Effect of inducible nitric oxide synthase on apoptosis in Candida-induced acute lung injury

Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxinitrite. We previously showed that the expression of iNOS and lung injury were suppressed by inhalation of a novel iNOS inhibitor, ONO-1714, in mice with Candida-induced ALI, and that nitric oxide produced by iNOS and apoptosis of epithelial cells were found to have a crucial role in Candida-induced ALI. In the present study, we investigated the effect of NO on the apoptosis of alveolar epithelial cells in Candida-induced ALI. Mice were pretreated by inhalation of ONO-1714 or saline (vehicle control of ONO-1714), and were given an intravenous injection of Candida albicans to induce ALI. After 24 h from injection of Candida albicans, we performed bronchoalveolar lavage and removed lung tissues. We assessed apoptosis on the basis of TUNEL staining and caspase 3 activity. Our results showed that apoptosis was suppressed by inhibition of iNOS-derived NO production by ONO-1714 inhalation. The augmented production of NO increased FasL, TNF-alpha, and mRNA production of Bax of lung that induced apoptosis of alveolar epithelial cells. Inhibition of iNOS-derived NO production by ONO-1714 inhalation ameliorated Candida-induced ALI and improved survival by suppressing apoptosis of alveolar epithelial cells.     

An Active Drimane-Type Lactone from <Emphasis Type="Italic">Polygonum jucundum</Emphasis> Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice Through TLR4-MAPKs Signaling Pathway

The herbs of Polygonum jucundum Lindex. (Polygonaceae) is a traditional Chinese medicine for inflammatory diseases. 2α-Hydroxyl-3β-angeloylcinnamolide (HAC), a drimane-type sesquiterpenoid, was the major active compound of the ethanol extract of P. jucundum which inhibited the production of inflammatory mediators. However, the biological mechanism of HAC for anti-inflammatory activity has not been reported. In the current study, we investigated whether HAC could suppress the production of inflammatory mediators in lipopolysaccharide (LPS)-induced acute lung injury in mice (ALI) through downregulation of Toll-like receptor 4 (TLR4) and activations of mitogen-activated protein kinases (MAPKs) and inducible protein nitric oxide synthase (iNOS). Moreover, our data indicated that HAC inhibits the overexpression of iNOS and TLR4 in LPS-treated RAW264.7, and also inhibits MAPK signal. These findings suggest that HAC shows anti-inflammatory effects in ALI mice through suppressing TLR4-mediated MAPK pathway in activated macrophages. In addition, six derivatives of HAC obtained by structure modification were investigated for their inhibitory effects on the production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), suggesting that the acetylation could increase the inhibition of HAC on TNF-α release in LPS-treated RAW264.7 cells. In summary, all these results showed that HAC may be a potential anti-inflammatory lead compound for the treatment of acute lung injury.     

An inhaled inducible nitric oxide synthase inhibitor reduces damage of Candida-induced acute lung injury

Excessive nitric oxide (NO) generated by inducible nitric oxide synthase (iNOS) aggravates acute lung injury (ALI) by producing peroxynitrite. We previously showed by immunostaining that the expression of iNOS was suppressed by inhalation of N(G)-nitro-L-arginine methyl ester in mice with Candida-induced ALI. This study tested the hypothesis that a novel iNOS inhibitor suppresses not only iNOS expression, but also iNOS messenger RNA (mRNA) production by interrupting a positive feedback loop at the time of NO production in Candida-induced ALI. Mice were pretreated by inhalation of saline or ONO-1714, a selective iNOS inhibitor, and were given an intravenous injection of Candida albicans to induce ALI. After inhalation of 1 mM aerosolized ONO-1714, the nitrite-nitrate concentration in bronchoalveolar lavage fluid (BALF) at 24 h was significantly lower than that after inhalation of saline. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) levels and neutrophils in BALF were decreased by inhalation of ONO-1714. Inhalation of ONO-1714 markedly suppressed nitrotyrosine production and inhibited the expression of iNOS mRNA as well as proteins in the lung. Survival was prolonged by inhalation of ONO-1714. We conclude that pretreatment with inhaled ONO-1714 suppresses the production of peroxinitrite and decreases oxidative stress associated with peroxinitrite in Candida-induced ALI.     

重组人血清白蛋白-硫氧还蛋白融合蛋白对流感病毒所致急性肺损伤小鼠的保护作用

目的:探讨重组人血清白蛋白(HSA)-硫氧还蛋白(Trx)融合蛋白(HSA-Trx)对流感病毒感染所致急性肺损伤(ALI)小鼠的保护作用。方法:利用毕赤酵母表达系统产生基因重组HAS-Trx融合蛋白,PR8(H1N1)流感病毒诱导ALI小鼠模型,设健康对照组、ALI组、ALI+Trx组和ALI+HSA-Trx组,每组10只。收集各组支气管肺泡灌洗液(BALF),计算细胞总数,测定肺泡中性粒细胞数量,考马斯亮蓝溶液法测定蛋白浓度,ELISA法测定BALF中干扰素γ(IFN-γ)含量。收集肺组织进行苏木精-伊红染色,免疫荧光法检测肺组织诱导型一氧化氮合酶(iNOS)、8-羟基脱氧鸟苷(8-OHdG)和3-硝基酪氨酸(NO2-Tyr)的表达。收集血浆,使用CR2000RC分析仪测量血浆中过氧化物浓度。结果:HSA-Trx治疗显著降低ALI小鼠BALF中总细胞、中性粒细胞数量和总蛋白的含量(P<0.05),降低肺组织中8-OHdG和NO2-Tyr水平和血浆中过氧化物浓度(P<0.05),但对肺组织iNOS和IFN-γ的表达无显著抑制效果(P>0.05)。结论:HSA-Trx能够抑制肺组织炎症反应和肺内一氧化氮的过量产生,这对流感病毒诱导的ALI小鼠具有一定的保护作用。     

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.     

Valproic Acid Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice

Valproic acid (VPA) has been shown to exert anti-inflammatory and antioxidant effects in a range of diseases including septic shock. However, the effects of VPA on lipopolysaccharide (LPS)-induced acute lung injury (ALI) remains not well understood. We found that VPA pretreatment attenuated the LPS-induced ALI, as evidenced by the reduced histological scores, myeloperoxidase activity, and wet-to-dry weight ratio in the lung tissues. This was accompanied by the downregulated nuclear factor kappa B (NF-κB) p65, nitric oxide, and inducible nitric oxide synthase in the lung tissues and the decreased levels of tumor necrosis factor alpha and interleukin-1β in the bronchoalveolar lavage fluid. Furthermore, VPA reduced the nuclear histone deacetylase (HDAC)3 expression whereas increased the cytoplasmic HDAC3 expression. Our results suggested that VPA attenuates the LPS-induced ALI via inhibiting the NF-κB activation probably through a mechanism depending on HDAC3 redistribution.     

Chamomile: an anti-inflammatory agent inhibits inducible nitric oxide synthase expression by blocking RelA/p65 activity

Chamomile has long been used in traditional medicine for the treatment of inflammation-related disorders. In this study we investigated the inhibitory effects of chamomile on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression, and explored its potential anti-inflammatory mechanisms using RAW 264.7 macrophages. Chamomile treatment inhibited LPS-induced NO production and significantly blocked IL-1β, IL-6 and TNFα-induced NO levels in RAW 264.7 macrophages. Chamomile caused reduction in LPS-induced iNOS mRNA and protein expression. In RAW 264.7 macrophages, LPS-induced DNA binding activity of RelA/p65 was significantly inhibited by chamomile, an effect that was mediated through the inhibition of IKKβ, the upstream kinase regulating NF-κB/Rel activity, and degradation of inhibitory factor-κB. These results demonstrate that chamomile inhibits NO production and iNOS gene expression by inhibiting RelA/p65 activation and supports the utilization of chamomile as an effective anti-inflammatory agent.     

硝基酪氨酸和诱导型一氧化氮合酶在大鼠胎粪吸入性肺损伤中表达的研究

目的：观察大鼠胎粪诱导肺损伤时肺组织硝基化酪氨酸和诱导型一氧化氮合酶（iNOS）表达的改变，探讨两者在此种损伤中的作用。 方法： 16只雄性SD大鼠，随机分为对照组和胎粪组，分别由气管插管注入生理盐水或20%胎粪生理盐水混悬液1 mL/kg。24 h后取材，观察支气管肺泡灌洗液（BALF）细胞计数，比色法检测肺组织匀浆髓过氧化物酶（MPO）活性、一氧化氮（NO）含量，Western blot法测定硝基酪氨酸和iNOS蛋白表达改变。 结果： 胎粪组BALF细胞计数、肺组织MPO活性、NO含量分别为(4.04±1.01)×109cells/L、(1.49±0.22)U/g wet lung tissue、(12.77±5.00)mmol/g protein，对照组BALF细胞计数、肺组织MPO活性、NO含量分别为(0.53±0.19)×109cells/L、(0.62±0.16)U/g wet lung tissue、(4.89±1.32)mmol/g protein，两组比较差异显著（均P<0.01）；Western blot结果显示胎粪组肺组织硝基酪氨酸和iNOS蛋白表达明显强于对照组，分别为0.46±0.19和1.49±0.60，与对照组（0.15±0.04和0.09±0.04）比较, 差异显著（均P<0.01）。 结论： 胎粪可诱导iNOS表达增强并产生过量的硝基酪氨酸，两者可能在胎粪性肺损伤发病机制中发挥重要作用。     

Anti-inflammatory effects of galangin on lipopolysaccharide-activated macrophages via ERK and NF-κB pathway regulation

Inflammation is the major symptom of the innate immune response to microbial infection. Macrophages, immune response-related cells, play a role in the inflammatory response. Galangin is a member of the flavonols and is found in Alpinia officinarum, galangal root and propolis. Previous studies have demonstrated that galangin has antioxidant, anticancer, and antineoplastic activities. However, the anti-inflammatory effects of galangin are still unknown. In this study, we investigated the anti-inflammatory effects of galangin on RAW 264.7 murine macrophages. Galagin was not cytotoxic to RAW 264.7 cells, and nitric oxide (NO) production induced by lipopolysaccharide (LPS)-stimulated macrophages was significantly decreased by the addition of 50?μM galangin. Moreover, galangin treatment reduced mRNA levels of cytokines, including IL-1β and IL-6, and proinflammatory genes, such as iNOS in LPS-activated macrophages in a dose-dependent manner. Galangin treatment also decreased the protein expression levels of iNOS in activated macrophages. Galangin was found to elicit anti-inflammatory effects by inhibiting ERK and NF-κB-p65 phosphorylation. In addition, galangin-inhibited IL-1β production in LPS-activated macrophages. These results suggest that galangin elicits anti-inflammatory effects on LPS-activated macrophages via the inhibition of ERK, NF-κB-p65 and proinflammatory gene expression.     

Inhibition of the induction of the inducible nitric oxide synthase in murine brain microglial cells by sodium salicylate.

The induction of the inducible nitric oxide synthase (iNOS) has been proposed to play a role in a variety of inflammatory diseases. Sodium salicylate (NaSal) is the most commonly used anti-inflammatory agent. We investigated whether NaSal can diminish the induction of iNOS in murine brain microglial cells. In primary cultures, interferon-gamma (IFN-gamma) or lipopolysaccharide (LPS) separately did not stimulate nitric oxide (NO) production, whereas IFN-gamma combined with LPS synergistically induced iNOS. NaSal inhibited both the production of NO and expression of iNOS in microglial cells. Synergy between IFN-gamma and LPS was mainly dependent on tumour necrosis factor-alpha (TNF-alpha) secretion as the increase of the induction of the iNOS by IFN-gamma plus LPS was associated with the increase of TNF-alpha secretion and IFN-gamma plus LPS-induced TNF-alpha secretion by microglial cells was decreased by the treatment with NaSal. These results suggest a possible use of NaSal in managing inflammation of the central nervous system through inhibition of the iNOS induction.     

N-palmitoylation of the radioprotective domain of interleukin-1 affords inhibition of LPS-induced nitric oxide generation

Interleukin-1β (IL-1β), a cytokine involved in homeostatic processes such as the immune system and inflammatory reactions, is a potent inducer of nitric oxide. The nonapeptide of human IL-1β (VQGEESNDK, position 163-171, specific radioprotective domain-SRD) has been shown to retain radioprotective, immunostimulatory, and adjuvant activities of the native molecule without any inflammatory and pyrogenic properties. Unlike the parent IL-1, SRD did not induce nitric oxide (NO) in control or irradiated RAW 264.7 cells nor did it affect inducible nitric oxide synthase (iNOS) as shown by ELISA based mRNA assay (Quantikine). A lipophillic derivative of the SRD (a palmitoyl residue at the amino terminus of the SRD) was synthesized (palmitoyl specific radioprotective domain, P-SRD) to find out if this structural derivatization would restore the NO-inducing ability of IL-1. Surprisingly, P-SRD not only did not induce NO, but significantly inhibited lipopolysaccharide (LPS) stimulated nitric oxide (NO) production. Quantikine studies indicated that P-SRD also inhibited iNOS in LPS stimulated macrophage cells, suggesting that decrease in NO production in the presence of P-SRD was the result of iNOS mRNA inhibition. These results indicate that N-palmitoylation of SRD may effectively ameliorate potentially fatal symptoms of LPS-induced endotoxemic hypotensive shock associated with IL-1 without inflammatory and pyrogenic toxic side effects.     

Pathophysiological mechanism of lung injury in patients with leptospirosis

AIMS: Acute lung injury (ALI) is a serious clinical problem. We investigated the pathogenetic mechanisms of ALI caused by leptospirosis. METHODS: The study included five cases of leptospirosis. We monitored the arterial pressure (AP) and heart rate (HR) and analysed the AP and HR variabilities for assessment of autonomic functions. Histopathological changes in the lung, brain, kidney, and liver were examined. In addition, we identified the expression of inducible nitric oxide synthase (iNOS) using immunohistochemical stain. RESULTS: Five patients associated with leptospirosis died of ALI. Before death, severe hypotension and bradycardia occurred. Spectral analysis of AP and HR variabilities indicated decreased sympathetic drive with increased parasympathetic activity. Pathological examinations revealed alveolar haemorrhage and necrotic lesions in various organs. Immunohistochemical stain disclosed iNOS activity in multiple organs. Biochemical determinations indicated hypoxia, hyperglycaemia, increased nitrite/nitrate, methyl guanidine and other factors. CONCLUSIONS: These changes suggest that leptospirosis causes severe hypotension and bradycardia accompanied by autonomic dysfunction. Finally, multiple organ failure and damage ensued. The pathogenesis of lung and organ injury may involve iNOS and NO production.     

Mycophenolate Mofetil Has Potent Anti-inflammatory Actions in a Mouse Model of Acute Lung Injury

Septic shock is a systemic inflammatory response syndrome, and it is the leading cause of death in intensive care units. Mycophenolate mofetil (MMF) is an immunosuppressant that has been shown to be effective in the treatment of various inflammatory diseases. In this study, the anti-inflammatory effect of MMF in a mouse model of acute lung injury (ALI) induced by lipopolysaccharide (LPS) was evaluated. ALI was induced by intrapleural injection of LPS (250 ng/cavity). The leukocyte migration, exudation, myeloperoxidase and adenosine deaminase activities, nitric oxide products, tumor necrosis factor alpha (TNF-α), and interleukin 1 beta (IL-1β) levels, as well as mRNA expression of TNF-α and IL-1β, were evaluated. This study showed that MMF significantly decreased all parameters studied in a manner comparable to treatment with dexamethasone. In conclusion, MMF has important anti-inflammatory effects that may be useful as an auxiliary treatment for septic shock.     

Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.     

N‐Palmitoylation of the Radioprotective Domain of Interleukin‐1 Affords Inhibition of LPS‐Induced Nitric Oxide Generation

Interleukin‐1β (IL‐1β), a cytokine involved in homeostatic processes such as the immune system and inflammatory reactions, is a potent inducer of nitric oxide. The nonapeptide of human IL‐1β (VQGEESNDK, position 163–171, specific radioprotective domain–SRD) has been shown to retain radioprotective, immunostimulatory, and adjuvant activities of the native molecule without any inflammatory and pyrogenic properties. Unlike the parent IL‐1, SRD did not induce nitric oxide (NO) in control or irradiated RAW 264.7 cells nor did it affect inducible nitric oxide synthase (iNOS) as shown by ELISA based mRNA assay (Quantikine). A lipophillic derivative of the SRD (a palmitoyl residue at the amino terminus of the SRD) was synthesized (palmitoyl specific radioprotective domain, P‐SRD) to find out if this structural derivatization would restore the NO‐inducing ability of IL‐1. Surprisingly, P‐SRD not only did not induce NO, but significantly inhibited lipopolysaccharide (LPS) stimulated nitric oxide (NO) production. Quantikine studies indicated that P‐SRD also inhibited iNOS in LPS stimulated macrophage cells, suggesting that decrease in NO production in the presence of P‐SRD was the result of iNOS mRNA inhibition. These results indicate that N‐palmitoylation of SRD may effectively ameliorate potentially fatal symptoms of LPS‐induced endotoxemic hypotensive shock associated with IL‐1 without inflammatory and pyrogenic toxic side effects.     

Alpinia katsumadai HAYATA Seed Extract Inhibit LPS-Induced Inflammation by Induction of Heme Oxygenase-1 in RAW264.7 Cells

In the present study, we investigated the effects of Alpinia katsumadai H_AYATA (Zingiberaceae) seed ethanolic extract (AKEE) and its three components on the production of inflammatory mediators and some potential underlying mechanisms in lipopolysaccharide (LPS)-induced inflammation RAW264.7 cells. The whole formula, AKEE, and three major component compounds were then evaluated for their effects on inflammation-related parameters using LPS-induced RAW264.7 cells. Production of namely nitric oxide (NO) and cytokine levels were measured by the Griess reagent and ELISA, respectively. To investigate the underlying mechanisms of anti-inflammatory activities of AKEE, protein expression of nitric oxide synthase (inducible nitric oxide synthase, iNOS), heme oxygenase-1 (HO-1), and nuclear factor-kappa B (NF-κB) were evaluated by western blot analysis. AKEE and the major group of compounds in AKEE (alpinetin, cardamonin, and pinocembrin) complement exert anti-inflammatory effects for NO and PGE₂ production. In addition, AKEE treatment significantly inhibited the LPS-induced production of interleukin-6 and tumor necrosis factor (TNF)-α, as well as the expression of iNOS. AKEE also induced HO-1 expression in RAW264.7 cells and inhibited the nuclear translocation of NF-κB by preventing degradation of the inhibitor kappa B-alpha. We also demonstrated that the effects of AKEE on TNF-α production were partially reversed by the HO-1 inhibitor tin protoporphyrin. These results indicate that AKEE and its major component may have anti-inflammatory activity via induction of HO-1 expression was partly responsible for the anti-inflammatory effects.     

NPS 2143, a selective calcium-sensing receptor antagonist inhibits lipopolysaccharide-induced pulmonary inflammation

NPS 2143, a novel and selective antagonist of calcium-sensing receptor (CaSR) has been reported to possess anti-inflammatory activity. In the present study, we examined the protective effect of NPS 2143 on lipopolysaccharide (LPS)-induced acute lung injury (ALI). NPS 2143 pretreatment significantly inhibited the influx of inflammatory cells and the expression of monocyte chemoattractant protein-1 (MCP-1) in the lung of mice with LPS-induced ALI. NPS 2143 decreased the levels of neutrophil elastase (NE) and protein concentration in the bronchoalveolar lavage fluid (BALF). NPS 2143 also reduced the production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the BALF and serum. In addition, NPS 2143 attenuated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), and increased the activation of AMP-activated protein kinase (AMPK) in the lung. NPS 2143 also downregulated the activation of nuclear factor-kappa B (NF-κB) in the lung. In LPS-stimulated H292 airway epithelial cells, NPS 2143 attenuated the releases of IL-6 and MCP-1. Furthermore, NPS 2143 upregulated the activation of AMPK and downregulated the activation of NF-κB. These results suggest that NPS 2143 could be potential agent for the treatment of inflammatory diseases including ALI.