INHIBITORY EFFECT OF RETINOIC ACID ON EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE IN L929 CELLS

Inflammation has been known to be associated with excess synthesis of nitric oxide (NO) by inducible NO synthase (iNOS). Retinoids have been reported to have anti-inflammatory activity, but the mechanism by which they can elicit this activity is poorly understood. The effects of retinoids on NO synthesis and iNOS gene expression in murine fibroblast L929 cells were examined. Treatment of the cells with interferon-γ resulted in excess NO synthesis and iNOS gene expression. All-trans-retinoic acid significantly inhibited NO synthesis and iNOS gene expression in a dose-dependent manner. Similarly, 9-cis-retinoic acid also inhibited NO synthesis, but retinol did not show any inhibitory effect on NO synthesis. These findings suggest that the modulation of iNOS gene expression is another possible pathway by which retinoids may function as anti-inflammatory agents.     

INHIBITORY EFFECT OF RETINOIC ACID ON EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE IN L929 CELLS

Inflammation has been known to be associated with excess synthesis of nitric oxide (NO) by inducible NO synthase (iNOS). Retinoids have been reported to have anti-inflammatory activity, but the mechanism by which they can elicit this activity is poorly understood. The effects of retinoids on NO synthesis and iNOS gene expression in murine fibroblast L929 cells were examined. Treatment of the cells with interferon-γ resulted in excess NO synthesis and iNOS gene expression. All-trans-retinoic acid significantly inhibited NO synthesis and iNOS gene expression in a dose-dependent manner. Similarly, 9-cis-retinoic acid also inhibited NO synthesis, but retinol did not show any inhibitory effect on NO synthesis. These findings suggest that the modulation of iNOS gene expression is another possible pathway by which retinoids may function as anti-inflammatory agents.     

Rhodiola sachalinesis induces the expression of inducible nitric oxide synthase gene by murine fetal hepatocytes (BNL CL.2)

We have examined the effect of the aqueous extract of Rhodiola sachalinensis root (RSE), a traditional herbal medicine, on nitric oxide (NO) synthesis in murine fetal hepatocytes (BNL CL.2) by measuring the stable end-product nitrite and the mRNA of inducible NO synthase (iNOS). Interferon-gamma (IFN-γ) by itself failed to induce NO synthesis in BNL CL.2 cells. RSE also did not elicit NO synthesis at concentrations up to 1000 μg/ml, but dose- and time-dependently induced NO synthesis in the presence of IFN-γ in BNL CL.2 cells. Whereas RSE or IFN-γ failed to induce detectable levels of iNOS mRNA, a combination of RSE and IFN-γ markedly induced iNOS mRNA in BNL CL.2 cells. Thus, we found that RSE triggered IFN-γ-primed BNL CL.2 cells to synthesize NO by inducing iNOS gene expression. The capability of RSE to induce NO synthesis might be related to the therapeutic efficacy of RSE on the liver diseases.     

Ex vivo all-trans retinoic acid modulates NO production and regulates IL-6 effect during rheumatoid arthritis: a study in Algerian patients

Rheumatoid arthritis (RA) is a chronic autoimmune disease. The pathophysiology of RA implicates several mediators such as nitric oxide (NO) and cytokines such as interleukin-6 (IL-6), which is deeply involved in the main characteristics of RA. Furthermore, all-trans retinoic acid (ATRA) is an active vitamin A derivative well-known to have diverse immunomodulatory actions. In our study, we investigated first, the ex vivo immunomodulatory potential of ATRA on NO pathway by peripheral blood mononuclear cells (PBMCs) from Algerian RA patients. Then, we assessed the possible regulatory effect of ATRA on NO production induced by IL-6. PBMCs isolated from active and inactive RA patients and healthy controls were cultured with different concentrations of IL-6 or/with ATRA. NO production was assessed using the Griess method. Inducible nitric oxide synthase expression and NF-κB activity were analyzed by immunofluorescence test. Our results revealed a high NO production during active RA. We noticed that while IL-6 induced a high NO production and iNOS expression, ATRA downregulated both. ATRA also inhibited nuclear NF-κB translocation. Interestingly, it seems that NO production mediated by IL-6 on PBMCs of RA patients is downregulated by ATRA. Taken together, our results highlight the immunomodulatory effect of ATRA on NO pathway in RA patients and its possible role in regulating IL-6-mediated NO production. All these findings suggest its potential therapeutic role during RA.     

Lack of inducible nitric oxide synthase activity in T cell clones and T lymphocytes from naive and Leishmania major-infected mice

Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is implicated in a number of immunological processes including killing of intracellular parasites, suppression of T cell proliferation, production of cytokines and destruction of tissue in autoimmune diseases. Considering that cytokine-activated mouse macrophages, fibroblasts and endothelial cells are potent producers of NO, we investigated whether T cells, as central participants in immune responses, can also be activated for the release of NO. Neither thymocytes nor type 1 or type 2 T helper cell clones generated significant amounts of nitrite (the stable end product of NO in culture supernatants) when stimulated by T cell mitogens, cytokines or antigen in the presence of irradiated antigen-presenting cells. Similarly, T cells freshly isolated from mice acutely infected with the intracellular pathogen Leishmania major did not produce NO upon restimulation in vitro. The lack of NO production was not due to the expression of enzymatically inactive iNOS, as we were unable to detect any iNOS protein in activated T helper clones or in freshly isolated T cells from infected mice by Western (protein) blot analysis. Finally, we tested whether iNOS expression in T cells might be restricted to a minor subpopulation and therefore only detectable on a single cell level. After immunofluorescence staining of lymph node or spleen cells from infected mice with antibodies against iNOS, F4/80- or Thy-1-antigen, macrophages, but no T cells, were found to express iNOS. Thus, we have no evidence that activated T helper cell clones or T cells from L. major-infected mice are high producers of NO.     

The Anti-Tumor Effect of Interleukin-12 is Enhanced by Mild (Fever-Range) Thermal Therapy

The cytokine interleukin 12 (IL-12) has resulted in notable anti-tumor activity in animal models and in patients and as a result there is considerable interest in learning how to maximize its therapeutic potential while at the same time reducing its known toxic side effects. Strategies which could maintain its effectiveness while permitting reduced dosage could be especially valuable. In this study we used BALB/c mice bearing CT26 tumors as a model for testing whether combining murine IL-12 with a mild (fever range) whole body hyperthermia protocol could result in such a strategy. Our data revealed that 100 ng of IL-12/mouse/day used in combination with FR-WBH was as effective as one in which 300 ng of IL-12/mouse/day was used alone. Importantly, the mice receiving the combination treatment exhibited fewer treatment related toxicities compared to those that received high dose IL-12 alone. Initiation of the IL-12 treatment immediately after FR-WBH induced the greatest anti-tumor effect. This effect does not appear to depend on differences in IL-12-induced IFN-γ, but may involve production of nitric oxide (NO), since treatment of mice with a NOS inhibitor, N^G-monomethyl-l-arginine (l-NMA), abolishes the additive anti-tumor effect of the combination treatment. Collectively, these data suggest that modification of physiological parameters in the host by mild fever-like thermal stimuli may be an effective and feasible adjuvant for cytokine-based immunotherapeutic strategies.     

补体C5b-9复合物诱导大鼠肾系膜细胞iNOS mRNA表达的实验研究

观察人血清补体C5b 9复合物对大鼠肾小球系膜细胞 (MC)表达诱生性一氧化氮合酶 (iNOS)mRNA的影响。方法 :首先提取人血清补体C5b 9复合物 ,然后用人C5b 9复合物刺激培养的大鼠MC ,检测MC在受C5b 9复合物刺激后3、6、2 4和 48h时iNOSmRNA的表达情况。同时检测其培养上清液中一氧化氮 (NO)代谢产物———硝酸根 (NO3- )和亚硝酸根(NO2- )含量的变化。结果 :用人C5b 9复合物刺激培养大鼠的肾MC能使其表达iNOSmRNA ,培养上清液中NO3- NO2- 含量也明显升高。人C5b 9复合物对MC的刺激作用能部分被相应的抗人C5b 9复合物抗体和RNA合成抑制剂———放线菌素D所抑制。结论 :人补体C5b 9复合物具有刺激大鼠肾MC合成NO的作用。     

iNOS expression in rat aorta is increased after spinal cord transection: a possible cause of orthostatic hypotension in man

Orthostatic hypotension commonly occurs in persons with spinal cord injury (SCI), limiting rehabilitation and independence. Findings of increased production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) after exposure to simulated microgravity suggest that increased iNOS expression contributes to OH in persons with SCI. To test this possibility, male Wistar rats underwent surgical transection of the spinal cord (T10) or sham-SCI surgery followed by euthanasia 3, 7 or 14 days later. Expression in thoracic aortic of inducible (iNOS), endothelial (eNOS) and neuronal (nNOS) NOS was then determined. In SCI rats, expression of iNOS mRNA was decreased at 3 days, had returned to normal levels of expression at 7 days and was increased at 14 days post-SCI (1.8-fold). In contrast, levels of eNOS mRNA were increased at 3 days (1.4-fold), then declined over time reaching levels by day 14 that were reduced compared to sham-SCI (0.23-fold). There were no significant effects of SCI on nNOS expression. These findings suggest a possible role for increased iNOS expression in the pathogenesis of OH in persons with SCI.     

11-羰基-β-乙酰乳香酸对小鼠黑素瘤细胞B16F10增殖的抑制作用

分析11-羰基-β-乙酰乳香酸(AKBA)对小鼠黑素瘤细胞系B16F10增殖和凋亡的影响,探讨其作用机制。AKBA是从天然乳香中提取的单体。通过CCK-8法测定AKBA在不同浓度及不同时间对B16F10细胞增殖的影响;以流式分析法检测AKBA对B16F10凋亡的作用;以Real-time PCR法检测细胞增殖及凋亡相关基因的表达;以Western blot法检测细胞增殖与凋亡相关的NF-κB信号通路的变化。结果显示,AKBA可以明显促进B16F10细胞的凋亡,抑制其增殖,并与NF-κB信号转导通路的活性有关。     

缺氧诱导因子-1在缺氧诱导一氧化氮合成酶基因表达中的作用

目的 研究缺氧诱导因子-1(HIF-1)在一氧化氮合成酶基因缺氧诱导反应中的作用。方法 体外合成具有HIF-1特异结合位点的DNA片段(红细胞生成素3'-增强子片段),借助脂质体,转入培养的鼠主动脉内皮细胞和肺微血管细胞,用半定量RT-PCR方法测定诱导型一氧化氮合成酶(iNOS)mRNA。结果 (1)大鼠主动脉内皮细胞、肺微血管内皮细胞在常氧下培养,有iNOS基因表达;(2)缺氧能诱导这两种细胞iNOS基因表达增加;(3)野生型EPO3'-增强子片段能阻断缺氧对内皮细胞iNOS基因表达的诱导作用,而突变片段则无此作用。结论 在iNOS基因序列中,可能存在EPO3'-端增强子片段,其参与内皮细胞的缺氧反应。     

菊米提取液对抗心肌缺血再灌注损伤

目的： 研究菊米提取液的抗心肌缺血作用及其机制。方法：采用离体大鼠心脏Langendorff灌流模型，观察心脏收缩功能和心肌梗死面积。结果：（1）菊米提取液(0.5、1.0、2.0 g/L)灌流心脏后，左室发展压（left ventricular developed pressure，LVDP）、最大左室收缩/舒张速率（maximal rate of increase/decline in left ventricular pressure，±dp/dtmax）和冠脉流量增高。（2）菊米提取液可浓度依赖性增加心肌组织一氧化氮合酶（nitric oxide synthase，NOS）活性和一氧化氮（nitric oxide，NO）含量。（3）在缺血前（预处理）或再灌注早期给予0.5 g/L菊米提取液，均可减轻缺血再灌注引起的收缩功能下降，并可缩小心肌梗死面积。预先给予L-NAME，可取消菊米对抗心肌缺血再灌注损伤。结论：菊米提取液具有强心作用。菊米提取液能对抗心肌缺血再灌注损伤，其机制可能依赖于NO途径。     

Anti-inflammatory effect of Mentha longifolia in lipopolysaccharide-stimulated macrophages: Reduction of nitric oxide production through inhibition of inducible nitric oxide synthase

Abstract

Mentha longifolia is an aromatic plant used in flavoring and preserving foods and as an anti-inflammatory folk medicine remedy. The present study assessed the effects of M. longifolia extracts, including essential oil and crude methanol extract and its fractions (ethyl acetate, butanol and hexane), on nitric oxide (NO) production and inducible NO synthase (iNOS) mRNA expression in lipopolysaccharide (LPS)-stimulated J774A.1 cells using real-time polymerase chain reaction (PCR). The cytotoxic effects of the extracts on the cells were examined and non-cytotoxic concentrations (<0.2?mg/ml) were used to examine their effects on NO production and iNOS mRNA expression. Only the hexane fraction that contained high levels of phenolic and flavonoid compounds at concentrations from 0.05–0.20?mg/ml significantly reduced NO production in LPS-stimulated cells (p?<?0.001). Real-time PCR analysis indicated the ability of this fraction at the same concentrations to significantly decrease iNOS as well as TNFα mRNA expression in the cells (p?<?0.001). All extracts were able to scavenge NO radicals in a concentration-dependent manner. At concentrations greater than 0.2?mg/ml, total radicals were 100% scavenged. In conclusion, M. longifolia possibly reduces NO secretion in macrophages by scavenging NO and inhibiting iNOS mRNA expression, and also decreases TNFα pro-inflammatory cytokine expression, thus showing its usefulness in the inflammatory disease process.     

牛磺酸锌对血管性痴呆小鼠学习和记忆能力的影响

目的:研究牛磺酸锌(taurine-zinc,TZC)对血管性痴呆(vascular dementia,VD)小鼠学习和记忆能力的影响及其相关机制。方法:将小鼠随机分为模型组、假手术组及TZC 50 mg/kg组、100 mg/kg和200 mg/kg给药组。给药组每天按10 m L/kg灌胃,假手术组和模型组给予等体积蒸馏水,灌胃14 d后,利用双侧颈总动脉阻断合并尾静脉放血法制备小鼠VD模型。ELISA检测脑组织中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)的水平;分光光度计法检测脑组织诱导型一氧化氮合酶(i NOS)活性和一氧化氮(NO)含量的变化;利用跳台和水迷宫实验观察小鼠的学习和记忆能力。结果:TZC 50 mg/kg、100 mg/kg和200 mg/kg组小鼠脑组织中TNF-α、IL-1β、i NOS和NO水平均降低。在水迷宫实验中,与模型组相比,TZC 100 mg/kg和200 mg/kg组的潜伏期缩短,TZC 50mg/kg、100 mg/kg和200 mg/kg组的错误次数减少;在跳台实验中,TZC 50 mg/kg、100 mg/kg和200 mg/kg组较模型组潜伏期延长,错误次数减少(P0.05)。结论:TZC对VD小鼠的学习和记忆能力有改善作用,其机制可能与降低脑组织TNF-α、IL-1β、i NOS和NO水平有关。     

Ellagic acid protects against LPS-induced acute lung injury through inhibition of nuclear factor kappa B,proinflammatory cytokines and enhancement of interleukin-10

Ellagic acid is a naturally occurring polyphenolic compound which is found in many fruits, nut galls and plant extracts. In the present study, we explored the ability of ellagic acid to modulate lipopolysaccharides (LPS) response using macrophage-mediated inflammatory conditions and acute lung injury (ALI). The data showed that ellagic acid reduced TNF-α, IL-6 and IL-1β secretions, enhance IL-10 production by LPS-stimulated RAW 264.7 macrophages in vitro. In murine ALI model, mice were treated with ellagic acid prior to LPS challenge. The data showed that ellagic acid possess a protective effect on LPS-induced ALI in mice. The underlying mechanism may be through shocking the NF-κB pathway to attenuate the nonspecific pulmonary inflammation induced by LPS administration.     

Immune stimulatory effects of Loranthi ramulus on macrophages through the increase of NO and TNF-α

The activation of macrophages by microorganisms plays an important role in host defense and immunopathology. Loranthi ramulus (LR) is commonly used as a traditional drug and health food in Korea. Here, we investigated the regulatory effects of LR on macrophage-mediated immune responses. Treatment of macrophages with LR resulted in the enhanced cell-surface expression of CD80, CD86 and major histocompatibility complex (MHC) class II, as well as the enhanced production of nitric oxide (NO) and tumor necrosis factor (TNF)-α, and also iNOS and TNF-α mRNA expression. These alterations of LR-treated cells were associated with the activation of NF-κB and mitogen-activated protein kinases (MAPKs). LR increased the phosphorylation of MAPKs (JNK, ERK1/2, p38 MAPK) and the activation of NF-κB in Raw 264.7 cells. These results suggest that LR has increased NO and TNF-α production through phosphorylation of all three MAPKs following IκBα degradation and NF-κB activation. In conclusion, our results demonstrate that LR can effectively promote the activation of macrophages, suggesting that LR may possess the potential to regulate immune responses.     

二十碳五烯酸联合视黄酸对HL-60细胞凋亡相关基因表达的影响

目的 研究二十碳五烯酸(EPA)是否协同视黄酸(RA)影响HL-60细胞的凋亡及相应分子机制。方法 流式细胞仪(FCM)检测细胞周期相分布以测定细胞增殖和凋亡功能；Western blot法分析bcl-2和caspase—3表达。结果 与单独应用相比，EPA和RA联合应用可增强HL-60细胞的调亡效应，以及下调节bcl—2和增强caspase-3基因表达。结论 EPA和RA联合应用对HL-60细胞凋亡的增强效应，可能与它们增强caspase-3和降低bcl—2基因表达相关。     

叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响

 目的: 观察叶酸对去卵巢大鼠抗氧化酶、一氧化氮合酶和一氧化氮的影响。方法: 40只3月龄健康雌性SD大鼠,随机分成5组:假手术组、去卵巢组、二乙基己烯雌酚(0.03 mg·kg^-1·d^-1)组、低剂量(5 mg·kg^-1·d^-1)叶酸组和高剂量(20 mg·kg^-1·d^-1)叶酸组。各组大鼠于术后1周开始灌胃给药,假手术组和去卵巢组给予蒸馏水,10周后,取L₅椎体和右股骨行骨密度(BMD)检测;测定血浆和骨匀浆总抗氧化能力(TAC)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、一氧化氮合酶(NOS)和一氧化氮(NO)水平。结果: 与假手术组比较,去卵巢组大鼠L₅椎体和股骨BMD显著降低(P < 0.01),血浆GSH-Px、NO和骨匀浆TAC、GSH-Px、NOS及NO水平明显降低(P < 0.01),MDA浓度升高显著(P < 0.01);与去卵巢组大鼠比较,高剂量叶酸组大鼠L₅椎体和股骨BMD增加(P < 0.01),骨匀浆TAC、GSH-Px、NOS和NO水平升高(P < 0.01),MDA浓度降低(P < 0.01),血浆GSH-Px和NO水平升高。结论: 去卵巢大鼠体内抗氧化酶、NOS和NO水平降低,氧化应激参与了去卵巢大鼠骨质疏松的发生;高剂量叶酸能提升去卵巢大鼠腰椎和股骨BMD,提高其体内抗氧化酶、NOS和NO水平,改善氧化应激,这可能是高剂量叶酸防治去卵巢大鼠骨质疏松的机制之一。