Decreased iNOS Expression Research Articles

EFFECTS OF ERYTHROPOIETIN ON ENDOTHELIAL PROGENITOR CELL TRANSPLANTATION AFTER MYOCARDIAL INFARCTION IN MICE

ObjectivesPrevious studies suggested that transplantation of endothelial progenitor cells (EPC) could improve cardiac function after myocardial infarction (MI). However, survival of transplanted EPC seems unsatisfactory. The purpose of the present...

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor-associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a(+) macrophages in M2-associated infectious pathologies. In late-stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll-like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL-4). LPS/dexamethasone/IL-4-induced Ms4a8a(+) BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF-κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL-4-stimulated Ms4a8a(+) BMDMs and in peritoneal macrophages from late-stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2-like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.

Effect of Shengmai injection (生脉注射液) on diaphragmatic contractility in doxorubicin-treated rats

To explore the diaphragmatic toxicity in doxorubicin (DOX)-treated rats and the related mechanisms, as well as the effects of Shengmai Injection (SMI, ) on the diaphragmatic dysfunction. Thirty Sprague-Dawley male rats were randomly divided into three groups: control, DOX-treated and DOX+SMI treated groups. DOX was given to rats in DOX and DOX+SMI groups in 6 equal doses [2.5 mg/kg, intraperitoneal injection (i.p.)], on alternate days, over a period of 2 weeks for a cumulative dose of 15 mg/kg. SMI was given to DOX+SMI rats in 12 doses (3 mL/kg, i.p.) for a period of 2 weeks before the administration of DOX and 2 weeks during the administration of DOX. The rats in the control group received equal volume of normal saline. Subsequently, the twitch and tetanic characteristics and force-frequency relationships, and the malondialdehyde (MDA) levels and the superoxide dismutase (SOD) activities, as well as the mRNA content and proteins of inducible nitric oxide synthase (iNOS) were determined. The DOX-treated rats had decreased the peak twitch tension (Pt), maximal tetanic tension (P0) and force-frequency relationship as compared with the control rats (P<0.01), while the diaphragm contractility in rats treated with SMI were significantly higher than that in DOX-treated rats (P<0.01). The DOX-treated rats had increased MAD levels and decreased SOD activities (P<0.05), and SMI decreased the MDA levels and increased the SOD activities in DOX-treated rats (P<0.05). Ultrastructure of diaphragm in the DOX-treated rats revealed typical alterations including fracture of diaphragm fibers, and edema and degeneration of mitochondria; these changes were relieved by SMI treatment. The mRNA content and protein of iNOS in DOX-treated rats were remarkably higher than those in control rats (P<0.01), while SMI decreased the mRNA expression level of iNOS in DOX-treated rats (P<0.05). Lipid peroxidation is responsible for DOX-induced diaphragm toxicity. SMI protects diaphragm muscles and their function from DOX impairment, and these beneficial effects may be somehow correlated with the decrease in expression of iNOS and lipid peroxidation.

Efficient Reprogramming of Human Cord Blood CD34+ Cells Into Induced Pluripotent Stem Cells With OCT4 and SOX2 Alone

The reprogramming of cord blood (CB) cells into induced pluripotent stem cells (iPSCs) has potential applications in regenerative medicine by converting CB banks into iPSC banks for allogeneic cell replacement therapy. Therefore, further investigation into novel approaches for efficient reprogramming is necessary. Here, we show that the lentiviral expression of OCT4 together with SOX2 (OS) driven by a strong spleen focus-forming virus (SFFV) promoter in a single vector can convert 2% of CB CD34(+) cells into iPSCs without additional reprogramming factors. Reprogramming efficiency was found to be critically dependent upon expression levels of OS. To generate transgene-free iPSCs, we developed an improved episomal vector with a woodchuck post-transcriptional regulatory element (Wpre) that increases transgene expression by 50%. With this vector, we successfully generated transgene-free iPSCs using OS alone. In conclusion, high-level expression of OS alone is sufficient for efficient reprogramming of CB CD34(+) cells into iPSCs. This report is the first to describe the generation of transgene-free iPSCs with the use of OCT4 and SOX2 alone. These findings have important implications for the clinical applications of iPSCs.

Mitochondrial dysfunction and activation of iNOS are responsible for the palmitate-induced decrease in adiponectin synthesis in 3T3L1 adipocytes

Mitochondrial dysfunction and endoplasmic reticulum (ER) stress are considered the key determinants of insulin resistance. Impaired mitochondrial function in obese animals was shown to induce the ER stress response, resulting in reduced adiponectin synthesis in adipocytes. The expression of inducible nitric oxide synthase (iNOS) is increased in adipose tissues in genetic and dietary models of obesity. In this study, we examined whether activation of iNOS is responsible for palmitate-induced mitochondrial dysfunction, ER stress, and decreased adiponectin synthesis in 3T3L1 adipocytes. As expected, palmitate increased the expression levels of iNOS and ER stress response markers, and decreased mitochondrial contents. Treatment with iNOS inhibitor increased adiponectin synthesis and reversed the palmitate-induced ER stress response. However, the iNOS inhibitor did not affect the palmitate-induced decrease in mitochondrial contents. Chemicals that inhibit mitochondrial function increased iNOS expression and the ER stress response, whereas measures that increase mitochondrial biogenesis (rosiglitazone and adenoviral overexpression of nuclear respiratory factor-1) reversed them. Inhibition of mitochondrial biogenesis prevented the rosiglitazone-induced decrease in iNOS expression and increase in adiponectin synthesis. These results suggest that palmitate-induced mitochondrial dysfunction is the primary event that leads to iNOS induction, ER stress, and decreased adiponectin synthesis in cultured adipocytes.

Ibuprofen-Glutathione Conjugate as Anti-inflammatory and Anti-apoptotic Agent in Rat Brain Infused with β Amyloid (1-40)

Background: To date it is proved that Alzheimer’s disease (AD) is characterized by a multifactorial etiology which comprises mitochondrial dysfunction, energy depletion, inflammation, and oxidative stress associated with glutathione depletion. All these factors are known to be impacted by beta amyloid protein (Aβ), which is responsible for the activation of amyloidogenic cascade. In the present work a rat intracerebroventricular Aβ(1-40) infusion model of early AD was employed to investigate the effects of Ibuprofen-Glutathione (IBU-GSH) conjugate on morphological modifications, Aβ plaque formation, apoptosis, learning, and memory performance. Methods: Water maze test was used to evaluate drug administration effects on spatial reference memory, immunohistochemistry was carried out to determine Aβ 1-40, iNOS and caspase-3 expression; semithin sections and ultrastructural analyses, by means of light and transmission electron microscopy, respectively, were realized to evaluate samples morphology; TUNEL analysis to identify apoptotic cells. Results and Conclusion: Results showed that in the Aβ+IBU-GSH conjugate treated group, long-term memory consolidation at day 12 was improved, while learning appeared slower respect to control. In Aβ-infused rats, a higher number of Aβ plaques, disorganized pyramidal pyknotic cells, apoptotic cells containing fragmented or swollen mitochondria, dilated blood vessels, and a low number of oligodendrocytes along with the high expression of inducible Nitric Oxide Synthase (iNOS) and caspase-3 were put in evidence. The inflammatory state seemed to be reversed by IBU-GSH treatment, as evidenced by a lower number of Aβ plaques and pyknotic cells, and reorganized neurons containing normal mitochondria; despite this, persistent dilated blood vessels, decreased iNOS expression, lower percentage of apoptosis, and weak caspase-3 levels were observed. Thus mitochondria involvement in the AD inflammatory state is here strongly suggested and neuro-protective and anti-apoptotic IBU-GSH conjugate effect could be useful to reduce the AD inflammatory state.

Nitric Oxide is Necessary for Diazoxide Protection Against Ischemic Injury in Skeletal Muscle.

Ischemia reperfusion injury (IR injury) is a common problem in clinical conditions. Researches have frequently revealed that ATP- sensitive potassium (KATP) channels and nitric oxide plays a role in protection against ischemic injury in skeletal muscle. The present study aimed at evaluating the possible link between this two pathways. Sixty-eight male wistar rats, were pretreated with saline, diazoxide (KATP opener; 45 mg/Kg, IP), glibenclamide (KATP inhibitor; 5 mg/Kg), or L-NAME (iNOS inhibitor; 20 mg/Kg, IP) before 3 h ischemia and 2 h reperfusion. Activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the level of malondialdehyde (MDA) and expression of iNOS were measured in muscle tissue. Tissue MDA content was significantly increased by IR (p < 0.001). Diazoxide significantly decreased the IR-induced elevation of tissue MDA level (p < 0.05) and Glibenclamide increased MDA (p < 0.05 vs. IR group). L-NAME inhibited the effect of diazoxide on decreasing MDA (p < 0.01 vs., diazoxide+IR group) and IR decreased the activity of SOD and CAT (p < 0.01), while pretreatment with diazoxide increased activity of SOD and CAT (p < 0.01). Glibenclamide decreased SOD and CAT activity after IR (p < 0.05). L-NAME pretreatment in diazoxide-treated rats abolished the effect of diazoxide on increasing the activity of SOD and CAT (p < 0.05 vs. Diaz+IR). Expression of iNOS was increased by IR (p < 0.01 vs. Sham group). Diazoxide significantly decreased iNOS expression after IR (p < 0.05 vs. IR). L-NAME significantly decreased iNOS expression after IR (p < 0.01) in diazoxide-treated rats (p < 0.01 vs. Diaz+IR). In conclusion, the results of present study suggested a NO dependent protective effect for diazoxide against muscle IR injury.

Nebivolol regulates eNOS and iNOS expressions and alleviates oxidative stress in cerebral ischemia/reperfusion injury in rats

AimsOxidative stress-induced cell damage is reported to contribute to the pathogenesis of cerebral ischemia/reperfusion injury. This study investigated the neuroprotective effect of nebivolol against cerebral ischemia/reperfusion insult in rats. Main methodsThe model adopted was that of surgically-induced forebrain ischemia, performed by means of bilateral common carotid artery occlusion for 1h, followed by reperfusion for 24h. The effects of 5 and 10mg/kg nebivolol, treated for 7days prior to ischemia/reperfusion insult, were investigated by estimating endothelial and inducible nitric oxide synthases (eNOS and iNOS) protein expressions and assessing oxidative stress-related biochemical parameters in the rat forebrain. Also, infarct volume measurement and histopathological study of the forebrain were examined. Key findingsAdministration of nebivolol increased eNOS expression with simultaneous decrease in iNOS expression in a dose dependent manner. Moreover, nebivolol inhibited ischemia/reperfusion-induced depletion of reduced glutathione level and decreased the elevated total nitric oxide end production and malondialdehyde levels, superoxide dismutase and lactate dehydrogenase activities. A notable finding is that catalase activity was not changed in response to either ischemia/reperfusion insult or nebivolol treatment. However, the results confirmed that nebivolol significantly reduced infarct volume and alleviated ischemia/reperfusion-induced histopathological changes. SignificanceThe present study demonstrates the neuroprotective effect of nebivolol against cerebral ischemia/reperfusion insult. Neuroprotection observed with nebivolol may possibly be explained by regulating eNOS and iNOS expressions and by inhibition of oxidative stress-induced injury. Thus, nebivolol may be considered as a potential candidate for treatment in patients who are prone to stroke.

The effects and molecular mechanisms of simvastatin on liver fibrosis in rats with non-alcoholic fatty liver disease

Objective To investigate the effects and molecular mechanism of simvastatin in liver fibrosis model of non-alcoholic fatty liver disease (NAFLD) in vivo and hepatic stellate cell in vitro.Methods Firstly,the rat liver fibrosis model of NAFLD was established by high-fat diet administration and intervened with simvastatin.The expression of endothelial nitric oxide synthase (eNOS),inducible nitric oxide synthase (iNOS) and Collagen Ⅰ at mRNA and protein level were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot.Secondly,quiescent phenotype of LX-2 cell line was induced by promoting adipocyte differentiation medium in vitro,and then the quiescent phenotype of LX-2 cell were treated with transforming growth factor β1(TGF-β1),Nitroso-L-arginine methyl ester (L-NAME) which was NOS inhibitor,simvastatin,TGFβ1 with simvastatin,and L-NAME with simvastatin separately.The changes of eNOS,iNOS,αsmooth muscle actin (α-SMA) and Collagen Ⅰ expressions at mRNA and protein level were determined by RT-PCR and Western blot.Results As modeling time extended,the expressions of eNOS in rat's liver tissue of model group at mRNA and protein level decreased gradually,however the expression of iNOS and Collagen Ⅰ at mRNA and protein level increased gradually,compared with normal control group and the differences were statistically significant (P ＜0.05 and 0.01).By 24weeks,the expressions of eNOS in rat's liver tissue of simvastatin group at mRNA and protein level were increased,the expression of iNOS at mRNA and protein level were decreased and the expression of Collagen Ⅰ at mRNA and protein level were decreased (all P ＜0.05).The expression of eNOS in rat's liver tissue of model group negatively correlated with the expression of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).The expression of iNOS positively correlated with that of Collagen Ⅰ at mRNA and protein level (all P ＜0.01).In LX-2 cell culture,L-NAME inhibited the activation of LX-2,reduced eNOS and iNOS expression and increased α-SMA and Collagen Ⅰ expression,consistent with the role of TGF-β1.Simvastatin could directly increase the eNOS expression both in quiescent and activated LX-2 cells,decrease iNOS expression,maintain quiescent phenotype and inhibit its activation.Conclusions Simvastatin ameliorated the genesis and progression of liver fibrosis by increasing eNOS expression in LX-2 cells and reducing iNOS,α-SMA and Collagen Ⅰ expression. Key words: Simvastatin; Liver cirrhosis; Nitric oxide synthase; Astrocytes; NG-nitroarginine methylester; Disease models, animal

Expression levels of cyclooxygenase-2, tumor necrosis factor-α and inducible NO synthase in placental tissue of normal and preeclamptic pregnancies

Objective: Although preeclampsia (PE) is one of the most important problems affecting pregnant women, etiologic factors in its development are still unclear. We aimed to investigate the expression levels of cyclooxygenase-2 (COX-2), tumor necrosis factor-α (TNF-α) and inducible NO synthase (iNOS) in preeclamptic and healthy control placentas. Patients and methods: Placental tissue samples were obtained after delivery from patients diagnosed with PE and from normal-term pregnants and analyzed for COX-2, TNF-α and iNOS expression by immunohistochemistry. Results: A strong expression of COX-2 was observed in syncytiotrophoblast cells of preeclamptic placentas, which was significantly higher than that of normal placentas (p = 0.005). A mild expression of TNF-α in both normal and preeclamptic syncytiotrophoblasts was seen (p = 0.435). In addition, a strong expression of iNOS in normal syncytiotrophoblasts was found, but the intensity of the iNOS expression was highly reduced in preeclamptic placentas (p = 0.001). No correlation was detected between COX-2, TNF-α and iNOS expression levels. Conclusion: The findings of a decrease of iNOS expression and an increase of COX-2 expression in placenta suggest the existence of functional roles of iNOS and COX-2 in the pathophysiology of PE, probably by contributing to the reduced placental blood flow and increased resistance to flow in the fetomaternal circulation.

Effects of hypoxia inducible factor-1 α siRNA on inducible nitric oxide synthase expression in HaCaT cells

To observe the effect of hypoxia inducible factor -1α (HIF-1α) small interfering RNA (siRNA) on the expression of HIF-1α and inducible nitric oxide synthase (iNOS) in HaCaT cells under hypoxia. HaCaT cells were divided into 4 groups: the normal control group (without any treatment), the hypoxia group (under hypoxia for 24 h), the liposome control group (HaCaT cells transfected with liposome before hypoxia treatment), the RNA interference group (HaCaT cells transfected with siRNA sequences then under hypoxia for 24 h). Real-time PCR and Western blot were utilized to determine HIF-1α and iNOS mRNA and protein expression in HaCaT cells. There was no significant difference of the mRNA expression of HIF-1α between the hypoxia group and the normoxia group (P>0.05), but the protein expressions of HIF-1α was increased in the hypoxic group than that in the normoxia group (P<0.05). Both the mRNA and protein expression of iNOS were increased in hypoxic conditions than that in the normoxia (P<0.05). Decreases were more significant in the mRNA and protein expression of HIF-1α and iNOS in the RNA interference group than that in the liposome control group in HaCaT cells (P<0.05). Hypoxia increased HIF-1α and iNOS expression in HaCaT cells and inhibition of HIF-1α expression decreased iNOS expression.

Comparative effects of recombinant human activated protein C and dexamethasone in experimental septic shock

To compare the effects of recombinant human activated protein C (APC) and glucocorticoids alone and in combination in non-anesthetized resuscitated septic shock induced by cecal ligation and puncture (CLP) on (a) survival, (b) hemodynamics, and (c) vascular reactivity. The effects of treatments on major cellular pathways likely implicated were also studied. Four hours after CLP, rats were continuously infused with either saline (10 ml/kg/h), saline + APC, saline + dexamethasone (Dexa), or saline + APC + Dexa. Eighteen hours after CLP, arterial pressure, cardiac output, nitrite/nitrate ratio, and lactate concentrations were measured. Aortic rings and mesenteric arteries were isolated and mounted in a myograph, after which arterial contractility and endothelium-dependent relaxation were measured in the presence or absence of nitric oxide synthase (NOS) or cyclooxygenase (COX) inhibitors. Protein expression was assessed by Western blotting. Aorta NO and superoxide anion content were measured by electron paramagnetic resonance. All treatments improved hemodynamic parameters and vascular reactivity and decreased lactate and nitrite/nitrate levels. In treated aorta and mesenteric arteries, contractility and endothelial dysfunction were improved. This effect was associated with an increase in the phosphorylated form of protein kinase B as well as an increase in COX vasodilatory pathways and a decrease in iNOS expression suggesting that these pathways are implicated in the vascular effect of the treatments. CLP was associated with a marked increase in aortic NO and superoxide anion content (p < 0.05), which were decreased by APC and Dexa and totally abolished by APC + Dexa (p < 0.01). Survival length was significantly increased by the APC-Dexa combination. Both APC and Dexa improve arterial contractility and endothelial dysfunction resulting from septic shock in rats. Moreover, their combination increased the length of survival. These findings provide important insights into the mechanisms underlying APC- and/or Dexa-induced improvements of arterial dysfunction during septic shock.

Caveolin-2-deficient mice show increased sensitivity to endotoxemia

Caveolin proteins are structural components of caveolae and are involved in the regulation of many biological processes. Recent studies have shown that caveolin-1 modulates inflammatory responses and is important for sepsis development. In the present study, we show that caveolin-1 and caveolin-2 have opposite roles in lipopolysaccharide (LPS)-induced sepsis using caveolin-deficient (Cav-1-/- and Cav-2-/-) mice for each of these proteins. While Cav-1-/- mice displayed delayed mortality following challenge with LPS, Cav-2-/- mice were more sensitive to LPS compared to wild-type (WT). With Cav-2-/- mice, this effect was associated with increased intestinal injury and increased intestinal permeability. This negative outcome was also correlated with enhanced expression of iNOS in epithelial intestinal cells, and enhanced production of nitric oxide (NO). By contrast, Cav-1-/- mice demonstrated a decrease in iNOS expression with decreased NO production, but no alteration in intestinal permeability. The differential expression of iNOS was associated with a significant increase of STAT-1 activation in these mice. Intestinal cells of Cav-2-/- mice showed increased phosphorylation of STAT-1 at tyrosine 701 compared to wild-type. However, Cav-1-/- mice-derived intestinal cells showed decreased levels of phosphorylation of STAT-1 at tyrosine 701. Since caveolin-2 is almost completely absent in Cav-1-/- mice, we conclude that it is not just the absence of caveolin-2 that is responsible for the observed effects, but that the balance between caveolin-1 and caveolin-2 is important for iNOS expression and ultimately for sepsis outcome.

Magnolol Attenuates the Lung Injury in Hypertonic Saline Treatment from Mesenteric Ischemia Reperfusion through Diminishing iNOS

Hypertonic saline (HTS) administration can decrease the inflammation following ischemia reperfusion. Magnolol is a potent antioxidant. The present study investigated whether combined treatment of magnolol and HTS could provide further protection in mesenteric ischemia reperfusion injury. Male C3H/HeOuJ mice were randomly segregated into the following groups: sham-operated (sham), vehicle treatment and mesenteric ischemia reperfusion (MSIR) (vehicle-treated), magnolol treatment and MSIR (magnolol-treated), HTS treatment and MSIR (HTS-treated), as well as co-administration of magnolol plus HTS and MSIR (combined-treated). In MSIR, mice were subjected to mesenteric ischemia for 60 min followed by reperfusion for 30 min. Lung injury was evaluated by lung edema (water ratio) and myeloperoxide (MPO) activity; RNA expression of inducible nitric oxide synthetase (iNOS), TNF-α, and IL-6 were assayed by real time RT-PCR. The formation of peroxynitrite in plasma was assayed by the peroxynitrite-dependent oxidation of dihydrorhodamine 123 (DHR 123) to rhodamine. Compared with those in the sham-treated group, lung edema and MPO activity, expressions of iNOS, TNF-α and IL-6, and plasma peroxynitrite were significantly increased in the vehicle-treated group. Significant attenuations of these parameters were found in the magnolol-treated or HTS-treated animals. Combined treatment of magnolol and HTS further suppressed the lung edema, iNOS, and TNF-α expressions, and plasma peroxynitrite, compared with the results of a single treatment of magnolol or HTS. Compared with single-agent use, co-administration of magnolol and HTS further decreases iNOS expression and plasma peroxynitrite as well as the degree of lung injury from MISR. These results may provide another treatment measure for post-injury immunomodulation.

N‐Acetylcysteine modulates acute lung injury induced by Pseudomonas aeruginosa in rats

1. In critically ill patients, Pseudomonas aeruginosa-induced pneumonia and the lung injury associated with infection are major causes of mortality. The aim of the present study was to evaluate the protective properties of N-acetylcysteine (NAC) in rats infected with P. aeruginosa and the role of nitric oxide synthases (NOS) protein in this process. 2. Pneumonia was induced in rats by infecting them with P. aeruginosa intratracheally. One group of rats was treated with NAC (150 mg/kg per day, i.p., for 7 days). An untreated group served as the control. Samples were collected both before (0 h) and after infection (24 h). Bacterial loads in lung tissue, the lung wet : dry (W/D) ratio and pulmonary vascular permeability were assessed. Total cell and polymorphonuclear leucocyte cell counts in bronchoalveolar lavage fluid were determined. The expression of inducible (i) NOS and endothelial (e) NOS protein was analysed and correlated with indices of lung injury using Pearson's correlation analysis. 3. Bacterial load, lung injury indices and NOS expression increased after infection. Pretreatment with NAC mitigated lung injury although it did not significantly change bacterial loads. Furthermore, NAC treatment increased eNOS protein expression, but decreased iNOS expression, in lung tissues after infection. The expression of iNOS protein was positively correlated with indices of lung injury, whereas there was a negative correlation between eNOS expression and lung injury indices. 4. N-Acetylcysteine modulated P. aeruginosa-induced lung injury in rats. The results suggest that this effect maybe due to regulation of iNOS and eNOS protein expression by NAC.

Synergistic anti‐inflammatory effects of combination of Nobiletin and Sulforaphane in lipopolysaccharide‐stimulated RAW 264.7 cells

Inflammation plays an important role in carcinogenesis of various organ sites. Increasing studies have been focused on identification of dietary components that can exert anti-cancer effects by inhibiting inflammation. Nobiletin (NBN) is a flavonoid found in citrus peels, and sulforaphane (SFN) is an organosulfur compound found in cruciferous vegetables. Herein, we investigated the anti-inflammatory effects of SFN and NBN in combination. The results showed that SFN/NBN co-treatments produced much stronger inhibitory effects on the produce of lipopolysaccharide (LPS)-induced NO compared to their individual treatments. These enhanced inhibitory effects were synergistic based on the isobologram analysis. Western blotting analysis showed SFN/NBN co-treatments synergistically decreased iNOS expression level. Real-time PCR analysis indicated low doses of SFN and NBN in combination could synergistically suppress LPS-induced increase in iNOS and COX-2 mRNA levels. Furthermore, the SFN/NBN co-treatments also synergistically induced mRNA expression of HO-1 and NQO-1, which may contributed to their anti-inflammatory effects. Overall our results demonstrated that SFN and NBN in combination produced synergistic effects in inhibiting LPS-induced inflammation reactions in RAW 264.7 cells. This study was supported in part by NIH (CA139174), CEAR pilot grant, and USDA Special Grant on Bioactive Foods.

Programmed death ligand 2 regulates arginase induction and modifies Trypanosoma cruzi survival in macrophages during murine experimental infection

The programmed death ligands 1 (PD-L1) and 2 (PD-L2) that bind to programmed death 1 (PD-1) have been involved in peripheral tolerance and in the immune escape mechanisms during chronic viral infections and cancer. However, there are no reports about the role of these molecules during Trypanosoma cruzi infection. We have studied the role of PD-L1 and PD-L2 in T. cruzi infection and their importance in arginase/inducible nitric oxide synthase (iNOS) balance in the immunomodulatory properties of macrophages (Mφ). In this work, we have demonstrated that expression of the PD-1/PD-L pathway is modified during T. cruzi infection on Mφs obtained from peritoneal cavity. The Mφs from T. cruzi-infected mice suppressed T-cell proliferation and this was restored when anti-PD-1 and anti-PD-L1 antibodies were added. Nevertheless, anti-PD-L2 antibody treatment did not re-establish T-cell proliferation. PD-L2 blockade on peritoneal cells from infected mice showed an increase in arginase expression and activity and a decrease in iNOS expression and in nitric oxide (NO) production. Additionally, interleukin-10 production increased whereas interferon-γ production was reduced. As a result, this microenvironment enhanced parasite proliferation. In contrast, PD-1 and PD-L1 blockage increased iNOS expression and NO production on peritoneal Mφs from T. cruzi-infected mice. Besides, PD-L2 knockout infected mice showed an increased in parasitaemia as well as in arginase activity, and a reduction in NO production. Taken together, our results demonstrate that PD-L2 is involved in the arginase/iNOS balance during T. cruzi infection having a protective role in the immune response against the parasite.

The role of tumour-derived iNOS in tumour progression and angiogenesis

Background:Progressive tumour growth is dependent on the development of a functional tumour vasculature and highly regulated by growth factors and cytokines. Nitric oxide (NO) is a free radical, produced both by tumour and host cells, and functions as a signalling molecule downstream of several angiogenic factors. Both pro- and antitumourigenic properties have been attributed to NO.Methods:The expression of the inducible isoform of NO synthase (iNOS) was knocked down in the C6 glioma cell line using constitutive expression of antisense RNA, and the effect of tumour-derived NO on tumour progression and angiogenesis was investigated.Results:Tumours in which iNOS expression was decreased displayed significantly reduced growth rates compared with tumours derived from parental C6 cells. Quantitative non-invasive magnetic resonance imaging and fluorescence microscopy of tumour uptake of Hoechst 33342, and haematoxylin and eosin staining, revealed significantly impaired vascular development and function in antisense iNOS tumours compared with control in vivo, primarily associated with the more necrotic tumour core. Decreased iNOS expression had no effect on tumour VEGF expression.Conclusion:Nitric oxide derived from tumour iNOS is an important modulator of tumour progression and angiogenesis in C6 gliomas and further supports the therapeutic strategy of inhibiting iNOS for the treatment of cancer.

Regulation of Inducible Nitric Oxide Synthase Expression in Bovine Granulosa Cells and Its Involvement in Follicular Dominance.

Understanding the paracrine events that regulate fertility in the cow is necessary not only because of the agricultural importance of this species, but also its potential use as a model for humans. Nitric oxide (NO), a free-radical gas, has been implicated in follicular growth in rodents, but the cow is an intriguing enigma: NO is produced by bovine granulosa cells and is regulated by FSH, but the enzymes responsible are reported to be absent. Three genes encode NO synthase proteins, one of which - iNOS - is widely considered to be regulated by extracellular ligands. We hypothesize that granulosa cells express the gene encoding iNOS, and that the expression of this gene is under hormonal regulation. The objectives of the present study were to determine the abundance of iNOS mRNA in growing and non-growing follicles in cattle, to determine the regulation of iNOS expression by FSH and growth factors, and to determine the action of NOS in bovine granulosa cells. The two largest follicles on days 1 to 5 of the first follicular wave of the cycle were collected from each pair of ovaries from six cows. Messenger RNA encoding iNOS was detected in granulosa and theca cells of bovine follicles by real-time PCR. The granulosa cells of dominant follicles contained higher levels of mRNA for iNOS compared with subordinate follicles (P<0.01). The regulation of iNOS expression was evaluated in a serum-free granulosa cell culture system. FSH (P<0.05) and IGF1 (P<0.01) stimulated E2 secretion iNOS mRNA abundance in a dose-dependent manner. Moreover, both FGF2 and EGF (P<0.01) decreased iNOS expression and E2 secretion in FSH and in IGF1-treated cells. Owing to the parallel changes in estradiol secretion and iNOS mRNA, we assessed the role of estradiol in the regulation of iNOS mRNA; replacement of estrogen precursor with a non-aromatizable androgen (DHT) or the addition of an estrogen receptor antagonist inhibited FSH-stimulated iNOS mRNA abundance, and addition of E2 alone stimulated NO production in vitro. To assess the role of endogenous iNOS, cells were treated with an iNOS-selective inhibitor, which resulted in increased FasL mRNA levels (P<0.01), induction of caspase-3 activation (P<0.01) and an increase in the incidence of cell death (P<0.05). In conclusion, bovine graulosa cells express iNOS, and increased iNOS mRNA levels are associated with growth of the dominant follicle. Gonadotropic factors stimulate iNOS mRNA levels through increased E2 secretion, and physiological levels of NOS activity may contribute to growth and survival of granulosa cells. Supported by NSERC Canada and CNPq Brazil. (poster)

Abstract 4506: Elevated expression of IL-1 induces iNOS/NO production and inhibits IL-1Ra synthesis leading to progression of metastatic melanoma

Abstract Aberrant expression of proinflammatory IL-1 α and β and the overproduction of nitric oxide (NO) have been shown to promote the progression of some tumors. In previous studies, we had confirmed that inducible nitric oxide synthase (iNOS) was constitutively expressed in most advanced melanomas and highly correlated with poor patient survival. In order to determine whether the imbalance of IL-1 and its natural competitor IL-1 receptor antagonist (IL-1Ra) contributes to the upregulation of iNOS in metastatic melanoma, we initiated an investigation of the expression profile of IL-1 system genes in melanoma and the linkage between IL-1 and iNOS/NO signaling pathways. Immunohistochemistry staining of 38 biopsy samples from metastatic melanoma patients showed that 33 samples highly expressed IL-1α and/or IL-1β. Only 5 samples were negative for both IL-1α and IL-1β expression. RT-PCR confirmed that melanoma cell lines A375, WM793, WM35, and SB2 constitutively expressed high levels of IL-1, IL-1 receptor I (IL-1RI), and iNOS. However, in the same cell lines, there was no detectable IL-1Ra mRNA, which is known to maintain IL-1 balance in normal skin tissues. Our study suggests that the imbalance of IL-1 and IL-Ra is widespread in metastatic melanoma. The engagement of IL-1 and IL-1RI can induce several kinase cascades leading to the activation of SAPK/JNK and NF-κB, and it is well known that transcription of iNOS can be regulated by NF-κB. In our studies, siRNA knockdown of either IL-1α or MyD88 (an important adaptor for IL-1RI signal pathway) in WM375 and A375 led to a dramatic decrease in the phoshorylation of SAPK/JNK and IκB. Furthermore, an 80% decrease of iNOS expression in WM793 was observed after the siRNA knockdown of MyD88. We confirmed that upregulated IL-1 signal pathway can activate NF-κB and the iNOS transcription. Treatment with 200 μM 7-Nitroindazole, a NO synthase inhibitor, not only reduced NO production, but also induced over 50% inhibition of cell growth and a marked increase in IL-1Ra mRNA in WM793 and A375. This indicates that a high level of NO can inhibit the IL-1Ra expression in melanoma cells. Based on our data, we proposed an inflammatory dysfunction model to explain the development of melanoma. It proposes that the constitutive expression of IL-1 can inhibit IL-1Ra expression mediated by the upregulation of iNOS/NO production in melanoma. The constant inhibition of IL-1Ra expression by NO results in a cycle of kinase transduction cascades, which drives NF-κB and its downstream inflammatory genes (including iNOS) constitutively expressed in melanoma. Our studies cast new insight into the mechanism of the pathology of metastatic melanoma. Selective inhibition of iNOS and increasing IL-1Ra levels are promising therapeutic strategies to suppress the aberrant IL-1 inflammation pathway in melanoma. Supported by MDACC SPORE in Melanoma CA093459. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4506.