Up-regulation of IRF3 is required for docosahexaenoic acid suppressing ferroptosis of cardiac microvascular endothelial cells in cardiac hypertrophy rat.

The molecular characteristics of ferroptosis in cardiac hypertrophy have been rarely studied. Especially, there have been no studies to investigate the regulatory mechanisms of docosahexaenoic acid (DHA) on ferroptosis in cardiac hypertrophy. This study was designed to determine the role of ferroptosis in microvascular injury, and investigate the contribution of DHA in suppressing ferroptosis and preventing pressure overload-mediated endothelial damage. Our results indicated that the expression of interferon regulating factor 3 (IRF3) was primarily inhibited by pressure overload and consequently caused endothelial ferroptosis. Nevertheless, administration of DHA increased IRF3 expression and provided a pro-survival advantage for the endothelial system in the context of pressure overload. Experimental studies clearly showed that inhibition of IRF3 down-regulated SLC7A11 expression, and the latter leaded to the increase in the activities of arachidonate 12-lipoxygenase, which obligated cardiac microvascular endothelial cells to undergo ferroptosis via augmenting lipid peroxides. Interestingly, DHA supplementation suppressed endothelial ferroptosis via up-regulation of IRF3. Taken together, our studies identified the IRF3-SLC7A11-arachidonate 12-lipoxygenase axis as a new pathway responsible for pressure overload-mediated microvascular damage via initiating endothelial ferroptosis. In contrast, DHA treatment up-regulated the expression of IRF3 and thus reduced cellular ferroptosis, conferring a protective advantage to the endothelial system in pressure overload. These findings revealed that targeting IRF3 might be a useful therapeutic strategy for cardioprotection in cardiac hypertrophy and heart failure.

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

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

Discovery of natural 15-LOX small molecule inhibitors from Chinese herbal medicine using virtual Screening, biological evaluation and molecular dynamics studies.

Chinese herbal medicines (CHM) are frequently used to treat different types of inflammatory diseases and 15-Lipoxygenase (15-LOX) is a critical target enzyme for treating various inflammatory diseases. In this study, natural 15-LOX inhibitors were identified in CHM using an approach of virtual screening combined with the biological assays. First, an in-house Chinese medicine database containing 360 compounds was screened using a virtual screening approach based on pharmacophore and molecular docking to uncover several novel potential 15-LOX inhibitors. Secondly, the inhibitory effect of virtual screening hits against the 15-LOX enzyme was validated in an in vitro enzyme inhibition assay. Then, a tumor necrosis factor-α (TNF-α) release assay was carried out to explore the anti-inflammatory response of the active compounds. Furthermore, molecular dynamics (MD) simulation and binding free energy calculation were applied to analyze the process of inhibitors binding and also compared the mode of binding of the inhibitors by using the Molecular Mechanics-Generalized Born Surface Area (MM/GBSA) method. Finally, licochalcone B and eriodictyol were confirmed as inhibitors of the 15-LOX enzyme with IC50 values of 9.67 and 18.99 µM, respectively. In vitro cell-based assay showed that licochalcone B and eriodictyol inhibited the release of TNF-α factor in RAW264.7 cells stimulated by lipopolysaccharides (LPS) in a dose-dependent manner. Molecular dynamics and binding free energy analysis showed that the two 15-LOX-ligand systems immediately attained equilibrium with almost 1 Å fluctuation, the calculated binding free energies were found around -18.89 and -12.96 kcal/mol for licochalcone B and eriodictyol, respectively. Thr412, Arg415, Val420, Thr429, Ile602 and Trp606 were the main amino acid residues for the inhibition of 15-LOX enzyme activity. The current study confirms that licochalcone B and eriodictyol are 15-LOX inhibitors and can suppress the release of the TNF-α factor in RAW264.7 cells stimulated by LPS, thus providing a basis for the follow-up research and development for 15-LOX inhibitors.

Lipoxygenase inhibitor ML351 dysregulated an innate inflammatory response leading to impaired cardiac repair in acute heart failure.

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.

Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.

An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) tissue status can lead to a significant formation of anti-inflammatory lipid mediators and effective reduction in inflammation and tissue injury in murine colitis. Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory bowel disease as well as in the formation of pro- and anti-inflammatory lipid mediators. To explore the role of Alox15 in the protective response found in fat1 transgenic mice with endogenously increased n-3 PUFA tissue status fat1 transgenic mice were crossed with Alox15-deficient animals and challenged in the dextran sulfate sodium (DSS)- and the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model. Transgenic fat1 mice rich in endogenous n-3 PUFAs were protected from colitis. However, additional systemic inactivation of the Alox15 gene counteracted this protective effect. To explore the molecular basis for this effect Alox15 lipid metabolites derived from n-3 PUFA were analyzed in the different mice. Alox15 deficiency suppressed the formation of n-3 PUFA-derived 15-hydroxy eicosapentaenoic acid (15-HEPE). In contrast, treating mice with intraperitoneal injections of 15S-HEPE protected wild-type mice from DSS- and TNBS-induced colitis. These data suggest that the anti-colitis effect of increased n-3 PUFA in the transgenic fat1 mouse model is mediated in part via Alox15-derived 15-HEPE formation.

Omega-6 DPA and its 12-lipoxygenase-oxidized lipids regulate platelet reactivity in a nongenomic PPARα-dependent manner.

Arterial thrombosis is the underlying cause for a number of cardiovascular-related events. Although dietary supplementation that includes polyunsaturated fatty acids (PUFAs) has been proposed to elicit cardiovascular protection, a mechanism for antithrombotic protection has not been well established. The current study sought to investigate whether an omega-6 essential fatty acid, docosapentaenoic acid (DPAn-6), and its oxidized lipid metabolites (oxylipins) provide direct cardiovascular protection through inhibition of platelet reactivity. Human and mouse blood and isolated platelets were treated with DPAn-6 and its 12-lipoxygenase (12-LOX)-derived oxylipins, 11-hydroxy-docosapentaenoic acid and 14-hydroxy-docosapentaenoic acid, to assess their ability to inhibit platelet activation. Pharmacological and genetic approaches were used to elucidate a role for DPA and its oxylipins in preventing platelet activation. DPAn-6 was found to be significantly increased in platelets following fatty acid supplementation, and it potently inhibited platelet activation through its 12-LOX-derived oxylipins. The inhibitory effects were selectively reversed through inhibition of the nuclear receptor peroxisome proliferator activator receptor-α (PPARα). PPARα binding was confirmed using a PPARα transcription reporter assay, as well as PPARα-/- mice. These approaches confirmed that selectivity of platelet inhibition was due to effects of DPA oxylipins acting through PPARα. Mice administered DPAn-6 or its oxylipins exhibited reduced thrombus formation following vessel injury, which was prevented in PPARα-/- mice. Hence, the current study demonstrates that DPAn-6 and its oxylipins potently and effectively inhibit platelet activation and thrombosis following a vascular injury. Platelet function is regulated, in part, through an oxylipin-induced PPARα-dependent manner, suggesting that targeting PPARα may represent an alternative strategy to treat thrombotic-related diseases.

Alox12/15 Deficiency Exacerbates, While Lipoxin A4 Ameliorates Hepatic Inflammation in Murine Alcoholic Hepatitis.

Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15-/- mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15-/- mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15-/- mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.

Tianma Gouteng granules decreases the susceptibility of Parkinson's disease by inhibiting ALOX15-mediated lipid peroxidation.

ETHNOPHARMACOLOGICAL RELEVANCE: Tianma Gouteng granules (TG), a clinical prescription of traditional Chinese medicine, has been clinically applied to treat Parkinson's disease (PD) in combination with Madopar, as included in the Chinese Pharmacopoeia (2015). TG has the potential to decrease the susceptibility of PD pharmacologically, however the mechanisms need detailed demonstration. AIM OF THE STUDY: To evaluate the pharmacological activities, as well as the possible mechanism of TG in diverse models of PD. MATERIALS AND METHODS: 6-OHDA-treated rats, MPTP-treated mice, and α-synuclein A53T overexpressed mice, were utilized as PD animal models. Rotarod, locomotor activity, inclined plane and traction tests were used for behavioral assessment. Immunohistochemistry was used for tyrosine hydrolase determination. Western blot were conducted for detection of 4-HNE and 15-lipoxygenase-1 (ALOX15). The interactions of ALOX15 with the components in TG were predicted by molecular docking approach. RESULTS: Lipid peroxidation was involved in dopaminergic neuron damage in 6-OHDA-induced rat models. In MPTP-treated mice, the inhibition of lipid peroxidation improved behavioral and pathological symptoms of PD. The lipid peroxidation-related protein, ALOX15 was found to be the key factor in PD process in diverse PD models including 6-OHDA-treated rats, MPTP-treated mice, and α-synuclein A53T overexpressed mice. TG treatment significantly relieved behavioral and pathological symptoms of MPTP-induced PD mouse models with a potential mechanism of alleviating ALOX15-induced lipid peroxidation. Moreover, the results of molecular docking analysis show that compounds in TG might have interactions with ALOX15. CONCLUSIONS: TG effectively improved the behavioral and dopaminergic neuron damage in diverse PD models. The mechanism of this action may be related to the direct inhibition of ALOX15 and the relief of lipid peroxidation.

A dual role of 12/15-lipoxygenase in LPS-induced acute renal inflammation and injury.

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 ±â€¯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.

Involvement of the Hydroperoxy Group in the Irreversible Inhibition of Leukocyte-Type 12-Lipoxygenase by Monoterpene Glycosides Contained in the Qing Shan Lu Shui Tea.

We have previously found two novel monoterpene glycosides, liguroside A and liguroside B, with an inhibitory effect on the catalytic activity of the enzyme leukocyte-type 12-lipoxygenase in the Qing Shan Lu Shui tea. Here, two new monoterpene glycosides, liguroside C and liguroside D which inhibit this enzyme, were isolated from the same tea. The spectral and chemical evidence characterized the structures of these compounds as (5E)-7-hydroperoxy-3,7-dimethyl-1,5-octadienyl-3-O-(α-l-rhamnopyranosyl)-(1''→3')-(4'''-O-trans-p-coumaroyl)-ß-d-glucopyranoside and (2E)-6-hydroxy-3,7-dimethyl-2,7-octadienyl-3-O-(α-l-rhamnopyranosyl)-(1''→3')-(4'''-O-trans-p-coumaroyl)-ß-d-glucopyranoside, respectively. These ligurosides, which irreversibly inhibited leukocyte-type 12-lipoxygenase, have a hydroperoxy group in the monoterpene moiety. Additionally, monoterpene glycosides had the same backbone structure but did not have a hydroperoxy group, such as kudingoside A and lipedoside B-III, contained in the tea did not inhibit the enzyme. When a hydroperoxy group in liguroside A was reduced by using triphenylphosphine, the resultant compound, kudingoside B, showed a lower inhibitory effect on the enzyme. These results strongly suggest the involvement of the hydroperoxy group in the irreversible inhibition of the catalytic activity of leukocyte-type 12-lipoxygenase by the monoterpene glycosides contained in the Qing Shan Lu Shui tea.

Emerging role of 12/15-Lipoxygenase (ALOX15) in human pathologies.

12/15-lipoxygenase (12/15-LOX) is an enzyme, which oxidizes polyunsaturated fatty acids, particularly omega-6 and -3 fatty acids, to generate a number of bioactive lipid metabolites. A large number of studies have revealed the importance of 12/15-LOX role in oxidative and inflammatory responses. The in vitro studies have demonstrated the ability of 12/15-LOX metabolites in the expression of various genes and production of cytokine related to inflammation and resolution of inflammation. The studies with the use of knockout and transgenic animals for 12/15-LOX have further shown its involvement in the pathogenesis of a variety of human diseases, including cardiovascular, renal, neurological and metabolic disorders. This review summarizes our current knowledge on the role of 12/15-LOX in inflammation and various human diseases.

Transcriptomic and lipidomic profiling of eicosanoid/docosanoid signalling in affected and non-affected skin of human atopic dermatitis patients.

Lipoxygenases (LOX) and cyclooxygenase (COX) are the main enzymes for PUFA metabolism to highly bio-active prostaglandins, leukotrienes, thromboxanes, lipoxins, resolvins and protectins. LOX and COX pathways are important for the regulation of pro-inflammatory or pro-resolving metabolite synthesis and metabolism for various inflammatory diseases such as atopic dermatitis (AD). In this study, we determined PUFAs and PUFA metabolites in serum as well as affected and non-affected skin samples from AD patients and the dermal expression of various enzymes, binding proteins and receptors involved in these LOX and COX pathways. Decreased EPA and DHA levels in serum and reduced EPA level in affected and non-affected skin were found; in addition, n3/n6-PUFA ratios were lower in affected and non-affected skin and serum. Mono-hydroxylated PUFA metabolites of AA, EPA, DHA and the sum of AA, EPA and DHA metabolites were increased in affected and non-affected skin. COX1 and ALOX12B expression, COX and 12/15-LOX metabolites as well as various lipids, which are known to induce itch (12-HETE, LTB4, TXB2, PGE2 and PGF2) and the ratio of pro-inflammatory vs pro-resolving lipid mediators in non-affected and affected skin as well as in the serum of AD patients were increased, while n3/n6-PUFAs and metabolite ratios were lower in non-affected and affected AD skin. Expression of COX1 and COX-metabolites was even higher in non-affected AD skin. To conclude, 12/15-LOX and COX pathways were mainly upregulated, while n3/n6-PUFA and metabolite ratios were lower in AD patients skin. All these parameters are a hallmark of a pro-inflammatory and non-resolving environment in affected and partly in non-affected skin of AD patients.

ALOX12 gene polymorphisms and serum selenium status in elderly osteoporotic patients.

BACKGROUND: Osteoporosis is a systemic bone disease which leads to a reduction in bone mass. Many studies have shown that up to 80% of bone mineral density (BMD) variations are attributed to genetic factors. Arachidonate 12-lipoxygenase enzyme, encoded by the ALOX12 gene, produces lipid peroxides as reactive oxygen species (ROS), leading to oxidative stress and the development of osteoporosis. Selenium (Se) is incorporated into selenoproteins, which may reduce the risk of osteoporosis. OBJECTIVES: We aimed to investigate the association of 2 ALOX12 single nucleotide polymorphisms (SNPs) and serum Se level with lumbar spine and femoral neck BMD among elderly individuals living in Amirkola, Iran. MATERIAL AND METHODS: The study consisted of 180 individuals aged ≥60 years (90 healthy and 90 osteoporotic patients). We examined the effect of 2 ALOX12 SNPs (rs2292350 and rs9897850), using the polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) on both BMD regions measured by dual energy X-ray absorptiometry (DXA). Serum Se level was measured using an atomic absorption spectrophotometer PGG990 AAS (PG Instruments Ltd., Luterworth, USA). RESULTS: The rs2292350 SNP showed a significant association with femoral neck BMD (p = 0.04). Moreover, in terms of serum Se level, a significant difference was found between the patient group (57.58 ±25.54 µg/L) and the control group (81.09 ±25.58 µg/L) (p < 0.001). In addition, individuals with higher serum Se levels also had higher BMD of the lumbar spine (r2 = 0.392; p < 0.001) and the femoral neck (r2 = 0.478; p < 0.001). CONCLUSIONS: The results suggested that genetic variation in ALOX12 might influence BMD variations in our recruited participants. As for the patients with lower serum Se levels, it was observed that serum Se deficiency was accompanied by some ALOX12 variation, contributing to the development of osteoporosis.

Intravasation of SW620 colon cancer cell spheroids through the blood endothelial barrier is inhibited by clinical drugs and flavonoids in vitro.

Mechanisms how colorectal cancer (CRC) cells penetrate blood micro-vessel endothelia and metastasise is poorly understood. To study blood endothelial cell (BEC) barrier breaching by CRC emboli, an in vitro assay measuring BEC-free areas underneath SW620 cell spheroids, so called "circular chemorepellent induced defects" (CCIDs, appearing in consequence of endothelial retraction), was adapted and supported by Western blotting, EIA-, EROD- and luciferase reporter assays. Inhibition of ALOX12 or NF-κB in SW620 cells or BECs, respectively, caused attenuation of CCIDs. The FDA approved drugs vinpocetine [inhibiting ALOX12-dependent 12(S)-HETE synthesis], ketotifen [inhibiting NF-κB], carbamazepine and fenofibrate [inhibiting 12(S)-HETE and NF-κB] significantly attenuated CCID formation at low µM concentrations. In the 5-FU-resistant SW620-R/BEC model guanfacine, nifedipine and proadifen inhibited CCIDs stronger than in the naïve SW620/BEC model. This indicated that in SW620-R cells formerly silent (yet unidentified) genes became expressed and targetable by these drugs in course of resistance acquisition. Fenofibrate, and the flavonoids hispidulin and apigenin, which are present in medicinal plants, spices, herbs and fruits, attenuated CCID formation in both, naïve- and resistant models. As FDA-approved drugs and food-flavonoids inhibited established and acquired intravasative pathways and attenuated BEC barrier-breaching in vitro, this warrants testing of these compounds in CRC models in vivo.

Inhibiting 12/15-lipoxygenase to treat acute stroke in permanent and tPA induced thrombolysis models.

12/15-Lipoxygenase (12/15-LOX) contributes to the brain damage after middle cerebral artery occlusion (MCAO) in the acute phase of stroke. The aim of this study was to investigate the effects of a 12/15-LOX inhibitor, LOXBlock-1(LB1), in mice using a FeCl3-induced permanent distal MCAO model and FeCl3-induced ischemia/thrombolysis with tPA. In order to induce permanent distal MCAO, 30% FeCl3 was used in C57BL6 mice. LB1 or DMSO treatments were applied intraperitoneally 2 h following MCAO. For FeCl3-induced ischemia/thrombolysis experiments, 10% FeCl3 was preferred so as to obtain reperfusion with tPA in CD1 mice. 4 h following ischemia either LB1 or DMSO and iv tPA was administered. Outcomes were NSS, weight loss, infarct volume, hemorrhage area and reperfusion rate. FeCl3-induced distal MCAO caused an increase in 12/15-LOX signal in the ischemic cortex with an increase in MDA2 and AIF immunoreactivity. LB1 treatment, applied 2 h after ischemia, significantly decreased the infarct volume at 24 h of permanent distal MCAO. Weight loss was also significantly reduced in LB1 treated group. Distal MCAO and tPA application with LB1 or DMSO showed that treatment significantly decreased the infarct volume and the hemorrhage area. The reperfusion rate in the LB1-treated group was surprisingly higher than in the DMSO group and NSS results were significantly improved. These data suggest that LB1 can be used as an adjuvant agent to tPA. This study not only shows the effects of LB1 treatment in distal MCAO but also confirms that FeCl3-induced MCAO model can be a useful tool to screen novel treatment options in stroke.

Deletion of 12/15-lipoxygenase accelerates the development of aging-associated and instability-induced osteoarthritis.

OBJECTIVE: 12/15-Lipoxygenase (12/15-LOX) catalyzes the generation of various anti-inflammatory lipid mediators, and has been implicated in several inflammatory and degenerative diseases. However, there is currently no evidence that 12/15-LOX has a role in osteoarthritis (OA). The aim of this study was to investigate the role of 12/15-LOX in the pathogenesis of OA. METHODS: The development of aging-associated and destabilization of the medial meniscus (DMM)-induced OA were compared in 12/15-LOX-deficient (12/15-LOX-/-) and wild-type (WT) mice. The extent of cartilage damage was evaluated by histology. The expression of OA markers was evaluated by immunohistochemistry and RT-PCR. Cartilage explants were stimulated with IL-1α in the absence or presence of the 12/15-LOX metabolites, 15-hydroxyeicosatetraenoic acids (15-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) or lipoxin A4 (LXA4), and the levels of matrix metalloproteinases-13 (MMP-13), Nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. The effect of LXA4 on the progression of OA was evaluated in wild type (WT) mice. RESULTS: The expression of 12/15-LOX in cartilage increased during the progression of DMM-induced OA and with aging in WT mice. Cartilage degeneration was more severe in 12/15-LOX-/- mice compared to WT mice in both models of OA, and this was associated with increased expression of MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs, aggrecanases (ADAMTS5), inducible NO synthases (iNOS), and mPGES-1. Treatment of cartilage explants with 12/15-LOX metabolites, suppressed IL-1α-induced production of MMP-13, NO and PGE2, with LXA4 being the most potent. Intra-peritoneal injection of LXA4 reduced the severity of DMM-induced cartilage degradation. CONCLUSIONS: These data suggest an important role of 12/15-LOX in the pathogenesis of OA. They also suggest that activation of this pathway may provide a novel strategy for prevention and treatment of OA.

Chemical Composition and Immuno-Modulatory Effects of Urtica dioica L. (Stinging Nettle) Extracts.

The purpose of this work was to determine the chemical profile of stinging nettle and to provide an insight into the mechanisms by which it ameliorates the immune response. Qualitative and quantitative liquid chromatography tandem mass spectrometry analyses indicated that phenolic acids (5-O-caffeoylquinic acid as dominant) and flavonol glycosides (rutin, isoquercitrin, and kaempferol 3-O-glucoside) are present in the aerial parts, while lignans (secoisolariciresinol, 9,9'-bisacetyl-neo-olivil and their glucosides) were detected in the root. Herb and root extracts expressed selective inhibition toward cyclooxygenase and lipoxygenase branches in human platelets: root extracts were better at inhibiting thromboxane production, while herb extracts were more specific toward inhibition of 12-lipoxygenase pathway. Stinging nettle extracts mildly increased monocyte chemoattractant protein-1 and growth-related oncogene release from nonstimulated intestinal epithelial cells, stimulating MyD88/NF-κB/p38 signaling, hence preserving the epithelial integrity and enhancing intestinal steady-state defense. Additionally, root extract reduced lipopolysaccharide-induced monocyte chemoattractant protein-1/growth-related oncogene secretion and cyclooxygenase-2 expression in intestinal epithelial cells, thus showing the potential protective effect against tissue damage caused by inflammation processes. These observations suggest that stinging nettle is an interesting candidate for the development of phytopharmaceuticals or dietary supplements for cotreatment of various inflammatory diseases, particularly inflammatory bowel diseases. Copyright © 2017 John Wiley & Sons, Ltd.

The Analgesic and Anxiolytic Effect of Souvenaid, a Novel Nutraceutical, Is Mediated by Alox15 Activity in the Prefrontal Cortex.

Pain and anxiety have a complex relationship and pain is known to share neurobiological pathways and neurotransmitters with anxiety. Top-down modulatory pathways of pain have been shown to originate from cortical and subcortical regions, including the dorsolateral prefrontal cortex. In this study, a novel docosahexaenoic acid (DHA)-containing nutraceutical, Souvenaid, was administered to mice with infraorbital nerve ligation-induced neuropathic pain and behavioral responses recorded. Infraorbital nerve ligation resulted in increased face wash strokes of the face upon von Frey hair stimulation, indicating increased nociception. Part of this response involves general pain sensitization that is dependent on the CNS, since increased nociception was also found in the paws during the hot plate test. Mice receiving oral gavage of Souvenaid, a nutraceutical containing DHA; choline; and other cell membrane components, showed significantly reduced pain sensitization. The mechanism of Souvenaid's activity involves supraspinal antinociception, originating in the prefrontal cortex, since inhibition of the DHA-metabolizing enzyme 15-lipoxygenase (Alox15) in the prefrontal cortex attenuated the antinociceptive effect of Souvenaid. Alox15 inhibition also modulated anxiety behavior associated with pain after infraorbital nerve ligation. The effects of Souvenaid components and Alox15 on reducing central sensitization of pain may be due to strengthening of a known supraspinal antinociceptive pathway from the prefrontal cortex to the periaqueductal gray. Together, results indicate the importance of the prefrontal cortex and DHA/Alox15 in central antinociceptive pathways and suggest that Souvenaid may be a novel therapeutic for neuropathic pain.

Total steroid and terpenoid enriched fraction from Euphorbia neriifolia Linn offers protection against nociceptive-pain, inflammation, and in vitro arthritis model: An insight of mechanistic study.

The plant Euphorbia neriifolia Linn has been successfully used for the management of acute inflammatory, arthritic, nociceptive pain and relieves the asthmatic symptom as a tribal folk medicine in India. The present study was conducted to evaluate the anti-inflammatory, analgesic, anti-arthritic activity from total steroid and terpenoid rich fractions derived from hydro-alcoholic extract of Euphorbia neriifolia stem (STF-HAENS). STF-HAENS fraction demonstrated 68.58±2.5% and 75.25±5.1% protection against acetic acid-induced pain and central neuropathic pain at 80mg/kg. It also showed 98.47% protection against acute inflammation at 100mg/kg with 1.7 fold higher protective activity than the standard drug. The fraction exhibited this efficacy via inhibition of proinflammatory cytokines TNF-α, IFN-γ, IL-12 and IL-6 by 74%, 81.26%, 92.10% and 93.4% respectively at 100µg/ml. It also showed dual inhibition of cyclooxygenase (COX) and lipooxygenase (LOX) activity in a dose-dependent manner that elicited the desired pharmacological action. The fraction downregulated nitric-oxide production from lipopolysaccharide (LPS) stimulated PBMC derived macrophages. The spectrophotometric analysis reveals the STF-HAENS induced ameliorative effect against heat-induced denaturation of BSA protein and exhibited significant antiproteinase activity. Our findings suggest that STF-HAENS could be used as an effective safe therapeutic agent for treatment of nociceptive pain, acute inflammation and arthritis.