呼吸道合胞病毒诱导一氧化氮产生的机制

目的探讨呼吸道合胞病毒(RSV)感染时一氧化氮(NO)的水平变化及其产生的调控机制,为临床治疗RSV疾病提供有益的思路。方法以RSV感染人肺上皮细胞A549细胞,并设立不同的感染时间(4h、8h、16h和24h),同时分别给予PDTC(核转录因子NF-κB的特异性抑制剂)或AG(诱导型一氧化氮合酶i NOS的特异性抑制剂)处理。收集各组细胞和细胞培养上清,用Western blot法检测细胞核内活性NF-κBp65蛋白的表达,半定量RT-PCR和免疫细胞化学法检测i NOS mR-NA和蛋白表达,硝酸还原酶法检测细胞培养上清中NO含量。结果RSV感染4h后,核内活性NF-κBp65蛋白、i NOS mR-NA和蛋白、细胞培养上清中NO含量均升高,各指标的变化与正常对照相比,差异均有显著性,并且与RSV感染存在时间依赖关系。加入PDTC后可明显抑制NF-κB活化,同时下调i NOS mRNA和蛋白表达。加入AG后,则明显降低细胞培养上清中NO含量。结论RSV感染可诱导产生大量的NO,其主要受i NOS的调节。NF-κB活化对i NOS基因表达具有重要的正调控作用。提示RSV诱导NO生成可能通过活化NF-κB所致。     

Expression of inducible nitric oxide synthase in cultured smooth muscle cells from rat mesenteric lymphatic vessels

OBJECTIVE: The objective was to devise a method for establishing cultures of rat mesenteric lymphatic vessel smooth muscle cells (LSMC) and to investigate if inducible nitric oxide synthase (iNOS) expression could be activated in LSMC treated with bacterial lipopolysaccharide (LPS). METHODS: LSMC were successfully grown from explanted rat lymphatic microvessels and maintained by subculture. Treatment of LSMC for 24 h with LPS (1-100 microg/mL) activated iNOS protein induction, associated with (1) assay of increased nitrite concentrations in the medium representing cellular nitric oxide synthesis, and (2) demonstration of iNOS in cell extracts by Western blotting. RESULTS: The protein synthesis inhibitor cycloheximide (10 microM) blocked both LPS-induced nitrite formation and iNOS protein expression in LSMC. 1400 W (1 microM), a selective iNOS inhibitor, prevented LPS-induced nitrite formation but not iNOS expression. As well as induction of iNOS by LPS, "constitutive" iNOS was present in some cultures, producing nitrite in amounts that were also subsequently reduced after cell treatment with 1400 W. CONCLUSION: Rat mesenteric LSMC produce nitrite and express iNOS in response to bacterial LPS. Cultured LSMC may provide a useful model for studying mechanisms of iNOS induction in relation to possible influences of iNOS upon lymphatic vessel function.     

Nitric oxide production by hepatocytes contributes to the inhibitory effect of endotoxin on insulin-like growth factor I gene expression

We tested whether endotoxin (lipopolysaccharide, LPS) inhibits IGF-I gene expression in hepatocytes and the possible role of Kupffer cells and nitric oxide (NO) in this effect. LPS decreased IGF-I mRNA in hepatocyte cultures and increased the nitrite + nitrate levels in the culture medium. Furthermore, there was a negative correlation between the IGF-I mRNA and the nitrite+nitrate levels. When hepatocytes were cocultured with Kupffer cells, the inhibitory effect of LPS on IGF-I mRNA was higher than in hepatocyte cultures, but the stimulatory effect on nitrite+nitrate was similar in both conditions. The exogenous NO donated by S-nitroso-n-acetyl-d,l-penicillamide also decreased the IGF-I gene expression in hepatocyte cultures. In addition, two specific inducible NO synthase (iNOS) inhibitors, l-N6-(1-iminoethyl)lysine (l-NIL) and aminoguanidine, prevented the effect of LPS on nitrite+nitrate levels and on IGF-I gene expression in hepatocyte cultures. These data indicate that iNOS-derived NO may cause downregulation of IGF-I gene expression in hepatocytes. However, in cocultures, the iNOS inhibitor l-NIL prevented the effect of LPS on nitrite+nitrate levels, but only attenuated the LPS-induced decrease in IGF-I gene expression. We conclude that in hepatocytes, LPS-induced decrease in IGF-I is mainly due to induction of iNOS, whereas in the presence of Kupffer cells LPS inhibits IGF-I through NO release and through other inhibitory pathways.     

iNOS/NO体系在慢性肝炎及肝硬化患者中的表达

目的:探讨内源性NO在慢性病毒性肝炎-肝硬化发展进程中的作用.方法:采用硝酸还原酶法比色测定外周静脉及门静脉血浆中iNOS活性,并用免疫组织化学和RT-PCR方法观察肝组织iNOS蛋白及RNA的表达.结果:在慢性肝炎患者及肝硬化患者门静脉血与外周血中iNOS活性与对照组相比均明显升高(F=102.793,25.052,P<0.01),且门静脉血iNOS活性增高更为明显.慢性肝炎组及肝硬化组中iNOS蛋白的灰阶值均低于正常对照组(F=46.796,P<0.05),表明iNOS表达增强.慢性肝炎组、肝硬化组iNOS mRNA的表达均分别显著高于正常对照组(F=26.832,P<0.01),且随着肝脏病变的加重表达逐渐增加.结论:iNOS/NO体系在慢性肝炎-肝硬化发生发展中起着保持血管舒张状态的重要作用.     

Diabetic ketosis activates lymphomonocyte-inducible nitric oxide synthase.

AIMS: Inappropriate production of nitric oxide (NO) may be responsible for the haemodynamic disturbances of diabetic ketoacidosis. We investigated whether this metabolic condition is associated with increased plasma nitrate (the stable oxidation product of NO) levels and NO synthase gene expression in lymphomonocytes. RESEARCH DESIGN AND METHODS: Plasma nitrate concentrations, lymphomonocyte-inducible nitric oxide synthase (iNOS) gene expression, tumour necrosis factor-alpha (TNF-alpha) and soluble thrombomodulin were measured in 11 Type 1 diabetic patients at baseline, during mild ketosis and after euglycaemia was re-established. RESULTS: During diabetic ketosis plasma nitrate concentrations were higher (18 (16-21) vs. 9 (7-11) micro mol/l; (95% lower-upper confidence interval) P < 0.05) than at baseline. At baseline lymphomonocyte iNOS mRNA expression and iNOS protein levels were undetectable, but in ketosis both were increased (both at P < 0.0001). After recovery from ketosis, NO3 concentration, iNOS mRNA, and iNOS expression (270 +/- 36%, mean +/- sd) decreased but not significantly. No significant changes were observed in either TNF-alpha or soluble thrombomodulin levels between the three conditions. CONCLUSIONS: Diabetic ketosis is associated with increased nitrate levels and the activation of iNOS expression in circulating lymphomonocytes, but it does not affect either the proinflammatory cytokine TNF-alpha or a marker of endothelial dysfunction such as thrombomodulin. Our data support the hypothesis that, during diabetic ketosis, alterations in NO homeostasis are present in circulating lymphomonocytes.     

大鼠海马脑片缺血-再灌注模型中一氧化氮及一氧化氮合酶的变化

目的 建立器官型海马脑片缺血-再灌注模型,以阐明一氧化氮和一氧化氮合酶(NOS)在脑缺血-再灌注中的作用。 方法 以12只出生7-8 d的Sprague-Dawley乳鼠制备海马脑片,根据培养时间随机分为4组,每组2个微孔膜,每个Millcell-CM微孔膜放置6张脑片,在培养液和气体界面上培养脑片,4组中每组36张脑片。培养14 d后,缺氧、缺糖30 min,再灌注12、24和36 h,用逆转录聚合酶链反应(RT-PCR)和Western印迹技术,分别检测脑片组织中一氧化氮、NOS的mRNA和蛋白质水平的变化;用改良镉还原法检测培养液中一氧化氮代谢产物亚硝酸盐含量;生化法榆测培养液中乳酸脱氢酶(LDH)水平。 结果 缺血-再灌注模型的脑片组织,诱导性的一氧化氮合酶(iNOS)和神经源性的一氧化氮合酶(nNOS)在mRNA和蛋白质水平均表达上调,培养液中一氧化氮代谢产物亚硝酸盐含量明显高于对照组,差异有显著意义(P<0.05)。同时,培养液中LDH的水平随再灌注时间的延长而升高。结论 一氧化氮及NOS在脑缺血-再灌注损伤中起着重要作用。     

Nitric oxide expression in airway epithelial cells in response to tubercle bacilli stimulation

Abstract In order to investigate the role of airway epithelial cells in pulmonary tuberculosis, inducible nitric oxide synthetase (iNOS) expression and nitric oxide (NO) production were studied in A549 cells. Peripheral blood mononuclear cells (PBMC) from normal volunteers were separated and cultured for 24h with LPS or tubercle bacilli (H37Rv, H37Ra). Thereafter, A549 cells were stimulated for another 24h with culture supernatant fluids of PBMC. iNOS messenger RNA (mRNA) expression was measured with Northern blot analysis and NO production was measured with the Griess reaction, which can measure nitrite concentration. iNOS mRNA expression and NO production were minimal in the control cells. iNOS mRNA expression and NO production were significantly increased with LPS ( P < 0.05) or tubercle bacilli ( P < 0.01) stimulation. However, there was no difference in iNOS mRNA expression and NO production between H37Rv and H37Ra stimulations. Interestingly, iNOS mRNA expression and NO production were greater in A549 cells stimulated with tubercle bacilli-conditioned media than in the cells stimulated with LPS-conditioned media. IL-1β, tumour necrosis factor-alpha and interferon gamma concentrations were increased in culture supernatant fluids of PBMC stimulated with tubercle bacilli. These findings suggest that airway epithelial cells may play a certain role in the pathogenesis of pulmonary tuberculosis by producing NO. However, the role of airway epithelial cells, regarding the virulence of tubercle bacilli, was not clear in this study.     

Nitric Oxide Inhibitors Ameliorate Indomethacin-Induced Enteropathy in Rats

The role of nitric oxide (NO) synthase inhibitors in indomethacin (INDO) -induced enteropathy was investigated in male Sprague-Dawley rats. Rats were subcutaneously administered 5% sodium bicarbonate (controls), two doses of INDO 7.5 mg/kg, and three different inducible NO synthase (iNOS) inhibitors at various concentrations 24 hr, apart; aminoguanidine (AG), guanidinoethyldisulfide (GED), and n-(3-aminomethyl)benzylacetamidine (1400W). Rats were killed four days after the initial injection and small intestinal mucosa was assayed for myeloperoxidase (MPO) activity and iNOS expression by western blot analysis. Serum nitrite/nitrate (NO_x) concentration was measured colorimetrically. INDO produced acute ulcers along the mesenteric border from the ileum to proximal jejunum. Rats treated with AG (25 and 50 mg/kg), GED (2.5 mg/kg), and 1400W (0.1 mg/kg) showed decreased total ulcer length and MPO activity by 51, 72, 53, and 61% and by 58, 88, 68, and 70%, respectively, compared to INDO alone. All inhibitors similarly reduced INDO-enhanced serum NO_x concentrations to its basal levels. Significant iNOS expression was detected in INDO-treated rats, but the inhibitors did not alter iNOS expression. Our data suggest that NO derived from iNOS may be a key factor in the pathogenesis of acute INDO-induced enteropathy in rats.     

Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats.

BACKGROUND: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. This study investigated the activity of nitric oxide synthase (NOS) in cirrhosis induced by bile duct-ligation (BDL) with NOS inhibitors. METHOD: Three days after operation, rats were randomized to receive aminoguanidine (AG, 25 mg/kg/day) or L-N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) for 21 days. RESULTS: Vascular NO production, which was increased in BDL cirrhotic rats, was reduced by 75% with AG but not L-NAME chronic administration. AG treatment attenuated liver damage, while L-NAME aggravated it. AG significantly suppressed inducible NOS (iNOS) expression in aorta of BDL rats at both mRNA and protein level, but much less efficient in reducing it in liver. In contrast, endothelial NOS (eNOS) expression was not markedly affected. Calcium-independent NOS activity, which was dramatically increased in aorta of BDL rats, was abolished by AG treatment. In liver, however, both calcium-dependent and -independent NOS activity were increased by AG treatment. CONCLUSION: Chronic administration of AG could reduce systemic NO levels as well as suppress iNOS expression and activity in aorta of BDL rats. It also improved liver function, possibly because of its ability to increase hepatic NOS activity, and to correct the systemic hemodynamic disorders by decreasing vascular NO production.     

脓毒症时各型一氧化氮合酶在大鼠心脏中的表达及其可能机制

目的研究脓毒症时各型一氧化氮合酶(NOS)在心脏中的损伤作用及其机制。方法成年雄性Wistar大鼠腹腔注射脂多糖(LPS)制备脓毒症模型。应用多导生理仪监测大鼠心功能变化;用分光光度计法测定大鼠心肌组织NOS的活性;用RT-PCR和Western blot对大鼠心肌组织各型NOS的表达进行半定量分析。结果给予LPS后6h大鼠心肌收缩和舒张功能受损下降,心肌中iNOS的活性明显升高,eNOS和nNOS(合称cNOS)活性减弱;RT-PCR和Western blot结果显示,给予LPS后cNOS的表达减少,给予LPS后iNOS表达量明显增加。结论脓毒症时,iNOS、nNOS和eNOS的表达和活性发生改变;心肌细胞上iNOS表达及活性升高,这些变化可能在心功能降低中发挥作用。     

Role of neuronal nitric oxide synthase and inducible nitric oxide synthase in intestinal injury in neonatal rats

AIM: To investigate the dynamic change and role of neuronal nitric oxide synthase (nNOS) and inducible nitric oxide synthase (iNOS) in neonatal rat with intestinal injury and to define whether necrotizing enterocolitis (NEC) is associated with the levels of nitric oxide synthase (NOS) in the mucosa of the affected intestine tissue. METHODS: Wistar rats less than 24 h in age received an intraperitoneal injection with 5 mg/kg lipopolysaccharide (IPS). Ileum tissues were collected at 1, 3, 6, 12 and 24 h following LPS challenge for histological evaluation of NEC and for measurements of nNOS and iNOS. The correlation between the degree of intestinal injury and levels of NOS was determined. RESULTS: The LPS-injected pups showed a significant increase in injury scores versus the control. The expression of nNOS protein and mRNA was diminished after LPS injection. There was a negative significant correlation between the nNOS protein and the grade of median intestinal injury within 24 h. The expression of iNOS protein and mRNA was significantly increased in the peak of intestinal injury. CONCLUSION: nNOS and iNOS play different roles in LPS-induced intestinal injury. Caution should be exerted concerning potential therapeutic uses of NOS inhibitors in NEC.     

肿瘤坏死因子α诱导脑血管内皮细胞产生病理性一氧化氮的实验研究

目的探讨肿瘤坏死因子α（TNF—α）作用于脑血管内皮细胞产生病理性一氧化氮（NO）的机制。方法体外培养肿瘤坏死因子受体（TNFR1）基因敲除的小鼠脑血管内皮细胞（BVEC／RI）和野生型小鼠脑血管内皮细胞（BVEC）,分别给予5ng／ml TNF-α刺激24h后,应用PCR技术、Western blot方法、硝酸还原酶法,测定两种细胞的诱导型一氧化氮合酶（iNOS）基因mRNA和蛋白表达量以及所分泌的一氧化氮（NO）含量。结果①给予TNF—α刺激后,野生型BVEC的iNOS mRNA表达增加,BVEC／RI的iNOS mRNA表达未出现明显变化。②给予TNF—α刺激后,野生型BVEC的iNOS蛋白表达量（0．91±0．08）高于未给予TNF-α的BVEC（0．15±0．02）,差异有统计学意义,P〈0．05;BVEC／RI的iNOS蛋白表达量（0．21±0．06）与未给予TNF—α的BVEC／RI（0．30±0．05）相比,差异无统计学意义,P〉0．05。③给予TNF-α刺激后,野生型BVEC的NO含量[（58．6±2．6）μxmol／L]高于未给予TNF—α的BVEC[（18．1±4．3）μmol／L],差异有统计学意义,P〈0．05;BVEC／RI的NO含量[（21．2±3．5）μmol／L]与未给予TNF-α的BVEC／RI[（16．9±3．4）μmol／L]相比,差异无统计学意义,P〉0．05。结论TNF—α可能通过作用于脑血管内皮细胞TNFR1增加iNOS表达,从而增加病理性NO产生。     

Differential inhibitory actions by glucocorticoid and aspirin on cytokine-induced nitric oxide production in vascular smooth muscle cells

Glucocorticoids and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory and immune diseases. Nitric oxide (NO) has a diversity of physiological functions, but its excess production has been implicated in the inflammatory process. The present study was designed to elucidate the mechanisms by which glucocorticoids and NSAIDs affect inducible nitric oxide synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMCs). Both interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha potently stimulated nitrite/nitrate (NOx) production with a concomitant expression of iNOS mRNA and protein as demonstrated by Northern and Western blot analysis, respectively. Both IL-1beta and TNF-alpha activated nuclear factor (NF)-kappaB as demonstrated by electrophoretic mobility shift assay. Dexamethasone, salicylate and aspirin, but not indomethacin, dose dependently inhibited cytokine-stimulated NOx production and iNOS protein expression. Dexamethasone decreased cytokine-induced NF-kappaB activation and iNOS mRNA expression, but neither salicylate nor aspirin affected NF-kappaB activation or iNOS mRNA expression. IL-1beta caused a rapid increase in phosphorylated IkappaB-alpha levels and subsequent transient decrease in IkappaB-alpha levels, an inhibitor of NF-kappaB, as revealed by Western blot analysis using specific antibodies for phosphorylated and nonphosphorylated IkappaB-alpha. These effects were blocked by pretreatment with dexamethasone. Aspirin dose dependently inhibited iNOS enzymatic activity, whereas salicylate and dexamethasone had limited effect. The present study demonstrates that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS expression and NO production in rat VSMCs, although potentially acting at multiple levels, is partly mediated by inhibition of NF-kappaB activation resulting from decreased phosphorylation and degradation of IkappaB-alpha, 2) both salicylate and aspirin inhibit cytokine-stimulated NO production at translational and/or posttranslational levels without affecting NF-kappaB- mediated iNOS gene expression, and 3) aspirin directly inhibits iNOS enzyme activity. These data suggest the differential inhibitory mechanisms of iNOS-mediated NO synthesis by glucocorticoids and NSAIDs in the vasculature.     

KE-298 and its active metabolite KE-758 suppress nitric oxide production by murine macrophage cells and peritoneal cells from rats with adjuvant induced arthritis.

OBJECTIVE: To analyze the effects of KE-298 and KE-758 on lipopolysaccharide (LPS) induced nitric oxide (NO) production by the RAW264.7 murine macrophage cell line, and the effect of KE-758 on spontaneous NO production by peritoneal cells from rats with adjuvant induced arthritis. METHODS: The amount of NO was determined using Griess reagents. The proteins for inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) were detected by Western blot, then mRNA for interferon-beta (IFN)-beta, IFN regulatory factor-1 (IRF-1), and iNOS were detected by RT-PCR. Degradation of iNOS mRNA was analyzed using Northern blot. Nuclear factor-kappa B (NF-kappa B) in nuclear extracts was determined by EMSA. Adjuvant arthritis in rats was induced by inoculating heat killed Mycobacterium butyricum s.c. in the tail. RESULTS: KE-298 and KE-758 suppressed NO production by LPS activated RAW264.7 cells by inhibiting iNOS gene expression. Neither LPS induced NF-kappa B activation nor degradation of iNOS mRNA was affected by KE-758 treatment. LPS induced IFN-beta and IRF-1 gene expression were markedly suppressed by KE-758. In rats with adjuvant induced arthritis, enhanced NO and iNOS production by cultured peritoneal cells and the development of arthritis were suppressed by KE-758. CONCLUSION: KE-758 suppressed LPS induced iNOS gene expression by murine macrophage cells by inhibiting IFN-beta/IRF-1 expression. The potential of KE-758 to inhibit iNOS production might partly explain its efficacy on adjuvant induced arthritis in rats.     

阿霉素对大鼠心肌诱导型一氧化氮合酶mRNA表达的影响

目的研究阿霉素(ADM)对大鼠心肌诱导型一氧化氮合酶(iNOS)mRNA表达的影响。方法将雄性wistar大鼠24只,随机分为ADM组和对照组,每组12只,ADM组按每次给ADM 2 mg/kg腹腔注射,隔日一次共6次;对照组给等体积的生理盐水腹腔注射。于实验第30 天,应用生化方法测定心肌组织中的一氧化氮(NO)水平及iNOS的活性;用TUNEL法检测心肌细胞的凋亡指数;用RT—PCR方法检测心肌组织iNOS mRNA的表达。结果与对照组比较ADM组大鼠心肌组织NO、iNOS活性增加(P< 0.01),心肌细胞凋亡指数及iNOS mRNA的表达均显著增高(P< 0.01)。结论ADM能诱导大鼠心肌细胞iNOS mRNA表达增加,使NO合成增多,引起细胞凋亡而参与对心肌的损害。