Effect of polyunsaturated fatty acids on the reactive oxygen and nitrogen species production by raw 264.7 macrophages

Background  

Polyunsaturated fatty acids (PUFAs) can affect various functions of the immune system including inflammatory responses. An oxidative burst of phagocytes accompanied by reactive oxygen species (ROS) and reactive nitrogen species (RNS) formation is one of the phagocyte functions that could be modulated by PUFAs.     

Bovine dialyzable leukocyte extract modulates the nitric oxide and pro-inflammatory cytokine production in lipopolysaccharide-stimulated murine peritoneal macrophages in vitro

Lipopolysaccharides (LPS) released from Gram-negative bacteria after infection initiate an exagerated response that leads to a cascade of pathophysiological events termed sepsis. Monocytes or macrophages produce many of the mediators found in septic patients. Targeting of these mediators, especially tumor necrosis factor (TNF)-alpha and nitric oxide (NO), has been pursued as a mean of reducing mortality in sepsis. Bovine dialyzable leukocyte extract (bDLE) is a dialysate of a heterogeneous mixture of low-molecular-weight substances released from disintegrated leukocytes of the blood or tissue lymphoid. In this study, to determine whether bDLE modulates NO and pro-inflammatory cytokine production, murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) before LPS (20 mg/mL) stimulation, and also LPS-stimulated murine peritoneal macrophages were treated with bDLE (0.05 or 0.5 U/mL) at 0, 4, 8, 12, and 24 hours. The bDLE significantly decreased NO production, and also decreased TNF-alpha and interleukin (IL)-6 but increased IL-10 production in LPS-stimulated murine peritoneal macrophages. Our results demonstrate that bDLE plays an important role in modulating TNF-alpha, IL-6, and NO production through IL-10, and this may offer therapeutic potential in clinical endotoxic shock.     

Polyvinylpyridine-N-oxide and carboxymethyl cellulose inhibit mineral dust-induced production of reactive oxygen species by human macrophages.

Polyvinylpyridine-N-oxide (PVPNO) and carboxymethyl cellulose (CMC) are known to inhibit the cytotoxic effects of quartz particles and chrysotile asbestos fibers, respectively. The effect of PVPNO and CMC on mineral dust-induced production of reactive oxygen species (ROS) by human monocyte-derived macrophages was studied using lucigenin-dependent chemiluminescence. Ten micrograms of PVPNO inhibited the ROS production induced by 100 micrograms of quartz completely, but caused only 25% inhibition of the response to 100 micrograms of chrysotile. Ten micrograms of CMC inhibited the chrysotile-induced ROS production to 70%, but had a weak effect on the responses to quartz. Neither PVPNO nor CMC caused any inhibition of the ROS responses to serum-opsonized zymosan. PVPNO was bound to both quartz and chrysotile, but no binding of CMC to either dust could be detected. Neither PVPNO nor CMC had any superoxide scavenging effect. In conclusion, PVPNO and CMC seem to selectively modify mineral dust surface properties, which are of importance in the induction of ROS production by phagocytes. These observations may be useful in describing the pathogenesis of mineral dust-induced diseases, and may also have therapeutic implications.     

Tocotrienols inhibit lipopolysaccharide-induced pro-inflammatory cytokines in macrophages of female mice

Background  

Inflammation has been implicated in cardiovascular disease, and the important role of proteasomes in the development of inflammation and other macrophage functions has been demonstrated. Tocotrienols are potent hypocholesterolemic agents that inhibit β-hydroxy-β-methylglutaryl coenzyme A reductase activity, which is degraded via the ubiquitin-proteasome pathway. Our objective was to evaluate the effect of tocotrienols in reducing inflammation. Lipopolysaccharide (LPS) was used as a prototype for inflammation in murine RAW 264.7 cells and BALB/c female mice.     

Absorption of individual fatty acids from long chain or medium chain triglycerides in very small infants

Using stool samples previously obtained for a nutritional balance study of very small infants, the intake, excretion, and percent absorption (%A) of individual fatty acids were quantitated by gas-liquid chromatography in two groups of patients; one receiving a formula providing fat as long chain triglycerides (LCT), the other providing 40% fat as medium chain triglyceride (MCT). The total fat intake for each group was 7.22 g/kg/day (MCT group), 6.02 g/kg/day (LCT group) and the absorption was 6.79 g/kg/day (MCT group) and 5.41 g/kg/day (LCT group). The %A of individual acids was comparable in both groups being greater than 99% for MCT and being lowest (62%) for C18:0 FA. As a consequence, infants in the MCT-fed group had a better total %A of fat, 95.2 vs 89.9% and absorbed more fat than the LCT group.     

Contrasting roles for reactive oxygen species and nitric oxide in the innate response to pulmonary infection with Streptococcus pneumoniae

The pulmonary innate response to low-dose bacterial challenge requires functioning alveolar macrophages (AM) but also subsequent macrophage apoptosis. To address the role of reactive oxygen species (ROS) and nitric oxide (NO) in AM apoptosis, sub-clinical Streptococcus pneumoniae infection was established in gp91(phox-/-) and inducible NO synthase deficient (iNOS(-/-)) mice. Both AM apoptosis and the number of macrophages containing apoptotic bodies are reduced in iNOS(-/-) as compared to control or gp91(phox-/-) mice. iNOS(-/-) mice recruit neutrophils and generate TNF-alpha to compensate for impaired AM competence but ROS deficiency has no apparent effect on AM function in this model.     

Modulation of nitric oxide and cytokines production by L-arginine in human gut mucosa

BACKGROUND: Arginine is a conditionally essential amino-acid with immuno-modulatory properties, mainly through the nitric oxide (NO) pathway. AIM: To assess the effects of arginine on intestinal production of pro- and anti-inflammatory cytokines and NO in human gut. METHODS: An enteral solution of arginine or a control solution of amino-acids was administered to 8 healthy volunteers on a randomized cross-over design. Duodenal biopsies were taken. Pro- (IL-6, IL-8) and anti-inflammatory (IL-4, IL-10) cytokines mRNA expression was assessed by RT-PCR. Other biopsies were cultured with 0.1, 0.5 or 2 mM arginine or control amino-acids, under basal or IL-1beta-induced inflammatory conditions. Interleukin-4, IL-6, IL-8 and IL-10 production was measured in culture supernatant by ELISA and NO production by Griess reaction. RESULTS: Arginine enhanced the production of NO under inflammatory conditions in a dose-dependent manner (P=0.03). IL-1beta increased the production of IL-8 and IL-6 (P<0.01). Arginine had no effect on pro- and anti-inflammatory cytokines production both under basal and inflammatory conditions. CONCLUSIONS: Arginine enhanced the production of NO but did not affect that of cytokines in inflammatory human gut. Further clinical studies are required to assess whether arginine-enhanced NO production plays a beneficial or deleterious effect in intestinal inflammation.     

Inhibition of nitric oxide and inflammatory cytokines in LPS-stimulated murine macrophages by resveratrol, a potent proteasome inhibitor

Background

Altered immune function during ageing results in increased production of nitric oxide (NO) and other inflammatory mediators. Recently, we have reported that NO production was inhibited by naturally-occurring proteasome inhibitors (quercetin, ??-tocotrienol, and riboflavin) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, and thioglycolate-elicited peritoneal macrophages from C57BL/6 mice. In a continuous effort to find more potent, non-toxic, commercially available, naturally-occurring proteasome inhibitors that suppress inflammation, the present study was carried out to describe the inhibition of NF-??B activation and NO, TNF-??, IL-6, IL-1??, and iNOS expression by trans-resveratrol, trans-pterostilbene, morin hydrate, and nicotinic acid in LPS-induced RAW 264.7 cells and thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.

Results

The present results indicate that resveratrol, pterostilbene, and morin hydrate caused significant inhibition (>70% to 90%; P?<?0.02) in the activities of chymotrypsin-like, trypsin-like, and post-acidic (post-glutamase) proteasome sites in RAW 264.7 cells at a dose of only 20 ??M. These compounds also inhibited the production of NO by RAW-264.7 cells stimulated with LPS alone (>40%; P?<?0.05), or LPS?+?interferon-?? (IFN-??; >60%; P?<?0.02). Furthermore, resveratrol, pterostilbene, morin hydrate, and quercetin suppressed secretion of TNF-?? (>40%; P?<?0.05) in LPS-stimulated RAW 264.7 cells, and suppressed NF-??B activation (22% to 45%; P?<?0.05) in LPS-stimulated HEK293T cells. These compounds also significantly suppressed LPS-induced expression of TNF-??, IL-1??, IL-6, and iNOS genes in RAW 264.7 cells, and also in thioglycolate-elicited peritoneal macrophages from C57BL/6 and BALB/c mice.

Conclusions

The present results clearly demonstrate that resveratrol and pterostilbene are particularly potent proteasome inhibitors that suppress expression of genes, and production of inflammatory products in LPS-stimulated RAW 264.7 cells, and macrophages from C57BL/6 and BALB/c mice. Resveratrol and pterostilbene which are present in grapes, blueberries, and red wine, have been implicated as contributing factors to the lower incidence of cardiovascular disease in the French population, despite their relatively high dietary fat intake. Consequently, it appears likely that the beneficial nutritional effects of resveratrol and pterostilbene are due at least in part, to their ability to inhibit NF-??B activation by the proteasome, thereby suppressing activation of pro-inflammatory cytokines and iNOS genes, resulting in decreased secretion of TNF-??, IL-1??, IL-6, and NO levels, in response to inflammatory stimuli. This is the first report demonstrating that resveratrol and pterostilbene act as proteasome inhibitors, thus providing a mechanism for their anti-inflammatory effects.     

活性氧对尘肺患者肺泡巨噬细胞凋亡信号转导途径的作用

目的 探讨活性氧(ROS)对Fas/FasL信号转导途径致肺泡巨噬细胞凋亡作用.方法 以中国北戴河煤炭工人疗养院无其他肺部疾病的Ⅰ期、Ⅱ期尘肺患者共45例为研究对象,将支气管肺泡灌洗液(BALF)中的肺泡巨噬细胞(AM)分两组培养,即对照组、超氧化物歧化酶(SOD)组.采用TUNEL技术检测AM凋亡情况,琼脂糖凝胶电泳法检测DNA片段,Western blot法检测AM的Fas、FasL、caspase 8、caspase 3表达水平.结果 SOD组的凋亡指数(9.50±2.76)显著低于对照组(19.18±2.83,P< 0.05);SOD组的Fas、FasL、caspase 8、caspase 3蛋白表达水平(分别为0.107±0.065、0.115±0.047、0.231±0.139、0.312±0.114)均低于对照组(分别为0.209±0.084、0.210±0.097、0.374±0.071、0.441±0.095,P<0.05);对照组出现凋亡细胞特征性"梯状带",而SOD组未出现.结论 Fas/FasL信号转导途径介导的AM凋亡可能与ROS上调Fas/FasL系统的表达有关.     

溴氰菊酯对PC12细胞活性氧生成的影响

目的 研究溴氰菊酯(DM)对大鼠肾上腺嗜铬细胞瘤(PC12)细胞活性氧(ROS)生成的影响.方法 对培养的PC12细胞分别进行处理:(1)终浓度为0、10和100 μmol/L的DM分别处理PC12细胞1、6和12 h(两因素析凶设计);(2)终浓度为0、10和100;μmol/L的DM分别处理PC12细胞1、6、24h或24、48 h;(3)PC12细胞分别在终浓度为10mmol/L乙酰半胱氨酸(NAC)预处理2 h、终浓度为500 μmol/L的DL-甲硫氨酸磺酰亚胺(BSO)及终浓度为40 μmol/L的叔丁基对苯二酚(tBHQ)预处理16 h,再给予10 μmol/LDM作用6ho所有处理结束时,用2',7'-二氯荧光黄双乙酸酯(DCFH-DA)探针法测定细胞ROS含量.结果 DM诱导ROS生成呈剂量和时间效应关系.10 μmol/L DM处理组PC12细胞增加ROS的DCF荧光强度,是溶剂对照组的2.24倍,差异有统计学意义(P<0.01).而分别用NAC、BSO及tBHQ预先处理PC12细胞均能明显降低DM所增加的ROS,分别是DM组的22%、62%、38%.差异均有统计学意义(P<0.05).结论 DM能诱导PC12细胞ROS生成增高,胞内巯基水平和抗氧化功能降低可能是ROS生成增高的影响因素.     

温石棉与吸烟对肺泡巨噬细胞产生一氧化氮的影响

为探讨石棉与吸烟对人体健康影响的机制，用Ｇｒｅｉｓｓ法测定了温石棉及香烟烟雾溶液分别与共同作用下肺泡巨噬细胞（ＡＭ）产生的ＮＯ终产物ＮＯ－２。结果显示：温石棉可使ＡＭ产生的ＮＯ－２明显增加，且有剂量－效应关系，但烟溶液不能使ＡＭ产生的ＮＯ－２增加或减少，在两者共同作用时，烟溶液反而抑制温石棉诱导ＡＭ产生ＮＯ－２。当测定ＡＭ的吞噬功能时，发现烟溶液可明显抑制ＡＭ的吞噬功能。提示：烟溶液对温石棉诱导ＡＭ产生ＮＯ具有抑制作用，这种作用主要是由于其抑制了ＡＭ吞噬温石棉的能力造成的     

Effects of oxysterols on cell viability, inflammatory cytokines, VEGF, and reactive oxygen species production on human retinal cells: cytoprotective effects and prevention of VEGF secretion by resveratrol

Background and aims

Oxysterols are assumed to play important roles in age-related macular degeneration, a major cause of blindness. So we characterized the cytotoxic, oxidative, inflammatory, and angiogenic activities of oxysterols (7β-hydroxycholesterol (7β-OH), 7-ketocholesterol (7KC), 25-hydroxycholesterol (25-OH)) in human retinal ARPE-19 cells, and evaluated the protective effects of resveratrol (Rsv: 1 μM), a polyphenol from red wine.

Methods

ARPE-19 cells were treated with 7β-OH, 7KC, or 25-OH (5–40 μg/mL; 24–48 h) without or with Rsv. Cell viability was determined using trypan blue and the MTT assay. Cell death was characterized by electron microscopy and in situ detection of activated caspases with fluorochrome-labeled inhibitors of caspases. Reactive oxygen species (ROS) production was measured with hydroethidine. ELISA methods and a cytometric bead assay were used to quantify cytokines involved in inflammation (IL-8, IL-1β, IL-6, IL-10, IL-12p70, TNF-α, MCP-1) and VEGF.

Results

7β-OH and 7KC triggered a caspase-independent cell death process associated with the presence of multilamellar cytoplasmic structures evocating phospholipidosis, increased ROS production, and IL-8 secretion. 7β-OH enhanced VEGF secretion. No cytotoxic effects were identified with 25-OH, which highly stimulated ROS production, MCP-1, and VEGF secretion. With oxysterols, no IL-10, TNF-α, and IL-12p70 secretion were detected. 25-OH induced IL-8 secretion through the MEK/ERK½ signaling pathway, and Rsv showed cytoprotective activities and inhibited VEGF secretion.

Conclusion

7β-OH, 7KC, and 25-OH have cytotoxic, oxidative, inflammatory, and/or angiogenic activities on ARPE-19 cells. As Rsv has some protective effects against oxysterol-induced cell death and VEGF secretion it could be valuable in ARMD treatment.     

Increased effectiveness of carbon ions in the production of reactive oxygen species in normal human fibroblasts

The production of reactive oxygen species (ROS), especially superoxide anions (O₂^·–), is enhanced in many normal and tumor cell types in response to ionizing radiation. The influence of ionizing radiation on the regulation of ROS production is considered as an important factor in the long-term effects of irradiation (such as genomic instability) that might contribute to the development of secondary cancers. In view of the increasing application of carbon ions in radiation therapy, we aimed to study the potential impact of ionizing density on the intracellular production of ROS, comparing photons (X-rays) with carbon ions. For this purpose, we used normal human cells as a model for irradiated tissue surrounding a tumor. By quantifying the oxidization of Dihydroethidium (DHE), a fluorescent probe sensitive to superoxide anions, we assessed the intracellular ROS status after radiation exposure in normal human fibroblasts, which do not show radiation-induced chromosomal instability. After 3–5 days post exposure to X-rays and carbon ions, the level of ROS increased to a maximum that was dose dependent. The maximum ROS level reached after irradiation was specific for the fibroblast type. However, carbon ions induced this maximum level at a lower dose compared with X-rays. Within ∼1 week, ROS decreased to control levels. The time-course of decreasing ROS coincides with an increase in cell number and decreasing p21 protein levels, indicating a release from radiation-induced growth arrest. Interestingly, radiation did not act as a trigger for chronically enhanced levels of ROS months after radiation exposure.     

The effects of chlorothalonil on oyster hemocyte activation: phagocytosis, reduced pyridine nucleotides, and reactive oxygen species production

The production of reactive oxygen species (ROS) by a putative NADPH oxidase-like enzyme system is thought to contribute to antimicrobial activity in oyster hemocytes. NADPH oxidase in vertebrate phagocytes generates superoxide anion from molecular oxygen and NADPH, which is then converted to additional ROS, including H2O2 and HOCl. The fungicide chlorothalonil (TCIN) is a thiol-reactive compound that binds to protein sulfhydryl groups, which can result in enzyme inactivation. NADPH oxidase, containing several sulfhydryl groups, is a potential target of TCIN. Previous studies have demonstrated that in vitro exposure of fish (Morone saxatilus) macrophages to TCIN (10-500 microg/L) suppressed immunostimulated ROS and baseline NAD[P]H concentration but did not inhibit phagocytosis; the production of NADPH in stimulated cells was decreased only at the highest concentration. In this study, we evaluated the effects of TCIN (10-500 microg/L) on oyster hemocyte functions. As with striped bass macrophages, in vitro exposure to TCIN suppressed hemocyte ROS production in a dose-dependent manner, but did not affect phagocytosis. In contrast to the striped bass data, baseline NAD[P]H concentration was relatively unaffected and immunostimulated NAD[P]H production was marginally suppressed at the higher exposure concentrations. Despite these minor differences, these results suggest that TCIN may also be inhibiting an NAD[P]H oxidase-like enzyme in oyster hemocytes.     

In vitro effects of a smokeless tobacco extract on the production of reactive oxygen species by human oral epidermal cells and rat hepatic mitochondria and microsomes, and peritoneal macrophages

The possible role of reaction oxygen species in the toxicity of smokeless tobacco was explored. In order to determine possible sources of reactive oxygen species in response to smokeless tobacco, rat peritoneal macrophages (3×10⁶/ml) and hepatic mitochondria and microsomes (1 mg protein/ml) from untreated female Sprague-Dawley rats were incubated with an aqueous smokeless tobacco extract (STE) (200 g/ml). STE resulted in rapid increases in chemiluminescence with maximum increases occurring at approximately 6 min for the macrophages and 8 min for mitochondria and microsomes. Maximum increases in chemiluminescence of 1.4-, 3.2-, and 3.1-fold relative to control values occurred for macrophages, mitochondria, and microsomes, respectively. Hepatic mitochondria and microsomes (1 mg protein/ml) from female Sprague-Dawley rats were incubated at 37°C for 60 min in the presence of 0–500 g/ml STE. Potential tissue damage was measured as lipid peroxidation, and dose-dependent increases of 1.1–2.4-fold occurred in mitochondria and microsomes. Pre-incubation with various oxygen free radical scavengers including superoxide dismutase (SOD) (100 g/ml), catalase (100 g/ml), SOD + catalase (100 g/ml each), mannitol (1.25 mmol/ml), and allopurinol (100 g/ml) inhibited STE (200 g/ml) induced lipid peroxidation by 15% to 70%. Previous studies in our laboratories strongly suggest that STE induces the production of oxygen free radicals which cause tissue-damaging effects. We therefore examined the cytotoxicity of STE by incubating cultured human oral epidermal carcinoma (KB) cells with STE, and assessing the release of the enzyme lactate dehydrogenase (LDH) into the media as an indicator of cellular membrane damage. The amount of LDH released by STE was both concentration- and time-dependent. The results demonstrate that oral cells, peritoneal macrophages, and hepatic mitochondria and microsomes produce reactive oxygen species following in vitro incubation with an aqueous extract of smokeless tobacco. Tissue damage in response to STE may occur as the result of reactive oxygen species production.     

Effects of soy pinitol on the pro-inflammatory cytokines and scavenger receptors in oxidized low-density lipoprotein-treated THP-1 macrophages

Pinitol, a methylated form of D-chiro-inositol, acts as a insulin mediator. We investigated the effects of soy pinitol on the factors involved in foam cell formation using differentiated THP-1 macrophages. Pinitol slightly inhibited the lipid-laden foam cell formation by oxidized low-density lipoprotein (oxLDL) in a dose-dependent manner. Tumor necrosis factor-alpha and monocyte chemoattractant protein-1 releases were significantly reduced by pinitol treatment (0.05-0.5 mM), whereas interleukin-1beta and interleukin-8 secretions were significantly reduced in low-dose pinitol (0.05 or 0.1 mM) and 0.5 mM pinitol-treated cells, respectively, compared to no pinitol-treated cells. Gene expressions of CD36 and CD68 were significantly down-regulated by 0.05-0.5 mM pinitol compared to the oxLDL-treated control cells. Matrix metalloproteinase-9 gene expression was significantly decreased in 0.05-0.5 mM pinitol-treated cells compared to the no pinitol-treated macrophages. We conclude that pinitol has some inhibitory effects on foam cell formation by reducing lipid accumulation, secretion, and expression of some cytokines and macrophage scavenger receptor expression via its insulin-like action.