In vitro effects of Actinobacillus pleuropneumoniae on inducible nitric oxide synthase and cyclooxygenase-2 in porcine alveolar macrophages

OBJECTIVE: To determine the amount of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) activity in alveolar macrophages in response to Actinobacillus pleuropneumoniae (APP) by determining nitric oxide (NO) and prostaglandin E2 (PGE2) concentrations. SAMPLE POPULATION: Freshly isolated porcine alveolar macrophages. PROCEDURE: Alveolar macrophages were incubated for 48 hours with APP (1 X 10(4) colony-forming units/mL), interleukin-1beta, (IL-1beta; 5 U/mL), tumor necrosis factor-alpha (TNFalpha; 500 U/mL), interferon-gamma (IFN-gamma, 100 U/mL), or lipopolysaccharide (LPS; 10 microg/mL). In a second experiment, alveolar macrophages were incubated with fresh medium (negative control), APP alone, or APP with 1 of the following: IL-1beta, TNF-alpha, or IFN-gamma. In a third experiment, alveolar macrophages were incubated with fresh medium (negative control), LPS (positive control), APP alone, or APP with 1 of the following: an iNOS inhibitor (3.3 microM), a COX-2 inhibitor (10 microM); or both the iNOS and COX-2 inhibitors. Supernatant was obtained at 0, 3, 6, 9, 12, 24, and 48 hours after treatment for determination of NO and PGE2 production. RESULTS: The addition of APP to alveolar macrophages resulted in significant increases in NO and PGE2 production. The addition of APP and IFN-gamma synergistically induced NO production. Inhibition of iNOS and COX-2 decreased NO and PGE2 production, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In vitro activation of alveolar macrophages by APP results in increased production of NO and PGE2. Nitric oxide and PGE2 production appears to be largely dependent on iNOS and COX-2 activity. Pharmacologic modulation of iNOS and COX-2 activity may represent a therapeutic target for pigs with pleuropneumonia.     

Upregulation of cyclooxygenase-2 expression in porcine macula densa with chronic nitric oxide synthase inhibition

The objective of this study was to investigate the effects of chronic inhibition of nitric oxide synthase (NOS) on cyclooxygenase-2 (COX-2) expression in the macula densa (MD) of swine, as well as the effects on expression of related proteins. Adult female Yucatan swine were given either tap water (control, n = 6) or water with N (G)-nitro-L-arginine methyl ester (L-NAME, 100 mg/liter, n = 5) for a minimum of 30 days. Duplicate samples of kidney were fixed or snap frozen. There was a significant (P = .0082) upregulation of COX-2 mRNA expression in the MD of L-NAME, as well as an apparent increase in COX-2 protein. Plasma renin activity also increased with L-NAME treatment (control, 0.34 ± 0.08 ng/ml; L-NAME, 1.26 ± 0.03 ng/ml; P = .00000003). There were no differences between groups in expression of either inducible NOS or renin protein or in serum electrolyte concentrations. In conclusion, with chronic inhibition of NOS, COX-2 in MD is upregulated, perhaps to compensate for loss of nitric oxide. Increases in COX-2 products may counteract renal arteriolar constriction and sustain renin release.     

Evidence of nitric oxide synthase 2 activity in swine naturally infected with Actinobacillus pleuropneumoniae

Evidence of nitric oxide synthase (NOS) 2 activity was determined by formation of nitrotyrosine (a reaction product of peroxynitrite) and by activation of poly(ADP-ribose) synthetase (PARS) in NOS2-expressed pleuropneumonic lungs from 20 pigs naturally infected with Actinobacillus pleuropneumoniae using immunohistochemistry. Intense immunostaining for nitrotyrosine residue was seen within the lung lesions from A. pleuropneumoniae-infected pigs, but it was minimal in the unaffected parts of the lung from A. pleuropneumoniae-infected pigs and in the normal lung from control pigs. Staining was especially strong in neutrophils and macrophages in the periphery of the lesions and within the alveolar spaces. There was close cell-to-cell correlation when serial sections were examined by immunohistochemistry for NOS2 and nitrotyrosine in each of the 20 lung samples. Expression of PARS was always present within inflammatory lesions but was minimal in the unaffected lung of A. pleuropneumoniae-infected pigs. Macrophages in alveolar spaces frequently exhibited strong staining for PARS. Colocalization of nitrotyrosine and PARS antigen was especially prominent in macrophages in the periphery of lesions. NOS2 expression in pleuropneumonic areas associated with protein nitrosation and PARS suggests that NOS2 is functionally active during infections caused by A. pleuropneumoniae.     

Dietary conjugated linoleic acid enhances spleen PPAR-gamma mRNA expression in broiler chicks

1. The anti-inflammatory effects of dietary conjugated linoleic acid (CLA) on broilers repeatedly challenged with lipopolysaccharide (LPS) were investigated. 2. Day-old broiler chicks were allotted into three treatment groups and fed on a control diet or diets containing 5.0 or 10.0 g CLA/kg diet. Six chicks from each treatment were injected with LPS (0.25 mg/kg body weight) at 16, 18 and 20 d of age. Splenic cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) activities, and prostaglandin E(2) (PGE(2)) and nitric oxide (NO) production as well as peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA expression were measured at 21 d of age. 3. Chicks fed 10.0 g CLA/kg diet had lower COX activities and PGE(2) production that the controls. Dietary CLA (10.0 g/kg) did not significantly diminish LPS-induced enhancement of COS-2 activity, inhibited the subsequent increase in PGE(2) production. 4. Regulation of COX-1 activity contributed to the difference in PGE(2) production. 5. CLA did not markedly attenuate the increase of iNOS activity and NO production caused by LPS challenge. Chicks fed CLA had lower iNOS activity and NO production than the controls. 6. Dietary CLA activated splenic PPAR-gamma mRNA expression and increased PPAR-gamma mRNA expression after LPS injection. 7. These results suggest that dietary CLA has immunomodulatory effects in the spleen by restricting basal PGE(2) and NO to lower levels and enhancing PPAR-gamma mRNA expression. During the inflammatory response, dietary CLA did not alleviate the increase in COX-2 and iNOS activities but enhanced PPAT-gamma mRNA expression.     

Nitric oxide synthase expression in foetal placentas of cows with retained fetal membranes

The objectives of this study were to investigate relationship of retained fetal membranes (RFM) to expression of NOS and NOS mRNA and to analyze pathohistological changes and the distribution of nitric oxide synthase (NOS) in foetal placentas of cows with RFM. Twenty cows were assigned to two groups, a control group (no retained fetal membranes, NRFM, n = 10) and a diseased group (RFM, n = 10). The endpoint method was used to detect the nitric oxide (NO) content and nitric oxide synthase (NOS) activity in foetal placental tissue fluid and the fluorescent quantitation PCR was used to measure the expression of NOS mRNA. Immunohistochemistry and hematoxylin-eosin staining were used to observe pathohistological changes. Tissue from RFM cows showed fibronecrosis of the chorionic villi, and a decreased number of trophoblastic cells. The majority of trophoblastic cells displayed vacuolar degeneration. Interstitium vessels were distended and congested. Expression of induced nitric oxide synthase (iNOS) protein and iNOS mRNA was significantly higher (P < 0.05) in the cytoplasm of placental villus trophoblastic cells in the RFM group. But expression of endothelial nitric oxide synthase (eNOS) protein and eNOS mRNA was significantly lower (P<0.05) in the RFM group. The NO content and NOS activity of cows with RFM were significantly higher (P < 0.05). A high expression of iNOS protein and iNOS mRNA in the cow foetal placenta could produce high content of NO, which might inhibit uterine contraction. So over expression of iNOS protein and iNOS mRNA might be an important agent of retained fetal membranes in cows, and it may be a potential diagnosis biomarker.     

Effects of nitric oxide and tumor necrosis factor-alpha on production of prostaglandin F2alpha and E2 in bovine endometrial cells

The purpose of this study was to determine whether nitric oxide (NO) mediates tumor necrosis factor (TNF)alpha influence on the bovine endometrium. TNFalpha influence on the bovine endometrium is limited to the stromal cells. Therefore, it was interesting to find out whether NO production by the stromal cells, stimulated by TNFalpha might influence the endometrial epithelium. Moreover, we investigated the intracellular mechanisms of TNFalpha- and NO-regulated prostaglandin (PG) F(2alpha) and PGE(2) synthesis. Epithelial and stromal cells from the bovine endometrium (Days 2-5 of the oestrous cycle) were separated by means of enzymatic dispersion and cultured for 6-7 days in 48-well plates. The confluent endometrial cells were exposed to a NO donor (S-NAP; 1-1000 microM) for 24 h. S-NAP strongly stimulated PGE(2) production in both bovine endometrial cell types (P<0.001). The effect of SNAP on PGF(2alpha) production was limited only to the stromal cells (P<0.05). To study the intracellular mechanisms of TNFalpha and NO action, stromal cells were incubated for 24 h with TNFalpha or S-NAP and with NO synthase (NOS) inhibitor (L-NAME; 10 microM) or an inhibitor of phosphodiesterase (IBMX; 10 microM). When the cells were exposed to TNFalpha in combination with NOS inhibitor (L-NAME), TNFalpha-stimulated PGs production was reduced (P<0.05). The inhibition of enzymatic degradation of cGMP by IBMX augmented the actions of S-NAP and TNFalpha on PGs production (P<0.05). The overall results suggest that TNFalpha augments PGs production by bovine endometrial stromal cells partially via induction of NOS with subsequent stimulation of NO-cGMP formation. NO also stimulates PGE(2) production in epithelial cells.     

Nitric oxide stimulates progesterone and prostaglandin E2 secretion as well as angiogenic activity in the equine corpus luteum

Cytokines and nitric oxide (NO) are potential mediators of luteal development and maintenance, angiogenesis, and blood flow. The aim of this study was to evaluate (i) the localization and protein expression of endothelial and inducible nitric oxide synthases (eNOS and iNOS) in equine corpora lutea (CL) throughout the luteal phase and (ii) the effect of a nitric oxide donor (spermine NONOate, NONOate) on the production of progesterone (P4) and prostaglandin (PG) E(2) and factor(s) that stimulate endothelial cell proliferation using equine luteal explants. Luteal tissue was classified as corpora hemorrhagica (CH; n = 5), midluteal phase CL (mid-CL; n = 5) or late luteal phase CL (late CL; n = 5). Both eNOS and iNOS were localized in large luteal cells and endothelial cells throughout the luteal phase. The expression of eNOS was the lowest in mid-CL (P < 0.05) and the highest in late CL (P < 0.05). However, no change was found for iNOS expression. Luteal explants were cultured with no hormone added or with NONOate (10(-5) M), tumor necrosis factor-α (TNFα; 10 ng/mL; positive control), or equine LH (100 ng/mL; positive control). Conditioned media by luteal tissues were assayed for P4 and PGE(2) and for their ability to stimulate proliferation of bovine aortic endothelial cells (BAEC). All treatments stimulated release of P4 in CH, but not in mid-CL. TNFα and NONOate treatments also increased PGE(2) levels and BAEC proliferation in CH (P < 0.05). However, in mid-CL, no changes were observed, regardless of the treatments used. These data suggest that NO and TNFα stimulate equine CH secretory functions and the production of angiogenic factor(s). Furthermore, in mares, NO may play a role in CL growth during early luteal development, when vascular development is more intense.     

富硒麦芽对实验性肝癌大鼠NO、NOS和脂质过氧化的影响

50只清洁级sD大鼠随机分为4组：对照组（5只）、模型组（15只）、富硒麦芽组（15只）和亚硒酸钠组（15只）。对照组和模型组饲喂大鼠基础饲料，饲料含硒量为0．1mg／kg；富硒麦芽组和亚硒酸钠组分别在基础饲料中补充富硒麦芽和亚硒酸钠，饲料含硒量为3．0mg／kg。除对照组外，其余3组采用0．01％二乙基亚硝胺（DEN）诱发大鼠肝癌16周，停止诱癌后继续饲养2周再处死全部大鼠，观察富硒麦芽对丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）、白蛋白（ALB）、总胆红素（TBIL）、一氧化氮（NO）、一氧化氮合成酶（NOS）、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH—Px）的影响。结果显示，富硒麦芽组大鼠血浆ALT、ALP、TBII—NO、MDA和肝组织NO、NOs、MDA显著低于模型组，全血和肝组织GSH—Px活性显著高于模型组；与亚硒酸钠组相比，富硒麦芽组大鼠血浆和肝组织的NO含量和NOS活性显著降低。试验表明富硒麦芽具有减轻肝脏损伤、延缓大鼠肝癌形成的能力，其作用机制之一可能为富硒麦芽能抑制NO的产生，清除体内大量过剩的氧自由基。减轻NO等自由基所致的脂质过氧化损伤。     

Balb/c小鼠肺组织一氧化氮及一氧化氮合酶的动态变化

为探讨Balb/c小鼠正常生理状况下肺组织中一氧化氮自由基含量以及一氧化氮合酶活性的动态变化,采用电子自旋共振法直接测定了一氧化氮自由基含量,采用分光光度计法测定了一氧化氮合酶的活性.结果表明:在第3,6,7,12天Balb/c小鼠肺组织的一氧化氮自由基含量、一氧化氮合酶活性均无显著性差异(P＞0.05).说明生理状态下,Balb/c小鼠肺组织一氧化氮自由基的产生维持动态平衡.     

Expression of cyclooxygenase-2 in swine naturally infected with Actinobacillus pleuropneumoniae

Cyclooxygenase-2 (COX-2) was detected and localized in 15 pigs with naturally occurring pleuropneumonia using a 437-base pair digoxigenin-labeled cDNA probe in an in situ hybridization protocol. Histopathologic changes in the acute stage were characterized by coagulative necrosis of lung parenchyma, hemorrhage, vascular thrombosis, edema, fibrin deposition, and infiltration of lung parenchyma by neutrophils and alveolar macrophages in nine pigs. In chronic lesions, a thick layer of granulation tissue surrounded foci of pulmonary necrosis in six pigs. All 15 pigs infected with Actinobacillus pleuropneumoniae, confirmed by bacterial isolation, had distinct positive hybridization signals for COX-2 in bronchial, bronchiolar epithelial cells, alveolar macrophages, neutrophils, and type I pneumocytes. COX-2 expression was detected primarily in neutrophils from pigs with acute lesions and primarily in alveolar macrophages from pigs with chronic lesions. The results suggest that a prostanoid product of COX-2 is an important component of the inflammatory response to acute and chronic A. pleuropneumoniae infection.     

The influence of the phase of the estrous cycle on sheep endometrial tissue response to lipopolysaccharide

The effect of lipopolysaccharide on ovine endometrial tissue was examined at estrus (follicular phase) and during the luteal phase. Endometrial tissues were cultured with 0, 1, or 10 microg/mL lipopolysaccharide. After 24 h, culture supernates were harvested and analyzed for PGF2alpha, PGE2, 6-keto-PGF1alpha, thromboxane B2 (TXB2), and cysteinyl-leukotrienes (leukotrienes) using EIA. Homogenates of endometrial tissue were analyzed for prostaglandin endoperoxidase-1 (PTGS-1), and -2 (PTGS-2) as well as Type-I, -II, and -III nitric oxide synthase (NOS) by Western analysis. Follicular phase tissue produced more PGF2alpha (P < 0.001), TXB2 (P < 0.001), and leukotrienes (P < 0.02) than luteal tissue. Lipopolysaccharide increased PGE2 (P < 0.001) and TXB2 (P < 0.02) production by endometrial tissue. Elevations in these eicosanoids were likely due to the measured increases in PTGS-2 (P = 0.002) because no changes in PTGS-1 (P = 0.54) or Type-I, -II, or -III NOS (P > or = 0.20) occurred in endometrial tissue following lipopolysaccharide exposure. These data suggest that the phase of the estrous cycle regulates prostaglandin production by immune-challenged endometrial tissue.     

Expression of secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound mucin (MUC4) in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae

The expression patterns of different secreted (MUC2, MUC5AC, MUC5B, and MUC6) and membrane-bound (MUC4) mucins were determined immunohistochemically in the lungs of pigs experimentally infected with Actinobacillus pleuropneumoniae. Forty-seven-week-old colostrum-deprived pigs were randomly allocated to infected (n=20) or control groups (n=20). Five infected and uninfected pigs were euthanized at 0, 6, 12, and 48 h post-inoculation (hpi). In the infected pigs, the expression of both types of mucins, which were invariably observed, was associated with bronchiolar and respiratory bronchiolar lesions. Strong positive mucin signals were seen on the surface of bronchiolar and respiratory bronchiolar epithelium with neutrophil infiltration. The mean mucin-positive area peaked at 6 hpi and decreased significantly to control levels by 48 hpi on the surface of the bronchiolar and respiratory bronchiolar epithelium. Further studies are needed to establish the functional relationship between mucin expression and the host defense mechanism against A. pleuropneumoniae in the lungs of infected pigs.     

Changes of Tissue Endothelin-1 and Nitric Oxide Synthase in a Sheep Model of Large Intestinal Obstruction

Large intestinal obstruction (LIO) in farm animals can cause a ischaemic necrosis of intestinal tissue, eventually leading to death. The roles of endothelin-1 (ET-1) and nitric oxide (NO) are not well understood in the process of LIO, but evidence suggests that endothelial-derived mediators may participate. In the present study, ET-1 concentration and total nitric oxide synthase (NOS) activity were measured in heart, liver, pancreas, lung and kidney in a model of LIO in sheep. Our data demonstrated that ET-1 concentration and NOS activity were altered, with significant increases of ET-1 in heart, lung and kidney and of NOS activity in pancreas and kidney, but a marked decline of NOS activity in liver (p < 0.05). It is postulated that these alterations in NOS activity and ET-1 concentration may contribute to the progressive loss of organ function, and finally lead to death in LIO in sheep.     

补中益气丸对糖尿病大鼠胃组织MUC1、COX、NOS表达变化及相互作用

观察补中益气丸对糖尿病大鼠胃组织MuC1、COX-1、COX-2、cNOS、iNOS不同病程表达变化及其相互作用的影响、大鼠随机分为正常组。模型组和中药组,采用STZ腹腔注射结合乙醇灌胃的方法建立糖尿病大鼠胃黏膜损伤模型．检测胃组织NO、PGE2、6-ketoPC-F1d、MuCl、COX-1、cOX2、cNOS和iNOs随病程的表达变化及其相互作用。结果显示,糖尿病大鼠出现胃黏膜损伤和胃肠功能紊乱,胃组织MUC1、COX2、iNOS发生显著变化．MUC1随病程进展逐渐降低,iNOs和COX-2表现为早期活性降低,晚期活性升高,PGE2、6-keto—PGF1d和NO随病程显著升高,相关分析发现．COX1与cNOS呈显著正相关,与iNOS呈著负相关。补中益气丸能调节MUC1、COX-2、iNOS的表达及其相互作用,阻止胃黏膜损伤。结果表明,STZ腹腔注射结合乙醇灌胃的方法可成功诱导糖尿病大鼠胃黏膜损伤模型,iNOS与COX2可能为相互协作和相互介导的关系,在糖尿病胃黏膜损伤方面起重要作用,补中益气丸通过升高MUC1、降低iNOS和COX-2的表达及其相互作用,阻止胃黏膜损伤和糖尿病的发展。     

Endometrial nitric oxide synthase activity in mares susceptible or resistant to persistent breeding‐induced endometritis and the effect of a specific iNOS inhibitor in vitro

Emerging research suggests that the nitric oxide system may play a role in persistent breeding‐induced endometritis (PBIE) in the mare. Differences in uterine nitric oxide (NO) levels between mares susceptible or resistant to PBIE and a dose‐dependent inhibitory effect of NO on uterine contractility have been demonstrated. The objectives of this study were to investigate the difference in total nitric oxide synthase (NOS) activity of the endometrium between susceptible and resistant mares and the effect of a specific inducible nitric oxide synthase (iNOS) inhibitor on the endometrial NOS activity in vitro. Six susceptible and six resistant mares were selected based on preset criteria and the results of an intrauterine challenge with killed spermatozoa during oestrus. Endometrial biopsy samples were collected 24 hr post‐challenge and cultured at 37°C for 24 hr in L‐arginine supplemented minimum essential medium with or without a specific iNOS inhibitor (1,400 W dihydrochloride, 1 mM). The medium and the cultured endometrial tissue were collected after 24 hr of culture and assayed for NO and total protein, respectively. Total NO content of the medium, normalized to endometrial tissue wet weight or total protein, was used as a measure of endometrial NOS activity. Non‐parametric tests were applied for statistical analysis. Susceptible mares had significantly greater endometrial NOS activity than resistant mares. The iNOS inhibitor treatment significantly reduced NOS activity in endometrial samples derived from susceptible and resistant mares. These findings provide a basis for in vivo testing of specific iNOS inhibitors as preventative or therapeutic options for PBIE in mares.     

Effects of prostaglandin F(2alpha) and nitric oxide on the secretory function of bovine luteal cells

The objective of the present study was to investigate the influence of prostaglandin F(2alpha) (PGF (2alpha)) and nitric oxide (NO) on production of steroids and PGs by culturing bovine luteal cells obtained from ovaries on days 8-12 of the estrous cycle with a nitric oxide (NO) donor (Spermine NONOate), and a NO synthase inhibitor (N(G)-nitro-L-arginine methyl ester dihydrochloride: L-NAME). When the cells were exposed for 24 h to PGF(2alpha) (10(-7)-10(-5) M), production of progesterone (P(4)) increased significantly at all doses used (P<0.05). Moreover, PGF(2alpha) stimulated PGF(2alpha) production (P<0.01), depressed testosterone (T) production (P<0.05), but did not affect synthesis of prostaglandin E(2) (PGE(2)). Spermine NONOate decreased P(4) production to 66%, 47% and 34% of the control concentration after treatment with 10(-5) M, 10(-4) M and 10(-3) M, respectively, but did not affect T production, and increased PGF(2alpha) synthesis (P<0.05) and PGE(2) (P<0.01) at all doses used. L-NAME increased production of P(4) (P<0.01) but did not affect (P>0.05) secretion of T, PGF(2alpha) and PGE(2). Estradiol-17beta (E(2)) was detectable on the level of sensitivity of assay and was not significantly altered by any treatments. The overall results suggest that PGF(2alpha) and NO produced locally in bovine CL play roles in the regulation of the secretory function of the bovine CL as auto/paracrine factors.     

Pulmonary lesions in guinea pigs experimentally infected with Actinobacillus pleuropneumoniae (A.p.) serovar 1

Pathological studies were carried out on the lungs of guinea pigs intratracheally inoculated with 4.6 x 10(6-8) colony forming units (CFU)/head of Actinobacillus pleuropneumoniae serovar 1. All animals in the highest dose group died within 24 hr post inoculation (hpi) and showed pulmonary lesions being hemorrhagic in nature while all animals in the lowest dose group were killed as scheduled at 11 days post inoculation (dpi) and showed only hyperplasia of peribronchial lymphoid tissues. In the middle dose group, two died within 24 hpi, two died at 9 dpi, and the remaining one was killed at 11 dpi. Two guinea pigs which died at 9 dpi showed fibrinonecrotic pleuropneumonia which is the most characteristic acute pulmonary lesion in swine, and has not yet been reproduced in laboratory animals up to the present time. This suggests that guinea pigs may be a useful laboratory animal for studying the pathogenesis of Actinobacillus pleuropneumoniae infection in swine.