Chitosan oligosaccharides suppress production of nitric oxide in lipopolysaccharide-induced N9 murine microglial cells in vitro

Chitosan oligosaccharides (COS) have been reported to exert many biological activities, such as antioxidant, antitumor and anti-inflammatory effects. In the present study, we examined the effect of COS on nitric oxide (NO) production in LPS induced N9 microglial cells. Pretreatment with COS (50?~?200?μg/ml) could markedly inhibit NO production by suppressing inducible nitric oxide synthase (iNOS) expression in activated microglial cells. Signal transduction studies showed that COS remarkably inhibited LPS-induced phosphorylation of p38 MAPK and ERK1/2. COS pretreatment could also inhibit the activation of both nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). In conclusion, our results suggest that COS could suppress the production of NO in LPS-induced N9 microglial cells, mediated by p38 MAPK and ERK1/2 pathways.     

Fluorescence spectrometric study on the interaction of tamibarotene with bovine serum albumin

The interaction between tamibarotene and bovine serum albumin (BSA) was studied using fluorescence quenching technique and ultraviolet–visible spectrophotometry. The results of experiments showed that tamibarotene could strongly quench the intrinsic fluorescence of BSA by a dynamic quenching mechanism. The apparent binding constant, number of binding site and corresponding thermodynamic parameters at different temperatures were calculated respectively, and the main interaction force between tamibarotene and BSA was proved to be hydrophobic force. Synchronous fluorescence spectra showed that tamibarotene changed the molecular conformation of BSA. When BSA concentration was 1.00 × 10^?6 mol L^?1, the quenched fluorescence ΔF had a good linear relationship with the concentration of tamibarotene in the range 1.00 × 10^?6 to 12.00 × 10^?6 mol L^?1 with the detection limit of 6.52 × 10^?7 mol L^?1. Copyright © 2010 John Wiley & Sons, Ltd.     

Relationship among managanese, arginase, and nitric oxide in childhood asthma

It has been demonstrated that the lowest intakes of manganese (Mn) were associated with more than a fivefold increased risk of bronchial reactivity. It was also known that nitric oxide (NO) production was found to be significantly higher in asthmatics. There is a reciprocal pathway between arginase and nitric oxide synthase (NOS) for NO production, and Mn is required for arginase activity and stability. We investigated plasma NO, arginase, and its cofactor Mn levels to evaluate this reciprocal pathway in patients with childhood asthma. Arginase activities and Mn and NO levels were measured in plasma from 31 patients with childhood asthma and 22 healthy control subjects. Plasma arginase activities and Mn concentrations were found to be significantly lower and NO levels were significantly higher found to be significantly lower and NO levels were significantly higher in patients with childhood asthma as compared to the control subjects. There was a significantly positive correlation between plasma Mn and arginase and negative correlations between arginase and NO values and Mn and NO values in patients with childhood asthma. These data indicate that the lower concentration of Mn could cause lower arginase activity and this could also upregulate NO production by increasingl-arginine content in patients with childhood asthma.     

Enhanced expression of neuronal nitric oxide synthase in islets of exercise-trained rats

It is well known that glucose-stimulated insulin secretion (GSIS) decreases after exercise training. In the present study, we investigated the effects of exercise training (9 weeks of running) on the activity of glucokinase (GK), the production of nitric oxide (NO), and the protein expressions of both glucose transporter-2 (GLUT-2) and NO synthase (NOS) in rat pancreatic islets. Exercise training significantly reduced GSIS, with decreases in GK activity and GLUT-2 protein expression. The NO releases and cGMP contents were higher in the islets of trained rats than in those of control rats. Exercise training enhanced cNOS activity, the protein expression of both neuronal nitric oxide synthase (nNOS) and calmodulin, and NADPH-cytochrome c reductase activity in the homogenates of islets. Thus, exercise training-induced reduction of GSIS would result from, at least in part, decreases in both glucose entry and the first step in glycolytic utilization of glucose. Moreover, exercise training could enhance the protein expression of nNOS, which in turn enhances two catalytic activities of nNOS, an NO production and a cytochrome c reductase activity.     

外源NO对缺铁豌豆幼苗生长以及光合作用的影响

以水培豌豆(Pisum sativum)品种‘陇碗一号’幼苗为材料,以硝普钠(SNP)为一氧化氮(NO)供体,研究外源NO对缺铁豌豆幼苗的生长及光合作用的影响。结果显示:0.6 mmol.L-1的外源SNP所产生的NO能够促进缺铁豌豆幼苗的生长,并促进叶绿素的合成,增强净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr),而使胞间CO2浓度(Ci)下降;同时,叶绿素荧光最大光化学量子产量(Fv/Fm)、实际光化学量子效率(Yield)和光化学淬灭系数(qP)均升高,在一定程度上缓解缺铁对豌豆幼苗叶片PSII反应中心的影响。外源NO对正常铁(100μmol.L-1Fe)处理下豌豆幼苗的生长和光合作用具有一定的抑制作用。研究表明,缺铁和正常铁处理的豌豆幼苗对NO的敏感性不同,适宜浓度的NO对缺铁下豌豆幼苗的生长和光合都具有一定的改善作用。     

Nitric oxide inhibits ultraviolet B-induced murine keratinocyte apoptosis by regulating apoptotic signaling cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.     

姜黄素对脂多糖激活的小胶质细胞iNOS 表达的抑制及抗氧化作用

用姜黄素预处理小胶质细胞株BV,1 h后加用脂多糖(200 ng/ml)进行刺激,通过MTT检测细胞活性;硝酸还原酶法检测细胞上清液中一氧化氮(NO)的含量;Western 印迹、免疫细胞化学染色检测细胞活化后形态及诱导型一氧化氮合酶(iNOS)蛋白的表达;瞬时转染和荧光素酶报告基因鉴定iNOS和NF-κB基因表达活性;SOD和GSH-Px检测姜黄素的抗氧化能力.结果证明,脂多糖可促使小胶质细胞活化,使iNOS和NF-κB基因表达活性显著增强;iNOS蛋白表达明显上调,NO释放增多;细胞内SOD和GSH-Px活性明显下降.而姜黄素(10 μmol/L)可以显著抑制活化后小胶质细胞NO的产生、iNOS蛋白的表达及iNOS-Luc、NF-κB-Luc的表达活性,其机制可能通过NF-κB的信号转导途径抑制iNOS的表达.姜黄素可通过提高细胞内SOD和GSH-Px的活性发挥抗氧化作用.     

An Investigation of Antioxidant and Anti-inflammatory Activities from Blood Components of Crocodile (Crocodylus siamensis)

Antioxidant and anti-inflammatory activities were found from Crocodylus siamensis (C. siamensis) blood. The 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radical scavenging, nitric oxide scavenging, hydroxyl radical scavenging and linoleic peroxidation assays were used to investigate the antioxidant activities of the crocodile blood. Results show that crocodile blood components had antioxidant activity, especially hemoglobin (40.58 % nitric oxide radical inhibition), crude leukocyte extract (78 % linoleic peroxidation inhibition) and plasma (57.27 % hydroxyl radical inhibition). Additionally, the anti-inflammatory activity of the crocodile blood was studied using murine macrophage (RAW 264.7) as a model. The results show that hemoglobin, crude leukocyte extract and plasma were not toxic to RAW 264.7 cells. Also they showed anti-inflammatory activity by reduced nitric oxide (NO) and interleukin 6 (IL-6) productions from lipopolysaccharide (LPS)-stimulated cells. The NO inhibition percentages of hemoglobin, crude leukocyte extract and plasma were 31.9, 48.24 and 44.27 %, respectively. However, only crude leukocyte extract could inhibit IL-6 production. So, the results of this research directly indicate that hemoglobin, crude leukocyte extract and plasma of C. siamensis blood provide both antioxidant and anti-inflammatory activities, which could be used as a supplementary agent in pharmaceutical products.     

Specific protein nitration in nitric oxide-induced apoptosis of human monocytes

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.     

Structural requirements of flavonoids for nitric oxide production inhibitory activity and mechanism of action

To clarify the structure-activity relationships of flavonoids for nitric oxide (NO) production inhibitory activity, we examined the inhibitory effects of 73 flavonoids on NO production in lipopolysaccharide-activated mouse peritoneal macrophages. Among those flavonoids, apigenin (IC(50)=7.7 microM), diosmetin (8.9 microM), and tetra-O-methylluteolin (2.4 microM), and hexa-O-methylmyricetin (7.4 microM) were found to show potent inhibitory activity, and the results suggested the following structural requirements of flavonoids: (1) the activities of flavones were stronger than those of corresponding flavonols; (2) the glycoside moiety reduced the activity; (3) the activities of flavones were stronger than those of corresponding flavanones; (4) the flavones and flavonols having the 4'-hydroxyl group showed stronger activities than those lacking the hydroxyl group at the B ring and having the 3',4'-dihydroxyl group; (5) the flavonols having the 3',4'-dihydroxyl group (catechol type) showed stronger activities than those having the 3',4',5'-trihydroxyl group (pyrogallol type); (6) the 5-hydroxyl group tended to enhance the activity; (7) methylation of the 3-, 5-, or 4'-hydroxyl group enhanced the activity; (8) the activities of isoflavones were weaker than those of corresponding flavones; (9) methylation of the 3-hydroxyl group reduced the cytotoxicity. In addition, potent NO production inhibitors were found to inhibit induction of inducible nitric oxide synthase (iNOS) without iNOS enzymatic inhibitory activity.     

一氧化氮在乙烯诱导蚕豆气孔关闭中的作用

以蚕豆为材料研究了一氧化氮(nitric oxide,NO)和乙烯对气孔运动的影响。结果表明,10μmol/L的NO供体硝普钠(sodium nitroprusside,SNP)以及0.04%的乙烯能明显诱导蚕豆气孔关闭,并且二者共同处理后,能够增强其促进气孔关闭的作用。乙烯合成抑制剂AVG可以减弱NO诱导气孔关闭的程度,NO清除剂c-PTIO和NR抑制剂NaN3也可减弱乙烯诱导气孔关闭的程度,而一氧化氮合酶(nitric oxide synthase,NOS)抑制剂L-NAME对乙烯诱导气孔关闭的作用不明显。推测,在调控蚕豆气孔关闭过程中,NO可能主要通过NR途径参与乙烯调控气孔关闭过程。     

Nitric oxide is involved in abscisic acid-induced antioxidant activities in Stylosanthes guianensis

Previous studies suggest that abscisic acid (ABA) stimulates the activities of antioxidant enzymes under normal and chilling temperature and enhanced chilling resistance in Stylosanthes guianensis. The objective of this study was to test whether nitric oxide (NO) is involved in the ABA-induced activities of the antioxidant enzymes in Stylosanthes guianensis due to its nature as a second messenger in stress responses. Plants were treated with NO donors, ABA, ABA in combination with NO scavengers or the nitric oxide synthase (NOS) inhibitor and their effects on the activity of antioxidant enzymes and NO production were compared. The results showed that ABA increased the activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The effect of ABA on antioxidant enzyme activities was suppressed by the NOS inhibitor, N(omega)-nitro-L-arginine (L-NNA), and the NO scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl3-oxide (PTIO). NO content increased after 5 h of ABA treatment. The NO-scavenger, PTIO, and the NOS-inhibitor, L-NNA, inhibited the accumulation of NO in ABA-treated Stylosanthes guianensis. NO donor treatment enhanced the activities of SOD, CAT, and APX. The results suggested that NO was involved in the ABA-induced activities of SOD, CAT, and APX in Stylosanthes guianensis. ABA triggered NO production that may lead to the stimulation of antioxidant enzyme activities.     

Abscisic acid improves drought tolerance of triploid bermudagrass and involves H2O2- and NO-induced antioxidant enzyme activities

Drought is a major limiting factor for turfgrass growth. Protection of triploid bermudagrass against drought stress by abscisic acid (ABA) and its association with hydrogen peroxide (H₂O₂) and nitric oxide (NO) were investigated. ABA treatment increased relative water content, decreased ion leakage and the percentage of dead plants significantly under drought stress. Superoxide dismutase (SOD) and catalase (CAT) activities increased in both ABA-treated and control plants, but more in ABA-treated plants, under drought stress. Malondialdehyde, an indicator of plant lipid peroxidation, was lower in ABA-treated plants than in control plants, indicating that ABA alleviated drought-induced oxidative injury. ABA treatment increased H₂O₂ and NO contents. ABA-induced SOD and CAT activities could be blocked by scavengers of H₂O₂ and NO, and inhibitors of H₂O₂ and NO generation. The results indicated that H₂O₂ and NO were essential for ABA-induced SOD and CAT activities. Both H₂O₂ and NO could induce SOD and CAT activities individually. SOD and CAT induced by H₂O₂ could be blocked by scavenger of NO and inhibitors of NO generation, while SOD and CAT induced by NO could not be blocked by scavenger of H₂O₂ and inhibitor of H₂O₂. The results revealed that ABA-induced SOD and CAT activities were mediated sequentially by H₂O₂ and NO, and NO acted downstream of H₂O₂.     

Pharmacological and genetical evidence supporting nitric oxide requirement for 2,4-epibrassinolide regulation of root architecture in Arabidopsis thaliana

Brassinosteroids (BRs) regulate various physiological processes, such as tolerance to stresses and root growth. Recently, a connection was reported between BRs and nitric oxide (NO) in plant responses to abiotic stress. Here we present evidence supporting NO functions in BR signaling during root growth process. Arabidopsis seedlings treated with BR 24-epibrassinolide (BL) show increased lateral roots (LR) density, inhibition of primary root (PR) elongation and NO accumulation. Similar effects were observed adding the NO donor GSNO to BR-receptor mutant bri1-1. Furthermore, BL-induced responses in the root were abolished by the specific NO scavenger c-PTIO. The activities of nitrate reductase (NR) and nitric oxide synthase (NOS)-like, two NO generating enzymes were involved in BR signaling. These results demonstrate that BR increases the NO concentration in root cells, which is required for BR-induced changes in root architecture.     

脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用

在乌拉坦麻醉的成年ＳＤ大鼠上,用玻璃微电极细胞外记录的方法,观察了脑室内注射一氧化氮供体及一氧化氮合酶抑制剂对室旁核大细胞自发电活动的作用。结果发现：脑室内注射一氧化氮供体硝普钠对下丘脑室旁核中的加压素神经元产生剂量依赖性抑制作用;脑室内注射一氧化氮合酶抑制剂对加压素神经元也产生抑制作用。上述两种药物对催产素神经元均无作用。这些结果提示：一氧化氮可能在调节加压素和催产素神经元活动中起着不同的作用。     

NOS-like-mediated nitric oxide is involved in Pinus thunbergii response to the invasion of Bursaphelenchus xylophilus

The content of NO and H(2)O(2) as well as the activities of nitric oxide synthase (NOS)-like and nitrate reductase (NR) were monitored in the needles of Pinus thunbergii infected by Bursaphelenchus xylophilus. The results showed that the content of NO increased significantly only 8?h after the invasion of B. xylophilus, while H(2)O(2) increased 12?h after invasion. NO donor SNP could promote and NO scavenger cPTIO could prevent the production of NO and H(2)O(2). The content of NO changed earlier than that of H(2)O(2). In addition, the symptoms appeared 9, 5 and 12?days, respectively, after the inoculation with B. xylophilus, SNP pre-treatment and cPTIO pre-treatment followed by B. xylophilus infection. After B. xylophilus infection, the content of NO in P. thunbergii changed fiercely more earlier than the appearance of external symptoms, which indicated that the content of NO was related with the appearance and the development of the symptoms. The treatment with L-NNA (NOS inhibitor) inhibited the content of NO significantly, whereas, Na(2)WO(4) (NR inhibitor) had no effect. The further analysis of NOS revealed that NO changed in consistent with cNOS activity. To sum up, NO, as the upstream signal molecule of H(2)O(2), was involved in the pine early response to the invasion of B. xylophilus and influenced the accumulation of the content of H(2)O(2). Moreover, NOS-like rather than NR was responsible for the endogenous NO generation, which was modulated by cNOS during the interaction between P. thunbergii and B. xylophilus. Key message NO is involved in early response of P. thunbergii to the invasion of B. xylophilus and NOS is the key enzyme responsible for NO generation in P. thunbergii.     

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Oxidative Damage Involves in the Inhibitory Effect of Nitric Oxide on Spore Germination of <Emphasis Type="Italic">Penicillium expansum</Emphasis>

The effects of nitric oxide (NO) on spore germination of Penicillium expansum were investigated and a possible mechanism was evaluated. The results indicated that NO released by sodium nitroprusside (SNP) significantly suppressed fungal growth. With the use of an oxidant sensitive probe and Western blot analysis, an increased level of intracellular reactive oxygen species (ROS) and enhanced carbonylation damage were detected in spores of P. expansum under NO stress. Exogenous superoxide dismutase (SOD) and ascorbic acid (Vc) could increase the resistance of the spore to the inhibitory effect of NO. The activities of SOD and catalase (CAT), as well as ATP content in spores under NO stress were also lower than those in the control. We suggest that NO in high concentration induces the generation of ROS which subsequently causes severe oxidative damage to proteins crucial to the process of spore germination of P. expansum.     

Nitric oxide induced by hydrogen peroxide mediates abscisic acid-induced activation of the mitogen-activated protein kinase cascade involved in antioxidant defense in maize leaves

* The role of nitric oxide (NO) and the relationship between NO, hydrogen peroxide (H(2)O(2)) and mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense in leaves of maize (Zea mays) plants were investigated. * Both ABA and H(2)O(2) induced increases in the generation of NO in mesophyll cells of maize leaves, and H(2)O(2) was required for the ABA-induced generation of NO. Pretreatment with NO scavenger and nitric oxide synthase (NOS) inhibitor substantially reduced the ABA-induced production of NO, and partly blocked the activation of a 46 kDa MAPK and the expression and the activities of several antioxidant enzymes induced by ABA. Treatment with the NO donor sodium nitroprusside (SNP) also induced the activation of the MAPK, and enhanced the antioxidant defense systems. * Conversely, SNP treatment did not induce the production of H(2)O(2), and pretreatments with NO scavenger and NOS inhibitor did not affect ABA-induced H(2)O(2) production. * Our results suggest that ABA-induced H(2)O(2) production mediates NO generation, which, in turn, activates MAPK and results in the upregulation in the expression and the activities of antioxidant enzymes in ABA signaling.     

白细胞介素—10对血管一氧化氮／一氧化氮合酶系统的影响

为探讨抗炎因子－－白细胞介素－10（IL－10）对大鼠主动脉一氧化氮（NO）／一氧化氮合酶（NOS）系统的影响,应用Griess试剂、^3H－瓜氨酸生成及蛋白免疫印迹杂交等方法,测定IL－10孵育对血管NO释放、NOS活性及表达的影响。结果发现细菌脂多糖（LPS）呈浓度领带性地激活诱导型NOS（iNOS）,促进NO生成。IL－10（10^-10-10^-8g/ml）呈浓度依赖性地上调内皮型NOS（eNOS）蛋白表达及其活性,但对iNOS活性及表达无明显影响,IL－10（10^-9-10^-8g/ml）显著抑制10μg/ml LPS诱导的NO生成和iNOS激活;而高浓度IL－10（10^-7g/ml）则上调iNOS的活性,对eNOS蛋白的表达知活性无明显影响。因此IL－10对NO／NOS系统具有双重影响,一方面可抑制炎症介质诱发的作为炎性物质的iNOS的表达及激活,另一方面可上调内皮源扩血管物质NO的释放。