Fos expression in neurons immunoreactive for neuronal nitric oxide synthase in the rat paraventricular nucleus after intraperitoneal injection of interleukin-1 beta

Double immunostaining for Fos and neuronal nitric oxide synthase (nNOS) was used to examine whether nNOS-immunoreactive neurons in the paraventricular hypothalamic nucleus (PVN) are activated to express Fos immunoreactivity by intraperitoneal injection of interleukin-1 beta (IL-1 beta) in the rat. Quantitative analysis revealed that some nNOS-positive PVN neurons are activated by IL-1 beta (4 microg/kg, i.p.) administration, but the majority of the IL-1 beta-activated PVN neurons do not express nNOS and are distributed mainly in the parvocellular part of the PVN.     

Eupartoium makinoi suppresses lipopolysaccharide-induced inducible nitric oxide synthase and cyclooxygenase-2 expression

Inflammation is involved in numerous diseases including cancer. Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) play important roles in the development of certain inflammatory diseases. Eupatorium makinoi, which belongs to a family of Asteraceae plants, is used medicinally in East Asia. We investigated the effects of an ethanol extract of E. makinoi (EEM) on nuclear factor-kappa B (NF-κB) activation and the expression of iNOS and COX-2 with lipopolysaccharide (TLR4 agonist) in murine macrophages. EEM suppressed NF-κB activation and iNOS and COX-2 expression induced by LPS. These results suggest that EEM may regulate TLR4 signalling pathways and this may be a useful strategy for anti-inflammatory therapies.     

Negative feedback regulation of lipopolysaccharide-induced inducible nitric oxide synthase gene expression by heme oxygenase-1 induction in macrophages

Heme oxygenase-1 (HO-1) is induced under infectious diseases in macrophages. We performed experiments using various gene deficient mouse-derived macrophages to determine a detailed induction mechanism of HO-1 by lipopolysaccharide (LPS) and the functional role of HO-1 induction in macrophages. LPS (1 microg/mL) maximally induced inducible nitric oxide synthase (iNOS) and HO-1 mRNAs in wild-type (WT) macrophages at 6h and 12h after treatment, respectively, and liberated tumor necrosis factor alpha (TNFalpha) from WT macrophages. LPS also induced iNOS and HO-1 in TNFalpha(-/-) macrophages, but not in iNOS(-/-) macrophages. Interestingly, although LPS strongly induced iNOS, it failed to induce HO-1 almost completely in nuclear-factor erythroid 2-related factor 2 (Nrf2)(-/-) macrophages. The LPS-induced iNOS gene expression was suppressed by pretreatment with HO-1 inducers, hemin and Co-protoporphyrin (CoPP), but not with HO-1 inhibitor, Sn-protoporphyrin in WT macrophages. In the Nrf2(-/-) macrophages, the ability of CoPP to induce HO-1 and its inhibitory effect on the LPS-induced iNOS gene expression were lower than seen in WT macrophages. The present findings suggest that HO-1 is induced via NO-induced nuclear translocation of Nrf2, and the enzymatic function of HO-1 inhibits the overproduction of NO in macrophages.     

In vivo expression of monokine and inducible nitric oxide synthase in experimentally induced pulmonary granulomatous inflammation. Evidence for sequential production of interleukin-1, inducible nitric oxide synthase, and tumor necrosis factor.

The present study examined the temporal pattern and localization of interleukin-1, tumor necrosis factor-alpha, and inducible nitric oxide synthase expression in lung tissue undergoing foreign body granuloma formation. Pulmonary granulomas were induced by the intratracheal injection of dextran beads into genetically high granuloma responder, carrying Bcgs (BALB/c), and low responder, carrying Bcgr (C3H/HeJ and DBA/2), mice. There was a pattern of sequential expression of these molecules in BALB/c mice. Thus, interleukin-1 alpha and inducible nitric oxide synthase were induced mostly in the cells accumulated around the beads and also in some bronchiolar epithelial cells during the early phase (1 to 3 days), whereas tumor necrosis factor-alpha was induced in the cells around the beads at the later resolution phase (3 to 7 days). By contrast, in low responder mice, an increase in the expression of interleukin-1 alpha and inducible nitric oxide synthase was detected in lung macrophages as well as in alveolar cells and bronchiolar epithelial cells on day 1, but that of tumor necrosis factor-alpha was not detected throughout the period. These results together with our previous findings on cytokine activity in granuloma extract suggest that interleukin-1 and nitric oxide produced by recruited macrophages may take part in the early, macrophage-dependent phase of granuloma formation whereas tumor necrosis factor-alpha may be more crucial as a mediator responsible for the difference in innate resistance or susceptibility to granuloma formation.     

Wortmannin enhances lipopolysaccharide-induced inducible nitric oxide synthase expression in microglia in the presence of astrocytes in rats

There is evidence that morphological alterations concerning deficiency or abundance of thyroid hormones (TH) may be mediated by brain-derived neurotrophic factor (BDNF). It has been demonstrated that the mRNA-expression of BDNF is increased after TH-treatment during the first postnatal weeks. After transient treatment of newborn rats with thyroxine mRNA expression, protein concentration and number and size of BDNF-immunopositive neurons were quantified in the medial septum/vertical diagonal Band of Broca (MS/vDB). The number and size of BDNF-immunopositive neurons were estimated in young (P10) and adult (4 months). The amount of mRNA and protein are significantly increased in TH-treated rats at P10 compared to control animals. TH-treated animals showed a significant decrease of BDNF-immunopositive cell numbers in the adulthood. The results demonstrate a correlated increase of BDNF mRNA and protein in the septum at P10 which is an important stage of differentiation processes in the septohippocampal system. These results provide further evidence that BDNF is a possible candidate for the mediation the TH effects in the MS/vDB.     

Temporal expression of pro-inflammatory cytokines and inducible nitric oxide synthase in experimental acute Chagasic cardiomyopathy.

To characterize the kinetics of myocardial cytokine and inducible nitric oxide synthase (iNOS) expression in acute Chagasic cardiomyopathy, we studied a rat model of acute Trypanosoma cruzi infection. Rats were euthanized 36 hours and 5, 10, and 15 days after infection, and hearts were collected for histology, mRNA, and protein analyses. Histological analysis of myocardium showed a progressive increase in the number of amastigotes and mononuclear inflammatory cells. Organisms were first detected 5 days after intraperitoneal inoculation as isolated nests and became numerous by day 15. Northern blot analysis of total RNA revealed no signal for interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha and a weak signal for IL-6 in control hearts. High levels of expression for the three genes were detected in the infected animals at 36 hours after infection. Although IL-1beta and IL-6 levels increased steadily up to 10 days, TNF-alpha levels were the highest at 5 days, remained high at 10 days, and declined thereafter. Western blot analysis showed similar results to that of mRNA expression. No signal was detected for iNOS in the controls, but both its mRNA and protein were found in the infected animals, with levels being highest at 15 days after infection. Immunohistochemistry revealed no iNOS immunoreactivity in uninfected animals, but intense iNOS staining was detected in blood vessels of infected animals, which decreased progressively with period of infection. Positive staining for iNOS in cardiomyocytes was first detected at 36 hours after infection (at a time when there was no histological inflammatory reaction), which steadily increased, being the highest at 15 days after infection. These results indicate that, in addition to mechanical damage by T. cruzi, substantial pro-inflammatory cytokine production within the myocardium is likely to participate in the pathophysiology of acute Chagasic cardiomyopathy.     

Inhibition of inducible nitric oxide synthase expression by interleukin-4 and interleukin-13 in human lung epithelial cells.

Oral feeding of proteins causes peripheral T-cell tolerance, as revealed by reduced delayed-type hypersensitivity (DTH) reactivity after immunization. This type of tolerance can be due both to passive T-cell anergy and active immunosuppression. Using ovalbumin-fed mice we studied whether putatively immunostimulatory cytokines could break this state of mucosal tolerance. Cytokines were administered locally at the site of attempted sensitization. It was found that neither interleukin-2 (IL-2), interferon-gamma (IFN-gamma) nor granulocyte-macrophage colony-stimulating factor (GM-CSF) could restore the response to immunization. In contrast, local administration of IL-12 at the site of attempted immunization resulted in full recovery of DTH reactivity. The dichotomy between the two Th1 stimulatory cytokines IFN-gamma and IL-12 was also reflected by different effects on ovalbumin-specific antibody isotypes. Although both IFN-gamma and IL-12 downregulated serum IgG1-levels in tolerant mice, suggesting decreased ovalbumin-specific Th2 function, only local administration of IL-12 led to increased serum IgG2a levels. These results support the view that potentiation of Th1 effector function is critical for reversal of mucosal tolerance.     

内皮型、诱导型一氧化氮合酶在乳腺癌中的表达

目的 :研究内皮型一氧化氮合酶 (eNOS)、诱导型一氧化氮合酶 (iNOS)在乳癌中表达及与淋巴结转移的关系。方法 :采用免疫组化S P法检测 60例乳癌中eNOS和iNOS的表达。结果 :eNOS和iNOS阳性在乳癌中表达率分别为 75 0 %和71 7%。在淋巴结转移组和无淋巴结转移组中eNOS阳性表达率分别为 66 7%和 83 3 % ,两组间差异无统计学意义 (χ2 =2 2 2 ,P >0 0 5) ,而iNOS在淋巴结转移和无转移组中阳性表达率分别为 53 3 %和 90 0 % ,两组间差异有统计学意义 (χ2 =9 93 ,P <0 0 1 )。结论 :内皮型、诱导型一氧化氮合酶在乳腺癌中高表达 ;iNOS的表达与乳腺癌的淋巴转移相关     

巨噬细胞诱导型一氧化氮合酶的表达调节机制

一氧化氮是一种重要的巨噬细胞免疫效应分子,它参与免疫调节和宿主防御反应.一氧化氮的生成主要由诱导型一氧化氮合酶调节,然而诱导型一氧化氮合酶表达的调节机制及信号通路尚不完全清楚.     

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。     

大鼠坐骨神经结扎后脊髓内诱导型一氧化氮合酶的表达

目的:研究坐骨神经结扎损伤后诱导型一氧化氮合酶(iNOS)在大鼠脊髓内的表达变化规律.方法:健康成年SD大鼠随机分为正常组、假手术组和坐骨神经结扎组.存活1、3、5、7、14、21、28 d后取腰4～6脊髓节段冷冻切片,用免疫组织化学方法结合图像分析技术检测脊髓内iNOS的表达变化,同时用免疫荧光双标染色技术检测14 d组中央管周围iNOS和神经肽Y(NPY)的共表达.结果:根据免疫阳性产物灰度值,损伤侧脊髓前角iNOS表达1 d明显升高,21 d达高峰,28 d接近正常,损伤后1～21 d损伤侧脊髓前角与对侧和正常组免疫阳性产物相比有统计学意义;损伤侧脊髓后角1 d iNOS表达增强,其他各组未见明显变化;损伤后中央管周围免疫阳性细胞数增多,3 d尤为明显,28 d恢复正常,免疫荧光双标染色显示14 d组iNOS和NPY在中央管周围共表达.结论:iNOS可能参与神经损伤后的再生过程及伤后神经性痛的凋节.     

HIV-1 induced decrease of nitric oxide production and inducible nitric oxide synthase expression during in vivo and in vitro infection

Nitric oxide (*NO) has been implicated in immunopathogenesis of HIV-1 infection. Initial reports using low sensitive techniques showed elevated levels of *NO in sera and tissues from seropositive patients. These results were not further supported using similar experimental approaches. To gain insight on *NO deregulation during HIV-1 infection, we used recently described fluorescent probes with enhanced sensitivity to assess *NO levels combined with iNOS mRNA expression in peripheral blood mononuclear cells (PBMC) from HIV-infected patients or after in vitro HIV-1 infection of normal cells. We demonstrate that PBMC from HIV-infected patients display a significant decrease of *NO production and iNOS mRNA expression. Results from in vitro infection showed that HIV-1 induces a significant decrease in *NO production and iNOS mRNA expression. Since *NO could play a role in some key processes like apoptosis, regulation of immune responses and viral replication, these results could help in elucidating HIV-1 immunopathogenesis.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression.

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Regional expression of inducible nitric oxide synthase in the kidney stimulated by lipopolysaccharide in the rat

The renal medulla contains more mRNA of the inducible isoform of nitric oxide synthase (iNOS) than the cortex, which may be important in preventing ischaemic injury, since blood flow and tissue oxygen tension are normally low in this region. We examined the effects of the bacterial endotoxin E. coli lipopolysaccharide (LPS) on renal function and regional expression of iNOS in male Sprague-Dawley rats. In six rats, glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were 0.95 +/- 0.09 ml min(-1) g(-1) and 3.36 +/- 0.20 ml min(-1) g(-1), respectively, and decreased significantly to 0.35 +/- 0.09 and 1.74 +/- 0.54 ml min(-1) g(-1), respectively, 1 h after administration of LPS. In an additional seven rats, GFR and ERPF were 0.91 +/- 0.07 and 2.97 +/- 0.30 ml min(-1) g(-1), respectively, 18 h after LPS administration; these values were similar to those in control rats. In all rats, arterial pressure was stable throughout all study periods. In control rats, immunoblot analysis revealed expression of the iNOS protein in the cortex and more pronounced expression in the medulla. In rats studied 18 h after LPS treatment, there was a striking increase in the iNOS expression in the outer medulla. Immunohistochemical examination in the LPS-treated rats showed limited iNOS immunostaining in the cortex, localised to the vascular endothelium and macula densa; however, intense and widespread staining was noted in the tubular and vascular structures of the outer medulla. These findings demonstrated a differential constitutive expression of iNOS protein in different regions of the rat kidney, and marked augmentation of iNOS expression in the outer medulla by LPS.     

Endothelial and inducible nitric oxide synthase expression in Venezuelan patients with pre-eclampsia

Preeclampsia (PE) is a disease that onsets in the second half of pregnancy. This condition is characterized by hypertension, proteinuria and, frequently, intrauterine growth restriction (IUGR). Nitric oxide (NO) regulates blood flow in the human placenta, it induces vasodilatation, inhibition of platelet aggregation and prevents adhesion of platelets to endothelial cells. In this work, nitrite levels were evaluated in the sera of peripheral blood of normal pregnant women (n = 46) and women with PE (n = 50); additionally, the expression of endothelial constitutive nitric oxide and inducible synthases (eNOS and iNOS, respectively) of placental tissues, were determined. An increased concentration of serum nitrites from patients with PE, in relation to normal pregnant women (150.64 +/- 8.94 vs 40.62 +/- 1.65 microM, p < 0.00001) was observed. An increased expression of nitric oxide synthases (eNOS and iNOS), in the placental tissues of (PE) patients, as compared to that of normal pregnant women (iNOS 4.29 +/- 1.51 vs 0.59 +/- 0.13; eNOS 1.78 +/- 0.74 vs 0.46 +/- 0.22, p < 0.005) was also observed. Our results show that there exists a relationship between serum nitrites concentration and the expression of eNOS and iNOS, as analyzed in protein extracts of placental tissues.     

Sphingomyelinase but not ceramide induces nitric oxide synthase expression in rat brain microglia

We have asked whether treatment of PC12 cells with cyclic adenosine monophosphate (cAMP) and epidermal growth factor (EGF) results, like treatment with cAMP and nerve growth factor (NGF), in irreversible neuronal differentiation characterized by irreversible neurite extension, loss of serum-dependence, and death by apoptosis after trophic factor withdrawal. Although EGF alone, unlike NGF, did not cause morphological differentiation or prevent cell death, synergy between a cAMP-mediated signal transduction pathway and a pathway activated by the EGF receptor tyrosine kinase resulted in the same irreversible differentiation. EGF/cAMP-differentiated cells required cAMP to survive, but NGF, through a TrkA-dependent mechanism, could substitute for cAMP. The cyclin-dependent kinase inhibitors olomoucine and roscovitine also promoted survival of the irreversibly differentiated cells, by a mechanism that must be determined, since cell death was not associated with nuclear (3)H-thymidine accumulation, an index of mitotic activity.