一氧化氮与肺纤维化的研究进展

肺纤维化是一组由多种因素引起的肺间质性病变,肺纤维化的发病机制迄今尚未完全清楚。近年来,发现在哺乳动物细胞的一氧化氮合酶催化合成的一氧化氮在肺纤维化的发生发展中发挥着重要的作用。因此,阐述一氧化氮与肺纤维化的关系,有着重要的理论意义和潜在的临床应用价值。     

Curcumin attenuates ovalbumin-induced airway inflammation by regulating nitric oxide

Curcumin has been strongly implicated as an anti-inflammatory agent, but the precise mechanisms of its action are largely unknown. In this study, we show that curcumin contributes to anti-inflammatory activity in the murine asthma model and lung epithelial cell A549 through suppression of nitric oxide (NO). To address this problem, curcumin was injected into the peritoneum of ovalbumin (OVA)-sensitized mice before the last allergen challenge. OVA challenge resulted in activation of the production of inducible nitric oxide (iNOS) in lung tissue, inflammatory cytokines, recruitment of eosinophils to lung airways, and airway hyper-responsiveness to inhaled methacholine. These effects of ovalbumin challenge were all inhibited by pretreatment of mice with curcumin. Furthermore, supplementation with curcumin in the A549 human airway epithelial cells decreased iNOS and NO production induced by IFN-γ. These findings show that curcumin may be useful as an adjuvant therapy for airway inflammation through suppression of iNOS and NO.     

Inhaled nitric oxide induced NOS inhibition and rebound pulmonary hypertension: a role for superoxide and peroxynitrite in the intact lamb

Previous in vivo studies indicate that inhaled nitric oxide (NO) decreases nitric oxide synthase (NOS) activity and that this decrease is associated with significant increases in pulmonary vascular resistance (PVR) upon the acute withdrawal of inhaled NO (rebound pulmonary hypertension). In vitro studies suggest that superoxide and peroxynitrite production during inhaled NO therapy may mediate these effects, but in vivo data are lacking. The objective of this study was to determine the role of superoxide in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy in vivo. In control lambs, 24 h of inhaled NO (40 ppm) decreased NOS activity by 40% (P<0.05) and increased endothelin-1 levels by 64% (P<0.05). Withdrawal of NO resulted in an acute increase in PVR (60.7%, P<0.05). Associated with these changes, superoxide and peroxynitrite levels increased more than twofold (P<0.05) following 24 h of inhaled NO therapy. However, in lambs treated with polyethylene glycol-conjugated superoxide dismutase (PEG-SOD) during inhaled NO therapy, there was no change in NOS activity, no increase in superoxide or peroxynitrite levels, and no increase in PVR upon the withdrawal of inhaled NO. In addition, endothelial NOS nitration was 18-fold higher (P<0.05) in control lambs than in PEG-SOD-treated lambs following 24 h of inhaled NO. These data suggest that superoxide and peroxynitrite participate in the decrease in NOS activity and rebound pulmonary hypertension associated with inhaled NO therapy. Reactive oxygen species scavenging may be a useful therapeutic strategy to ameliorate alterations in endogenous NO signaling during inhaled NO therapy.     

Ultrastructural localization of nitric oxide synthase and endothelin in coronary and pulmonary arteries of newborn rats

This is the first report on the ultrastructural distribution of nitric oxide synthase and endothelin immunoreactivities in the coronary and pulmonary arteries of newborn Wistar rats. The distribution of nitric oxide synthase and endothelin was investigated using pre-embedding peroxidase-antiperoxidase immunocytochemistry. In both arteries examined, positive labelling for nitric oxide synthase was localized both in the endothelium and smooth muscle, whereas positive labelling for endothelin was localized in the endothelium exclusively. In the coronary artery, approximately 80% and 55% of the endothelial cells examined were positive for nitric oxide synthase and endothelin, respectively, whereas in the pulmonary artery, 77% and 60% of the endothelial cells were positive for nitric oxide synthase and endothelin, respectively. These findings indicate that nitric oxide synthase and endothelin are colocalized in some of the endothelial cells of the newborn rat. In the endothelium, nitric oxide synthase and endothelin immunoreactivities were distributed throughout the cell cytoplasm and in association with the membranes of intracellular organelles. In smooth muscle, a relationship of nitric oxide synthase immunoreactivity to endoplasmic reticulum was observed in the pulmonary artery. In summary, in the newborn rat, endothelial cells of the coronary and pulmonary artery are rich in nitric oxide synthase (neuronal isoform) and endothelin, and it is suggested therefore that they may be substantially involved in vasomotor control of the cardiac and pulmonary circulation during early stages of postnatal development.     

Nitric oxide/cGMP protects endothelial cells from hypoxia-mediated leakiness

Leakiness of the endothelial bed is attributed to the over-perfusion of the pulmonary bed, which leads to high altitude pulmonary edema (HAPE). Inhalation of nitric oxide has been successfully employed to treat HAPE patients. We hypothesize that nitric oxide intervenes in the permeability of the pulmonary macrovascular endothelial bed to rectify the leaky bed under hypoxia. Our present work explores the underlying mechanism of 'hypoxia-mediated' endothelial malfunction by using human umbilical cord-derived immortalized endothelial cells, ECV-304, and bovine pulmonary artery primary endothelial cells. The leakiness of the endothelial monolayer was increased by two-fold under hypoxia in comparison to cells under normoxia, while optical tweezers-based tethering assays reported a higher membrane tension of endothelial cells under hypoxia. Phalloidin staining demonstrated depolymerization of F-actin stress fibers and highly polarized F-actin patterns in endothelial cells under hypoxia. Nitric oxide, 8-Br-cGMP and sildenafil citrate (phosphodiesterase type 5 inhibitor) led to recovery from hypoxia-induced leakiness of the endothelial monolayers. Results of the present study also suggest that 'hypoxia-induced' cytoskeletal rearrangements and membrane leakiness are associated with the low nitric oxide availability under hypoxia. We conclude that nitric oxide-based recovery of hypoxia-induced leakiness of endothelial cells is a cyclic guanosine monophosphate (cGMP)-dependent phenomenon.     

Developmental changes in the response of murine cerebellar granule cells to nitric oxide

Nitric oxide is a diffusible messenger that plays a multitude of roles within the nervous system including modulation of cell viability. However, its role in regulating neuronal survival during a defined period of neurodevelopment has never been investigated. We discovered that expression of the messenger RNA for both neuronal and endothelial nitric oxide synthase increased in the early postnatal period in the cerebellum in vivo, whilst the expression of inducible nitric oxide synthase remained constant throughout this time in development. Whilst scavenging of nitric oxide was deleterious to the survival of early postnatal cerebellar granule neurons in vitro, this effect was lost in cultures derived at increasing postnatal ages. Conversely, sensitivity to exogenous nitric oxide increased with advancing postnatal age. Thus, we have shown that as postnatal development proceeds, cerebellar granule cells alter their in vitro survival responses to both nitric oxide inhibition and donation, revealing that the nitric oxide's effects on developing neurons vary with the stage of development studied. These findings have important consequences for our understanding of the role of nitric oxide during neuronal development.     

Detection of nitrous oxide in the neuronal nitric oxide synthase reaction by gas chromatography-mass spectrometry

Using headspace gas chromatography-mass spectrometry, we detected significant amounts of nitrous oxide in the reaction products of the monooxygenase reaction catalyzed by neuronal nitric oxide synthase. Nitrous oxide is a dimerization product of nitroxyl anion; its presence in the reaction products indicates that the nitroxyl anion is a product of the neuronal nitric oxide synthase-catalyzed reaction.     

神经型一氧化氮合酶的活性调控

一氧化氮是重要的信使分子,在生物体内参与众多生理及病理过程。生物体内存在着复杂的一氧化氮合酶活性调控机制以精确调控一氧化氮的生成。在神经系统中,一氧化氮主要由神经型一氧化氮合酶催化生成。神经型一氧化氮合酶的活性主要受到翻译后水平上钙离子和钙调蛋白的调控,其调控方式包括二聚化、多位点的磷酸化和去磷酸化,以及主要由PDZ结构域介导的蛋白质-蛋白质相互作用。一氧化氮本身对其合酶的活性具有负反馈调控作用。近年来的研究提示,细胞质膜上的脂筏微区在神经性一氧化氮合酶的活性调控中也起到重要的调节作用。     

Chronic hypoxia attenuates nitric oxide-dependent hemodynamic responses to acute hypoxia

Alterations in the nitric oxide (NO) pathway have been implicated in the pathogenesis of chronic hypoxia-induced pulmonary hypertension. Chronic hypoxia can either suppress the NO pathway, causing pulmonary hypertension, or increase NO release in order to counteract elevated pulmonary arterial pressure. We determined the effect of NO synthase inhibitor on hemodynamic responses to acute hypoxia (10% O(2)) in anesthetized rats following chronic exposure to hypobaric hypoxia (0.5 atm, air). In rats raised under normoxic conditions, acute hypoxia caused profound systemic hypotension and slight pulmonary hypertension without altering cardiac output. The total systemic vascular resistance (SVR) decreased by 41 +/- 5%, whereas the pulmonary vascular resistance (PVR) increased by 25 +/- 6% during acute hypoxia. Pretreatment with N(omega)-nitro-L-arginine methyl ester (L-NAME; 25 mg/kg) attenuated systemic vasodilatation and enhanced pulmonary vasoconstriction. In rats with prior exposure to chronic hypobaric hypoxia, the baseline values of mean pulmonary and systemic arterial pressure were significantly higher than those in the normoxic group. Chronic hypoxia caused right ventricular hypertrophy, as evidenced by a greater weight ratio of the right ventricle to the left ventricle and the interventricular septum compared to the normoxic group (46 +/- 4 vs. 28 +/- 3%). In rats which were previously exposed to chronic hypoxia (half room air for 15 days), acute hypoxia reduced SVR by 14 +/- 6% and increased PVR by 17 +/- 4%. Pretreatment with L-NAME further inhibited the systemic vasodilatation effect of acute hypoxia, but did not enhance pulmonary vasoconstriction. Our results suggest that the release of NO counteracts pulmonary vasoconstriction but lowers systemic vasodilatation on exposure to acute hypoxia, and these responses are attenuated following adaptation to chronic hypoxia.     

Combined inhaled nitric oxide and inhaled prostacyclin during experimental chronic pulmonary hypertension.

Inhaled nitric oxide (NO) and inhaled prostacyclin (PGI2) produce selective reductions in pulmonary vascular resistance (PVR) through differing mechanisms. NO decreases PVR via cGMP, and PGI2 produces pulmonary vasodilation via cAMP. As a general pharmacological principle, two drugs that produce similar effects via different mechanisms should have additive or synergistic effects when combined. We designed this study to investigate whether combined inhaled NO and PGI2 therapy results in additive effects during chronic pulmonary hypertension in the rat. Monocrotaline injected 4 wk before study produced pulmonary hypertension in all animals. Inhaled NO (20 parts/million) reversibly and selectively decreased pulmonary artery pressure (Ppa) with a mean reduction of 18%. Four concentrations of PGI2 were administered via inhalation (5, 10, 20, and 80 microg/ml), both alone and combined with inhaled NO. Inhaled PGI2 alone decreased Ppa in a dose-dependent manner with no change in mean systemic arterial pressure. Combined inhaled NO and PGI2 selectively and significantly decreased Ppa more did than either drug alone. The effects were additive at the lower concentrations of PGI2 (5, 10, and 20 microg/ml). The combination of inhaled NO and inhaled PGI2 may be useful in the management of pulmonary hypertension.     

Malignant glioma progression and nitric oxide

Glioblastoma multiforme, the most common of the malignant gliomas, carries a dismal prognosis in spite of the most aggressive therapy and recent advances in molecular pathways of glioma progression. Although it has received relatively little attention in the setting of malignant gliomas, nitric oxide metabolism may be intimately associated with the disease process. Interestingly, nitric oxide has both physiological roles (e.g., neurotransmitter-like activity, stimulation of cyclic GMP), and pathophysiological roles (e.g., neoplastic transformation, tumor neovascularization, induction of apoptosis, free radical damage). Moreover, whether nitric oxide is neuroprotective or neurotoxic in a given disease state, or whether it enhances or diminishes chemotherapeutic efficacy in malignant neoplasia, is unresolved. This review discusses the multifaceted activity of nitric oxide with particular reference to malignant gliomas.     

软体动物的一氧化氮及其合酶的研究进展

一氧化氮作为一种重要的信息分子,参与调节软体动物的嗅觉、运动、取食、机体防御及学习行为。本文从生理、生化、形态定位以及信号转导几方面综述了有关软体动物一氧化氮及其合酶的最新研究进展。     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Could nasal nitric oxide help to mitigate the severity of COVID-19?

The nasal cavity and turbinates play important physiological functions by filtering, warming and humidifying inhaled air. Paranasal sinuses continually produce nitric oxide (NO), a reactive oxygen species that diffuses to the bronchi and lungs to produce bronchodilatory and vasodilatory effects. Studies indicate that NO may also help to reduce respiratory tract infection by inactivating viruses and inhibiting their replication in epithelial cells. In view of the pandemic caused by the novel coronavirus (SARS-CoV-2), clinical trials have been designed to examine the effects of inhaled nitric oxide in COVID-19 subjects. We discuss here additional lifestyle factors such as mouth breathing which may affect the antiviral response against SARS-CoV-2 by bypassing the filtering effect of the nose and by decreasing NO levels in the airways. Simple devices that promote nasal breathing during sleep may help prevent the common cold, suggesting potential benefits against coronavirus infection. In the absence of effective treatments against COVID-19, the alternative strategies proposed here should be considered and studied in more detail.     

Protection of lung tissue against ischemia/reperfusion injury by preconditioning with inhaled nitric oxide in an in situ pig model of normothermic pulmonary ischemia

Topical administration of nitric oxide (NO) by inhalation is currently used as therapy in various pulmonary diseases, but preconditioning with NO to ameliorate lung ischemia/reperfusion (I/R) injury has not been fully evaluated. In this study, we investigated the effects of NO inhalation on functional pulmonary parameters using an in situ porcine model of normothermic pulmonary ischemia. After left lateral thoracotomy, left lung ischemia was maintained for 90 min, followed by a 5h reperfusion period (group I, n = 7). In group II (n = 6), I/R was preceded by inhalation of NO (10 min, 15 ppm). Animals in group III (n = 7) underwent sham surgery without NO inhalation or ischemia. In order to evaluate the effects of NO preconditioning, lung functional and hemodynamic parameters were measured, and the zymosan-stimulated release of reactive oxygen species in arterial blood was determined. Animals in group I developed significant pulmonary I/R injury, including pulmonary hypertension, a decreased pO(2) level in pulmonary venous blood of the ischemic lung, and a significant increase of the stimulated release of reactive oxygen species. All these effects were prevented, or the onset (release of reactive oxygen species) was delayed, by NO inhalation. These results indicate that preconditioning by NO inhalation before lung ischemia is protective against I/R injury in the porcine lung.     

Inhibition of Nitric Oxide Synthase with 7-Nitroindazole does not Modify Early Metabolic Recovery Following Focal Cerebral Ischemia in Rats

Nitric oxide has been strongly implicated in the development of tissue infarction in response to focal cerebral ischemia. Nitric oxide and its derivatives can inhibit components of the electron transport chain, providing a likely target for these substances in ischemic and post-ischemic brain. Lactate content is increased during post-ischemic reperfusion in tissue destined to become infarcted, consistent with impairment of mitochondrial respiration. To investigate the possible involvement of nitric oxide in generating these changes, we have tested the effect of 7-nitroindazole, a nitric oxide synthase (NOS) inhibitor, on the content of lactate and other metabolites during early reperfusion following temporary focal ischemia. This treatment inhibited total NOS by approximately 50%. However, the treatment did not significantly affect the marked increases in lactate in post-ischemic brain nor did it alter the recovery of other energy-related metabolites. These findings indicate that inhibition of oxidative metabolism is probably not the primary site of the deleterious effects of nitric oxide and derivatives during early post-ischemic reperfusion. Special issue dedicated to John P. Blass.     

Nitric oxide produced by iNOS is associated with collagen synthesis in keloid scar formation

Nitric oxide (NO) has emerged as an important mediator of many physiological functions. Recent reports have shown that NO participates in the wound healing process, however, its role in keloid formation remains unclear. This study aimed to investigate the effect of NO on keloid fibroblasts (KF) and to determine the levels of inducible nitric oxide synthase (iNOS) expression in clinical specimens of keloid. Scar tissue from seven keloid patients with matched perilesion skin tissue controls was studied for inducible nitric oxide synthase expression and location. In addition, primary keloid and normal scar skin fibroblast cultures were set up to investigate the effects of NO in inducing collagen type I expression. Inducible nitric oxide synthase expression, and NO production were elevated in keloid scar tissues but not in matched perilesion skin tissues. Furthermore, exposure of KF to exogenous NO resulted in increased expression of collagen type I in a dose-dependent manner. NO exposure also induced time-course dependent collagen I expression that peaked at 24h in KF. Taken together, these results indicate that excess collagen formations in keloid lesion may be attributed to iNOS overexpression.     

Nitric Oxide in Systemic and Pulmonary Hypertension

Endothelium-derived nitric oxide (NO) is an important gas molecule in the regulation of vascular tone and arterial pressure. It has been considered that endothelial dysfunction with impairment of NO production contributes to a hypertensive state. Alternatively, long-term hypertension may affect the endothelial function, depress NO production, and thereby reduce the dilator action on vasculatures. There were many studies to support that endothelium-dependent vasodilatation was impaired in animals and humans with long-term hypertension. However, results of some reports were not always consistent with this consensus. Recent experiments in our laboratory revealed that an NO synthase inhibitor, N^G-nitro-L-arginine monomethyl ester (L-NAME) caused elevation of arterial pressure (AP) in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). The magnitude of AP increase following NO blockade with L-NAME was much higher in SHR than WKY. In other experiments with the use of arterial impedance analysis, we found that L-NAME slightly or little affected the pulsatile hemodynamics including characteristic impedance, wave reflection and ventricular work. Furthermore, these changes were not different between SHR and WKY. The increase in AP and total peripheral resistance (TPR) following NO blockade in SHR were significantly greater than those in WKY, despite higher resting values of AP and TPR in SHR. In connection with the results of other studies, we propose that heterogeneity with respect to the involvement of NO (impairment, no change or enhancement) in the development of hypertension may exist among animal species, hypertensive models and different organ vessels. Our study in SHR provide evidence to indicate that the effects of basal release of NO on the arterial pressure and peripheral resistance are not impaired, but enhanced in the hypertensive state. The increase in NO production may provide a compensatory mechanism to keep the blood pressure and peripheral resistance at lower levels. The phenomenon of enhanced NO release also occurs in certain type of pulmonary hypertension. We first hypothesized that a decrease in NO formation might be responsible for the pulmonary vasoconstriction during hypoxia. With the measurement of NO release in the pulmonary vein, we found that ventilatory hypoxia produced pulmonary hypertension accompanying an increase in NO production. Addition of NO inhibitor (L-NAME), blood or RBC into the perfusate attenuated or abolished the NO release, while potentiating pulmonary vasoconstriction. During hypoxia, the increased NO formation in the pulmonary circulation similarly exerts a compensatory mechanism to offset the degree of pulmonary vasoconstriction.     

Changes of renal function and structure in rats exposed to cold

The kidney appears to play a crucial role in both initiating and maintaining the high blood pressure in cold-induced hypertension (CIH). The aim of the present study was to evaluate the changes of renal function and structure in rats exposed to cold for 2, 4 and 6 weeks. Systolic blood pressure increased significantly after 2 weeks of cold exposure and was maintained throughout the whole experiment. Upregulation of angiotensin type 1 receptor (AT₁R) expression was seen in the vascular zone and distal tubule after 4 and 6 weeks of cold exposure. This was accompanied by an increase in malondialdehyde (MDA) levels and decreases in superoxide dismutase (SOD), nitric oxide synthase (NOS) activities and nitric oxide (NO) content in kidney. Structural changes were also observed in glomeruli, tubules and arteries in cold-treated rats. These results suggest that upregulation of kidney AT₁R plays a critical role in the development of CIH, and its interaction with oxidative stress, NO and NOS may be involved in changes of renal function and structure.     

{Different Signalling Pathways Mediate the Opposite Effects of Endogenous Versus Exogenous Nitric Oxide on Hydroperoxide Toxicity in CHP100 neuroblastoma Cells

The results presented in this study indicate that the toxic response brought about by increasing concentrations of tert-butylhydroperoxide in CHP100 cells was mitigated significantly by exogenously added nitric oxide donors via a cyclic GMP-independent mechanism. In contrast with these results, endogenous nitric oxide generated by the Ca2+-mobilizing agent caffeine was found to increase hydroperoxide toxicity. Under these conditions, nitric oxide was not directly toxic to the cells. Rather, nitric oxide was found to promote the caffeine-mediated release of Ca2+ from ryanodine-sensitive Ca2+ stores via a cyclic GMP-independent mechanism. Release of the cation from ryanodine-sensitive Ca2+ stores was causally linked with the caffeine/nitric oxide-mediated enhancement of tert-butylhydroperoxide toxicity. It is concluded that endogenous and exogenous nitric oxide activate diverging signalling pathways independent of cyclic GMP formation and causing opposite effects on the toxic response evoked by tert-butylhydroperoxide in CHP100 cells.