Miyabenol A inhibits LPS-induced NO production via IKK/IkappaB inactivation in RAW 264.7 macrophages: possible involvement of the p38 and PI3K pathways

The anti-inflammatory effect of miyabenol A, a stilbene isolated from Vitis thunbergii, on lipopolysaccaride (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages was studied. Miyabenol A inhibited NO production (EC 50: 2.7 muM) and iNOS protein and mRNA expression in a parallel concentration-dependent manner. LPS-evoked NF-kappaB nuclear translocation and associated IkappaB degradation were abrogated by miyabenol A treatment. Phosphorylations of IKKalpha/beta, ERK1/2, JNK p38 MAPK, and Akt were observed in LPS-stimulated cells; nevertheless, miyabenol A selectively blocked IKKalpha/beta, p38, and Akt phosphorylation. Furthermore, LPS-stimulated IKKalpha/beta and Akt phosphorylation was abolished by p38 inhibitor SB203580. Wortmannin (a PI3K inhibitor) also attenuated LPS-induced IKKalpha/beta phosphorylation, although to a less extent than SB203580, but failed to affect p38 phosphorylation. These observations suggested that PI3K/Akt might lie downstream of p38 MAPK to coregulate LPS-induced IKKalpha/beta phosphorylation. Taken together, miyabenol A acted via interfering with p38 MAPK-related signal pathways to down-regulate IKK/IkappaB activation and NO production.     

Suppression effect of soy isoflavones on nitric oxide production in RAW 264.7 macrophages

Genistein, daidzein, and glycitein, as primary isoflavones in soybeans, are reported to have beneficial effects on atherosclerosis, chronic inflammatory diseases, and cancers that are conducted by nitric oxide (NO) injury. The objectives of this study were to investigate the effects and mechanisms of these soy isoflavones on the inducible nitric oxide synthase (iNOS) system in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Genistein, daidzein, and glycitein dose-dependently suppress NO production (IC(50) = 50 microM) in supernatants of LPS-activated macrophages as measured on the basis of nitrite accumulation. In addition, direct inhibition of iNOS activity, determined by means of the conversion of L-[(3)H]arginine to L-[(3)H]citrulline, and markedly reduced iNOS protein and mRNA levels, evaluated by means of Western blot and RT-PCR, respectively, were found in homogenates of LPS-activated cells treated with each isoflavone. Moreover, genistein was found to have a greater inhibitory effect on NO production but no significant effect on iNOS activity or protein and gene expression to daidzein and glycitein. These observations reveal that the suppression of NO production by genistein, daidzein, and glycitein might be due to the inhibition of both the activity and expression of iNOS in LPS-activated macrophages. The result suggests that soy isoflavones might attenuate excessive NO generation at inflammatory sites.     

Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-gamma-activated RAW 264.7 macrophages

Flavonoids have been reported to lower oxidative stress and possess beneficial effects on cardiovascular diseases and chronic inflammatory diseases associated with nitric oxide (NO). Common phenolic compounds, including phenolic acids, flavonols, isoflavones, and anthocyanins, present in fruits were investigated for their effects on NO production in LPS/IFN-gamma-activated RAW 264.7 macrophages. Phenolic compounds at the range of 16-500 microM that inhibited NO production by > 50% without showing cytotoxicity were the flavonols quercetin and myricetin, the isoflavone daidzein, and the anthocyanins/anthocyanidins pelargonidin, cyanidin, delphinidin, peonidin, malvidin, malvidin 3-glucoside, and malvidin 3,5-diglucosides. Anthocyanins had strong inhibitory effects on NO production. Anthocyanin-rich crude extracts and concentrates of selected berries were also assayed, and their inhibitory effects on NO production were significantly correlated with total phenolic and anthocyanin contents. This is the first study to report the inhibitory effects of anthocyanins and berry phenolic compounds on NO production.     

Effects of anthocyanins and other phenolic compounds on the production of tumor necrosis factor alpha in LPS/IFN-gamma-activated RAW 264.7 macrophages

Flavonoids have been reported to demonstrate their benefits in lowering oxidative stress and beneficial effects on cardiovascular and chronic inflammatory diseases. Common phenolic compounds, including phenolic acids, flavonols, isoflavones, and anthocyanins, present in fruits, vegetables, and grains were investigated for their effects on the production of tumor necrosis factor alpha (TNF-alpha) in LPS/IFN-gamma-activated RAW 264.7 macrophages. Gallic acid and (+)-catechin showed small but significant effects, whereas chlorogenic acid had no effect on TNF-alpha production. The flavonol quercetin inhibited TNF-alpha production, but kaempferol and myricetin induced the secretion of TNF-alpha. The isoflavone genistein was an inhibitor of TNF-alpha, whereas daidzein induced TNF-alpha production. Glycosylation of genistein changed its inhibitory effects to TNF-alpha induction, and glycosylation of daidzein had no effect on its activity. Anthocyanidins/anthocyanins and anthocyanin-rich extracts induced TNF-alpha production and acted as modulators of the immune response in activated macrophages. This is the first study to report the effects of anthocyanins and berry extracts on TNF-alpha production.     

Echinacea species and alkamides inhibit prostaglandin E(2) production in RAW264.7 mouse macrophage cells

Inhibition of prostaglandin E(2) (PGE(2)) production in lipopolysaccharide-stimulated RAW264.7 mouse macrophage cells was assessed with an enzyme immunoassay following treatments with Echinacea extracts or synthesized alkamides. Results indicated that ethanol extracts diluted in media to a concentration of 15 microg/mL from E. angustifolia, E. pallida, E. simulata, and E. sanguinea significantly inhibited PGE2 production. In further studies, PGE2 production was significantly reduced by all synthesized alkamides assayed at 50 microM, by Bauer alkamides 8, 12A analogue, and 14, Chen alkamide 2, and Chen alkamide 2 analogue at 25 microM and by Bauer alkamide 14 at 10 microM. Cytotoxicity did not play a role in the noted reduction of PGE2 production in either the Echinacea extracts or synthesized alkamides. High-performance liquid chromatography analysis identified individual alkamides present at concentrations below 2.8 microM in the extracts from the six Echinacea species (15 microg/mL crude extract). Because active extracts contained <2.8 microM of specific alkamide and the results showed that synthetic alkamides must have a minimum concentration of 10 microM to inhibit PGE2, it is likely that alkamides may contribute toward the anti-inflammatory activity of Echinacea in a synergistic or additive manner.     

Seed priming improves seedling emergence and reduces oxidative stress in Nigella sativa under soil moisture stress

Nigella sativa seeds have multiple industrial and pharmaceutical uses for the diseases treatment such as bronchitis, rheumatism, high blood pressure, cough, eczema, inflammation, and influenza. Although semi-arid areas are suitable for production of annual medicinal plants because they receive favorable amounts of solar radiations, the establishment of these plants' seedling requires frequent irrigations because their seeds are small and the climate is hot at the time of planting. When the irrigation sources are limited, poor establishment and inadequate production will result. Therefore, in order to improve nitrogen (N) in N. sativa seedling establishment, different effects of seed priming treatments were evaluated under drought stress. Different levels of soil moisture (irrigation after 11, 22, 33, and 44 mm of evaporation as no water stress, mild water stress, moderate water stress, and severe water stress, respectively) and seed priming treatments [no priming, potassium nitrate (KNO₃), zinc sulfate (ZnSO₄), polyethylene glycol 6000, and gibberellic acid (GA)] were evaluated as the main plot and subplot factors, respectively. The results showed that under mild water stress condition, seed priming significantly increased proline content and the highest percentage of emergence was obtained with ZnSO₄. Furthermore, the highest amount of soluble protein was observed in the KNO₃ treatment and the catalase enzyme content increased in all priming treatments, except polyethylene glycol treatment. Under moderate water stress condition, the KNO₃ treatment raised the rate and percentage of emergence and the seedling length. However, under severe water stress condition, the highest percentage of emergence was achieved by GA and ZnSO₄ treatments. Moreover, hydropriming and GA produced the highest proline content and seedling dry weight. Under moderate and severe water stress conditions, the hydropriming and ZnSO₄ treatments showed a significant increase in catalase activity. It can be concluded that priming of N. sativa seeds with ZnSO₄ and GA can alleviate drought stress in the early stages of seedling growth by increasing the antioxidants in the seedlings. Farmers in semi-arid regions can prime N. sativa seeds with these compounds before planting to increase production of this valuable medicinal plant and reduce water consumption.     

Proteomic analysis of pycnogenol effects in RAW 264.7 macrophage reveals induction of cathepsin D expression and enhancement of phagocytosis

Pycnogenol, polyphenolic compounds extracted from the pine bark, is beneficial for human health. To understand more of its effects, the present study is to explore the protein expression pattern induced by pycnogenol in RAW 264.7 cells. Global analysis using two-dimensional gel electrophoresis indicated that treatment with pycnogenol induces upregulation of four proteins, whose identities were revealed by mass spectrometry as cathepsin D, keratinocyte lipid-binding protein, proteasome subunit alpha type 1, and annexin IV. The pycnogenol effect displayed a time- and concentration-dependent manner. Unlike pycnogenol, N-acetyl cysteine and vitamin C had no effect on cathepsin D expression. Further studies showed that cathepsin D induction is correlated with an increase of lysosomal staining and enhancement of phagocytosis. These results reveal the novel effects of pycnogenol on protein expression and phagocytic functions and illustrate the advantage of proteomics-based strategy in unveiling the molecular basis of phytochemicals.     

Inhibition of prostaglandin E(2) production by anti-inflammatory hypericum perforatum extracts and constituents in RAW264.7 Mouse Macrophage Cells

Hypericum perforatum (Hp) is commonly known for its antiviral, antidepressant, and cytotoxic properties, but traditionally Hp was also used to treat inflammation. In this study, the anti-inflammatory activity and cytotoxicity of different Hp extractions and accessions and constituents present within Hp extracts were characterized. In contrast to the antiviral activity of Hp, the anti-inflammatory activity observed with all Hp extracts was light-independent. When pure constituents were tested, the flavonoids, amentoflavone, hyperforin, and light-activated pseudohypericin, displayed anti-inflammatory activity, albeit at concentrations generally higher than the amount present in the Hp extracts. Constituents that were present in the Hp extracts at concentrations that inhibited the production of prostaglandin E(2) (PGE(2)) were pseudohypericin and hyperforin, suggesting that they are the primary anti-inflammatory constituents along with the flavonoids, and perhaps the interactions of these constituents and other unidentified compounds are important for the anti-inflammatory activity of the Hp extracts.     

Litter decomposition reduces either N2O or NO production in strongly acidic coniferous and broad-leaved forest soils under anaerobic conditions

Purpose

The beneficial effect to the environment of nitrate (NO₃ ^?) removal by denitrification depends on the partitioning of its end products into nitrous oxide (N₂O), nitric oxide (NO), and dinitrogen (N₂). However, in subtropical China, acidic forest mineral soils are characterized by negligible denitrification capacity and thus reactive forms of N could not be effectively converted to inert N₂, resulting in a negative environmental consequence. In this study, the influences of C input from litter decomposition on denitrification rate and its gaseous products under anoxic conditions in the acidic coniferous and broad-leaved forest soils in subtropical China were investigated using the acetylene (C₂H₂) blockage technique in the laboratory.

Materials and methods

The coniferous and broad-leaved forest soils with and without litter addition were incubated under anaerobic conditions for 244 h. There were three treatments for each forest soil including addition of 0.5 and 1% corresponding litter (gram of litter per gram of soil) and the control without addition of litter.

Results and discussion

The results showed that litter addition into the broad-leaved forest soil had no effect on average rates of denitrification (calculated as the sum of NO, N₂O, and N₂), whereas in the coniferous forest soil, the addition resulted in a significant increase in average denitrification rate. In the broad-leaved forest soil, both rates of litter addition decreased the production of NO but increased the production of N₂, and high rates of litter addition into the coniferous forest soil promoted the reduction of N₂O to N₂.

Conclusions

Increased decomposition of litter in the forest soils could effectively reduce N₂O and NO production through denitrification under anaerobic conditions.     

Effects of lanthanum on growth,element uptake,and oxidative stress in rice seedlings

We assessed the effects of increasing amounts of lanthanum (La) in solution on growth, nutrient metal accumulation, and the expression of oxidative stress in rice seedlings. The La concentration in shoots increased with La³⁺ supply and differentially affected the uptake of nutrient elements. Hormetic effects were observed on seed germination and biomass accumulation with increasing trends up to 0.1 mM La³⁺. Higher La concentrations were associated with an increase in malondialdehyde and H₂O₂ and a decline in chlorophyll, soluble proteins, and photosynthetic activity. We conclude that La supply at low rates may be beneficial to rice, while at higher rates, La induces oxidative stress.     

Soil NO3-N production and immobilization affected by NH4-N,glycine, and NO3-N addition in different forest types in Shikoku,southern Japan

The characteristics of production and immobilization of NO₃-N were evaluated for soils from four forest types in Kochi Prefecture, southern Japan. Net NO₃-N production during the laboratory incubation differed among the soils from the four forest types, being high under Japanese cedar (Cryptomeria japonica D. Don) and deciduous hardwood, and negligible under Japanese red pine (Pinus densiflora Sieb. et Zucc.) and hinoki cypress (Chamaecyparis obtusa Endlicher). Nitrification under Japanese cedar and hardwood was mainly autotrophic based on the fact that nitrification was inhibited by acetylene or nitrapyrin, and was not affected by cycloheximide. Net NO₃-N production in these soils increased by glycine addition, but did not increase appreciably by NH₄Cl addition. However, net NO₃-N production increased after the addition of CaCO₃ with NH₄Cl. These results indicate that the substrate of nitrification is NH₃ rather than NH₄ ⁺ and that the added NH₄ ⁺ is not utilized by nitrifiers at low pH values. With NO₃-N addition to soils under red pine and hinoki cypress, immobilization of NO₃-N was observed followed by rapid production of NH₄-N. These findings suggested that mobile NO₃-N can be converted to less mobile NH₄-N by the activities of soil microorganisms. This microbial process may play an important role in retaining nitrogen within forest ecosystems where the potential of N loss is high due to the high precipitation in the area.     

A commercial extract of fruits and vegetables, Oxxynea, acts as a powerful antiatherosclerotic supplement in an animal model by reducing cholesterolemia, oxidative stress, and NADPH oxidase expression

The effects of fruit and vegetable extract (Oxxynea) on plasma cholesterol, early atherosclerosis, cardiac production of superoxide anion, and NAD(P)H oxidase expression were studied in an animal model of atherosclerosis. Thirty six hamsters were divided into two groups of 18 and fed an atherogenic diet for 12 weeks. They received by gavage either water or Oxxynea in water at a human dose equivalent of 10 fruits and vegetables per day. Oxxynea lowered plasma cholesterol and non-HDL cholesterol, but not HDL-cholesterol, and increased plasma antioxidant capacity. It also strongly reduced the area of aortic fatty streak deposition by 77%, cardiac production of superoxide anion by 45%, and p22phox subunit of NAD(P)H oxidase expression by 59%. These findings support the view that chronic consumption of antioxidants supplied by fruits and vegetables has potential beneficial effects with respect to the development of atherosclerosis. The underlying mechanism is related mainly to inhibiting pro-oxidant factors and improving the serum lipid profile.     

Antioxidant and iron-binding properties of curcumin, capsaicin, and S-allylcysteine reduce oxidative stress in rat brain homogenate

Research demonstrates that antioxidants and metal chelators may be of beneficial use in the treatment of neurodegenerative diseases, such as Alzheimer's disease (AD). This study investigated the antioxidant and metal-binding properties of curcumin, capsaicin, and S-allylcysteine, which are major components found in commonly used dietary spice ingredients turmeric, chilli, and garlic, respectively. The DPPH assay demonstrates that these compounds readily scavenge free radicals. These compounds significantly curtail iron- (Fe2+) and quinolinic acid (QA)-induced lipid peroxidation and potently scavenge the superoxide anion generated by 1 mM cyanide in rat brain homogenate. The ferrozine assay was used to measure the extent of Fe2+ chelation, and electrochemistry was employed to measure the Fe3+ binding activity of curcumin, capsaicin, and S-allylcysteine. Both assays demonstrate that these compounds bind Fe2+ and Fe3+ and prevent the redox cycling of iron, suggesting that this may be an additional method through which these agents reduce Fe2+-induced lipid peroxidation. This study demonstrates the antioxidant and metal-binding properties of these spice ingredients, and it is hereby postulate that these compounds have important implications in the prevention or treatment of neurodegenerative diseases such as AD.     

Chondroitin sulfate extracted from ascidian tunic inhibits phorbol ester-induced expression of Inflammatory factors VCAM-1 and COX-2 by blocking NF-kappaB activation in mouse skin

Inflammatory factors are known to play a key role in promoting tumorigenesis; therefore, it is a promising strategy to inhibit the inflammation for cancer prevention. The current study was performed to investigate the potential effects of chondroitin sulfate (CS) extracted from ascidian tunic on the expression of inflammatory factors induced by treatment with 12- O-tetradecanoylphorbol-13-acetate (TPA) and to elucidate the underlying molecular mechanism of CS action in mouse skin inflammation. TPA was topically applied to the shaven backs of ICR mice with or without CS (1 or 2 mg) for 4 h. The results demonstrated that CS suppressed TPA-induced edema and reduced the expression of cyclooxygenase-2, vascular cell adhesion molecule-1, and Akt signaling in mouse skin. These studies suggest that CS from ascidian tunic may be developed as an effective natural anti-inflammatory agent.     

Boron stress,oxidative damage and antioxidant protection in potato cultivars (Solanum tuberosum L.)

Six potato cultivars grown in Turkey in boron-prone areas and differing in their tolerance towards high boron were studied to reveal whether boron causes oxidative stress. To assess stress level, chlorophyll fluorescence and growth parameters were measured. Oxidative damage was assessed as malondialdehyde level, and antioxidant protection was evaluated as ascorbate (AA), dehydroascorbate, reduced glutathione (GSH) and oxidized glutathione amounts and superoxide dismutase, catalase, ascorbate peroxidase (APX) and glutathione reductase (GR) activities. High boron stress affected photosynthesis negatively in a threshold-dependent manner and inhibited growth. No pronounced changes in oxidation of lipids occurred in any cultivar. Activation of APX suggested the involvement of an ascorbic acid–reduced glutathione cycle in the protection against oxidative stress caused by high boron. Efficient work of this antioxidant system was probably hindered by boron complexation with NAD(P)⁺/NAD(P)H and resulted in the inhibition of GR and a decrease in AA and GSH. Hence, oxidative stress associated with high boron is a secondary component of boron toxicity which arises from metabolic changes caused by boron interference with major metabolites. Potato cultivars tolerate excess boron stress well and show damage only in very high boron concentrations. The potato cvs best suited for high boron soils/breeding purposes are cvs Van Gogh and Agria.

Abbreviations: AA: ascorbic acid; APX: ascorbate peroxidase; CAT: catalase; DHA: dehydroascorbic acid; DHAR: dehydroascorbate reductase; DTNB: 5; 5′-dithiobis-2-nitrobenzoic acid; DTT: dithiotreitol; F_v/F_m: photosynthetic efficiency at the dark-adapted state; GR: glutathione reductase; GSH: reduced glutathione; GSSG: oxidized glutathione; MDA: malondialdehyde; ROS: reactive oxygen species; SOD: superoxide dismutase; TCA: trichloroacetic acid     

Consumption of a novel dietary formula of plant sterol esters of canola oil fatty acids, in a canola oil matrix containing 1,3-diacylglycerol, reduces oxidative stress in atherosclerotic apolipoprotein E-deficient mice

The antiatherogenic properties of a novel dietary formula (PS-CO) of plant sterol esters of fatty acids, produced by enzymatic interesterification of plant sterols with canola oil (CO), in a CO matrix containing 1,3-diacylglycerol, were evaluated in apolipoprotein E-deficient mice. PS-CO consumption strongly tended to lower total plasma cholesterol levels by 21%, compared to the placebo group. Blood triglycerides were reduced by 38% and 36% compared to CO and placebo-fed mice, respectively. Serum lipid peroxide levels were lowered following PS-CO administration by 62% and 63%, compared to CO and placebo administration, respectively. Unlike CO supplementation, PS-CO consumption preserved serum paraoxonase (PON1) activity. Mouse peritoneal macrophages from PS-CO-fed mice exhibited reduced cellular uptake of oxidized-LDL compared to those from placebo-fed mice and demonstrated a tendency toward a decreased capability to release superoxide anions. These findings indicate that PS-CO supplementation is beneficial in reducing serum lipid levels, and serum and macrophage oxidative stress, thus contributing to the reduction in atherogenic risk factors.     

Effects of cocoa extract on glucometabolism, oxidative stress, and antioxidant enzymes in obese-diabetic (Ob-db) rats

In this present study, we investigated the effects of cocoa extract containing polyphenols and methylxanthines prepared from cocoa powder on the biochemical parameters of obese-diabetic (Ob-db) rats. Obese-diabetic (Ob-db) rats were developed using a high-fat diet (49% fat, 32% carbohydrate, and 19% protein from total energy, kcal) for 3 months, followed by a low dose (35 mg/kg body weight) streptozotocin (STZ) injection. Cocoa extract (600 mg/kg body weight/day) was given to the rats for 4 weeks. The results indicated that there were no significant differences in fasting plasma glucose and insulin level after 4 weeks of cocoa extract administration. Oral glucose tolerance test revealed that cocoa supplementation in Ob-db rats significantly (p < 0.05) reduced plasma glucose at 60 and 90 min compared to unsupplemented Ob-db rats. Plasma free fatty acid and oxidative stress biomarker (8-isoprostane) were significantly (p < 0.05) reduced after cocoa supplementation. Superoxide dismutase activity was enhanced in Ob-db compared to that in nonsupplemented rats. However, no change was observed in catalase activity. The results showed that cocoa supplementation had an effect on postprandial glucose control but not for long term (4 weeks). Moreover, cocoa supplementation could reduce circulating plasma free fatty acid and 8-isoprostane and may enhance the antioxidant defense system.     

Silicon alleviates nickel toxicity in cotton seedlings through enhancing growth,photosynthesis, and suppressing Ni uptake and oxidative stress

Cotton (Gossypium hirsutum L.) is a well-known and economically most beneficial crop worldwide while nickel (Ni) toxicity is a widespread problem in crops grown on Ni-contaminated soils. We investigated the response of silicon (Si) in cotton under Ni stress with respect to growth, biomass, gas exchange attributes, enzymatic activities, and Ni uptake and accumulation. For this, plants were grown in hydroponics for 12 weeks with three levels of Ni (0, 50, and 100 µM) in the presence or absence of 1 mM Si. Results showed that Ni significantly reduced the plant growth, biomass, gas exchange attributes, and pigment contents while Si application mitigated these adverse effects under Ni stress. Nickel stress significantly decreased antioxidant enzymes’ activities while increased malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and electrolyte leakage (EC) in leaves and roots. The application of Si enhanced the activities of antioxidant enzymes and reduced MDA, H₂O₂, and EC in plants. Nickel application significantly increased Ni concentration and accumulation in leaf, stem, and roots while Si application significantly decreased Ni in these plant parts. The present study indicates that Si could improve cotton growth under Ni stress by lowering Ni uptake and reactive oxygen species (ROS) and by increasing antioxidant enzymes activities.