Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

Immunostimulating effects of a sulfated galactan from Codium fragile

A pyruvylated sulfated galactan from Codium fragile is a highly ramified polysaccharide consisting of 3-linked, 3,6-linked, and non-reducing terminal d-galactose with pyruvate and sulfate groups; the glycan exerts anti-herpes simplex virus type 2 effects in vitro and in vivo. This particular polysaccharide was found to stimulate the production of nitric oxide by inducing iNOS at the mRNA and protein levels. In addition, the polysaccharide also induced several cytokine mRNA expressions such as IL-1β, IL-6, IL-10 and TNF-α. Therefore, it appears that the sulfated galactan might possess the immunostimulating effects via activation of macrophages.     

Macrophage cell line RAW264.7 but not P-388D1 is an appropriate in vitro-model for studying oxidative burst as well as cytokine production in context of fatty acid enrichment

Oxidative burst and cytokines synthesis by macrophages is a crucial point for successful pathogen defense. However, macrophage cell lines commonly used in inflammatory research differ in their responses to external stimuli. Thus, there is the necessity to carefully characterize the cells before experimental usage. In this study we investigated the applicability of two widely-used macrophage cell lines, RAW264.7 and P-388D1, for studying oxidative burst and cytokine synthesis. Cells were tested for NADPH oxidase activity, iNOS-mRNA levels, and the release of NO, TNF-α, IL-6 and IL-10. Stimulation of RAW264.7 triggered oxidative burst as well as synthesis of TNF-α, IL-6 and IL-10. In contrast, following stimulation P-388D1 produced TNF-α and IL-6 only. Our findings confirm the relevance of cell line selection for reliability of in vitro-experiments. Moreover, the results approve RAW264.7 cells to be a suitable model to investigate the modulation capability of macrophages e.g. in context of fatty acid supplementation.     

The relationship between structural properties and activation of RAW264.7 and natural killer (NK) cells by sulfated polysaccharides extracted from Astragalus membranaceus roots

The aim of the present study to isolate the water-soluble polysaccharide from Astragalus membranaceus roots (AMP) and investigate the structural effects on RAW 264.7 murine macrophage and natural killer (NK) cells. AMP mainly consists of carbohydrates (66.2 %), proteins (11.8 %), and sulfates (18.0 %) with minor level of uronic acid (2.0 %). The structural modification was carried out by removal of protein and sulfate from AMP through the deproteination and desulfation. After deproteination (DP), the protein content was decreased from 11.8 % to 5.4 %. Similarly, the sulfate content of desulfated AMP (DS) was decreased from 18.0%–8.1%. AMP and DP could stimulate RAW264.7 cells to produce nitric oxide (NO) and up-regulate mRNA expression through NF-κB and MAPKs pathways. However, DS showed a considerably lower level of NO production than AMP and DP, suggesting that DS could not stimulate RAW264.7 cells. AMP and its derivatives significantly increased the natural killer cells (NK cell) proliferation (113.1%–128.7%) and cytotoxicity against HeLa cells (37.4%–55.5%). However, DS showed the lowest level of NK cells activation through the expression of IFN-γ, TNF-α, Granzyme-B, and NKp44. These results suggest that sulfate groups of AMP might play a crucial role in the RAW264.7 cells and NK cells activation.     

Phytosterol from the Callus of Stevia Rebaudiana Bertoni

We investigated the effect of 17β-estradiol (E2) on the expression of daintain/AIF-1, a marker of activated macrophages, in RAW264.7. E2 upregulated the protein and mRNA levels of daintain/AIF-1 in similar manners under physiological concentrations of 10^?11 M to 10^?7 M. The application of ICI 182,780, an estrogen receptor (ER) antagonist, attenuated E2-induced daintain/AIF-1 production, suggesting the involvement of ER in this process.     

Medium-throughput ESR detection of superoxide production in undetached adherent cells using cyclic nitrone spin traps

Abstract

Spin trapping with cyclic nitrones coupled to electron spin resonance (ESR) is recognized as a specific method of detection of oxygen free radicals in biological systems, especially in culture cells. In this case, the detection is usually performed on cell suspensions, which is however unsuitable when adhesion influences free radical production. Here, we performed ESR detection of superoxide with four spin traps (5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DEPMPO; 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline N-oxide, DIPPMPO; (4R*, 5R*)-5-(diisopropyloxyphosphoryl)-5-methyl-4-[({[2-(triphenylphosphonio)ethyl]carbamoyl}oxy)methyl]pyrroline N-oxide bromide, Mito-DIPPMPO; and 6-monodeoxy-6-mono-4-[(5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide)-ethylenecarbamoyl-(2,3-di-O-methyl) hexakis (2,3,6-tri-O-methyl)]-β-cyclodextrin, CD-DIPPMPO) directly on RAW 264.7 macrophages cultured on microscope coverslip glasses after phorbol 12-myristate 13-acetate (PMA) stimulation. Distinct ESR spectra were obtained with each spin trap using this method. CD-DIPPMPO, a recently published phosphorylated cyclic nitrone bearing a permethylated β-cyclodextrin moiety, was confirmed as the most specific spin trap of the superoxide radical, with exclusive detection of the superoxide adduct. ESR detection performed on cells attached to coverslips represents significant advances over other methods in terms of simplicity, speed, and measurement under near-physiological conditions. It thus opens the way for numerous applications, such as medium-throughput screening of antioxidants and reactive oxygen species (ROS)-modulating agents.     

Induction of extracellular ATP mediates increase in intracellular thioredoxin in RAW264.7 cells exposed to low-dose γ-rays

We previously showed that low doses (0.25-0.5 Gy) of γ-rays elevated thioredoxin (Trx-1) in various organs of mice after whole-body irradiation. Also, it is reported that extracellular ATP, which is released in response to various stresses, regulates the expression of intracellular antioxidants through activation of P2 receptors. We have recently found that low-dose γ-rays induce ATP release from the exposed cells. However, it is not yet clear whether the radiation-induced extracellular ATP modulates the cellular redox balance. Here, we investigated whether γ-ray irradiation-induced release of extracellular ATP contributes to the induction of the cellular antioxidant Trx-1, using mouse macrophage-like RAW264.7 cells. Irradiation with γ-rays or exogenously added ATP increased the expression of Trx-1, and in both cases the increase was blocked by pretreatment with an ectonucleotidase, apyrase. Then, the involvement of ATP-dependent reactive oxygen species (ROS) generation in the increase in antioxidant capacity was examined. ATP stimulation promoted the generation of intracellular ROS and also increased Trx-1 expression. The increase in Trx-1 expression was significantly suppressed by pretreatment of the cells with antioxidants. In conclusion, the γ-ray irradiation-induced release of extracellular ATP may, at least in part, contribute to the production of ROS via purinergic signaling, leading to promotion of intracellular antioxidants as an adaptive response to an oxidative stress.