Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

Antioxidative effects of Panax notoginseng saponins in brain cells

Oxidative stress resulting from accumulation of reactive oxygen species (ROS) is involved in cell death associated with neurological disorders such as stroke, Alzheimer's disease and traumatic brain injury. Antioxidant compounds that improve endogenous antioxidant defenses have been proposed for neural protection. The purpose of this study was to investigate the potential protective effects of total saponin in leaves of Panax notoginseng (LPNS) on oxidative stress and cell death in brain cells in vitro. Lactate dehydrogenase (LDH) assay indicated that LPNS (5 μg/ml) reduced H₂O₂-induced cell death in primary rat cortical astrocytes (23 ± 8% reduction in LDH release vs. control). Similar protection was found in oxygen and glucose deprivation/reoxygenation induced SH-SY5Y (a human neuroblastoma cell line) cell damage (78 ± 7% reduction vs. control). The protective effects of LPNS in astrocytes were associated with attenuation of reactive oxygen species (ROS) accumulation. These effects involved activation of Nrf2 (nuclear translocation) and upregulation of downstream antioxidant systems including heme oxygenase-1 (HO-1) and glutathione S-transferase pi 1 (GSTP1). These results demonstrate for the first time that LPNS has antioxidative effects which may be neuroprotective in neurological disorders.     

Protective role against hydrogen peroxide and fibroblast stimulation via Ce-doped TiO2 nanostructured materials

BackgroundCerium oxide (CeO₂) and Ce-doped nanostructured materials (NMs) are being seen as innovative therapeutic tools due to their exceptional antioxidant effects; nevertheless their bio-applications are still in their infancy.MethodsTiO₂, Ce–TiO₂ and CeO₂–TiO₂ NMs were synthesized by a bottom-up microemulsion-mediated strategy and calcined during 7 h at 650 °C under air flux. The samples were compared to elucidate the physicochemical characteristics that determine cellular uptake, toxicity and the influence of redox balance between the Ce^3 +/Ce^4 + on the cytoprotective role against an exogenous ROS source: H₂O₂. Fibroblasts were selected as a cell model because of their participation in wound healing and fibrotic diseases.ResultsCe–TiO₂ NM obtained via sol–gel reaction chemistry of metallic organic precursors exerts a real cytoprotective effect against H₂O₂ over fibroblast proliferation, while CeO₂ pre-formed nanoparticles incorporated to TiO₂ crystalline matrix lead to a harmful CeO₂–TiO₂ material. TiO₂ was processed by the same pathways as Ce–TiO₂ and CeO₂–TiO₂ NM but did not elicit any adverse or protective influence compared to controls.ConclusionsIt was found that the Ce atoms source and its concentration have a clear effect on material's physicochemical properties and its subsequent influence in the cellular response. It can induce a range of biological reactions that vary from cytotoxic to cytoprotective.General significanceEven though there are still some unresolved issues and challenges, the unique physical and chemical properties of Ce-based NMs are fascinating and versatile resources for different biomedical applications.     

Asymmetric synthesis of novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluation of their anti-inflammatory activity

AimsTo design and synthesize novel N-(1-phenyl-2,3-dihydroxypropyl)arachidonylamides and evaluate their analgesic and anti-inflammatory potential.Main methodsThe murine macrophage cell line RAW 264.7 has been widely used as a model for inflammatory responses in vitro. Our model consists of cultured monolayers of RAW 264.7 cells in which media concentrations of 15-deoxy-Δ^13,14-PGJ₂ (PGJ) are measured by ELISA following LPS (10 ng/ml) stimulation and treatment with 0.1, 0.3, 1.0, 3.0 and 10 μM concentrations of the compounds.Key findingsOur data indicate that several of our compounds have the capacity to increase production of PGJ and may also increase the occurrence of programmed cell death (apoptosis).SignificanceThus these agents are potential candidates for the therapy of conditions characterized by ongoing (chronic) inflammation and its associated pain.     

pO2-dependent NO production determines OPPC activity in macrophages

Stimulated macrophages produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) using molecular O₂, L-arginine, and NADPH. Exposure of macrophages to hypoxia decreases NO production within seconds, suggesting substrate limitation as the mechanism. Conflicting data exist regarding the effect of pO₂ on NADPH production via the oxidative pentose phosphate cycle (OPPC). Therefore, the present studies were developed to determine whether NADPH could be limiting for NO production under hypoxia. Production of NO metabolites (NOx) and OPPC activity by RAW 264.7 cells was significantly increased by stimulation with lipopolysaccharide (LPS) and interferon γ (IFNγ) at pO₂ ranging from 0.07 to 50%. OPPC activity correlated linearly with NOx production at pO₂ > 0.13%. Increased OPPC activity by stimulated RAW 264.7 cells was significantly reduced by 1400 W, an iNOS inhibitor. OPPC activity was significantly increased by concomitant treatment of stimulated RAW 264.7 cells with chemical oxidants such as hydroxyethyldisulfide or pimonidazole, at 0.07 and 50% O₂, without decreasing NOx production. These results are the first to investigate the effect of pO₂ on the relationship between NO production and OPPC activity, and to rule out limitations in OPPC activity as a mechanism by which NO production is decreased under hypoxia.     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.     

Synthesis and PGE2 production inhibition of 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives

3,4-Diphenyl-substituted 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione derivatives were synthesized and evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells. Both 1H-furan-2,5-dione and 1H-pyrrole-2,5-dione rings as main scaffolds were easily obtained using one of three synthetic methods. Among the compounds investigated, 1H-3-(4-sulfamoylphenyl)-4-phenyl-pyrrole-2,5-dione (6l) showed a strong inhibitory activity (IC₅₀ = 0.61 μM) of PGE₂ production.     

Flavonoid glycosides from Barringtonia acutangula

Using various chromatographic separation techniques, ten flavonoid glycosides, including six new compounds namely barringosides A?F (1–6), were isolated from a methanol extract of the Barringtonia acutangula leaves. The structure elucidation was confirmed by spectroscopic analyses, including 1D and 2D NMR, and HR ESI MS. Their inhibitory effects on LPS-induced NO production in RAW264.7 cells were also evaluated. Among the isolated compounds, quercetin 3-O-β-d-(6-p-hydroxybenzoyl)galactopyranoside (9) showed significant effect with an IC₅₀ of 20.00 ± 1.68 µM. This is the first report of these flavonoid glycosides from Barringtonia genus and their inhibition on LPS-induced NO production in RAW264.7 cells was reported here for the first time.     

Neohesperidin dihydrochalcone down-regulates MyD88-dependent and -independent signaling by inhibiting endotoxin-induced trafficking of TLR4 to lipid rafts

Fulminant hepatic failure (FHF) is a lethal clinical syndrome characterized by the activation of macrophages and the increased production of inflammatory mediators. The purpose of this study was to investigate the effects of neohesperidin dihydrochalcone (NHDC), a widely-used low caloric artificial sweetener against FHF. An FHF experimental model was established in mice by intraperitoneal injection of D-galactosamine (d-GalN) (400 mg/kg)/lipopolysaccharides (LPS) (10 μg/kg). Mice were orally administered NHDC for 6 continuous days and at 1 h before d-GalN/LPS administration. RAW264.7 macrophages were used as an in vitro model. Cells were pre-treated with NHDC for 1 h before stimulation with LPS (10 μg/ml) for 6 h. d-GalN/LPS markedly increased the serum transaminase activities and levels of oxidative and inflammatory markers, which were significantly attenuated by NHDC. Mechanistic analysis indicated that NHDC inhibited LPS-induced myeloid differentiation factor 88 (MyD88) and TIR-containing adapter molecule (TRIF)-dependent signaling. Transient transfection of TLR4 or MyD88 siRNA inhibited the downstream inflammatory signaling. This effect could also be achieved by the pretreatment with NHDC. The fluorescence microscopy and flow cytometry results suggested that NHDC potently inhibited the binding of LPS to TLR4 in RAW264.7 macrophages. In addition, the inhibitory effect of NHDC on LPS-induced translocation of TLR4 into lipid raft domains played an important role in the amelioration of production of downstream pro-inflammatory molecules. Furthermore, the activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) by NHDC inhibited TLR4 signaling. In conclusion, our results suggest that NHDC attenuates d-GalN/LPS-induced FHF by inhibiting the TLR4-mediated inflammatory pathway, demonstrating a new application of NHDC as a hepatoprotective agent.     

Rubiyunnanins C-H, cytotoxic cyclic hexapeptides from Rubia yunnanensis inhibiting nitric oxide production and NF-κB activation

Six new (rubiyunnanins C–H, 1–6) and five known (7–11) cyclic hexapeptides were isolated from the roots of Rubia yunnanensis (Franch.) Diels. The structures and stereochemistry of 1–6 were established by extensive spectroscopic analyses and chemical methods. All compounds (1–11) not only exhibited cytotoxic activities against a panel of eleven cancer cell lines with IC₅₀ values ranging from 0.001 to 56.24 μM, but also exerted inhibitory activities against nitric oxide (NO) production in LPS and IFN-γ-induced RAW 264.7 murine macrophages with IC₅₀ values ranging from 0.05 to 12.68 μM. Furthermore, this is the first time it is being reported that compounds 2 and 7–10 significantly inhibited TNF-α-induced NF-κB activation in HEK-293-NF-κB luciferase stable cells with IC₅₀ values of 35.07, 0.03, 1.69, 12.64 and 1.18 μM, respectively.     

Advantage of menadione-catalyzed chemiluminescent assay for the determination of viable mammalian cell number

A chemiluminescent assay composed of TCPO [bis(2,4,6-trichlorophenyl)oxalate] and harmless rhodamine B is proposed to be superior in the determination of menadione-catalyzed hydrogen peroxide (H₂O₂) production by viable mammalian cells to that composed of TCPO and harmful pyrene [Anal. Biochem. 207 (1992) 255–260]. In tests, the proposed assay showed that the measurable concentration of H₂O₂ and the viable cell number ranged from 10^?9 to 10^?3 M and from 2 × 10² to 2 × 10⁶ cells/100 μl/well in the presence of 10% bovine serum, respectively. The measuring time was approximately 10 min. On the other hand, the measurable cell numbers by the colorimetric WST-1 and MTT assays requiring several hours ranged only from 10³ to 10⁴ cells/100 μl/well and from 10⁴ to 10⁵ cells/100 μl/well, respectively. The cytotoxicity of sodium dodecyl sulfate was also observed at intervals of 1 min by the proposed assay, but not by the above colorimetric assays.     

Role of the membrane in the formation of heme degradation products in red blood cells

AimsRed blood cells (RBCs) have an extensive antioxidant system designed to eliminate the formation of reactive oxygen species (ROS). Nevertheless, RBC oxidant stress has been demonstrated by the formation of a fluorescent heme degradation product (excitation (ex) 321 nm, emission (em) 465 nm) both in vitro and in vivo. We investigated the possibility that the observed heme degradation results from ROS generated on the membrane surface that are relatively inaccessible to the cellular antioxidants.Main methodsMembrane and cytosol were separated by centrifugation and the fluorescence intensity and emission maximum were measured. The effect on the maximum emission of adding oxidized and reduced hemoglobin to the fluorescent product formed when hemin is degraded by hydrogen peroxide (H₂O₂) was studied.Key findings90% of the fluorescent heme degradation products in hemolysates are found on the membrane. Furthermore, these products are not transferred from the cytosol to the membrane and must, therefore, be formed on the membrane. We also showed that the elevated level of heme degradation in HbCC cells that is attributed to increased oxidative stress was found on the membrane.SignificanceThese results suggest that, although ROS generated in the cytosol are neutralized by antioxidant enzymes, H₂O₂ generated by the membrane bound hemoglobin is not accessible to the cytosolic antioxidants and reacts to generate fluorescent heme degradation products. The formation of H₂O₂ on the membrane surface can explain the release of ROS from the RBC to other tissues and ROS damage to the membrane that can alter red cell function and lead to the removal of RBCs from circulation by macrophages.     

Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity

BackgroundHorseradish peroxidase (HRP) catalyzes H₂O₂ dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H₂O₂-induced inactivation, have been investigated.MethodsHRP reaction with H₂O₂ was studied by following H₂O₂ depletion, O₂ evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow.ResultsNitroxide protects HRP against H₂O₂-induced inactivation. The rate of H₂O₂ dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H₂O₂. The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO) > 4-OH-TPO > 3-carbamoyl proxyl > 4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III.ConclusionsNitroxide catalytically protects HRP against inactivation induced by H₂O₂ while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex.General SignificanceNitroxides catalytically protect heme proteins against inactivation induced by H₂O₂ revealing an additional role played by nitroxide antioxidants in vivo.     

Diarylheptanoids from the fruits of Amomum kravanh and their inhibitory activities of nitric oxide production

Two new diarylheptanoids with a tetrahydropyran ring, kravanhol A (1) and kravanhol B (2), along with one known diarylheptanoid renealtin A (3) were isolated from the fruits of Amomum kravanh. The structures of compounds 1 and 2 were established by analysis of spectroscopic data and their absolute configurations were determined by Mosher's method and CD experiments. Compound 2 showed inhibitory effect on nitric oxide production in lipopolysaccharide-activated RAW264.7 macrophages with an IC₅₀ value of 38.9 ± 1.8 μM.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Exploration of 2-benzylbenzimidazole scaffold as novel inhibitor of NF-κB

For finding the novel inhibitor of nuclear factor κB activity, a series of benzimidazole derivatives were rationally designed, synthesized and systematically studied for their in vitro activities against LPS induced NF-κB inhibition in RAW 264.7 cells using the SEAP assay based on the flexible chalcone JSH ((E)-1-(2-hydroxy-6-(isopentyloxy)phenyl)-3-(4-hydroxy phenyl)prop-2-en-1-one) which was previously reported. Although most of the benzimidazole derivatives showed strong inhibitory activity in low micromolar potency, 2-(4-methoxybenzyl)-1H-benzo[d]imidazole (3m; IC₅₀ = 1.7 μM) and 2-(2-methoxybenzyl)-1H-benzo[d]imidazole (3n; IC₅₀ = 2.4 μM) showed the best inhibition. The structure activity relationship revealed that 2-benzylbenzimidazole scaffold with hydrogen bonding acceptor on phenyl ring appears as a pharmacophore.     

Antioxidant activity and protection of human umbilical vein endothelial cells from hydrogen peroxide-induced injury by DMC,a chalcone from buds of Cleistocalyx operculatus

The aim of this work was to study the antioxidant activity and the protective effect of 2′,4′-dihydroxy-6′-methoxy-3′,5′-dimethylchalcone (DMC), the main compound from the buds of Cleistocalyx operculatus, on human umbilical vein endothelial cells against cytotoxicity induced by H₂O₂. The antioxidant activities of DMC were measured by ABTS assay, ferric reducing antioxidant power (FRAP) and hydroxyl radical scavenging activity, and protective effects of DMC on human umbilical vein endothelial cells against cytotoxicity induced by H₂O₂ were tested. DMC was found to have high ABTS radical scavenging activity (176.5 ± 5.2 μmol trolox equivalents/500 μmol DMC) and strong ferric reducing antioxidant power (213.3 ± 5.8 μmol trolox equivalents/500 μmol DMC). In addition, DMC scavenged the hydroxyl radicals, with IC₅₀ values of 243.7 ± 6.3 μM, slightly lower than the reference antioxidant ascorbic acid (ASC). Moreover, DMC could protect the human umbilical vein endothelial cells against H₂O₂-induced cytotoxicity by decrease intracellular and extracellular ROS levels, reduction in catalase (CAT) activity and increment in malondialdehyde (MDA) level. These results suggested that DMC has the potential to be used in the therapy of oxidative damage.