Inhibitory effect of the coffee diterpene kahweol on carrageenan-induced inflammation in rats

Previous studies reported that kahweol, a coffee-specific diterpene, inhibits cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in cultured lipopolysaccharide-activated macrophages. The aim of this study was to confirm the anti-inflammatory effects of kahweol by examining its effect on the inflammatory response induced by carrageenan in a rat using an acute air pouch inflammation model. Kahweol significantly reduced the levels of the inflammatory process markers in the air pouch, such as the volume of exudates, the amount of protein and the number of leukocytes and neutrophils. The levels of nitrite, TNF-alpha and prostaglandin E2 (PGE2) were also markedly lower in the air pouch of the kahweol-treated animals than in the controls. Immunoblot analysis showed that kahweol reduced the COX-2 and iNOS expression level in the exudate cells. The histological examination showed that there was a lower inflammatory response in the pouch tissues from the kahweol-treated animals. In addition, kahweol significantly reduced the paw edema induced by carrageenan and also markedly reduced the level of PGE2 production in the inflamed paw. These results suggest that kahweol has significant anti-inflammatory effects in vivo, which might be due to the inhibition of iNOS and COX-2 expression in the inflammatory sites.     

Mercaptoethylguanidine attenuates inflammation in bacterial meningitis in rabbits

Reactive oxygen and nitrogen species participate in the inflammatory process during meningitis. Among them, superoxide, nitric oxide (NO), and their reaction product peroxynitrite exert cytotoxic effects. Mercaptoethylguanidine (MEG) exerts beneficial effects in in vivo inflammatory conditions by scavenging peroxynitrite and inhibiting the inducible NO synthase. This study was designed to investigate whether MEG may attenuate inflammation and brain injury in experimental meningitis. Meningitis increased nitrite/nitrate, and protein content in the cerebrospinal fluid (CSF). In the brain tissue high levels of malondialdehyde and formation of nitrotyrosine indicated lipid peroxidation and nitrosative stress, respectively. Myeloperoxidase activity was increased indicating accumulation of neutrophils into the brain parenchyma. Treatment with MEG decreased nitrite/nitrate levels whereas it did not affect the bacterial clearance from the CSF. Furthermore, treatment with MEG markedly reduced brain tissue levels of myeloperoxidase and malondialdehyde. These data demonstrate that MEG could have a therapeutic role in meningitis.     

Adenovirus infection increases iNOS and peroxynitrite production in the lung

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.     

Antifibrotic role of inducible nitric oxide synthase.

Long-term treatment in rats with l-NAME, an isoform-non-specific inhibitor of nitric oxide synthase (NOS), leads to fibrosis of the heart and kidney, suggesting that nitric oxide (NO) may play a role in preventing tissue fibrosis. In this process, a likely target of NO is the quenching of reactive oxygen species (ROS) through peroxynitrite formation, and one possible source for this NO is inducible NOS (iNOS). Using Peyronie's disease (PD) tissue from both human specimens and from a rat model of PD as the source of fibrotic tissue, we investigated if NO derived from iNOS could act as such an antifibrogenic defense mechanism by determining whether: (a) tunical ROS and iNOS are increased in PD; and (b) the long-term inhibition of iNOS activity decreases the NO/ROS balance in the tunica albuginea thereby promoting collagen deposition. It was determined that in the human PD plaque, iNOS mRNA and protein, ROS, collagen, and the peroxynitrite marker, nitrotyrosine, were all increased in comparison to the normal tunica. In the rat model of PD, the fibrotic plaque also showed significant increases in iNOS mRNA and protein, nitrotyrosine, ROS as measured by heme oxygenase-1, and collagen when compared with the normal control tunica. When a selective inhibitor of iNOS, L-NIL, was given to rats with the PD-like plaque, this resulted in a decrease in nitrotyrosine levels but intensified ROS levels and collagen deposition. These data demonstrate that: (a) iNOS induction occurs in both the human and rat PD fibrotic plaque; and (b) that the NO derived from iNOS appears to counteract ROS formation and collagen deposition. Because the inhibition of iNOS activity leads to a decrease in the NO/ROS ratio, thereby favoring the development of fibrosis, it is proposed that iNOS induction in this tissue may be a protective mechanism against fibrosis and abnormal wound healing.     

Anti-inflammatory agents and substance P depletion in experimental ileitis

To understand the interactions between substance P and gut inflammation, changes in substance P levels were evaluated in a chronic model of ileitis in response to three anti-inflammatory agents with distinct mechanisms of action. The agents were the prostaglandin E(1) analogue misoprostol (30 mug/kg, s.c., b.i.d.), the nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mug/ml in drinking water) and the leumedin, N-(fluorenyl-9-methoxycarbonyl)-L-leucine (NPC 15199, 10 mg/kg, s.c.). Ileitis was induced by a transmural injection of trinitrobenzene sulphonic acid (TNBS 30 mg/kg in 50% ethanol) into the distal ileum of guinea-pigs. All anti-inflammatory therapies were introduced after TNBS administration and continued until day 7, when guinea-pigs were killed. Ileal substance P levels were measured by radioimmunoassay, and granulocyte infiltration was quantified by myeloperoxidase (MPO) activity. Protein and nitrite (an index of nitric oxide formation) levels in a luminal saline lavage were quantified in all groups. TNBS ileitis caused a marked reduction in ileal substance P content and increased MPO activity, protein and nitrite secretion. The nitric oxide synthase inhibitor, L-NAME, completely restored all parameters to baseline. Misoprostol attenuated the granulocyte infiltration and exacerbated protein leak but had no effect on substance P levels. In contrast, NPC 15199 had no effect on granulocyte infiltration but normalized substance P levels and protein leak. Only L-NAME and NPC 15199 blocked the TNBS induced increase in nitrite levels. These results suggest that the regulation of granulocyte infiltration in this model is unrelated to changes in substance P levels. Inhibition of nitric oxide synthase was the most effective therapeutic strategy in TNBS ileitis but the precise interactions between nitric oxide and the enteric nervous system during inflammatory states remain to be defined.     

Peroxynitrite formation and decreased catalase activity in autoimmune MRL-lpr/lpr mice

BACKGROUND: (MRL)-lpr/lpr mice spontaneously develop autoimmune disease characterized by arthritis and glomerulonephritis. Nitric oxide is postulated to play a role in the disease pathogenesis, as mice treated with the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine (NMMA) show markedly reduced manifestations of the disease. The purpose of this study was to examine the role of peroxynitrite in disease development in MRL-lpr/lpr mice. MATERIALS AND METHODS: We examined kidney extracts from control and MRL-lpr/lpr mice for nitrotyrosine by immunoblot with a rabbit polyclonal anti-nitrotyrosine antibody. Catalase activity was determined spectrophotometrically or by activity staining of native polyacrylamide gels. In some experiments, we studied the ability of peroxynitrite and other agents to modify purified catalase in vitro. RESULTS: Kidney extracts from diseased mice had elevated levels of nitrotyrosine, and decreased levels of catalase activity and protein, relative to control mice. MRL-lpr/lpr mice treated with NMMA in vivo had decreased levels of nitrotyrosine, and demonstrated a partial restoration of both catalase activity and protein levels. Treatment of catalase in vitro with peroxynitrite or tetranitromethane at pH 8.0 resulted in protein nitration and a decrease in catalase activity. 1,3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, also decreased the activity of catalase. CONCLUSIONS: These observations suggest that peroxynitrite formation, with an associated decrease in catalase activity and general decrease in antioxidant enzyme activity, may result in increased levels of hydrogen peroxide and other oxidants that can contribute to the pathogenesis of disease in MRL-lpr/lpr mice.     

Plasma levels of nitrites, PGF1α and nitrotyrosine in LPS-treated rats: functional and histochemical implications in aorta

We investigated the effects of lipopolysaccharide (LPS) administration on plasma nitrite, nitrotyrosine and 6-keto prostaglandin F1alpha, (PGF1alpha) levels and the related resultant changes in function and histochemistry of aorta in rats. Plasma nitrite and PGF1alpha nitrotyrosine levels were analysed after 5 mg/kg intravenous LPS was administered to rats compared with those in non-treated rats. The distribution of nitrotyrosine in the aorta was studied immunohistochemically. The contractile responses of aortic rings to phenylephrine (PE) from both the LPS-treated and control rats were studied in the organ baths. There were increases in plasma nitrite, PGF1alpha, and nitrotyrosine concentrations of LPS-treated rats compared to non-treated rats. Immunoreactivity of nitrotyrosine residues were detected in the endothelial and smooth muscle cells in LPS-treated but not in control rat aorta. The contractile responses to PE of the LPS-treated rat aortic rings were significantly reduced as compared with those of control rat's. Incubation of the aortic rings from LPS-treated rats with cyclooxygenase inhibitor indomethacine or with a combination of indomethacine and nitric oxide synthase inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) increased the contractile responses to the levels observed in control rats suggesting that both prostanoids and particularly nitric oxide (NO) are involved in the reduced contractile responses in LPS-treated rats. These results supported the view that LPS might cause an increment in both NO and PGI2 levels. This increase in the NO and PGI2 levels may be responsible from the reduction in responses of aorta to contractile agents in LPS-treated rats. Increased peroxynitrite formation in LPS-treated rats may lead to nitration of the tyrosil residues of the proteins in the aorta.     

Effects of inducible nitric oxide synthase inhibitors on asthma depending on administration schedule

The effectiveness of two inducible nitric oxide synthase (iNOS) inhibitors on allergic airway inflammation was investigated under different administration schedules. Rats sensitized to ovalbumin (OVA) were exposed to OVA for 3 consecutive days. Both iNOS inhibitors showed markedly different effects between two pretreatment schedules: pretreatment before each of three OVA exposures S1 and before the third exposure alone S2. S1 pretreatment resulted in higher pulmonary resistance than triple OVA alone. This potentiation was associated with increased eosinophil infiltration and malondialdehyde levels in the lungs, which were suppressed by superoxide dismutases (SODs) but not by methylprednisolone. However, the S2 administration of both iNOS inhibitors completely suppressed the airway response. Administration by schedule S1 completely suppressed plasma nitrite and nitrate levels, but that by S2 caused only a slight suppression. The triple OVA exposures resulted in the upregulation of iNOS in alveolar macrophages and arginase activity, Mn- and Cu/Zn-SOD expression, and nitrotyrosine and lipid peroxide deposition in the airway. However, inhibitors administered by schedule S1 suppressed this upregulation, but further potentiated nitrotyrosine, which in turn was inhibited by SOD. Although iNOS inhibitors may be beneficial for asthma, repeated administration may be detrimental because of extensive reduction of NO and downregulation of SOD.     

Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.     

Nitrotyrosinylation,remodeling and endothelial‐myocyte uncoupling in iNOS,cystathionine beta synthase (CBS) knockouts and iNOS/CBS double knockout mice

Increased levels of homocysteine (Hcy), recognized as hyperhomocysteinemia (HHcy), were associated with cardiovascular diseases. There was controversy regarding the detrimental versus cardio protective role of inducible nitric oxide synthase (iNOS) in ischemic heart disease. The aim of this study was to test the hypothesis that the Hcy generated nitrotyrosine by inducing the endothelial nitric oxide synthase, causing endothelial‐myocyte (E‐M) coupling. To differentiate the role of iNOS versus constitutive nitric oxide synthase (eNOS and nNOS) in Hcy‐mediated nitrotyrosine generation and matrix remodeling in cardiac dysfunction, left ventricular (LV) tissue was analyzed from cystathionine beta synthase (CBS) heterozygote knockout, iNOS homozygote knockout, CBS?/+/iNOS?/? double knockout, and wild‐type (WT) mice. The levels of nitrotyrosine, MMP‐2 and ‐9 (zymographic analysis), and fibrosis (by trichrome stain) were measured. The endothelial‐myocyte function was determined in cardiac rings. In CBS?/+ mice, homocysteine was elevated and in iNOS?/? mice, nitric oxide was significantly reduced. The nitrotyrosine and matrix metalloproteinase‐9 (MMP‐9) levels were elevated in double knockout and CBS?/+ as compared to WT mice. Although MMP‐2 levels were similar in CBS?/+, iNOS?/?, and CBS?/+/iNOS?/?, the levels were three‐ to fourfold higher than WT. The levels of collagen were similar in CBS?/+ and iNOS?/?, but they were threefold higher than WT. Interesting, the levels of collagen increased sixfold in double knockouts, compared to WT, suggesting synergism between high Hcy and lack of iNOS. Left ventricular hypertrophy was exaggerated in the iNOS?/? and double knockout, and mildly increased in the CBS?/+, compared to WT mice. The endothelial‐dependent relaxation was attenuated to the same extent in the CBS?/+ and iNOS?/?, compared to WT, but it was robustly blunted in double knockouts. The results concluded that homocysteine generated nitrotyrosine in the vicinity of endothelium, caused MMP activation and endothelium‐myocyte uncoupling. The generation of nitrotyrosine was independent of iNOS. J. Cell. Biochem. 106: 119–126, 2009. © 2008 Wiley‐Liss, Inc.     

Irradiation of the rabbit cornea with UVB rays stimulates the expression of nitric oxide synthases-generated nitric oxide and the formation of cytotoxic nitrogen-related oxidants

Until now, the role of nitric oxide (NO) in cornea irradiated with UVB rays remains unknown. Therefore, we investigated nitric oxide synthase isomers (NOS), enzymes that generate NO, nitrotyrosine (NT), a cytotoxic byproduct of NO, and malondialdehyde (MDA), a byproduct of lipid peroxidation, in rabbit corneas repeatedly irradiated with UVB rays (312 nm, 1x daily for 6 days, the dose per day 1.01 J/cm2) using immunohistochemical methods. The biochemical measurement of nitrite and nitrate has been used for the indirect investigation of NO concentration in the aqueous humor. Results show that in contrast to normal corneas, where of the NOS isomers only endothelial nitric oxide synthase (NOS3) was expressed in a significant amount (in the epithelium and endothelium), in irradiated corneas all NOS isomers (also brain nitric oxide synthase, NOS1, and inducible nitric oxide synthase, NOS2) as well as an indirect measure of ONOO-formation and MDA were gradually expressed, first in the epithelium, the endothelium and the keratocytes beneath the epithelium and finally in the cells of all corneal layers and the inflammatory cells that invaded the corneal stroma. This was accompanied by an elevated concentration of NO in the aqueous humor. In conclusion, repeated irradiation with UVB rays evoked the stimulation of NO production, peroxynitrite formation (demonstrated by NT residues) and lipid peroxidation (evaluated by MDA staining).     

Neutral sphingomyelinase inhibition decreases ER stress-mediated apoptosis and inducible nitric oxide synthase in retinal pigment epithelial cells

Endoplasmic reticulum (ER) stress and excessive nitric oxide production via the induction of inducible nitric oxide synthase (NOS2) have been implicated in the pathogenesis of ocular diseases characterized by retinal degeneration. Previous studies have revealed the sphingomyelinase/ceramide pathway in the regulation of NOS2 induction. Thus, the objective of this study was to determine the activity of the sphingomyelinase/ceramide pathway, assess nitric oxide production, and examine apoptosis in human retinal pigment epithelial (RPE) cells undergoing ER stress. Sphingomyelinase (SMase) activity; nuclear factor κB (NF-κB) activation; NOS2, nitrite/nitrate, and nitrotyrosine levels; and apoptosis were determined in cultured human RPE cell lines subjected to ER stress via exposure to tunicamycin. Induction of ER stress was confirmed by increased intracellular levels of ER stress markers including phosphorylated PKR-like ER kinase, C/EBP-homologous protein, and 78-kDa glucose-regulated protein. ER stress increased nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, and nitrotyrosine formation and caused apoptosis in RPE cell lines. Inhibition of neutral SMase (N-SMase) activity via GW 4869 treatment caused a significant reduction in nuclear translocation of NF-κB, NOS2 expression, nitrite/nitrate levels, nitrotyrosine formation, and apoptosis in ER-stressed RPE cells. In conclusion, N-SMase inhibition reduced nitrative stress and apoptosis in RPE cells undergoing ER stress. Obtained data suggest that NOS2 can be regulated by N-SMase in RPE cells experiencing ER stress.     

Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury

The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.     

Age alters cerebrovascular inflammation and effects of estrogen

In young adult females, estrogen treatment suppresses the cerebrovascular inflammatory response; this is mediated in part via NF-kappaB, a key regulator of inflammatory genes. To examine whether age modifies effects of estrogen on vascular inflammation in the brain, female rats, 3 and 12 mo of age, were ovariectomized; half were treated with estrogen for 4 wk. Cerebral blood vessels were isolated from the animals at 4 and 13 mo of age. Inflammation was induced by LPS, either injected in vivo or incubated with isolated vessels ex vivo. Basal levels of cytoplasmic NF-kappaB were significantly higher in cerebral vessels of young rats, but the ratio of nuclear to cytoplasmic levels was greater in middle-aged animals. LPS exposure increased nuclear NF-kappaB DNA binding activity, protein levels of inducible nitric oxide synthase and cyclooxygenase-2, and production of nitric oxide and PGE(2) in cerebral vessels. All effects of LPS were markedly greater in vessels from the older animals. Estrogen significantly inhibited the LPS-induced increase in NF-kappaB DNA binding activity in cerebral vessels from animals at both ages. In 4-mo-old rats, estrogen also significantly suppressed LPS induction of inducible nitric oxide synthase and cyclooxygenase-2 proteins, as well as production of nitric oxide and PGE(2). In contrast, in 13-mo-old females, estrogen did not significantly affect these indexes of cerebrovascular inflammation. Thus the protective, anti-inflammatory effect of estrogen on cerebral blood vessels that is observed in young adults may be attenuated in aged animals, which exhibit a greater overall cerebrovascular response to inflammatory stimuli.