ABSTRACT
BACKGROUND: Dengue virus (DENV) is considered one of the most important pathogens in the world causing 390 million infections each year. Currently, the development of vaccines against DENV presents some shortcomings and there is no antiviral therapy available for its infection. An important challenge is that both treatments and vaccines must be effective against all four DENV serotypes. Nordihydroguaiaretic acid (NDGA), isolated from Larrea divaricata Cav. (Zygophyllaceae) has shown a significant inhibitory effect on a broad spectrum of viruses, including DENV serotypes 2 and 4. PURPOSE: We evaluated the in vitro virucidal and antiviral activity of NDGA on DENV serotype 1 (DENV1), including the study of its mechanism of action, to provide more evidence on its antiviral activity. METHODS: The viability of viral particles was quantified by the plaque-forming unit reduction method. NDGA effects on DENV1 genome and viral proteins were evaluated by qPCR and immunofluorescence, respectively. Lysosomotropic activity was assayed using acridine orange and neutral red dyes. RESULTS: NDGA showed in vitro virucidal and antiviral activity against DENV1. The antiviral effect would be effective within the first 2 h after viral internalization, when the uncoating process takes place. In addition, we determined by qPCR that NDGA decreases the amount of intracellular RNA of DENV1 and, by immunofluorescence, the number of cells infected. These results indicate that the antiviral effect of NDGA would have an intracellular mechanism of action, which is consistent with its ability to be incorporated into host cells. Considering the inhibitory activity of NDGA on the cellular lipid metabolism, we compared the antiviral effect of two inhibitors acting on two different pathways of this type of metabolism: 1) resveratrol that inhibits the sterol regulatory element of binding proteins, and 2) caffeic acid that inhibits the 5-lipoxygenase (5-LOX) enzyme. Only caffeic acid produced an inhibitory effect on DENV1 infection. We studied the lysosomotropic activity of NDGA on host cells and found, for the first time, that this compound inhibited the acidification of cell vesicles which would prevent DENV1 uncoating process. CONCLUSION: The present work contributes to the knowledge of NDGA activity on DENV. We describe its activity on DENV1, a serotype different to those that have been already reported. Moreover, we provide evidence on which stage/s of the viral replication cycle NDGA exerts its effects. We suggest that the mechanism of action of NDGA on DENV1 is related to its lysosomotropic effect, which inhibits the viral uncoating process.
Subject(s)
Dengue Virus , Acridine Orange/pharmacology , Antiviral Agents/pharmacology , Arachidonate 5-Lipoxygenase/genetics , Caffeic Acids , Coloring Agents/pharmacology , Dengue Virus/physiology , Masoprocol/pharmacology , Neutral Red/pharmacology , RNA , Resveratrol/pharmacology , Serogroup , Sterols/pharmacology , Viral Proteins , Virus ReplicationABSTRACT
5-Lipoxygenase (5-LO) is an enzyme required for the production of leukotrienes and lipoxins and interferes with parasitic infections. In vitro, Toxoplasma gondii inhibits leukotriene B4 (LTB4) production, and mice deficient in 5-LO are highly susceptible to infection. The aim of this study was to investigate the effects of the pharmacological inhibition of the 5-LO pathway and exogenous LTB4 supplementation during experimental toxoplasmosis. For this purpose, susceptible C57BL/6 mice were orally infected with T. gondii and treated with LTB4 or MK886 (a selective leukotriene inhibitor through inhibition of 5-LO-activating protein [FLAP]). The parasitism, histology, and immunological parameters were analyzed. The infection decreased 5-LO expression in the small intestine, and treatment with MK886 reinforced this reduction during infection; in addition, MK886-treated infected mice presented higher intestinal parasitism, which was associated with lower local interleukin-6 (IL-6), interferon gamma (IFN-γ), and tumor necrosis factor (TNF) production. In contrast, treatment with LTB4 controlled parasite replication in the small intestine, liver, and lung and decreased pulmonary pathology. Interestingly, treatment with LTB4 also preserved the number of Paneth cells and increased α-defensins expression and IgA levels in the small intestine of infected mice. Altogether, these data demonstrated that T. gondii infection is associated with a decrease in 5-LO expression, and on the other hand, treatment with the 5-LO pathway product LTB4 resulted in better control of parasite growth in the organs, adding to the knowledge about the pathogenesis of T. gondii infection.
Subject(s)
Parasites , Toxoplasma , Toxoplasmosis , Animals , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Leukotriene B4 , Lipoxygenase , Mice , Mice, Inbred C57BL , Parasites/metabolismABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Pyrus pashia Buch ham ex. D. Don (Kainth) fruit from the Himalayan region is traditionally consumed by native people in the form of decoctions for various clinical conditions including inflammatory diseases. However, scientific studies on the biofunctional properties of Kainth fruits are still scarce. AIM OF THE STUDY: The study is aimed to investigate the anti-inflammatory effects of Kainth fruit extracts using in vitro and in vivo inflammation models. MATERIAL AND METHODS: Free, esterified and bound fractions from the Kainth ethanolic extracts were prepared for determining the anti-inflammatory effect. The levels of 5-LOX and COX-2 were determined in vitro. The protein levels of cytokines (IL-6, TNF-α & IL-10) were quantitated by ELISA method in lipopolysaccharide-stimulated RAW macrophages. Also, the anti-inflammatory potential of the Kainth fruit extracts was determined using the carrageenan-induced mice paw edema model. The bioaccessibility of Kainth fruit extracts was measured using a simulated in vitro digestion system (salivary, gastric and intestinal). RESULTS: The Kainth fruit extracts were partially purified to yield free, esterified and bound phenolics. Free and bound phenolics of Kainth fruits inhibited 5-Lipoxygenase, Cyclooxygenase-2 activities and pro-inflammatory cytokines (Interleukin-6 and tumour necrosis factor-α) expression in vitro. Also, oral administration of these extracts to the carrageenan-injected mice showed an anti-inflammatory effect by decreasing the pro-inflammatory cytokines and reducing the cellular infiltration in paw tissues. Also, both the extracts showed better bioavailability and bioaccessibility in in vitro and in vivo studies. CONCLUSIONS: The results indicated that free and bound phenolics from Kainth fruits that are rich in catechin, epicatechin, arbutin and chlorogenic acid exhibited anti-inflammatory effects and could potentially be used to treat inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Fruit/chemistry , Inflammation/drug therapy , Plant Extracts/pharmacology , Polyphenols/pharmacology , Pyrus/chemistry , Animals , Anti-Inflammatory Agents/chemistry , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Carrageenan/toxicity , Cell Survival/drug effects , Edema/chemically induced , Edema/drug therapy , Lipopolysaccharides/toxicity , Mice , Phytotherapy , Plant Extracts/chemistry , Polyphenols/chemistry , RAW 264.7 CellsABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Flavonoids are the main components of the traditional Chinese medicine Anemarrhenae Rhizoma (dried rhizome of Anemarrhena asphodeloides Bge.), which has been reported to possess activity against inflammation and tumor. AIM OF STUDY: Regulation of the arachidonic acid (AA) cascade through cyclooxygenase (COX) and lipoxygenase (LOX) represent the two major pathways to treat inflammatory of benign prostatic hyperplasia (BPH). In this study, Anemarrhenae Rhizoma flavonoids and its main compounds (mangiferin, neomangiferin and isomangiferin) were investigated for effects on AA metabolism. METHODS: Ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) was used to monitor AA metabolites in BPH rats and in PC-3 cells. COX-2 and 5-LOX protein and mRNA levels were measured by Western blot and qPCR, respectively, along with histopathological assessment of prostate tissues. RESULTS: Treatment with flavonoids significantly ameliorated BPH-associated prostate inflammation and inhibited the expression of COX-2 and 5-LOX at the protein and mRNA levels. Quantitative metabolomic analysis of blood plasma showed flavonoids treatment decreased AA levels and its metabolites associated with the COX and LOX pathways. Further exploration of the flavonoid compounds mangiferin, neomangiferin and isomangiferin showed they inhibited AA metabolism to varying degrees in PC-3 cell cultures. CONCLUSION: Anemarrhenae Rhizoma flavonoids act to inhibit BPH-related inflammation in vivo and in vitro by targeting AA metabolism and interfering with COX and LOX pathways. The identification of mangiferin, neomangiferin and isomangiferin as anti-inflammatory components suggests flavonoids interventions represent a promising therapeutic approach for BPH.
Subject(s)
Anemarrhena/chemistry , Flavonoids/pharmacology , Inflammation/drug therapy , Prostatic Hyperplasia/drug therapy , Animals , Arachidonate 5-Lipoxygenase/genetics , Chromatography, High Pressure Liquid , Cyclooxygenase 2/genetics , Disease Models, Animal , Drugs, Chinese Herbal/pharmacology , Flavonoids/isolation & purification , Humans , Male , Metabolomics , PC-3 Cells , Rats , Rats, Sprague-Dawley , Rhizome , Tandem Mass SpectrometryABSTRACT
This study investigated the role 5-lypoxigenase (5-LO) on alveolar socket healing in aged female mice treated with zoledronic acid (ZL). Forty 129/Sv female mice (64-68 weeks old), 20 wild type (WT) and 20 5-LO knockout (5LOKO) were equally distributed according to ZL treatment: WT Control, WT ZL, 5LOKO Control, and 5LOKO ZL. ZL groups were treated with an intraperitoneal injection of 250 µg/Kg of ZL, while controls were treated with saline. Treatments were administered once a week, starting four weeks before surgery for tooth extraction and until 7 and 21 days post-surgery. Mice were euthanized for a comprehensive microscopic analysis (microCT, histomorphometry and immunohistochemistry). WT ZL mice presented intense inflammatory infiltrate (7 days), delayed bone formation (21 days), reduced collagenous matrix quality, and a deficiency in Runx-2 + , TRAP + , and macrophages as compared to controls. 5LOKO ZL animals presented decreased number of Runx-2 + cells in comparison to 5LOKO Control at 7 days, but no major changes in bone healing as compared to WT or 5LOKO mice at 21 days. The knockout of 5LO favored intramembranous bone healing in aged female mice, with a direct impact on inflammatory response and bone metabolism on the development of ONJ-like lesions.
Subject(s)
Arachidonate 5-Lipoxygenase/deficiency , Tooth Socket/drug effects , Wound Healing/drug effects , Zoledronic Acid/administration & dosage , Age Factors , Animals , Arachidonate 5-Lipoxygenase/genetics , Biomarkers , Disease Models, Animal , Female , Gene Expression , Immunohistochemistry , Mice , Mice, Knockout , Tooth Extraction/adverse effects , Tooth Extraction/methods , Tooth Socket/diagnostic imaging , Tooth Socket/pathology , Treatment Outcome , X-Ray MicrotomographyABSTRACT
Cervical cancer is a common public health issue with high morbidity worldwide. Paeonol (Pae) has been recognized as a traditional Chinese medicine used for the treatment of various cancer types. However, whether Pae could exert a protective effect on cervical cancer remains to be investigated. The aim of the present study was to explore the role of Pae in cervical cancer cells and identify the potential mechanism. Cell Counting Kit8 and colonyformation assays were conducted to test the proliferation of HeLa cells. Additionally, wound healing and transwell assays were used to detect the migratory and invasive abilities of cells. The plasmid that overexpressed 5lipoxygenase (5LO) or control vector was constructed and transfected into the cells. Subsequently, flow cytometry was used to monitor the apoptotic rate of cells. The expression levels of apoptosisassociated proteins and 5LO were detected using western blot analysis. Reverse transcriptionquantitative PCR analysis detected the expression of 5LO. Pae inhibited the proliferation, invasion and migration of HeLa cells, promoted cell apoptosis and downregulated the expression of 5LO. Overexpression of 5LO, however, attenuated these effects. Thus, Pae could inhibit the proliferation, migration and invasion, as well as promote apoptosis of HeLa cells by regulating the expression of 5LO.
Subject(s)
Acetophenones/pharmacology , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Arachidonate 5-Lipoxygenase/metabolism , Cell Movement/drug effects , Arachidonate 5-Lipoxygenase/genetics , Cell Proliferation/drug effects , HeLa Cells , HumansABSTRACT
Clinical studies using a range of omega-3 supplements have yielded conflicting results on their efficacy to control inflammation. Omega-3 fatty acids are substrate for the formation of potent immune-protective mediators, termed as specialized pro-resolving mediators (SPM). Herein, we investigated whether observed differences in the potencies of distinct omega-3 supplements were linked with their ability to upregulate SPM formation. Using lipid mediator profiling we found that four commercially available supplements conferred a unique SPM signature profile to human macrophages, with the overall increases in SPM concentrations being different between the four supplements. These increases in SPM concentrations were linked with an upregulation of macrophage phagocytosis and a decreased uptake of oxidized low-density lipoproteins. Pharmacological inhibition of two key SPM biosynthetic enzymes 5-Lipoxygenase or 15-Lipoxygenase reversed the macrophage-directed actions of each of the omega-3 supplements. Furthermore, administration of the two supplements that most potently upregulated macrophage SPM formation and reprogrammed their responses in vitro, to APOE-/- mice fed a western diet, increased plasma SPM concentrations and reduced vascular inflammation. Together these findings support the utility of SPM as potential prognostic markers in determining the utility of a given supplement to regulate macrophage responses and inflammation.
Subject(s)
Atherosclerosis/prevention & control , Dietary Supplements , Fatty Acids, Omega-3/administration & dosage , Leukotrienes/biosynthesis , Lipoxins/biosynthesis , Macrophages/drug effects , Prostaglandins/biosynthesis , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/genetics , Apolipoproteins E/immunology , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 15-Lipoxygenase/immunology , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/immunology , Atherosclerosis/etiology , Atherosclerosis/immunology , Atherosclerosis/metabolism , Diet, Western/adverse effects , Fatty Acids, Omega-3/metabolism , Female , Gene Expression , Humans , Leukotrienes/immunology , Lipoproteins, LDL/antagonists & inhibitors , Lipoproteins, LDL/pharmacology , Lipoxins/immunology , Lipoxygenase Inhibitors/pharmacology , Macrophages/cytology , Macrophages/immunology , Male , Mice , Mice, Knockout, ApoE , Phagocytosis/drug effects , Primary Cell Culture , Principal Component Analysis , Prostaglandins/immunologyABSTRACT
BACKGROUND: Modulation of the arachidonic acid (AA) cascade via 5-lipoxygenase (5-LOX) and cyclooxygenase-2 (COX-2) represent the two major pathways for treatments of inflammation and pain. The design and development of inhibitors targeting both 5-LOX and COX-2 has gained increasing popularity. As evidenced, 5-LOX and COX-2 dual targeted inhibitors have recently emerged as the front runners of anti-inflammatory drugs with improved efficacy and reduced side effects. Natural products represent a rich resource for the discovery of dual targeted 5-LOX and COX-2 inhibitors. By combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS), an efficient method was developed to identify spirostanol glycosides and furostanol glycosides as the 5-LOX/COX-2 dual inhibitors from saponins extract of Anemarrhenae Rhizoma (SEAR). METHODS: A highly efficient method by combining affinity ultrafiltration and high-performance liquid chromatography-mass spectrometry (AUF-LC-MS) was first developed to screen and characterize the 5-LOX/COX-2 dual targeted inhibitors from SEAR. The structures of compounds in the ultrafiltrate were characterized by high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). In addition, in vitro 5-LOX/COX-2 inhibition assays and their dual expression in vivo were performed to confirm the inhibitory activities of the compounds screened by AUF-LC-MS. Molecular docking studies with the corresponding binding energy were obtained which fit nicely to both 5-LOX and COX-2 protein cavities and in agreement with our affinity studies. RESULTS: A total of 5 compounds, timosaponin A-II, timosaponin A-III, timosaponin B-II, timosaponin B-III and anemarrhenasaponin I, were identified as potential 5-LOX/COX-2 dual targeted inhibitors with specific binding values > 1.5 and IC50 ≤ 6.07 µM. CONCLUSION: The present work demonstrated that spirostanol glycoside and furostanol glycoside were identified as two novel classes of dual inhibitors of 5-LOX/COX-2 enzymes by employing a highly efficient screening method of AUF-LC-MS. These natural products represent a novel class of anti-inflammatory agents with the potential of improved efficacy and reduced side effects.
Subject(s)
Anemarrhena/chemistry , Cyclooxygenase 2 Inhibitors/pharmacology , Glycosides/chemistry , Lipoxygenase Inhibitors/pharmacology , Spirostans/chemistry , Sterols/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Arachidonate 5-Lipoxygenase/chemistry , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Chromatography, High Pressure Liquid , Cyclooxygenase 2/chemistry , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cyclooxygenase 2 Inhibitors/chemistry , Drug Evaluation, Preclinical , Glycosides/pharmacology , Inflammation/drug therapy , Lipoxygenase Inhibitors/chemistry , Mass Spectrometry , Molecular Docking Simulation , Rats , Rhizome/chemistry , Saponins/chemistry , Saponins/pharmacology , Spirostans/pharmacology , Steroids/chemistry , Steroids/pharmacology , Sterols/pharmacology , UltrafiltrationABSTRACT
Macrophage polarization switches during the course of inflammation along with the lipid mediators released. We investigated the lipid mediator formation in human monocyte-derived macrophages during in vitro differentiation and pathogen stimulation. For this, peripheral blood monocytes were differentiated into M1 (CSF-2/IFNγ) or M2 (CSF-1/IL-4) macrophages followed by stimulation with the toll-like receptor (TLR) ligands zymosan (TLR-2), Poly(I:C) (TLR-3) or bacterial lipopolysaccharides (TLR-4) mimicking fungal, viral and bacterial infection, respectively. Expression of enzymes involved in lipid mediator formation such as 5- and 15-lipoxygenases (LO), the 5-LO activating protein and cyclooxygenase-2 (COX-2) was monitored on mRNA and protein level and lipid mediator formation was assessed. In addition, cytokine release was measured. In vitro differentiation of human peripheral blood monocytes to M1 and M2 macrophages considerably attenuated 5-LO activity. Furthermore, while TLR-2 and -4 stimulation of M1 macrophages primarily triggered pro-inflammatory cytokines and lipid mediators, persistent stimulation (16 h) of human M2 macrophages induced a coordinated upregulation of 5- and 15-LO-2 expression. This was accompanied by a marked increase in IL-10 and monohydroxylated 15-LO products in the conditioned media of the cells. After additional stimulation with Ca2+ ionophore combined with supplementation of arachidonic, eicosapentaenoic and docosahexaenoic acid these cells also released small amounts of SPM such as lipoxins and resolvins. From this we conclude that activation of TLR-2 or -4 triggers the biosynthesis of pro-inflammatory 5-LO and COX-2 derived lipid mediators in human monocyte-derived M1 macrophages while persistent stimulation of M2 macrophages induces a shift towards pro-resolving 15-LO derived oxylipins.
Subject(s)
Arachidonate 15-Lipoxygenase/metabolism , Arachidonate 5-Lipoxygenase/metabolism , Macrophages/metabolism , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/metabolism , Arachidonate 15-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/genetics , Cells, Cultured , Cyclooxygenase 2/metabolism , Cytokines/metabolism , Humans , Lipid Metabolism , Lipopolysaccharides/pharmacology , Macrophages/drug effectsABSTRACT
BACKGROUND: Adapted ketogenic diet (AKD) and caloric restriction (CR) have been suggested as alternative therapeutic strategies for inflammatory, hyperproliferative and neurodegenerative diseases. Pro-inflammatory eicosanoids have been implicated in the pathogenesis of multiple sclerosis since they augment vascular permeability and induce leukocyte migration into the brain. We explored the impact of ketogenic diets on gene expression of biosynthetic enzymes for pro- (ALOX5, COX1, COX2) and anti-inflammatory (ALOX15) eicosanoids in patients with relapsing-remitting multiple sclerosis. METHODS: 60 adults were prospectively recruited for this six months randomized controlled trial and the impact of dietary treatment on the Multiple Sclerosis Quality of Life-54 index (ClinicalTrials.gov (NCT01538355) has previously been published. Here we explored 24 patients (8 controls, 5 on CR and 11 on AKD). For statistical analysis we combined the two diet groups to a single pooled treatment group. FINDINGS: Inter-group comparison indicated that expression of the pro-inflammatory ALOX5 in the pooled treatment group was significantly (pâ¯<â¯0.05) reduced when compared with the control group. Moreover, intra-group comparison (same individuals before and after dietary treatment) suggested significantly impaired expression of other pro-inflammatory enzymes, such as COX1 (pâ¯<â¯0.001) and COX2 (pâ¯<â¯0.05). Finally, pretreatment cross-group analysis revealed a significant positive correlation between expression of pro-inflammatory ALOX5 and COX2 and an inverse correlation of ALOX5 and COX1 expression with the MSQoL-54 index. INTERPRETATION: Ketogenic diets can reduce the expression of enzymes involved in the biosynthesis of pro-inflammatory eicosanoids. Pharmacological interference with eicosanoid biosynthesis might constitute a strategy supplementing current therapeutic approaches for MS.
Subject(s)
Diet, Ketogenic , Gene Expression Regulation , Lipoxygenase/genetics , Multiple Sclerosis/etiology , Multiple Sclerosis/metabolism , Prostaglandin-Endoperoxide Synthases/genetics , Adolescent , Adult , Arachidonate 5-Lipoxygenase/genetics , Biomarkers , Child , Diet, Carbohydrate-Restricted , Diet, Ketogenic/adverse effects , Female , Humans , Inflammation Mediators/metabolism , Lipoxygenase/metabolism , Male , Middle Aged , Multiple Sclerosis/pathology , Prostaglandin-Endoperoxide Synthases/metabolism , Quality of Life , Recurrence , Young AdultABSTRACT
Recent evidence suggests that specialized proresolving lipid mediators (SPMs) generated from docosahexaenoic acid (DHA) can modulate the vascular injury response. However, cellular sources for these autacoids within the vessel wall remain unclear. Here, we investigated whether isolated vascular cells and tissues can produce SPMs and assessed expression and subcellular localization of the key SPM biosynthetic enzyme 5-lipoxygenase (LOX) in vascular cells. Intact human arteries incubated with DHA ex vivo produced 17-hydroxy DHA (17-HDHA) and D-series resolvins, as assessed by liquid chromatography-tandem mass spectrometry. Addition of 17-HDHA to human arteries similarly increased resolvin production. Primary cultures of human saphenous vein endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) converted 17-HDHA to SPMs, including resolvin D1 (RvD1) and other D-series resolvins and protectins. This was accompanied by a rapid translocation of 5-LOX from nucleus to cytoplasm in both ECs and VSMCs, potentially facilitating SPM biosynthesis. Conditioned medium from cells exposed to 17-HDHA inhibited monocyte adhesion to TNF-α-stimulated EC monolayers. These downstream effects were partially reversed by antibodies against the RvD1 receptors ALX/FPR2 and GPR32. These results suggest that autocrine and/or paracrine signaling via locally generated SPMs in the vasculature may represent a novel homeostatic mechanism of relevance to vascular health and disease.-Chatterjee, A., Komshian, S., Sansbury, B. E., Wu, B., Mottola, G., Chen, M., Spite, M., Conte, M. S. Biosynthesis of proresolving lipid mediators by vascular cells and tissues.
Subject(s)
Docosahexaenoic Acids/pharmacology , Endothelial Cells/metabolism , Lipid Metabolism/physiology , Myocytes, Smooth Muscle/metabolism , Antibodies , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Cells, Cultured , Cytokines/metabolism , Docosahexaenoic Acids/genetics , Docosahexaenoic Acids/metabolism , Gene Expression Regulation/physiology , Humans , Inflammation/metabolism , Leukocytes/physiology , Molecular Structure , Protein Transport/physiology , Receptors, Formyl Peptide/genetics , Receptors, Formyl Peptide/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Lipoxin/genetics , Receptors, Lipoxin/metabolismABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of the stem bark of Ficus platyphylla (FP) have been used in traditional the Nigerian medicine to treat psychoses, depression, epilepsy, pain and inflammation. Previous studies have revealed the analgesic and anti-inflammatory effects of FP in different assays including acetic acid-induced writhing, formalin-induced nociception, and albumin-induced oedema. PURPOSE/METHODS: In this study, we assessed the effects of the standardised extract of FP on hot plate nociceptive threshold and vocalisation threshold in response to electrical stimulation of the tail root in order to confirm its acclaimed analgesic properties. We also investigated the molecular mechanisms underlying these effects, with the focus on opiate receptor binding and the key enzymes of eicosanoid biosynthesis, namely cyclooxygenase (COX) and 5-lipoxygenase (5-LO). RESULTS: FP (i) increased the hot plate nociceptive threshold and vocalisation threshold. The increase in hot plate nociceptive threshold was detectable over a period of 30min whereas the increase in vocalisation threshold persisted over a period of 90min. (ii) FP showed an affinity for µ opiate receptors but not for δ or κ opiate receptors, and (iii) FP inhibited the activities of COX-2 and 5-LO but not of COX-1. CONCLUSIONS: We provided evidence supporting the use of FP in Nigerian folk medicine for the treatment of different types of pain, and identified opioid and non-opioid targets. It is interesting to note that the dual inhibition of COX-2 and 5-LO appears favourable in terms of both efficacy and side effect profile.
Subject(s)
Analgesics/pharmacology , Analgesics/therapeutic use , Ficus , Pain/drug therapy , Plant Extracts/pharmacology , Plant Extracts/therapeutic use , Animals , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolism , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/metabolism , Electric Stimulation , HEK293 Cells , Hot Temperature/adverse effects , Humans , Male , Methanol/chemistry , Mice , Plant Bark/chemistry , Receptors, Opioid/genetics , Receptors, Opioid/metabolism , Solvents/chemistry , Vocalization, Animal/drug effectsABSTRACT
Vitamin E forms are substantially metabolized to various carboxychromanols including 13'-carboxychromanols (13'-COOHs) that are found at high levels in feces. However, there is limited knowledge about functions of these metabolites. Here we studied δT-13'-COOH and δTE-13'-COOH, which are metabolites of δ-tocopherol and δ-tocotrienol, respectively. δTE-13'-COOH is also a natural constituent of a traditional medicine Garcinia Kola. Both 13'-COOHs are much stronger than tocopherols in inhibition of pro-inflammatory and cancer promoting cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX), and in induction of apoptosis and autophagy in colon cancer cells. The anticancer effects by 13'-COOHs appeared to be partially independent of inhibition of COX-2/5-LOX. Using liquid chromatography tandem mass spectrometry, we found that 13'-COOHs increased intracellular dihydrosphingosine and dihydroceramides after short-time incubation in HCT-116 cells, and enhanced ceramides while decreased sphingomyelins during prolonged treatment. Modulation of sphingolipids by 13'-COOHs was observed prior to or coinciding with biochemical manifestation of cell death. Pharmaceutically blocking the increase of these sphingolipids partially counteracted 13'-COOH-induced cell death. Further, 13'-COOH inhibited dihydroceramide desaturase without affecting the protein expression. In agreement with these mechanistic findings, δTE-13'-COOH significantly suppressed the growth and multiplicity of colon tumor in mice. Our study demonstrates that 13'-COOHs have anti-inflammatory and anticancer activities, may contribute to in vivo anticancer effect of vitamin E forms and are promising novel cancer prevention agents.
Subject(s)
Arachidonate 5-Lipoxygenase/genetics , Colonic Neoplasms/drug therapy , Cyclooxygenase 2/genetics , Vitamin E/metabolism , Animals , Apoptosis/drug effects , Autophagy/drug effects , Chromans/administration & dosage , Colonic Neoplasms/genetics , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclooxygenase 2 Inhibitors/administration & dosage , Garcinia kola/metabolism , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , Humans , Mice , Sphingolipids/metabolism , Tocopherols/metabolismABSTRACT
We have previously shown the influence of leukotrienes (LTs) on reproductive functions in vivo: LTB4 is luteotrophic and supports corpus luteum function inducing PGE2 and progesterone (P4) secretion, whereas LTC4 is luteolytic and stimulates PGF2α secretion in cattle. The aim of this study was to examine expression and production profiles of LTs and their actions in the endometrium. LT receptors (LTB4R for LTB4 and CysLTR2 for LTC4), 5-lipoxygenase (LO), 12-LO synthase (LTCS) and LTA4 hydrolase (LTAH) mRNA and protein expression, as well as LT production were measured in bovine endometrial tissue during the luteal phases of the oestrous cycle. The action of LTs on uterine function was studied by measuring the level of PGs after stimulating uterine slices with LTs on Days 8-10 of the cycle. Expression of 5-LO and LTB4R mRNA and protein were highest on Days 2-4 of the cycle, while CysLTR2 and LTCS were highest on Days 16-18 (P<0.05). LTB4 concentration was highest on Days 2-4 of the cycle, whereas the greatest LTC4 level was on Days 16-18 (P<0.05). Both LTB4 and C4 increased the content of PGE2 and F2α in endometrial slices at a dose of 10(-7)M (P<0.05). In summary, mRNA expression and activation of receptors for LTB4 and production occur in the first part of the cycle, whereas LTC4 and its receptors predominate at the end of the cycle. The 12-LO and 5-LO pathways are complementary routes of LT production in the bovine uterus.
Subject(s)
Arachidonate 5-Lipoxygenase/metabolism , Endometrium/metabolism , Glutathione Transferase/metabolism , Leukotrienes/metabolism , Luteal Phase/metabolism , Receptors, Leukotriene B4/metabolism , Receptors, Leukotriene/metabolism , Abattoirs , Animals , Animals, Inbred Strains , Arachidonate 5-Lipoxygenase/genetics , Cattle , Dairying , Endometrium/enzymology , Estrous Cycle/metabolism , Female , Gene Expression Profiling/veterinary , Gene Expression Regulation, Enzymologic , Glutathione Transferase/genetics , Leukotriene B4/metabolism , Leukotriene C4/metabolism , Poland , Prostaglandins/agonists , Prostaglandins/metabolism , RNA, Messenger/metabolism , Receptors, Leukotriene/agonists , Receptors, Leukotriene/genetics , Receptors, Leukotriene B4/agonists , Receptors, Leukotriene B4/genetics , Tissue Culture Techniques/veterinaryABSTRACT
The present study focused on the investigation of the inhibition of cyclooxygenase-2 and nuclear factor kappa B1 gene expression, nitric oxide production, leukotriene biosynthesis (5-lipoxygenase), and cyclooxygenase-1 and cyclooxygenase-2 enzymes of Onopordum acanthium, and the isolation and identification of its active compounds. From the chloroform soluble part of the MeOH extract prepared from aerial parts, lignans [pinoresinol (1), syringaresinol (2), and medioresinol (3)] and flavonoids [hispidulin (4), nepetin (5), apigenin (6), and luteolin (7)] were isolated by a combination of different chromatographic methods. The structures of the compounds were determined by means of mass spectrometry and 1D- and 2D-nuclear magnetic resonance spectroscopy, and by comparison of the spectral data with literature values. Extracts of different polarity and the isolated compounds obtained from the aerial parts, together with those previously isolated from the roots of the plant [4ß,15-dihydro-3-dehydrozaluzanin C (8), zaluzanin C (9), 4ß,15,11ß,13-tetrahydrozaluzanin C (10), nitidanin diisovalerianate (11), 24-methylenecholesterol (12), and 13-oxo-9Z,11E-octadecadienoic acid (13)], were evaluated for their inhibitory effects on cyclooxygenase-2 and nuclear factor kappa B1 gene expression, inducible nitric oxide synthase, 5-lipoxygenase, and cyclooxygenase-1 and cyclooxygenase-2 enzymes in in vitro assays. It was found that O. acanthium extracts exert strong inhibitory activities in vitro and some lignans, flavonoids, and sesquiterpenes may play a role in these activities. 4ß,15-Dihydro-3-dehydrozaluzanin C and zaluzanin C at 20 µM were the most active constituents tested against lipopolysaccharide/interferon-γ-induced nitric oxide production (100.4 ± 0.5â% and 99.4 ± 0.8â%) in the inhibition of cyclooxygenase-2 (98.6 ± 0.2â% and 97.0 ± 1.1â%) and nuclear factor kappa B1 gene expression (76.7 ± 7.3â% and 69.9 ± 3.4â%). Furthermore, it was shown that these inhibitory effects are not due to cytotoxicity of the compounds.
Subject(s)
Cyclooxygenase Inhibitors/pharmacology , NF-kappa B p50 Subunit/genetics , Nitric Oxide/metabolism , Onopordum/chemistry , Plant Extracts/pharmacology , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Cell Line/drug effects , Cyclooxygenase 1/genetics , Cyclooxygenase 1/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cyclooxygenase 2 Inhibitors/pharmacology , Cyclooxygenase Inhibitors/chemistry , Drug Evaluation, Preclinical/methods , Gene Expression Regulation/drug effects , Humans , Lipoxygenase Inhibitors/chemistry , Lipoxygenase Inhibitors/pharmacology , Molecular Structure , NF-kappa B p50 Subunit/antagonists & inhibitors , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Plant Extracts/chemistry , Plants, Medicinal/chemistryABSTRACT
Obese women with type 2 diabetes mellitus (T2DM) have more inflammation in their subcutaneous white adipose tissue (sWAT) than age-and-BMI similar obese women with normal glucose tolerance (NGT). We aimed to investigate whether WAT fatty acids and/or oxylipins are associated with the enhanced inflammatory state in WAT of the T2DM women. Fatty acid profiles were measured in both subcutaneous and visceral adipose tissue (vWAT) of 19 obese women with NGT and 16 age-and-BMI similar women with T2DM. Oxylipin levels were measured in sWAT of all women. Arachidonic acid (AA) and docosahexaenoic acid (DHA) percentages were higher in sWAT, but not vWAT of the T2DM women, and AA correlated positively to the gene expression of macrophage marker CD68. We found tendencies for higher oxylipin concentrations of the 5-LOX leukotrienes in sWAT of T2DM women. Gene expression of the 5-LOX leukotriene biosynthesis pathway was significantly higher in sWAT of T2DM women. In conclusion, AA and DHA content were higher in sWAT of T2DM women and AA correlated to the increased inflammatory state in sWAT. Increased AA content was accompanied by an upregulation of the 5-LOX pathway and seems to have led to an increase in the conversion of AA into proinflammatory leukotrienes in sWAT.
Subject(s)
Arachidonate 5-Lipoxygenase/analysis , Arachidonic Acid/analysis , Diabetes Mellitus, Type 2/enzymology , Docosahexaenoic Acids/analysis , Inflammation Mediators/analysis , Inflammation/enzymology , Obesity, Morbid/enzymology , Signal Transduction , Subcutaneous Fat/enzymology , 5-Lipoxygenase-Activating Proteins/genetics , Adult , Antigens, CD/genetics , Antigens, Differentiation, Myelomonocytic/genetics , Arachidonate 5-Lipoxygenase/genetics , Diabetes Mellitus, Type 2/diagnosis , Diabetes Mellitus, Type 2/genetics , Dipeptidases/genetics , Female , Humans , Inflammation/diagnosis , Intra-Abdominal Fat/enzymology , Leukotrienes/analysis , Middle Aged , Netherlands , Obesity, Morbid/diagnosis , Obesity, Morbid/genetics , Obesity, Morbid/surgery , Up-RegulationABSTRACT
The 5-lipoxygenase product 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is the most powerful human eosinophil chemoattractant among lipid mediators and could play a major pathophysiological role in eosinophilic diseases such as asthma. Its actions are mediated by the OXE receptor, orthologs of which are found in many species from humans to fish, but not rodents. The unavailability of rodent models to examine the pathophysiological roles of 5-oxo-ETE and the OXE receptor has substantially hampered progress in this area. As an alternative, we have explored the possibility that the cat could serve as an appropriate animal model to investigate the role of 5-oxo-ETE. We found that feline peripheral blood leukocytes synthesize 5-oxo-ETE and that physiologically relevant levels of 5-oxo-ETE are present in bronchoalveolar lavage fluid from cats with experimentally induced asthma. 5-Oxo-ETE (EC50, 0.7nM) is a much more potent activator of actin polymerization in feline eosinophils than various other eicosanoids, including leukotriene (LT) B4 and prostaglandin D2. 5-Oxo-ETE and LTB4 induce feline leukocyte migration to similar extents at low concentrations (1nM), but at higher concentrations the response to 5-oxo-ETE is much greater. Although high concentrations of selective human OXE receptor antagonists blocked 5-oxo-ETE-induced actin polymerization in feline granulocytes, their potencies were about 200 times lower than for human granulocytes. We conclude that feline leukocytes synthesize and respond to 5-oxo-ETE, which could potentially play an important role in feline asthma, a common condition in this species. The cat could serve as a useful animal model to investigate the pathophysiological role of 5-oxo-ETE.
Subject(s)
Arachidonic Acids/pharmacology , Asthma/metabolism , Eosinophils/drug effects , Neutrophils/drug effects , Actins/genetics , Actins/metabolism , Allergens/immunology , Animals , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Arachidonic Acids/biosynthesis , Asthma/chemically induced , Asthma/genetics , Asthma/immunology , Benzeneacetamides/pharmacology , Benzothiazoles/pharmacology , Bronchoalveolar Lavage Fluid/cytology , Cats , Chemotaxis/drug effects , Chemotaxis/immunology , Cynodon/chemistry , Cynodon/immunology , Disease Models, Animal , Eosinophils/metabolism , Eosinophils/pathology , Female , Gene Expression , Humans , Leukotriene B4/pharmacology , Male , Neutrophils/metabolism , Neutrophils/pathology , Polymerization , Primary Cell Culture , Prostaglandin D2/pharmacology , Receptors, Eicosanoid/antagonists & inhibitors , Receptors, Eicosanoid/genetics , Receptors, Eicosanoid/metabolismABSTRACT
Cardiac hypertrophy has been considered as an important risk factor of morbidity and mortality. It is characterized as thickening of ventricle wall of the heart and consequent reduction in the contracting ability of the heart to pump the blood. Hyperglycemia-induced reactive oxygen species act as major mediators of diabetic complications. Inflammation plays an essential role in the development of diabetic cardiac hypertrophy. Selenium has been shown to induce insulin-like and anti-inflammatory effects in human and experimental animals. But, its mechanism of action has not been elucidated. Hence, in order to probe into its mechanism at molecular level, we designed an experiment to study the effect of selenium as sodium selenite in streptozotocin-induced diabetic rats. The rats were divided into four groups and maintained as follows: (1) controls, (2) sodium selenite-treated controls, (3) diabetic, and (4) sodium selenite-treated diabetic rats. Duration of the experiment was 30 days. Selenium supplementation enhanced the streptozotocin-induced reduction in the activities of antioxidant enzymes, decreased the serum glucose level, glycated hemoglobin content, concentration of high-sensitivity C-reactive protein, levels of lipid peroxidation products, as well as inflammatory parameters. Decrease in the phospholipase activity by selenium supplementation also contributed to the downregulation of leukotriene pathway. It also downregulated the expressions of nuclear transcription factor κB (NFκB), lipoxygenase, cyclooxygenase, 5-lipoxygenase-activating protein, and receptor for leukotriene B4. Hence, selenium decreased the production of reactive oxygen species and inhibited the activation of NFκB-mediated transcription of pro-inflammatory mediators which resulted in the downregulation of leukotriene pathway in diabetic cardiac hypertrophy.
Subject(s)
Diabetes Mellitus, Experimental/prevention & control , Diabetic Cardiomyopathies/prevention & control , Leukotrienes/metabolism , Oxidative Stress/drug effects , Signal Transduction/drug effects , Sodium Selenite/pharmacology , Analysis of Variance , Animals , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Blood Glucose/metabolism , C-Reactive Protein/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetic Cardiomyopathies/genetics , Diabetic Cardiomyopathies/metabolism , Down-Regulation/drug effects , Gene Expression/drug effects , Glycated Hemoglobin/metabolism , Lipid Peroxidation/drug effects , Male , NF-kappa B/genetics , NF-kappa B/metabolism , Oxidative Stress/physiology , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/physiology , Sodium Selenite/administration & dosage , Trace Elements/administration & dosage , Trace Elements/pharmacologyABSTRACT
Diabetic cardiomyopathy, a disorder of the heart muscle in diabetic patients, is one of the major causes of heart failure. Since diabetic cardiomyopathy is now known to have a high prevalence in the asymptomatic diabetic patient, prevention at the earliest stage of development by existing molecules would be appropriate in order to prevent the progression of heart failure. In this study, we investigated the protective role of multiple antioxidants (MA), on cardiac dysfunction and cardiac cell apoptosis in streptozotocin (STZ)-induced diabetic rat. Diabetic cardiomyopathy in STZ-treated animals was characterized by declined systolic, diastolic myocardial performance, oxidative stress and apoptosis in cardiac cells. Diabetic rats on supplementation with MA showed decreased oxidative stress evaluated by the content of reduced levels of lipid per-oxidation and decreased activity of catalase with down-regulation of heme-oxygenase-1 mRNA. Supplementation with MA also resulted in a normalized lipid profile and decreased levels of pro-inflammatory transcription factor NF-kappaB as well as cytokines such as TNF-α, IFN-γ, TGF-ß, and IL-10. MA was found to decrease the expression of ROS-generating enzymes like xanthine oxidase, monoamine oxidase-A along with 5-Lipoxygenase mRNA and/or protein expression. Further, left ventricular function, measured by a microtip pressure transducer, was re-established as evidenced by increase in ±dp/dtmax, heart rate, decreased blood pressure, systolic and diastolic pressure as well as decrease in the TUNEL positive cardiac cells with increased Bcl-2/Bax ratio. In addition, MA supplementation decreased cell death and activation of NF-kappaB in cardiac H9c2 cells. Based on our results, we conclude that MA supplementation significantly attenuated cardiac dysfunction in diabetic rats; hence MA supplementation may have important clinical implications in terms of prevention and management of diabetic cardiomyopathy.
Subject(s)
Antioxidants/pharmacology , Diabetes Mellitus, Experimental/prevention & control , Diabetic Cardiomyopathies/prevention & control , Heart Failure/prevention & control , Heart/drug effects , Myocytes, Cardiac/drug effects , Animals , Arachidonate 5-Lipoxygenase/genetics , Arachidonate 5-Lipoxygenase/metabolism , Blood Pressure/drug effects , Catalase/genetics , Catalase/metabolism , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/physiopathology , Diabetic Cardiomyopathies/physiopathology , Gene Expression/drug effects , Heart/physiopathology , Heart Failure/physiopathology , Heart Rate/drug effects , Heme Oxygenase (Decyclizing)/genetics , Heme Oxygenase (Decyclizing)/metabolism , Humans , Lipid Peroxidation/drug effects , Male , Monoamine Oxidase/genetics , Monoamine Oxidase/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , NF-kappa B/genetics , NF-kappa B/metabolism , Oxidative Stress/drug effects , Rats , Rats, Wistar , Streptozocin , Xanthine Oxidase/genetics , Xanthine Oxidase/metabolismABSTRACT
SCOPE: Feeding a diet supplemented with 10% (w/w) black and brown rice brans inhibited growth of transplanted tumors in mice. METHODS AND RESULTS: Black and brown rice brans from Oryza sativa LK1-3-6-12-1 and Chuchung cultivars each contained 21 compounds characterized by GC/MS. Mice fed diets with added rice brans for 2 weeks were intracutaneously inoculated with CT-26 mouse cancer cells and fed the same diet for two additional weeks. Tumor mass was 35 and 19% lower in the black and brown bran-fed groups, respectively. Tumor inhibition was associated with increases in cytolytic activity of splenic natural killer (NK) cells; partial restoration of nitric oxide production and phagocytosis in peritoneal macrophages; increases in released tumor necrosis factor-α, IL-1ß, and IL-6 from macrophages; increases in infiltration of leukocyte into the tumor; and reduction in angiogenesis inside the tumor. Proangiogenic biomarkers vascular endothelial growth factor, cyclooxygenase-2 (COX-2), and 5-lipoxygenase (5-LOX) were also reduced in mRNA and protein expression. ELISA of tumor cells confirmed reduced expression of COX-2 and 5-LOX. Reduced COX-2 and 5-LOX expression downregulated vascular endothelial growth factor and inhibited neoangiogenesis inside the tumors. CONCLUSION: Induction of NK activity and macrophages and inhibition of angiogenesis seem to contribute to tumor regression.