5(S)-HpETE] and then an epoxide from 5(S)-HpETE to form leukotriene A4, from a single polyunsaturated fatty acid. This work investigates the kinetic mechanism of these two processes and the role of ATP in their activation. Specifically, it was determined that epoxidation of 5(S)-HpETE (dehydration of the hydroperoxide) has a rate of substrate capture (Vmax/Km) significantly lower than that of AA hydroperoxidation (oxidation of AA to form the hydroperoxide); however, hyperbolic kinetic parameters for ATP activation indicate a similar activation for AA and 5(S)-HpETE. Solvent isotope effect results for both hydroperoxidation and epoxidation indicate that a specific step in its molecular mechanism is changed, possibly because of a lowering of the dependence of the rate-limiting step on hydrogen atom abstraction and an increase in the dependency on hydrogen bond rearrangement. Therefore, changes in ATP concentration in the cell could affect the production of 5-LOX products, such as leukotrienes and lipoxins, and thus have wide implications for the regulation of cellular inflammation.

Abstract Background Genetic variation in the genes ALOX5 (arachidonate 5-lipoxygenase), ALOX5AP (arachidonate 5-lipoxygenase-activating protein) and LTA4H (leukotriene A4 hydrolase) has previously been shown to contribute to the risk of MI (myocardial infarction) in Caucasian and African American populations. All genes encode proteins playing a role in the synthesis of the pro-inflammatory leukotriene B mediators, possibly providing a link between MI and inflammation. The aim of the present study was to investigate whether these associations could be confirmed in the study of China MI patients. The study included 401 Han Chinese MI patients and 409 controls. Six tag single nucleotide polymorphisms (SNPs)—ALOX5 rs12762303 and rs12264801, ALOX5AP rs10507391, LTA4H rs2072512, rs2540487 and rs2540477—were selected. SNP genotyping was performed by an improved multiplex ligation detection reaction assay. Results The rs2540487 genotype was associated with the risk of MI in overdominant model (P = 0.008). rs12762303 and rs10507391 SNPs were significantly associated with lipid levels in MI patients (P 0.05 for all). Conclusions Taken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate atherogenic processes and the risk of MI in Chinese.

Background and objectives Ischemic events, such as ischemic heart disease and stroke, are the number one cause of death globally. Ischemia prevents blood, carrying essential nutrients and oxygen, from reaching tissues, leading to cell and tissue death, and eventual organ failure. While humans are relatively intolerant to ischemic events, other species, such as marine mammals, have evolved a unique tolerance to chronic ischemia/reperfusion during apneic diving. To identify possible molecular features of an increased tolerance for apnea, we examined changes in gene expression in breath-holding dolphins. Methodology Here, we capitalized on the adaptations possesed by bottlenose dolphins (Tursiops truncatus) for diving as a comparative model of ischemic stress and hypoxia tolerance to identify molecular features associated with breath holding. Given that signals in the blood may influence physiological changes during diving, we used RNA-Seq and enzyme assays to examine time-dependent changes in gene expression in the blood of breath-holding dolphins. Results We observed time-dependent upregulation of the arachidonate 5-lipoxygenase (ALOX5) gene and increased lipoxygenase activity during breath holding. ALOX5 has been shown to be activated during hypoxia in rodent models, and its metabolites, leukotrienes, induce vasoconstriction. Conclusions and implications The upregulation of ALOX5 mRNA occurred within the calculated aerobic dive limit of the species, suggesting that ALOX5 may play a role in the dolphin's physiological response to diving, particularly in a pro-inflammatory response to ischemia and in promoting vasoconstriction. These observations pinpoint a potential molecular mechanism by which dolphins, and perhaps other marine mammals, respond to the prolonged breath holds associated with diving. [ABSTRACT FROM AUTHOR]

Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, liquid chromatography/mass spectrometry lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low-pathogenicity strain X31/H3N2 induced a proinflammatory response followed by a distinct anti-inflammatory response; infection by the high-pathogenicity strain PR8/H1N1 resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5-lipoxygenase metabolites correlated with the pathogenic phase of the infection, whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-hydroxyoctadecadienoic acid, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009-2011 influenza seasons.

The 5- and 12/15-lipoxygenase (LOX) isozymes have been implicated to contribute to disease development in CNS disorders such as Alzheimer's disease. These LOX isozymes are distinct in function, with differential effects on neuroinflammation, and the impact of the distinct isozymes in the pathogenesis of Parkinson's disease has not as yet been evaluated. To determine whether the isozymes contribute differently to nigrostriatal vulnerability, the effects of 5- and 12/15-LOX deficiency on dopaminergic tone under naïve and toxicant-challenged conditions were tested. In naïve mice deficient in 5-LOX expression, a modest but significant reduction (18.0% reduction vs. wildtype (WT)) in striatal dopamine (DA) was detected (n=6-8 per genotype). A concomitant decline in striatal tyrosine hydroxylase (TH) enzyme was also revealed in null 5-LOX vs. WT mice (26.2%); however, no changes in levels of DA or TH immunoreactivity were observed in null 12/15-LOX vs. WT mice. When challenged with the selective dopaminergic toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), WT mice showed a marked reduction in DA (31.9%) and robust astrocytic and microglial activation as compared to saline-treated animals. In contrast, null 5-LOX littermates demonstrated no significant striatal DA depletion or astrogliosis (as noted by Western blot analyses for glial acidic fibrillary protein (GFAP) immunoreactivity). In naïve null 12/15-LOX mice, no significant change in striatal DA values was observed compared to WT, and following MPTP treatment, the transgenics revealed striatal DA reduction similar to the challenged WT mice. Taken together, these data provide the first evidence that: (i) LOX isozymes are involved in the maintenance of normal dopaminergic function in the striatum and (ii) the 5- and 12/15-LOX isozymes contribute differentially to striatal vulnerability in response to neurotoxicant challenge.

We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

Fusion protein RUNX1-ETO (AML1-ETO, RUNX1-RUNX1T1) is expressed as the result of the 8q22;21q22 translocation [t(8;21)], which is one of the most common chromosomal abnormalities found in acute myeloid leukemia. RUNX1-ETO is thought to promote leukemia development through the aberrant regulation of RUNX1 (AML1) target genes. Repression of these genes occurs via the recruitment of the corepressors N-COR and SMRT due to their interaction with ETO. Mechanisms of RUNX1-ETO target gene upregulation remain less well understood. Here we show that RUNX1-ETO9a, the leukemogenic alternatively spliced transcript expressed from t(8;21), upregulates target gene Alox5, which is a gene critically required for the promotion of chronic myeloid leukemia development by BCR-ABL. Loss of Alox5 expression reduces activity of RUNX1-ETO9a, MLL-AF9 and PML-RARα in vitro. However, Alox5 is not essential for the induction of leukemia by RUNX1-ETO9a in vivo. Finally, we demonstrate that the upregulation of Alox5 by RUNX1-ETO9a occurs via the C₂H₂ zinc finger transcription factor KLF6, a protein required for early hematopoiesis and yolk sac development. Furthermore, KLF6 is specifically upregulated by RUNX1-ETO in human leukemia cells. This identifies KLF6 as a novel mediator of t(8;21) target gene regulation, providing a new mechanism for RUNX1-ETO transcriptional control.

Ethnopharmacological Relevance: Suo-Quan-Wan (SQW), a traditional Chinese prescription, has been used for hundreds of years to alleviate overactive bladder (OAB) symptoms such as frequent and nocturnal urination. However, limited modern research on OAB therapeutic targets has hindered the use and development of SQW., Aim of the Study: This study aimed to investigate the biological mechanisms and key targets of SQW on OAB in spontaneously hypertensive rats (SHR) using an integrated analysis of network pharmacology, transcriptome and metabolome., Methods: Rats were divided into five groups: model group (SHR), control group (WKY), darifenacin group, high dose (SQWH) and low dose (SQWL) group. Urodynamic parameters and histological examination were detected. Network pharmacology, transcriptome, and metabolome were used to screen for disease gene targets, differential mRNA, and differential metabolites, respectively. The biological targets and mechanisms of SQW for OAB were analyzed. Western blotting was performed to verify the proteins of key differential targets., Results: Urodynamics revealed a significant decrease in storage parameters in SHR. After SQW treatment, the inter-contraction interval, voided volume and bladder capacity increased by 2-3 times, as well as bladder compliance. Additionally, SQW improved the pathological changes in the urinary tract epithelium and the detrusor layer of the bladder in SHR. Metabolomic results showed an increase in arachidonic acid (AA) and cyclic adenosine monophosphate (cAMP) in plasma, suggesting the involvement of arachidonic acid metabolism and purine metabolism in SQW treatment. The downregulation of cytochrome P450 1B1 (CYP1B1), thromboxane-A synthase (TBXAS1), polyunsaturated fatty acid 5-lipoxygenase (ALOX5), and cAMP-specific 3',5'-cyclic phosphodiesterase 4B (PDE4B) were confirmed through topological analysis and Venn analysis of omics data and network pharmacology. These proteins affected the metabolism of AA and cAMP, respectively, and consequently affected downstream proteins, such as transient receptor potential (TRP) cation channel proteins (e.g. TRPV1, TRPA1, and TRPM8), myosin light chain kinase (MLCK), and the phosphorylation of myosin regulatory light chain (p-MLC)., Conclusion: This study initially elucidated the importance of AA and cAMP in the treatment of SQW, indicating the AA-CYP1B1/TBXAS1/ALOX5-TRPA1/TRPV1/TRPM8 and cAMP-PDE4B-MLCK-p-MLC pathways as the important pathways in SQW-treated SHR bladder in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Background: Non-steroidal anti-inflammatory drugs, cyclooxygenase (COX)-2 selective inhibitors, have been explored for prevention and treatment of several inflammatory chronic conditions including arthritis, and cancer. However, the long-term use of these drugs is associated with gastrointestinal, renal, and cardiovascular side effects. Later, COX/5-lipoxygenase (5-LOX) dual inhibitors (eg, licofelone) have been developed but did not enter into the market from the clinical trails due to COX-1/2 inhibition-associated side effects. Hence, targeting microsomal prostaglandin E synthase-1 (mPGES-1) and 5-LOX can be an ideal approach while sparing COX-1/2 activities for development of the next generation of anti-inflammatory drugs with better efficacy and safety. Materials and Methods: In silico (molecular modelling) studies were used to design a mPGES-1/5-LOX dual inhibitory and COX-1/2 sparing lead molecule licofelone analogue-9 (LFA-9) by modifying the pharmacophore of licofelone. In vitro cell-free enzymatic (mPGES-1, 5-LOX, COX-1/2) assays using fluorometric/colorimetric methods and cell-based assays (LPS-induced PGE2, LTB4, and PGI2 productions from macrophages) using ELISA technique, isothermal calorimetry, and circular dichroism techniques were performed to determine the mPGES-1/5-LOX inhibitory efficacy and selectivity. Anti-inflammatory efficacy of LFA-9 was evaluated using a carrageenan (inflammogen)-induced rat paw edema model. Infiltration/expression of CD68 immune cells and TNF-α in paw tissues were evaluated using confocal microscope and immunoblot analysis. Anti-cancer effect of LFA-9 was evaluated using colon spheroids in vitro. Results: LFA‐9 inhibited mPGES-1/5-LOX and their products PGE2 and LTB4, spared COX-1/2 and its product PGI2. LFA‐9 bound strongly with human mPGES‐1/5‐LOX enzymes and induced changes in their secondary structure, thereby inhibited their enzymatic activities. LFA-9 inhibited carrageenan-induced inflammation (70.4%) in rats and suppressed CD68 immune cell infiltration (P ≤ 0.0001) and TNF-α expression. LFA-9 suppressed colon tumor stemness (60.2%) in vitro through inhibition of PGE2 (82%) levels. Conclusion: Overall study results suggest that LFA-9 is a mPGES-1/5-LOX dual inhibitor and showed anti-inflammatory and colorectal cancer preventive activities, and warranted detailed studies. [ABSTRACT FROM AUTHOR]

Eicosanoids and other bioactive lipid mediators are indispensable regulators of biological processes, as demonstrated by the numerous inflammatory diseases resulting from their dysregulation, including cancer, hyperalgesia, atherosclerosis, and arthritis. Despite their importance, a robust strategy comparable with gene or protein array technology for comprehensively analyzing the eicosanoid metabolome has not been forthcoming. We have developed liquid chromatography-tandem mass spectrometry methodology that quantitatively and comprehensively analyzes the eicosanoid metabolome and utilized this approach to characterize eicosanoid production during experimental Lyme arthritis in mice infected with the bacterium Borrelia burgdorferi. Eicosanoids were extracted throughout infection from the joints of Lyme arthritis-resistant and -susceptible mice and subjected to lipidomic profiling. We identified temporal and quantitative differences between these mouse strains in the production of eicosanoids, which correlated with differences in arthritis development. The eicosanoid biosynthetic enzyme cyclooxygenase (COX)-2 has been implicated in the regulation of Lyme arthritis pathology, and subsequent lipidomic profiling of B. burgdorferi-infected COX-2(-/-) mice identified reductions not only in COX-2 products but, surprisingly, also significant off-target reductions in 5-lipoxygenase metabolites. Our results demonstrate the utility of a comprehensive lipidomic approach for identifying potential contributors to disease pathology and may facilitate the development of more precisely targeted treatment strategies.

Cytochrome P-450 2C45 (CYP2C45) is the most highly expressed cytochrome P-450 isoform in chicken liver, and may play an important role in avian liver biology. However, information regarding the function of CYP2C45 in fatty liver is generally limited. The aim of this study was to investigate the role of CYP2C45 during the development of goose fatty liver. Our result indicated that the transcription of CYP2C45, together with PK and ALOX5, was increased in goose liver upon overfeeding for 19 D (P < 0.05). In goose primary hepatocytes, CYP2C45 RNA expression was also upgraded by the treatment with various chemicals like insulin, the fatty acids, and PPAR agonists (P < 0.05). We also found that both CYP2C45 overexpression and troglitazone treatment could increase the expression of pyruvate kinase (PK) and arachidonate 5-lipoxygenase (ALOX5), and furthermore, showed that the up-regulation of PK and ALOX5 induced by troglitazone could be suppressed by small interfering RNAs targeting CYP2C45 (P < 0.05). These findings suggest that fatty acids treatment and the overfeeding can induce the up-regulation of CYP2C45 expression possibly via PPARγ and that the induction of PK and ALOX5 in goose fatty liver is at least partially attributed to fatty acid-induced expression of CYP2C45. Thus, our data provides an insight into the mechanism by which glycolysis and arachidonic acid metabolism are modulated in goose fatty liver. [ABSTRACT FROM AUTHOR]

Eicosanoids have been implicated in a vast number of devastating inflammatory conditions, including arthritis, atherosclerosis, pain, and cancer. Currently, over a hundred different eicosanoids have been identified, with many having potent bioactive signaling capacity. These lipid metabolites are synthesized de novo by at least 50 unique enzymes, many of which have been cloned and characterized. Due to the extensive characterization of eicosanoid biosynthetic pathways, this field provides a unique framework for integrating genomics, proteomics, and metabolomics toward the investigation of disease pathology. To facilitate a concerted systems biology approach, this review outlines the proteins implicated in eicosanoid biosynthesis and signaling in human, mouse, and rat. Applications of the extensive genomic and lipidomic research to date illustrate the questions in eicosanoid signaling that could be uniquely addressed by a thorough analysis of the entire eicosanoid proteome.

Arachidonic acid is released by phospholipase A(2) and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist-stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca(2+) influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca(2+) and TLR pathways was detected and discovered to be independent of NF-kappaB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A(2)-mediated eicosanoid production work through two distinct pathways.

Abstract Purpose: To investigate the participation of cysteinyl leukotrienes in the pathophysiology of oral mucositis. Methods: Oral mucositis was induced in hamsters using 5-fluorouracil (5-FU; 60 and 40 mg/kg; i.p., on days 1 and 2, respectively, and with excoriations in jugal mucosa on day 4). Montelukast (10, 20, or 40 mg/kg/d; gavage), MK886 (3 mg/kg/d, i.p.), or saline or celecoxib (7.5 mg/kg/d; i.p.) was administered 1 h prior to 5-FU and daily, until the fourth (MK886) or tenth day, when the animals were euthanized and their jugal mucosa was collected for macroscopic, histopathological, and immunohistochemical evaluation. Results: Neither montelukast nor MK-886 prevented the oral mucositis induced by 5-FU, as observed by histopathological evaluation. In addition, we did not find significant differences in the expression of inducible nitric oxide synthase-2, cyclooxygenase-2, or interleukin (IL)-1β between the experimental and control groups. However, we did observe a significant decrease in tumor necrosis factor (TNF)-α expression for all doses of montelukast; we also observed a significant decrease in IL-10 with 40 mg/kg/d and MK 886. Conclusions: Cysteinyl leukotrienes do not play an important role in experimental oral mucositis induced by 5-FU. There is a modulating action specifically on TNF-α.

With the acceleration of aging society, delaying aging or promoting healthy aging has become a major demand for human health. 5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid into leukotrienes (LTs), which is a potent mediator of the inflammatory response. Previous studies showed that abnormal activation of 5-LOX and overproduction of LTs are closely related to the occurrence and development of aging-related inflammatory diseases. Therefore, inhibiting 5-LOX activation is a possibly potential strategy for treating age-related diseases. In this paper, the latest research progress in 5-LOX activation, 5-LOX in mediating aging-related diseases and its small molecule inhibitors is briefly reviewed to provide scientific theoretical basis and new ideas for the prevention and treatment of aging-related inflammatory diseases.

Background: Intrahepatic cholangiocarcinoma (ICC) is poorly treated due to the presence of an inhibitory immune microenvironment. Tumor-associated macrophages (TAM) are an important component of TME. ALOX5 is an important lipid metabolism enzyme in cancer progression, but the mechanism by which it regulates TAM to promote ICC progression is unknown. The aim of this study was to investigate the potential mechanism of TAM regulation by ALOX5 and the translational effect of targeting ALOX5., Methods: In this study, we investigated the association between the spatial localization of epithelial cells and TAMs by combining scRNA-seq analysis with multiplex immunofluorescence analysis. Through bulk sequencing analysis and spatial analysis, lipid metabolism genes closely related to TAM infiltration were screened. In vitro co-culture model was constructed to verify that ALOX5 and its downstream metabolite LTB4 promote M2 macrophage migration. Bulk sequencing after co-culture combined with single-cell analysis was performed to identify key pathways for up-regulation of M2 macrophage migration. Finally, the effect of CSF1R inhibitor (PLX3397) combined with ALOX5 inhibitor (Zileuton) in vivo was investigated by by xenograft tumor formation experiment in nude mice., Results: ALOX5 in ICC cells was a key lipid metabolism gene affecting the infiltration of M2 macrophages in TME. Mechanically, LTB4, a metabolite downstream of ALOX5, recruited M2 macrophages to migrate around tumor cells by binding to BLT1/BLT2 and activating the PI3K pathway, which ultimately lead to the promotion of ICC progression. Targeting CSF1R in combination with ALOX5 inhibitor effectively reduced tumor volume and M2 macrophage infiltration abundance., Conclusion: In ICC, LTB4, a metabolite secreted by ALOX5 of epithelial cells, binded to BLT1/BLT2 on TAM surface to activate PI3K pathway and promote TAM migration, thus promoting ICC progression. Targeting CSF1R in combination with ALOX5 inhibitor for ICC is a promising combination therapy modality., (© 2023. The Author(s).)

Background: The aim of this study was to determine how the levels of peptide and protein-based biomarkers in cerebrospinal fluid change in bacterial, tuberculous, and aseptic meningitis, and to determine the success of these agents in distinguishing between different types of infectious meningitis., Methods: The levels of arachidonate-5-lipoxygenase, S100 calcium-binding protein B, defensin-α 1, and glial fibrillary acidic protein in cerebrospinal fluid samples from 20 tuberculosis, 40 bacterial, 25 aseptic meningitis patients, and 55 control groups were measured and compared using an enzyme-linked immunosorbent assay., Results: The mean age of the patients was 37.9 ± 14.4 years. The parameter that contributed the most to the differential diagnosis of the infectious meningitis groups was S100 calcium-binding protein B. The S100 calcium-binding protein B levels were significantly higher in the tuberculous meningitis group than in the other groups, and arachidonate-5-lipoxygenase levels were significantly higher in the tuberculous meningitis and bacterial meningitis groups (P < .05)., Conclusion: This study showed that cerebrospinal fluid arachidonate-5-lipoxygenase, and S100 calcium-binding protein B levels may differ in bacterial, aseptic, and tuberculous meningitis, and the results obtained may be quite effective as important potential biomarkers in the differential diagnosis of different types of meningitis., Competing Interests: The authors have no funding and conflicts of interest to disclose., (Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc.)

Simplified analogues of the myxobacterial polyketide ajudazol were obtained by synthesis and evaluated for their biological activities. Potent simplified 5-lipoxygenase inhibitors were identified. Moreover, strong antiproliferative and apoptotic activities were observed in brain cancer cell lines at low nano- to micromolar concentrations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We report here small molecules consisting of dichlorophenyl substituted oxindole that is further tagged with pyrrole/indole moieties. These molecules were designed on the basis of the analysis of binding mode of 5-LOX with arachidonic acid and zileuton. The molecules traverse the active site pocket of the enzyme that otherwise hosts AA and zileuton. Moreover, with a provision of derivatization at pyrrole/indole-N, the physico-chemical properties of the molecules can be adjusted. Appreciable 5-LOX inhibitory activities of the compounds in sub-micromolar range were observed and their aqueous solubility, binding with human serum albumin and stability in blood plasma and liver microsomes were checked. The Michaelis-Menten constants obtained during the binding of the compounds with 5-LOX indicated competitive binding of the compounds with the enzyme. Overall, the combination of molecular modelling and experimental studies identified promising molecules against inflammatory diseases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Abstract Arachidonate 5-lipoxygenase (ALOX5) is an essential enzyme for the biosynthesis of leukotrienes, which are pro-inflammatory and anti-inflammatory mediators. In this study, the ALOX5 paralog of the big-belly seahorse (Hippocampus abdominalis; HaALOX5) was identified from our transcriptome database, and then molecularly and functionally characterized to determine its oxygenation capability and expression under pathogenic stress. The coding sequence of HaALOX5 consisted of 2025 bp and encoded a protein of 674 amino acids in length. Sequence and phylogenetic tree analysis of HaALOX5 revealed a close relationship with its corresponding teleost HaALOX5 counterparts. Structure prediction detected an N-terminal regulatory C2-like domain and a C-terminal catalytic domain, which are the two main functional domains in ALOX5 enzymes. Quantitative PCR showed that HaALOX5 was expressed in all the analyzed tissues at different magnitudes. The highest expression was detected in the intestine and stomach. In blood cells, the liver and the intestine, HaALOX5 transcripts were significantly elevated at many post injection time points, when immune challenged with lipopolysaccharide, polyinosinic:polycytidylic acid, and Streptococcus iniae , indicating its contribution to post immune defense mechanisms in the seahorse. Highlights • Identified and characterized arachidonate 5-lipoxygenase from big-belly seahorse. • The HaALOX5 gene was constitutively expressed in healthy fish tissues. • HaALOX5 gene expression pattern found to be correlated with the immune stresses. • Recombinant HaALOX5 protein showed in-vitro arachidonic acid oxygenase activity. [ABSTRACT FROM AUTHOR]

Highlights • ALOX5 rs4987105 polymorphism is associated with T2D. • ALOX5 CC risk genotype is linked to lower vitamin D and increased CRP levels. • IL-1β + calcitriol treatment increased ALOX5, LTA4H and LTB4R1 mRNA expression. • ALOX5 andLTA4H mRNA expression were increased in T2D patients compared to HC. Abstract The arachidonate 5-lipoxygenase (ALOX5) pathway has been implicated in chronic inflammatory disease which may be influenced by vitamin D due to vitamin D response elements (VDRE). We investigated an ALOX5 polymorphism (rs4987105) in patients with type 2 diabetes (T2D) and the in vitro effects of calcitriol (1,25(OH) 2 D 3) on ALOX5 metabolism in monocytes of T2D patients and healthy controls (HC). 533 T2D and 473 HC were genotyped for the rs4987105 polymorphism. In addition, the 25(OH)D 3 and 1,25(OH) 2 D 3 plasma levels were measured in both cohorts. Further C-reactive protein (CRP) was determined in T2D patients. Our results demonstrate, that genotype CC and the allele C of ALOX5 rs4987105 polymorphism were more frequent in T2D compared to HC (OR = 1.44; 95% CI: 1.12–1.84; p < 0.05). Lower levels of both vitamin D metabolites (p < 0.0001 respectively) were found in the CC genotyped T2D patients compared to CC genotyped HC. In addition, CC genotyped T2D patients had higher levels of CRP compared to CT and TT genotyped T2D patients, (p < 0.01). In order to evaluate the impact of calcitriol in primary isolated monocytes, we isolated monocytes of 20 T2D patients and 20 HC. The cells were treated with 1,25(OH) 2 D 3 and interleukin-1beta (IL-1β) for 24 h. The following genes were analysed for expression changes: ALOX5, leukotriene A4 hydrolase (LTA4H), leukotriene B4 receptor type 1 (LTB4R1) and CD14. Treatment with IL-1β+1,25(OH) 2 D 3 increased ALOX5, LTA4H and LTB4R1 and CD14 mRNA in both T2D patients and HC (p < 0.0001, respectively). In addition, IL-1β+1,25(OH) 2 D 3 treatment led to higher ALOX5, LTA4H and CD14 mRNA levels in T2D patients compared to HC (p < 0.001, p < 0.05, p ≤ 0.05, respectively). In conclusion, ALOX5 rs4987105 allele C confers susceptibility to T2D, lower vitamin D metabolites and higher CRP levels complement this association. Additionally, IL-1β+1,25(OH) 2 D 3 treatment on, ALOX5, LTA4H and CD14 mRNA indicate a diabetes specific modulation. These findings identify a novel pathway in T2D potentially amenable for individualized therapeutic targeting. [ABSTRACT FROM AUTHOR]

Background: Genetic variation in the genes ALOX5 (arachidonate 5-lipoxygenase), ALOX5AP (arachidonate 5-lipoxygenase-activating protein) and LTA4H (leukotriene A4 hydrolase) has previously been shown to contribute to the risk of MI (myocardial infarction) in Caucasian and African American populations. All genes encode proteins playing a role in the synthesis of the pro-inflammatory leukotriene B mediators, possibly providing a link between MI and inflammation. The aim of the present study was to investigate whether these associations could be confirmed in the study of China MI patients. The study included 401 Han Chinese MI patients and 409 controls. Six tag single nucleotide polymorphisms (SNPs)—ALOX5 rs12762303 and rs12264801, ALOX5AP rs10507391, LTA4H rs2072512, rs2540487 and rs2540477—were selected. SNP genotyping was performed by an improved multiplex ligation detection reaction assay. Results: The rs2540487 genotype was associated with the risk of MI in overdominant model (P = 0.008). rs12762303 and rs10507391 SNPs were significantly associated with lipid levels in MI patients (P < 0.006–0.008). Several SNPs interacted with alcohol consumption, cigarette smoking, and hypertension to modify TC, TG, LDL-C and CRE levels, and the risk of MI (P < 0.0017 for all). No association between the SNPs of LT pathway and susceptibility to MI was found (P > 0.05 for all). Conclusions: Taken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate atherogenic processes and the risk of MI in Chinese. [ABSTRACT FROM AUTHOR]

The specific intracellular sites at which enzymes act to generate arachidonate-derived eicosanoid mediators of inflammation are uncertain. We evaluated the formation and function of cytoplasmic lipid bodies. Lipid body formation in eosinophils was a rapidly (

5-Lipoxygenase-activating protein (FLAP) is an 18-kDa integral membrane protein required, in peripheral cells, for the activation of 5-lipoxygenase (5-LO) and for the resulting synthesis of leukotrienes from arachidonic acid. In the brain, the leukotrienes have been implicated in several pathophysiological events and in the electrophysiological effect of somatostatin, yet the cellular origin and role of these messenger molecules are still poorly understood. In the present study, we used reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunohistochemistry to demonstrate that 5-LO and FLAP are expressed in various regions of the rat brain, including hippocampus, cerebellum, primary olfactory cortex, superficial neocortex, thalamus, hypothalamus, and brainstem. Highest levels of expression were observed in cerebellum and hippocampus. In the latter we demonstrate the colocalization of 5-LO and FLAP in CA1 pyramidal neurons. Moreover, electrophysiological experiments show that selective inhibition of FLAP with the compound MK-886 (0.25-1 microM) prevents the somatostatin-induced augmentation of the hippocampal K+ M-current. Our results provide necessary evidence for the presence and signaling role of 5-LO and FLAP in central neurons and strongly support their proposed participation in somatostatin-receptor transmembrane signaling.